CZ309934B6 - A smart spherical nanotherapeutics with a broad-spectrum antiviral effect and a method of its production - Google Patents

Abstract

Hollow spherical nanoparticles with macro-openings with an outside diameter 200 and 4000 nm, particle macro-opening diameter ranging from 150 to 900 nm, 0 to 100 macro-diffusion openings with a dimeter ranging from 5 to 149 nm, diffusion channels with an effective area of 0.4 to 9 nm2 of each diffusion channel, with a scaffold formed by a covalently bound polymer from three-dimensionally cross-linked saccharide units forming 1 to 12 isocentric polysaccharide layers, with the method, degree as well as a type of cross-linking controlling the speed of biodegradation (metabolism) of nanoparticles in the organisms and thus forming the so-called temporal nanolock and with the inside nanoparticle wall being covalently modified by viricides (virucides) or, in general, with the substances with an antiviral or virus-inactivating effect, is prepared by polycondensing the saccharide units with cross-linkers. By binding the viricides on the inner wall of the nanoparticle scaffold a viral-trap type antiviral nanotherapeutics is created.

Description

Intelligent spherical nanotherapeutic agent with broad-spectrum antiviral effect and its production method

Field of technology

The invention relates to spherical hollow nanoparticles (Fig. 1) with an outer diameter of between 200 and 4000 nm and their production method. Nanoparticles contain two or more macropores II. with a diameter of 150 to 900 nm, 0 to 100 macrodiffusion holes III. with a diameter of 5 to 149 nm and diffusion openings of skeleton IV. with an effective area of 0.4 to 9 nm ² . The skeleton of nanoparticles is formed by a covalently bound polymer of three-dimensionally cross-linked saccharide units V. forming 1 to 12 isocentric layers of polysaccharide VI. and the structure of saccharide units, the method, degree, and type of cross-linking control the rate of biodegradation (metabolization) of nanoparticles in the organism, forming the so-called time nanolock VII. The inner wall of the nanoparticles is covalently modified with viricides (virucides) or generally with substances with an antiviral effect through linkers or spacers VIII.

Current state of the art

Viruses are extremely serious intracellular pathogens responsible for a remarkable array of diseases with usually a rather poor prognosis. Their specific reproductive (life) cycle and extraordinary variability make them undoubtedly the biggest opponent of non-invasive medicine in recent decades, because the question of universal therapy for viral diseases has not yet been satisfactorily resolved. Existing approaches use either immunization of the organism (vaccination) or so-called antiviral drugs, antiviral chemotherapeutic agents. Vaccination, which is considered to be currently the most effective and widespread method of combating viral diseases, is not always or not at all effective for a number of viral diseases and, in addition, is burdened by a significant occurrence of very serious side effects that can lead to the death of the patient (Tyring S.K.: Chapter 4. Vaccines and Immunotherapies in Antiviral Agents, Vaccines and Immunotherapies CRC Press (2005)). In addition, vaccination is linked to a specific pathogen strain, and in the case of mutation of the pathogen, the above-mentioned shortcomings can be multiplied.

The development of antiviral drugs is fundamentally difficult (Clercq E.D.: Highlights in antiviral drug research: antivirals at the horizon Medicinal Research Reviews (2012), DOI: 10.1002/med.21256; Antivirals: past, present and future. Biochemical Pharmacology 85(6), 727 (2013); “A

Cutting-Edge View on the Current State of Antiviral Drug Development ” Medicinal Research Reviews (2013), DOI: 10.1002/med.21281). A virus is an intracellular pathogen, i.e. any intervention of the antiviral drug in the life cycle of the virus inside the affected cell can lead to an intervention in the vital functions of the affected cell with subsequent fatal consequences. In such a case, the antiviral behaves cytotoxicly, and cytotoxicity is the principle common feature of all non-targeted antivirals. Current approaches to antiviral drug development are based on the elementaryization of the virus's reproductive cycle and the knowledge that every virus needs a host cell to synthesize vital proteins. In each phase of the reproductive cycle, processes can be identified that can be slowed down or blocked (virostatics) and thereby slow down or block the reproductive cycle of the virus. According to the effect on the phase of the reproductive cycle of the virus, it is possible to divide virostatics into ten main classes (Field H.J., Vere Hodge R.A.: Antiviral Agents, in Desk Encyclopedia of General Virology, Academic Press (2010)), while intracellularly active blockers and inhibitors of the synthesis of important proteins, which form a decisive group, with the mutation of the virus, their effect is reduced or completely lost.

In addition to the development of vaccines and virostatics, alternative approaches are also being developed, among which strategies focused on the so-called inactivation of the virus in the extracellular environment are undisputedly dominant. The motto of these approaches is to eliminate the virus before it can reach the cell. The approaches have been notoriously used in vitro for several decades in decontamination and disinfection (viricides, virucides) and show the extreme dependence of the success of the viricidal effect of the inactivation agent on the type of virus inactivated. For example, non-encapsulated viruses of the ssRNA and ssDNA class are resistant to commonly used viricides

- 1 CZ 309934 B6 completely or critically resistant. The situation has been clearly mapped repeatedly, as decontamination and disinfection are of extraordinary importance in the entire pharmaceutical and biopharmaceutical industry (Sofer G.: "Virus Inactivation in the 1990s - and into the 21st Century", "Part 1: Skin, Bone, and Cells", BioPharm 18 (6/2002); Part 3a: "Plasma and Plasma Products (Heat and Solvent/Detergent Treatments)" BioPharm 28 (9/2002); Part 4: ..Culture Media, Biotechnology Products, and Vaccines" BioPharm International 50 (1/2003); Part 5: "Disinfection" BioPharm International 44 (2/2003); Part 6: ..Inactivation Methods Grouped by Virus" BioPharm International 42 (4/2003)). In vivo use, however, is hindered by the cytotoxicity of viricides resulting from antivirally effective doses in the body (very low so-called Selectivity action index, SI).

A controversial area of development of virus inactivation agents is the group of antimicrobial peptides widely studied in the last two decades (Jenssen H, e.a.: Peptide Antimicrobial Agents Clinical Microbiology Reviews 19(3), 491 (2006); Paiva A.D., e.a.: Antimicrobial Peptides Produced by Microorganisms in Progress in Inflammation Research (Hiemstra P.S., Zaat S.A.J.; eds.), p. 53, Springer (2013); Espitia P.J.P, e.a.: Bioactive Peptides: Synthesis, Properties, and Applications in the Packaging and Preservation of Food Comprehensive Reviews in Food Science and Food Safety 187 (2012); Giuliani A., e.a.: Antimicrobial peptides: an overview of a promising class of therapeutics Central European Journal of Biology 2(1), 1 (2007)). Despite the original highly unrealistic expectations, even relatively short-term therapeutic use of this group of drugs can lead to the emergence of resistance in dangerous bacterial pathogens (Anaya-López J.L., e.a.: Bacterial resistance to cationic antimicrobial peptides Critical Reviews in Microbiology 39(2), 180 (2013) ; Hale J.D.F.:

Bacterial Resistance to Antimicrobial Peptides in Bacterialpathogenesis (Locht C., Simonet M.; eds.), p. 305, Horizon Scientific Press (2012)) and significantly endanger the native immune system of the human organism (Habets M.G.J.L., e.a.: Therapeutic antimicrobial peptides may compromise natural immunity Biol. Lett. 8(3), 416 (2012)), which can have an overwhelming impact especially in the case of modulated human defensins. The seriousness of such findings is underlined by the fact that the emergence of resistance to bacterial pathogens would occur as a result of the therapeutic use of the peptide as a non-specific antiviral agent. Moreover, synthetic antimicrobial peptides with longer chains can cause severe and difficult to predict immune responses, and immunogenicity (and eventual antigenicity) must be limited by chemical modification (Van Regenmortel M.H.V.: Antigenicity and Immunogenicity of Synthetic Peptides Biologicals 29, 209-213 (2001); Lien S., et al.: Therapeutic peptides TRENDS in Biotechnology 21(12), 556 (2003)). However, despite these shortcomings, antimicrobial peptides (antiviral peptides or peptide antivirals) represent a very attractive area of research (e.g. WO 2013036622, EP 17105182, EP 2073829, US 8129499, US 2013364667, etc.), and therefore it is not surprising that other modifications are presented, which can contribute to higher therapeutic efficacy (Brogden N.K., e.a.: Will new generations of modified antimicrobial peptides improve their potential as pharmaceuticals? International Journal of Antimicrobial Agents 38, 217 (2011)).

However, other approaches have been proposed to inactivate the virus. In addition to chemical modifiers of the capsid or virion proteins (F. Kaesermann et.a.: Virus inactivation and protein modifications by ethyleneimines Antiviral Research 52, 33-41 (2001); Broo K. et.a.: Viral capsid mobility: A dynamic conduit for inactivation PNAS 98(5) , 2274-2277 (2001)), which cannot be applied in view of extreme cytotoxicity in vivo, the approaches of supramolecular chemistry and biotechnology are worth attention. Komatsu patented (JP 2009263274) and later described (Komatsu T.: Protein-based nanotubes for biomedical applications Nanoscale 4, 1910 (2012); Komatsu T. e.a.: Virus Trap in Human Serum Albumin Nanotube J. Am. Chem. Soc. 133, 3246-3248 (2011)) the concept of nanotubes made of blood albumin, which can be modified on the inner wall with specific antigens and thus achieve the capture of the Hepatitis B virus inside the tubes. Another virus trap principle was proposed by Livneh (WO 2013030831). It uses a polystyrene basket as a mechanical sieve, in which a cell dummy is placed. Diwan (US 2010008938) described the self-assembly of structures from functionalized polymers as antivirally effective preparations, and his derived concept Nanoviricide™ (NanoViricides, Inc., USA) should also be mentioned, which is widely

- 2 CZ 309934 B6 publicized. Other nanotechnological approaches focus only on the use of nanoparticles to encapsulate antivirals (Lembo D. e.a.: Nanoparticulate delivery systems for antiviral drugs Antiviral Chemistry et Chemotherapy 21, 53 (2010); Jain K.K.: The Handbook of Nanomedicine, Chapter 6 Nanodevices for Medicine and Surgery , Humana Press 2008), e.g. from the group of self-assembly systems as protectors of encapsulated peptides (Zhang J., e.a.: Antiviral peptide nanocomplexes as a potential therapeutic modality for HIV/HCV co-infection Biomaterials 34(15), 3846 (2013)).

Immobilization of viricides on polymer supports is one of the currently studied solutions for in vitro applications. Similar to the case of bacterial pathogens (Siedenbiedel F. e.a.: Antimicrobial Polymers in Solution and on Surfaces: Overview and Functional Principles (Review)Polymers 4, 46 (2012)) it turns out that the effectiveness of these "two-dimensional viricides" is up to several orders of magnitude higher than in the case of free viricides. It is probably related to the high activity (active concentration) of the viricide at the surface of the polymer. Of the important viricides, free highly linear (Spoden G.A., e.a.: "Polyethylenimine Is a Strong Inhibitor of Human Papillomavirus and Cytomegalovirus Infection" Antimicrob. Agents Chemother. 56(1), 75 (2012)) and branched polyethylenimines functionalized on lipophilic quaternary ammonium groups (Larson A.M., e.a.: Decreasing Herpes Simplex Viral Infectivity in Solution by Surface-Immobilized and Suspended N,N-Dodecyl,methyl-polyethylenimine Pharm. Res. 30, 25 (2013); Hydrophobic Polycationic Coatings Disinfect Poliovirus and Rotavirus Solutions." Biotechnology and Bioengineering 108 (3), 720 (2011); Hydrophobic Polycationic Coatings Disinfect Poliovirus and Rotavirus Solutions Biotechnology and Bioengineering, 108(3), 720 (2011); Hsu, B.B. et al. '"Mechanism of Inactivation of Influenza Viruses by Immobilized Hydrophobic Polycations." Proceedings of the National Academy of Sciences 108 (1), 61-66 (2011); Hsu B.B.: Investigation of microbicidal activity of surface-immobilized hydrophobic polycations, MIT, Dissertation 2011; Klibanov A.M.: "Permanently microbicidal materials coatings" J. Mater. Chem. 17, 2479 (2007); Haldar J. e.a.: “Polymeric coatings that inactivate both influenza virus and pathogenic bacteria” Proc. Natl. Acad. Sci. US 103, 17667 (2006); Preparation, application, and testing of permanently antibacterial and antiviral coatings” Nature Protocols 2, 2412 (2007); Hydrophobic polycationic coatings inactivate wildtype and zanamivir-and/or oseltamivir-resistant human and avian influenza viruses ” Biotechnol. Lett. 30, 475 (2008)) and quaternary ammonium salts (Park D., e.a.: Antiviral and Antibacterial Polyurethanes of Various Modalities Appl. Biochem. Biotechnol. 169, 1134 (2012); Tuladhar E. e.a.: Different Virucidal Activities of Hyperbranched Quaternary Ammonium Coatings on Poliovirus and Influenza Virus Appl. Environ. Microbiol. 78(7), 2456 (2012); Tsao I-F. e.a.: "Removal and Inactivation of Viruses by a Surface-Bonded Quaternary Ammonium Chloride", Chapter 13 in "In Downstream Processing and Bioseparation" ACS Symposium Series p. 250 (1990); Interaction of Infectious Viral Particles with a Quaternary Ammonium Chloride (QAC) Surface Biotechnology and Bioengineering 34, 639 (1989)).

The functions of the above-described therapeutic agents for viral diseases are burdened by a number of shortcomings, among which the dependence of the antiviral effect on the type of pathogen (virus) and, in many cases, the strain of the pathogen dominates. In addition, attempts at antivirals built on a nanomaterial matrix are associated with a priori limits that prevent in vivo therapeutic use of the concept. These are mainly toxic by-products of the metabolism of supramolecular particles, or limited biodegradability of supramolecular particles (both: WO 2013030831), or very limited stability of the supramolecular matrix in vivo (JP 2009263274), or limited applicability of antivirals in the vascular or lymphatic system resulting from the possibility of clogging capillaries as due to the inappropriate shape of supramolecular particles for laminar flow in the organism (9 micron length nanotubes: JP 2009263274). From a technological point of view, there are also very significant limitations of the proposed technology for the production of supramolecular matrix in higher batches (JP 2009263274). A critical point of the Nanoviricide™ concept (NanoViricides, Inc., USA) is the natural possibility of interaction of the antiviral drug with other cells that express the relevant antigens, as selectivity occurs exclusively through receptor interaction. In addition, the Nanoviricide™ concept exhibits all the shortcomings of self-assembly systems (see e.g.: Abdelwahed W,

- 3 CZ 309934 B6

e.a.: Advanced Drug Delivery Reviews 58, 1688 (2006)), which fundamentally limit the possibilities of long-term storage in particular.

It is surprising how little attention the described supramolecular (nanotechnological) solutions aimed at therapeutic practice pay to the attributes of nanotoxicology in vivo, although this is a critical parameter for the success of the whole concept. The mentioned solutions are aimed at intravenous applications, where the effectiveness is determined by the time of recirculation in the vascular system (BertrandN. e.a.: The journey of a drug-carrier in the body: An anatomo-physiological perspective Journal of Controlled Release 161, 152 (2012)), because the effect against the virus is possible only in the event of an effective collision, and the probability of such a collision increases with time. The recirculation time is limited by the mechanical parameters of the nanoparticle (shape, size, flexibility, etc.) and the physicochemical parameters of the nanoparticle surface (surface charge, type and 3D geometry of surface functional groups, etc.), because during recirculation in the vascular system, the organism tries one of available ways to get rid of xenobiotics. The solution described by Livneh (WO 2013030831) is based on a lipophilic polystyrene basket with a diameter of hundreds to thousands of nm with rounded edges, which is the ideal shape, size and surface composition for the phagocytosis process induced by the high affinity of the skeleton to opsonins (Dobrovolskaia M.A. e.a.: Immunological properties of engineered nanomaterials Nature Nanotechnology 2, 469 (2007); Chou L.Y.T. e.a.: Strategies for the intracellular delivery of nanoparticles Chem. Soc. Rev. 40, 233 (2011); Daum N. e.a.: Novel approaches for drug delivery systems in nanomedicine: effects of particle design and shape” Nanomedicine and Nanobiotechnology 4, 52 (2012)). In the description of the solution, Livneh states that the skeleton of the polystyrene basket is modified with PEG to suppress immunogenesis ("immuno-isolation"), but the method of such a solution is not described in the patent solution examples and Livneh only refers to the solution described by Byun (Surface-grafted polystyrene beads with comb-like poly(ethylene glycol) chains: preparation and biological application. Macromol Biosci. 4(5), 512 (2004)), but designed for mechanically very resistant spherical polystyrene particles. Moreover, it can be considered verified that the modification of the PEG nanoparticle surface is not completely immunologically neutral and can cause serious pseudoallergenic reactions (Chahan-Khan A., e.a.: Complement activation following first exposure to pegylated liposomal doxorubicin (Doxil(R)): possible role in hypersensitivity reactions Ann. Oncol. 14, 1430 (2003)). Similarly, the Nanoviricide™ system, which uses self-assembly polymer systems (US 2010008938) functionalized with antigens, is naturally burdened by immunogenicity associated with the therapeutically undesirable so-called ABC effect (Dobrovolskaia M.A. e.a.: Immunological properties of engineered nanomaterials Nature Nanotechnology 2, 469 (2007) ) and from the point of view of eventual phagocytosis, surface functional groups carrying a charge are also very problematic.

If the supramolecular matrix (nanoparticle) is not smoothly metabolizable, there is a risk of serious secondary effects of the application (nanotoxicological effects), if, on the other hand, it is unstable, repeated doses are needed to reach an effective therapeutic level, while the effectiveness of the doses can decrease significantly over time (ABC effect).

The essence of the invention

The invention relates to spherical hollow nanoparticles with two or more macroholes (Fig. 2), where the outer diameter i of the nanoparticle is between 200 and 4000 nm, where the diameter of the macrohole ii of the nanoparticle is in the range of 150 to 900 nm, where the skeleton of the nanoparticle contains 0 to 100 macrodiffuse holes iii with a diameter in the range of 5 to 149 nm, where the skeleton of the nanoparticle contains diffusion channels iv with an effective area of 0.4 to 9 nm ² of each of the diffusion channels,

- 4 CZ 309934 B6 where the skeleton of the nanoparticle is formed by a covalently bound polymer of three-dimensionally cross-linked saccharide units v forming 1 to 12 isocentric layers of polysaccharide vi, where the method, degree and type of cross-linking v control the rate of biodegradation (metabolism) of the nanoparticles in the organism and thus form the so-called time nanolock vii, where the inner wall of the nanoparticles is covalently modified by viricides (virucides) or, in general, by substances with an antiviral or virus-inactivating effect, and the binding of these compounds to the inner wall of the nanoparticle skeleton takes place via linkers or spacers, or the inner wall of the nanoparticle itself has these effects viii.

The skeleton of the nanoparticle v is formed by a covalently bound three-dimensionally cross-linked monodisperse polymer of the structure I (Fig. 3), where the index nn is 0 to 1000, where the index mm is 0 to (nn-1), where Αξ is the structural unit of the polymer (ξ is the index nn or mm), where any branch Α _ψ ( _ψ is the index of a given branch between two Aξ) can be terminated independently of other branches, or it can also continue without branching, where each structural unit Aξ is formed independently of any other structural unit Aξ by structure II ( Fig. 4), where ii to is are independently 0 to 100,000, where the symbol "*" denotes one or two or three bonds to any one or any two or three other structural units B, _P ( _φ is an index from the range 1 to 6), any structural unit Αξ, and each structural unit Βφ is formed by the skeleton of a saccharide or modified saccharide of formula III (Fig. 5), where w is 0 or 1, where one of the substituents R1 and R2 is H, where the other of the substituents R1 and R2 is either a binding site "*" for binding to another structural unit Bp' ( _φ · is an index from the range 1 to 6), or is formed by a fragment of the general formula IV (Fig. 6), or is formed by a fragment of the general formula V (Fig. 7), where the symbol "*" in both cases indicates a bond to the skeleton of the Bp unit, where g is 0 or 1, where Z is the crosslinking unit of the formula VI (Fig. 8) , in which k1 through k6 are independently 0 or 1 or 2 or 3 or 4 or 5 or 6, in which k7 is 0 or 1,

- 5 CZ 309934 B6 in which ks is 0 or 1, in which one of the substituents X ^s and Υ ^δ (where ^δ is an index from the range 1 to 6) is H and the other is formed either by a fragment of general formula IV or general formula V, where the symbol "*" in both cases indicates a bond to the backbone of the cross-linking unit of formula VI, in which Qi is carbonyl or methylene, in which Q2 is carbonyl or methylene, or is formed by a fragment of the general formula VII (Fig. 9), where j is 0 or 1, where k is 0 or 1, where the symbol G stands for either O or NH, where Linker is a structural fragment of a linker or spacer, where Antivir is a structural fragment of a compound with a viricidal (virucidal) or antiviral or virus-inactivating effect, where the symbol "* " means binding to the skeleton of the unit B, _P , where one of the substituents R3 and R4 is H, where the second of the substituents R3 and R4 is either a binding site "*" for binding to another structural unit Β _φ ·, or is formed by a fragment of the general formula IV or is formed by a fragment of the general formula V or is formed by a fragment of the general formula VII, where one of the substituents R5 and R6 is H, where the other of the substituents R5 and R6 is either a binding site "*" for binding to another structural unit Bp', or is formed by a fragment of the general formula IV or is formed by a fragment of the general formula V or is formed by a fragment of the general formula VII, where one of the substituents R7 and R8 is H, where the other of the substituents R7 and R8 is either a binding site "*" for binding to another structural unit Bp', or is formed by a fragment of the general formula IV or is formed by a fragment of the general formula V or is formed by a fragment of the general formula VII, where one of the substituents R9 and R10 is H, where the other of the substituents R9 and R10 is either a binding site "*" for binding to another structural unit Bp', or is formed by a fragment of the general formula IV or is formed by a fragment of the general formula V or is formed by a fragment of the general formula VII, "Linker" in the formula VII is formed by oligomers or polymers of the general formula VIII (Fig. 10) type of polyethylene oxide (PEG), polyglycolate (PGA), polylactate (PLA), polyamide structures and their combinations, where n1 is 0 to 3, where n2 is 0 to 500, where n3 is 0 to 40, where n4 is 0 to 500,

- 6 CZ 309934 B6 where n5 is 0 to 40, where n6 is 0 to 500, where ηg is 0 to 3, where ns is 0 to 5, where ng is 0 or 1, where Mi is either carbonyl (CO) or methylene (CH2) or ethylidene (CH3CH), where M2 forms a fragment from the group CO, CH2, CH3CH, NH or O, where M3 forms a fragment from the group CO, CH2, CH3CH, NH or O, where M4 forms a fragment from the group CO, CH2, CH3CH , NH or O, where M5 is a fragment from the group CO, CH2, CH3CH, NH or O, where M6 is either O or NH, where M7 is a fragment from the group CO, CH2, CH3CH, NH or O, where Ms is either CO or CH2 or CH3CH.

"Antiviral" in formula VII is an antiviral fragment of a compound with a viricidal (virucidal) or antiviral or virus-inactivating effect from the group:

a) linear or branched polyethyleneimines with an Mr of less than 25 kDa or their N-alkylated or N-polyalkylated derivatives or quaternary ammonium salts derived by N-heteropolyalkylation with aliphatic and alicyclic C1 to C18 alkyls, or

b) quaternary ammonium salts with aliphatic and alicyclic C1 to C1s alkyls, or

c) cationic and anionic antimicrobial peptides of the α-helix, β-sheet, cyclic β-sheet, β-loop, or free configuration, and their combination.

Spherical hollow nanoparticles with macroholes of the above-mentioned macroscopic arrangement and skeleton structure I are prepared in one of three possible design variants, each variant consisting of several successive stages.

In the first stage of the first variant of embodiment IX (Fig. 11), the surfactant provided with a hydrophilic sulfo group is treated with an alkane in a polar solvent. The sulfo group is in the form of a free acid or an alkali metal salt. The interaction of the steric blocker creates a macroscopic structure in which the steric blocker is partially embedded in the sphere skeleton formed by micelles from alkane with surface sulfo groups. Phase one can be done by first acting on the steric blocker with an alkane and then forming micelles by the action of a surfactant in a polar solvent, or by adding a steric blocker to the formed micelles from the alkane and a surfactant in a polar solvent. In a preferred embodiment, a mixture of alkane and surfactant is first treated with a solvent and then a steric blocker is added.

The surfactant is an aliphatic, alicyclic or aliphatic chain-substituted aromatic sulfonic acid or its salt with an alkali metal cation of a chain length corresponding to C10 to C45 or a mixture of these surfactants. In a preferred embodiment, the surfactant or their mixture has an HLB index (Li. C., e.a.: Colloids and Surfaces A: Physicochem. Eng. Aspects 356, 71 (2010)) in the range of 9.3 to 11.9 and is in the reaction mixture present in a concentration of 2 to 15 wt.%

An alkane is an aliphatic or alicyclic hydrocarbon with a length corresponding to C16 to C60 or a mixture of these hydrocarbons, preferably a technical paraffin with a transition temperature parameter of

- 7 CZ 309934 B6 range of 35 °C to 80 °C and a melting point in the range of 35 °C to 80 °C. The alkane concentration in the mixture is chosen in the range of 5 to 40% by weight, preferably 20% by weight.

According to this invention, the solvent of phase one of embodiment IX is water, methanol, N,Ndimethylformamide, NN-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, Nmethylmorpholine N-oxide and their mixtures, in a preferred embodiment for the application of green chemistry principles, the solvent is water and a mixture of methanol, ethanol, 2-propanol, 2-methyl-2-propanol, N,Ndimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide with water. If the embodiment requires it, the ionic strength of the solvent is increased by adding 0.01 to 36% by weight. inorganic salts from the group sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium nitrate, potassium nitrate, ammonium sulfate, ammonium chloride, sodium iodide, potassium iodide, sodium bromide, potassium bromide, lithium chloride, lithium bromide, lithium iodide, or salts of organic acids from the group sodium acetate, potassium acetate, or with the addition of tetramethylammonium chloride.

Formation of micelles, i.e. the nano-/micro-emulsification process is carried out according to this invention by effective mixing in the temperature range of 0 °C to 135 °C, at temperatures when all components of the reaction system are liquid, which is a temperature higher than the temperature parameter of the transition and the melting point of the alkane . Preferably with technical paraffin with a melting point of 55 °C at a temperature between 85 and 95 °C.

The steric blocker is added to the mixture in a stoichiometric ratio in the range of 0.8 to 1.3 (blocker:micelle), preferably in a ratio in the range of 0.9 to 1.0 for each macrohole or macrodiffusion hole in the nanoparticle skeleton.

After the formation and stabilization of micelles with steric blockers, the mixture is cooled to a temperature lower than the melting point of the alkane, resulting in the formation of a suspension of spherical nanoparticles equipped with steric blockers. In a preferred embodiment, the cooling is carried out to a temperature 40°C lower than the melting point of the alkane.

If a surface-active substance in the form of a salt was used, it is converted at the end of phase 1 of design IX to free acid by the action of 2% wt. aqueous hydrochloric or sulfuric acids and the nanoparticles are thoroughly washed with water and dried under vacuum. The content of free sulfo groups per g of dry matter of nanoparticles is determined by titration with sodium hydroxide.

In the second stage of the first variant of embodiment IX, the nanoparticles prepared in the first stage are treated with a monosaccharide or oligosaccharide or polysaccharide or their modified forms or a mixture of the former in polar protic or aprotic solvents. As a result of electrostatic interactions, the saccharide will be sorbed on the surface of the nanoparticle and the saccharide skeleton will be oriented in such a way that the amino group of the saccharide converted to an ammonium cation is electrostatically bound to the sulfonate anion and points radially inside the sphere, while the saccharide skeleton is radially outside the sphere. At the site where the steric blocker is present, sulfo groups are not present and therefore sorption of the saccharide will not occur. After sorption of the saccharide, cross-linking of the adsorbed saccharide units is carried out.

The first variant of embodiment IX requires the presence of at least one strongly basic amino group IV in the skeleton of carbohydrate unit III. In a preferred embodiment, the nanoparticles prepared in the first stage are treated with a monosaccharide from the group of glucosamine, 1-glucosylamine, galactosamine, mannosamine, fructosamine, a disaccharide from the group of chitobiose, maltosamine, aminodeoxysucrose and lactosamine or with the polysaccharide chitosan or a mixture of these saccharides.

Sorption is carried out by adding a stoichiometric amount of dissolved saccharide calculated from the content of free sulfo groups to the nanoparticle suspension with very gentle stirring to avoid mechanical damage to the shape of the nanoparticles. The solvent for sorption is water, methanol, N,N- 8 CZ 309934 B6 dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, Nmethylmorpholine N-oxide and their mixtures.

The cross-linking is carried out by the cross-linking component after or without separation of the solvent used for sorption. The degree of cross-linking is governed by the type and stoichiometric ratio between the nanoparticle and the cross-linking component.

Cross-linking is carried out at a concentration of nanoparticles in the range of 0.1 to 20% by weight. in the mixture, preferably in a concentration of 1 to 3% of nanoparticles in the mixture, when the statistical average distance between the nanoparticles in the mixture is roughly 1 micron and the risk of intercorpuscular interaction is thus limited to a minimum (linking of two or more nanoparticles by covalent bonds).

In the third stage of the first version of IX, the number of layers of the skeleton of the future hollow nanosphere is increased as needed for the nanoparticles prepared in the second stage. Each additional layer of the skeleton is prepared by re-treating the nanoparticles with a cross-linking component and subsequently with an excess of saccharide. The newly formed linked carbohydrate units on the skeleton of the first layer of the nanosphere are cross-linked by repeating the cross-linking process from the second phase. By repeating the third phase, the number of layers can be increased arbitrarily. Depending on the type of cross-linking component used, and therefore also the type of functional group that forms the cross-linking of the nanosphere skeleton, it is possible to eliminate the charge of the skeleton. In the case of using active esters or ketenes for cross-linking, free carboxyls are present in the skeleton, which are undesirable from the point of view of further stages of nanoparticle processing and also clinical applications (see below). Free unwanted carboxyls are therefore converted to adequate carbinols. In a preferred embodiment, the reduction is carried out by the action of sodium borohydride in tetrahydrofuran in the presence of iodine at temperatures in the range of -15 to 0 °C.

In the fourth stage of the first variant of embodiment IX, the steric blocker and the alkane core are removed from the nanoparticles prepared in the third stage. To do this, the skeleton of nanoparticles from phase 3 is first treated in the cold with an aqueous alkali solution, and the nanoparticles treated in this way are subsequently extracted with non-polar solvents, preferably cyclohexane. In another embodiment, the nanoparticles from phase 3 are first extracted with non-polar solvents to remove the blocker and part of the core from the alkane, then the thus-modified nanoparticles are treated in the cold with an aqueous alkali solution, and the thus-modified nanoparticles are then extracted with non-polar solvents. The steric blocker is isolated from the extract by nanofiltration and recycled - see IX.

In the fifth phase of the first version of IX, the nanoparticles prepared in the fourth phase are modified with the inner wall of the nanospheres with allocated amino groups, or the skeleton from the fourth phase IX is used directly for clinical use. Modification of the inner wall, i.e. its functionalization is carried out according to scheme X (Fig. 12), where the symbols REAKT and REAKT' denote the reactive groups formyl or carboxyl and its other reactive forms from the group of active ester, mixed anhydride and carbonyl halide, either by the action of the reactive form of the linker-antiviral VII conjugate on amino groups of the inner wall of the nanospheres, or by the action of a bifunctional linker and subsequent reaction of the reactive form of the linker-nanosphere conjugate with a compound with a viricidal (virucidal) or antiviral or virus-inactivating effect.

To directly modify the amino groups of the inner wall, either the reaction (so-called peptide coupling) of the amino groups of the inner wall of the nanosphere skeleton with the active ester of the linker-antiviral VII conjugate is used, or the reaction with the linker-antiviral conjugate containing an aldehyde group on the linker side in the presence of borohydride. The reduction of aldimine to a secondary amine is carried out following the completion of the condensation of the amino groups of the inner wall of the nanospheres with an aldehyde or with an advantageous in situ during the condensation. The reduction is carried out in C1 to C4 alcohols or their mixtures with water at temperatures up to 25 °C to eliminate the possible reduction of ester bonds, if they are present in the nanosphere skeleton. In a preferred embodiment, the reduction is carried out at 0 °C in methanol. TO

- 9 CZ 309934 B6 reduction of the aldimine to a secondary amine, a reagent from the group sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, potassium borohydride, potassium cyanoborohydride or potassium triacetoxyborohydride is used.

In the case of modification of the amino groups of the inner wall of the nanospheres by peptide coupling, the procedure is also carried out in such a way that the skeleton of the nanoparticles from phase 4 is treated with a stoichiometric amount of the reactive form of the linker-antiviral VII conjugate. In a preferred embodiment, an excess of 5 to 10% mol. of the in situ generated active ester in solution in methanol or aqueous methanol by the effect of 4-(4,6dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride or tetrafluoroborate or hexafluorophosphate.

To modify the amino groups of the inner wall through the reactive form of the linker nanosphere conjugate, the reaction of the amino groups of the nanosphere with an excess of 10 to 10,000 mol% is used. of a difunctional linker of the bis-active ester type in a high-concentration solution and the resulting conjugate with active ester functional groups is allowed to react with a compound with a viricidal (virucidal) or antiviral or virus-inactivating effect containing reactive amino groups.

In another embodiment, the procedure is such that the skeleton of nanoparticles from phase 4 is acted upon with a stoichiometric amount or, in a preferred embodiment, with an excess of 5 to 100% mol. of a heterodifunctional linker of the active ester type in a high-concentration solution and the resulting conjugate with active ester functional groups is allowed to react with a compound with a viricidal (virucidal) or antiviral or virus-inactivating effect containing reactive amino groups.

In another embodiment, a heterodifunctional linker of the α,C')-dialdehyde-mono(dimethyl acetal) type is used to modify the amino groups of the inner wall. The reaction of the reactive aldehydic group of the linker with the amino group of the nanosphere is carried out in methanol and the reduction of the resulting aldimine to a secondary amine is carried out after the condensation of the amino groups of the inner wall of the nanospheres with aldehyde or with an advantageous in situ during condensation is completed. The reduction is carried out in C1 to C4 alcohols or their mixtures with water at temperatures up to 25 °C to eliminate the possible reduction of ester bonds, if they are present in the nanosphere skeleton. In a preferred embodiment, the reduction is carried out at 0 °C in methanol. To reduce the aldimine to a secondary amine, a reagent from the group sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, potassium borohydride, potassium cyanoborohydride or potassium triacetoxyborohydride is used. The resulting conjugate is converted to the reactive form of the nanosphere-linker conjugate by the action of lithium tetrafluoroborate in acetonitrile containing 2% water, while the basicity of the amino groups present is blunted by the action of 0.6 to 1 stoichiometric equivalent of tetrafluoroboric acid. The reaction takes place at laboratory temperature. The resulting reactive form of the nanosphere-linker X conjugate is treated with a compound with a virucidal (virucidal) or antiviral or virus-inactivating effect containing reactive amino groups, and the conversion to an amine is carried out again by reducing the resulting aldimine following the completion of the condensation of the amino groups of the inner wall of the nanospheres with an aldehyde or with an advantageous in situ during condensation. The reduction is carried out in C1 to C4 alcohols or their mixtures with water at temperatures up to 25 °C to eliminate the possible reduction of ester bonds, if they are present in the nanosphere skeleton. In a preferred embodiment, the reduction is carried out at 0 °C in methanol. To reduce the aldimine to a secondary amine, a reagent from the group sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, potassium borohydride, potassium cyanoborohydride or potassium triacetoxyborohydride is used.

In the first stage of the second variant of embodiment XI (Fig. 13), the starting point is the structural unit of a saccharide, which is equipped with a lipophilic group and thus forms a surface-active substance, which is acted upon by an alkane in a polar solvent. The interaction of the steric blocker creates a macroscopic structure in which the steric blocker is partially embedded in the sphere skeleton formed by micelles from alkane with surface groups of saccharide. Phase one can be done by first treating the steric blocker with an alkane and then forming micelles with a surfactant in a polar solvent, or adding a steric blocker to the micelles formed from the alkane and surfactant in a polar solvent. In an advantageous embodiment, it is first acted upon

- 10 CZ 309934 B6 solvent for the mixture of alkane and surface-active substance, and then a steric blocker is added.

The surfactant in the first stage of the second embodiment XI is a monosaccharide or disaccharide or oligosaccharide or polysaccharide covalently bound to one or more lipophilic aliphatic, alicyclic or aliphatic chain substituted aromatic groups with a chain length corresponding to C10 to C45 or a mixture of these saccharides. In a preferred embodiment, the surfactant is a saccharide of general formula III, where w is 0 or 1, where either one of the substituents R1 to R10 in formula III is formed by one of the fragments of general formula XII (Fig. 14), where the symbol "*" denotes a bond to skeleton III, where K is O or NH, where e is 0 to 40, where Q is O or CH2, where J is H or NO2, where the remaining substituents from the group R1 to R10 in formula III form hydrogen or one of the substituents IV or V, or where any pair of substituents from the group R1 to R10 outside the pair of the same carbon atoms of the skeleton III is formed by one of the fragments of the general formula XIII (Fig. 15), where the symbol "*" indicates a bond to the skeleton III, where e is 0 to 21, where Q is O or CH 2 , where J is H or NO 2 , where the remaining substituents from the group R 1 to R 10 in formula III form hydrogen or any of the substituents IV or V.

The lipophilic group may be attached via an ether linkage or as an N-amino derivative, with between 0 and 99% of the saccharide having a lipophilic ether-linked function and between 1 and 100% of the saccharide having a lipophilic N-alkylamine function in the reaction mixture. In a preferred embodiment, the reaction mixture contains between 1 and 100% of a saccharide with a lipophilic function XII, where the symbol K is NH.

In a preferred embodiment, the surfactant or their mixture has an HLB index (Li. C., e.a.: Colloids and Surfaces A: Physicochem. Eng. Aspects 356, 71 (2010)) in the range of 9.3 to 11.9 and is in the reaction mixture present in a concentration of 2 to 15 wt.% In a preferred embodiment, the N-amino derivative is quaternized by adding a stoichiometric amount of sulfuric or hydrochloric acid, thereby increasing the micellar strength of the saccharide derivative.

In a preferred embodiment, the surface-active saccharide with a lipophilic ether-bound function is a saccharide derivative from the group of glucose, galactose, mannose, fructose, maltose, cellobiose, lactose, dextrose, sucrose, maltodextrin, dextran, cellulose, amylose, amylopectin, glycogen or a mixture of these saccharides. A surface-active saccharide with a lipophilic N-alkylamine function is a saccharide derivative from the group glucosamine, 1-glucosylamine, galactosamine, mannosamine, fructosamine, chitobiose, maltosamine, lactosamine, aminodeoxysucrose or chitosan or a mixture of these saccharides.

An alkane is an aliphatic or alicyclic hydrocarbon with a length corresponding to C16 to C60 or a mixture of these hydrocarbons, preferably a technical paraffin with a transition temperature parameter in the range of 35°C to 80°C and a melting point in the range of 35°C to 80°C. The alkane concentration in the mixture is chosen in the range of 5 to 40% by weight, preferably 20% by weight.

- 11 CZ 309934 B6

According to this invention, the solvent of phase one of embodiment XI is water, methanol, N,Ndimethylformamide, NN-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, Nmethylmorpholine N-oxide and their mixtures, in a preferred embodiment for the application of green chemistry principles, the solvent is water and a mixture of methanol, ethanol, 2-propanol, 2-methyl-2-propanol, N,Ndimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide with water. If the embodiment requires it, the ionic strength of the solvent is increased by adding 0.01 to 36% by weight. inorganic salts from the group sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium nitrate, potassium nitrate, ammonium sulfate, ammonium chloride, sodium iodide, potassium iodide, sodium bromide, potassium bromide, lithium chloride, lithium bromide, lithium iodide or salts organic acids from the group sodium acetate, potassium acetate or with the addition of tetramethylammonium chloride.

Formation of micelles, i.e. the nano-/micro-emulsification process is carried out according to this invention by effective mixing in the temperature range of 0 °C to 135 °C, at temperatures when all components of the reaction system are liquid, which is a temperature higher than the temperature parameter of the transition and the melting point of the alkane . Preferably with technical paraffin with a melting point of 55 °C at a temperature between 85 and 95 °C.

The steric blocker is added to the mixture in a stoichiometric ratio in the range of 0.8 to 1.3 (blocker:micelle), preferably in a ratio in the range of 0.9 to 1.0 for each macrohole or macrodiffusion hole in the nanoparticle skeleton. Carbohydrate units are not present at the site of the nanosphere surface where the steric blocker is present.

After the formation and stabilization of micelles with steric blockers, the mixture is cooled to a temperature lower than the melting point of the alkane, resulting in the formation of a suspension of spherical nanoparticles equipped with steric blockers. In a preferred embodiment, the cooling is carried out to a temperature 40°C lower than the melting point of the alkane.

If a surface-active substance in the form of a salt was used, at the end of phase 1, embodiment XI is converted to a free base by the action of 2% wt. of aqueous sodium hydroxide and the nanoparticles are thoroughly washed with water and dried under vacuum.

In the second phase of the second variant of embodiment XI, the nanoparticles prepared in the first phase are treated with a cross-linking component, which creates the first layer of the nanosphere skeleton. The degree of cross-linking is governed by the type and stoichiometric ratio between the nanoparticle and the cross-linking component.

Cross-linking is carried out at a concentration of nanoparticles in the range of 0.1 to 20% by weight. in the mixture, preferably in a concentration of 1 to 3% of nanoparticles in the mixture, when the statistical average distance between the nanoparticles in the mixture is roughly 1 micron and the risk of intercorpuscular interaction is thus limited to a minimum (linking of two or more nanoparticles by covalent bonds).

In the third stage of the second variant of embodiment XI, the number of layers of the skeleton of the future hollow nanosphere is increased as needed for the nanoparticles prepared in the second stage. Each additional layer of the skeleton is prepared by re-treating the nanoparticles with a cross-linking component and subsequently with an excess of saccharide. The newly formed linked carbohydrate units on the skeleton of the first layer of the nanosphere are cross-linked by repeating the cross-linking process from the second phase. By repeating the third phase, the number of layers can be increased arbitrarily. Depending on the type of cross-linking component used, and therefore also the type of functional group that forms the cross-linking of the nanosphere skeleton, it is possible to eliminate the charge of the skeleton. In the case of using active esters or ketenes for cross-linking, free carboxyls are present in the skeleton, which are undesirable from the point of view of further stages of nanoparticle processing and also clinical applications (see below). Free unwanted carboxyls are therefore converted to adequate carbinols. In a preferred embodiment, the reduction is carried out by the action of sodium borohydride in tetrahydrofuran in the presence of iodine at temperatures in the range of -15 to 0 °C.

In the fourth stage of the second embodiment, XI is removed from the nanoparticles prepared in the third stage

- 12 CZ 309934 B6 steric blocker and alkane core. For this, the nanoparticle skeleton from phase 3 is extracted with non-polar solvents, preferably cyclohexane. Subsequently, the lipophilic chain is removed from the nanosphere skeleton hydrogenolytically or photochemically. In another preferred embodiment, the skeleton of the nanospheres is built from surface-active saccharides with lipophilic chains XII and XIII, where J stands for NO2, and the procedure is to photochemically cleave the bond between the lipophilic chain XII or XIII and the saccharide skeleton (III) and then the nanoparticles they extract with a non-polar solvent from the steric blocker, the alkane core and the residues of the lipophilic chain after photolysis. The steric blocker is isolated from the extract by nanofiltration and recycled - see XI.

In the fifth phase of the second variant of XI, the nanoparticles prepared in the fourth phase are modified with the inner wall of the nanospheres with allocated amino groups, or the skeleton from the fourth phase of XI is used directly for clinical use. The reaction conditions are adapted to the representation (surface density) of amino groups on the inner surface of the nanospheres, which results from the content of a surface-active saccharide with a lipophilic N-alkylamine function (see phase 1 of the second version) in the process of building the nanoparticle skeleton. The surface density is determined acidimetrically.

Modification of the inner wall, i.e. its functionalization is carried out according to scheme X, where the symbols REAKT and REAKT' denote the reactive groups formyl or carboxyl and its other reactive forms from the group of active ester, mixed anhydride and carbonyl halide, either by the action of the reactive form of the linker-antiviral VII conjugate on the amino groups of the inner wall of the nanospheres, or by the action of a bifunctional linker and subsequent reaction of the reactive form of the linker-nanosphere conjugate with a compound with a viricidal (virucidal) or antiviral or virus-inactivating effect.

To directly modify the amino groups of the inner wall, either the reaction (so-called peptide coupling) of the amino groups of the inner wall of the nanosphere skeleton with the active ester of the linker-antiviral VII conjugate is used, or the reaction with the linker-antiviral conjugate containing an aldehyde group on the linker side in the presence of borohydride. The reduction of aldimine to a secondary amine is carried out following the completion of the condensation of the amino groups of the inner wall of the nanospheres with an aldehyde or with an advantageous in situ during the condensation. The reduction is carried out in C1 to C4 alcohols or their mixtures with water at temperatures up to 25 °C to eliminate the possible reduction of ester bonds, if they are present in the nanosphere skeleton. In a preferred embodiment, the reduction is carried out at 0 °C in methanol. To reduce the aldimine to a secondary amine, a reagent from the group sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, potassium borohydride, potassium cyanoborohydride or potassium triacetoxyborohydride is used.

In the case of modification of the amino groups of the inner wall of the nanospheres by peptide coupling, the procedure is also carried out in such a way that the skeleton of the nanoparticles from phase 4 is treated with a stoichiometric amount of the reactive form of the linker-antiviral VII conjugate. In a preferred embodiment, an excess of 5 to 10% mol. of the in situ generated active ester in solution in methanol or aqueous methanol by the effect of 4-(4,6dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride.

To modify the amino groups of the inner wall through the reactive form of the nanosphere linker conjugate, the reaction of the amino groups of the nanosphere with an excess of 10 to 10,000 mol% is used. of a difunctional linker of the bis-active ester type in a high-concentration solution and the resulting conjugate with active ester functional groups is allowed to react with a compound with a viricidal (virucidal) or antiviral or virus-inactivating effect containing reactive amino groups.

In another embodiment, the procedure is such that the skeleton of nanoparticles from phase 4 is acted upon with a stoichiometric amount or, in a preferred embodiment, with an excess of 5 to 100% mol. of a heterodifunctional linker of the active ester type in a high-concentration solution and the resulting conjugate with active ester functional groups is allowed to react with a compound with a viricidal (virucidal) or antiviral or virus-inactivating effect containing reactive amino groups.

In another embodiment, a heterodifunctional linker of the α,C')-dialdehyde-mono(dimethyl acetal) type is used to modify the amino groups of the inner wall. Reaction of the reactive aldehyde group of the linker with

- 13 CZ 309934 B6 with the amino group of the nanospheres is carried out in methanol and the reduction of the resulting aldimine to a secondary amine is carried out following the completion of the condensation of the amino groups of the inner wall of the nanospheres with aldehyde or with an advantageous in situ during condensation. The reduction is carried out in C1 to C4 alcohols or their mixtures with water at temperatures up to 25 °C to eliminate the possible reduction of ester bonds, if they are present in the nanosphere skeleton. In a preferred embodiment, the reduction is carried out at 0 °C in methanol. To reduce the aldimine to a secondary amine, a reagent from the group sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, potassium borohydride, potassium cyanoborohydride or potassium triacetoxyborohydride is used. The resulting conjugate is converted to the reactive form of the nanosphere-linker conjugate by the action of lithium tetrafluoroborate in acetonitrile containing 2% water, while the basicity of the amino groups present is blunted by the action of 0.6 to 1 stoichiometric equivalent of tetrafluoroboric acid. The reaction takes place at laboratory temperature. The resulting reactive form of the nanosphere-linker X conjugate is treated with a compound with a virucidal (virucidal) or antiviral or virus-inactivating effect containing reactive amino groups, and the conversion to an amine is carried out again by reducing the resulting aldimine following the completion of the condensation of the amino groups of the inner wall of the nanospheres with an aldehyde or with an advantageous in situ during condensation. The reduction is carried out in C1 to C4 alcohols or their mixtures with water at temperatures up to 25 °C to eliminate the possible reduction of ester bonds, if they are present in the nanosphere skeleton. In a preferred embodiment, the reduction is carried out at 0 °C in methanol. To reduce the aldimine to a secondary amine, a reagent from the group sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, potassium borohydride, potassium cyanoborohydride or potassium triacetoxyborohydride is used.

The third variant of the XIV design (Fig. 16) is a special case of the first variant. It is technologically the shortest and simplest version of the production of nanoantivirals of the type of spherical hollow nanoparticles with macroholes, but it is bound by several principled limitations.

The third version of XIV is based on the same principle as the first version of IX. In the first stage of embodiment XIV, the surfactant provided with a hydrophilic sulfo group is treated with an alkane in a polar solvent. The sulfo group of the surfactant is in the form of a free acid or an alkali metal salt. The interaction of the steric blocker creates a macroscopic structure in which the steric blocker is partially embedded in the sphere skeleton formed by micelles from alkane with surface sulfo groups. Phase one can be done by first acting on the steric blocker with an alkane and then forming micelles by the action of a surfactant in a polar solvent, or by adding a steric blocker to the formed micelles from the alkane and a surfactant in a polar solvent. In a preferred embodiment, a mixture of alkane and surfactant is first treated with a solvent and then a steric blocker is added.

The surfactant is an aliphatic, alicyclic or aromatic sulfonic acid or its salt with an alkali metal cation of a chain length corresponding to C10 to C45 or a mixture of these surfactants. In a preferred embodiment, the surfactant or their mixture has an HLB index (Li. C., e.a.: Colloids and Surfaces A: Physicochem. Eng. Aspects 356, 71 (2010)) in the range of 9.3 to 11.9 and is in the reaction mixture present in a concentration of 2 to 15 wt.%

An alkane is an aliphatic or alicyclic hydrocarbon with a length corresponding to C16 to C60 or a mixture of these hydrocarbons, preferably a technical paraffin with a transition temperature parameter in the range of 35°C to 80°C and a melting point in the range of 35°C to 80°C. The alkane concentration in the mixture is chosen in the range of 5 to 40% by weight, preferably 20% by weight.

According to this invention, the solvent of phase one of embodiment XIV is water, methanol, NNdimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, Nmethylmorpholine N-oxide and their mixtures, in a preferred embodiment for the application of green chemistry principles, the solvent is water and a mixture of methanol, ethanol, 2-propanol, 2-methyl-2-propanol, N,Ndimethylformamide, NN-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide with water. If the embodiment requires it, the ionic strength of the solvent is increased by adding 0.01 to 36% by weight. inorganic salts from the group sodium chloride, potassium chloride, sodium sulfate, sulfate

- 14 CZ 309934 B6 potassium, sodium nitrate, potassium nitrate, ammonium sulfate, ammonium chloride, sodium iodide, potassium iodide, sodium bromide, potassium bromide, lithium chloride, lithium bromide, lithium iodide, or salts of organic acids from the group sodium acetate, acetate potassium, or by the addition of tetramethylammonium chloride.

Formation of micelles, i.e. the nano-/micro-emulsification process is carried out according to this invention by effective mixing in the temperature range of 0 °C to 135 °C, at temperatures when all components of the reaction system are liquid, which is a temperature higher than the temperature parameter of the transition and the melting point of the alkane . Preferably with technical paraffin with a melting point of 55 °C at a temperature between 85 and 95 °C.

The steric blocker is added to the mixture in a stoichiometric ratio in the range of 0.8 to 1.3 (blocker:micelle), preferably in a ratio in the range of 0.9 to 1.0 for each macrohole or macrodiffusion hole in the nanoparticle skeleton.

After the formation and stabilization of micelles with steric blockers, the mixture is cooled to a temperature lower than the melting point of the alkane, resulting in the formation of a suspension of spherical nanoparticles equipped with steric blockers. In a preferred embodiment, the cooling is carried out to a temperature 40°C lower than the melting point of the alkane.

If a surfactant was used in the form of a salt, it is converted at the end of phase 1 of embodiment XIV to a free acid by the action of 2% wt. aqueous hydrochloric or sulfuric acids and the nanoparticles are thoroughly washed with water and dried under vacuum. The content of free sulfo groups per g of dry matter of nanoparticles is determined by titration with sodium hydroxide.

In the second phase of the third variant of embodiment XIV, the nanosphere matrix with surface sulfo groups prepared in the first phase of the third variant of embodiment XIV is treated with saccharide units of general formula III, where w is 0 or 1, where one of the substituents R1 to R10 in formula III is formed by one of the fragments of the general formula XV (Fig. 17) and where the remaining substituents from the group R1 to R10 in the formula III form hydrogen or one of the substituents IV or V, while in the fragments XV there are:

index a1 between 0 and 3, index a2 between 0 and 2, index a3 between 0 and 30,

Z1, Z2 and Z3 independently of each other are aliphatic or alicyclic alkyl or alkyl substituted by an aromatic skeleton corresponding to the total chain length of C1 to C18, where the symbol "*" denotes a bond to skeleton III.

These saccharide units of the type of modified monosaccharides, oligosaccharides and polysaccharides contain either quaternary ammonium groups or tertiary amino groups capable of quaternization. Due to the influence of electrostatic interactions, when this saccharide unit is in contact with the nanospherical matrix with surface sulfo groups prepared in the first stage of the third variant of embodiment XIV, the sorption of the saccharide on the surface of the nanoparticle and the defined orientation of the saccharide skeleton will occur so that the amino groups of the saccharide unit in the form of ammonium cations are electrostatically bound to the sulfonate anions and point toward the surface of the sphere, while the saccharide backbone radially outside the sphere. In the place where the steric blocker is present, sulfo groups are not present, and therefore saccharide sorption will not occur.

Due to the need to preserve the possibility of cross-linking of carbohydrate units, it is necessary that

- 15 CZ 309934 B6 distance (and distribution) of saccharide hydroxyls between each pair of saccharide units corresponded to the future arrangement of the skeleton after cross-linking. If the carbohydrate units are placed too far away, intermolecular reactions (polycondensation) will not occur and cross-linking will not occur at all or only partially. Therefore, and this is a fundamental limitation of the methodology, it is necessary to take into account the area on the surface of the nanosphere matrix with surface sulfo groups prepared in the first stage of the third variant of the XIV embodiment, which will be occupied by the skeleton of the XV fragment due to the interaction. In a preferred embodiment, in the case of monosaccharides as saccharide units, one therefore works with XV fragments with indices ai and a2 of 0 or 1, i.e. with few cations.

To carry out crosslinking, it is necessary that the total charge of the nanoparticle is equal to zero. The stoichiometric amount of saccharide units for sorption is therefore calculated from the determined number of sulfo groups (see the conclusion of phase one).

In a preferred embodiment, the saccharide unit consists of a saccharide derivative from the group glucosamine, galactosamine, mannosamine, chitobiose, maltosamine, lactosamine, fructosamine, aminodeoxysucrose, 1-glucosylamine, chitosan, glucose, galactose, mannose, fructose, maltose, cellobiose, lactose, dextrose, sucrose, maltodextrin, dextran, cellulose, amylose, amylopectin, glycogen or a mixture of these carbohydrates.

Adsorption is performed by adding a stoichiometric amount of a dissolved saccharide derivative with very gentle mixing to avoid mechanical damage to the shape of the nanoparticles. The solvent for sorption is water, methanol, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, N-methylmorpholine N-oxide and their mixtures.

The cross-linking is carried out by the cross-linking component after or without separation of the solvent used for sorption. The degree of cross-linking is governed by the type and stoichiometric ratio between the nanoparticle and the cross-linking component.

Cross-linking is carried out at a concentration of nanoparticles in the range of 0.1 to 20% by weight. in the mixture, preferably in a concentration of 1 to 3% of nanoparticles in the mixture, when the statistical average distance between the nanoparticles in the mixture is roughly 1 micron and the risk of intercorpuscular interaction is thus limited to a minimum (linking of two or more nanoparticles by covalent bonds).

Phases 3 and 4 of the third variant of embodiment XIV are analogous to phases 3 and 4 of the first variant of embodiment IX, with the difference that phase 4 already produces the resulting nanovirotic product.

The steric blocker is a key three-dimensional element of the technology. It defines the diameter of the future macrohole and macrodiffusion hole of the nanoparticle by preventing the allocation of saccharide units on or near the surface of the nanosphere and during the construction of the nanoparticle skeleton, it prevents the crosslinking components in the crosslinking process at the location of the future macrohole or macrodiffusion hole. A steric blocker is a polymer nanoparticle whose shape, size and surface arrangement of functional groups corresponds to the end use in the production process of spherical hollow nanoparticles with precisely defined macroholes. Three types of steric blockers XVI can be used in the process of producing nanoparticles (Fig. 18).

1. Polystyrene spherical nanoparticles XVI-(a). The advantage of this type of blocker is a very low price and excellent availability of fractions with a narrow distribution of particles. The steric blocker can be used "as is", or it can be hydrophobized by the effect of non-polar solvents from the hexane, heptane, cyclohexane group by standing together in the mixture for 10 minutes to 24 hours at room temperature. Before adding to the micellar system XVI-(b), the steric blocker particles must be vacuum dried from the solvent. After hydrophobization, the micelles stabilize more quickly. The main disadvantage of this type of blocker is the low rate of establishment of the micellar equilibrium and the unfavorable distribution of the intensity (depth) of the blocker's embedding into the XVI-(b) micelle. This type of steric blocker therefore produces the widest distribution of diameter of macroholes or macrodiffuse holes.

- 16 CZ 309934 B6

2. Binary anisotropic nanoparticles XVI-(c) polystyrene-alkane. These systems have been studied in detail in recent years (Kim S.-H., e.a.: Synthesis and Assembly of Colloidal Particles with Sticky Dimples J. Am. Chem. Soc. 134, 16115 (2012); Yamagami T., e.a.: Preparation of hemispherical polymer particles via phase separation induced by microsuspension polymerization Colloid. Polym. Sci., DOI 10.1007/s00396-012-2625-y (2012); Wang Y., e.a.: Emulsion Interfacial Synthesis of Asymmetric Janus Particles Macromolecules 44, 3787 (2011) )) and form the basis of steric blocker technology.

The steric blocker XVI-(c) can be prepared either by the process of anisotropization of polystyrene spherical nanoparticles swollen in an alkane, or by a mixed emulsion or suspension, by polymerization of styrene in the presence of an alkane and a surfactant.

Anisotropization of polystyrene spherical nanoparticles is done by mixing dry polystyrene nanoparticles at a temperature higher than the melting point of the alkane but higher than 60 °C and lower than the glass transition point of polystyrene (95 °C) in an excess of the alkane and at this temperature and with stirring for 4 to 24 hours. Then, at the same temperature, they are sharply sucked off, possibly centrifuged from the excess alkane, and the temperature of the mixture is allowed to drop to laboratory temperature in water or aqueous ethanol (60% by weight) within 60 to 120 minutes. In a preferred embodiment, the operation is performed in situ, the mixture of polystyrene nanospherical particles and alkane is suspended at a suitable temperature from the range between 60°C and the boiling point of ethanol in 60 to 85% by weight. aqueous ethanol or 60 to 90% wt. aqueous 2-propanol or 60 to 90% aqueous 2-methyl-2-propanol and mix until coagulation of all the alkane with the polymer, but for at least 6 hours. The mixture is then cooled to room temperature or lower (depending on the melting point of the alkane) during an hour with very gentle stirring, and the anisotropic particles of the steric blocker XVI-(c) are filtered. It is used in a high dilution of 0.1 to 0.5% by weight. particles to prevent coagulation of the alkane hemispheres during cooling.

The alkane for the process of anisotropization of polystyrene spherical nanoparticles is an aliphatic or alicyclic hydrocarbon with a length corresponding to C16 to C60 or a mixture of these hydrocarbons, in a preferred embodiment technical paraffin with a transition temperature parameter in the range of 35 °C to 80 °C and a melting point in the range of 35 °C to 80 °C.

Particles of steric blocker XVI-(c) are prepared by emulsion or suspension polymerization according to Yamagami T., e.a.: Preparation of hemispherical polymer particles via phase separation induced by microsuspension polymerization Colloid. Polym. Sci., DOI 10.1007 s00396-0122625-y (2012) with the choice of an alkane with a higher melting point, preferably technical paraffin with a transition temperature parameter in the range of 35 °C to 65 °C and a melting point in the range of 35 ° C to 65 °C. The resulting anisotropic particles must be separated according to size by permeation chromatography.

The main advantage of the method of anisotropization of polystyrene spherical nanoparticles compared to the method of polymerization of styrene in the presence of an alkane is the preservation of the distribution of the used polystyrene nanoparticles at the input, i.e. at the end of the process, the unequivocal mechanical parameters (especially the active diameter) of the blocker are known.

Binary anisotropic nanoparticle type steric blocker XVI-(c) polystyrene-alkane is a cheap and very effective steric blocker for all methods of producing spherical hollow nanoparticles with one or more well-defined macroholes and macrodiffusion holes. It contains a highly lipophilic hemisphere made of the same material as the micelle core. By applying this type of steric blocker, a narrow distribution of macroholes and macrodiffusion holes is achieved, which exactly copies the distribution of the polystyrene nanoparticles used. 1 to 30 minutes XVI-(d) is sufficient to establish equilibrium when the blocker is added as the last part of the micelle-forming system.

3. Unilaterally hydrophilized spheroidal polystyrene nanoparticle XVI-(i). It is a type of steric blocker with an enhanced hydrophilic side and modified geometry of the lipophilic side to

- 17 CZ 309934 B6 reduction of the surface tension of the adduct from process XVI-(j). It is prepared by two independent methods. It is based on the binary anisotropic nanoparticle XVI-(c) polystyrene-alkane. In one embodiment XVI-(e), the anisotropic nanoparticles are treated at -15°C to 0°C with a 70% by weight perchloric acid mixture. and 0.3 to 0.5 wt.% of sodium chlorate per nanoparticle mass (adapted from Curtis A.S.G., e.a.: Adhesion of Cells to Polystyrene Surfaces J. Cell Biol. 97, 1500 (1983)). The hydroxylated polystyrene hemisphere thus generated is reduced with the sodium borohydride iodine system in triglyme at -15 °C in order to eliminate the carboxyls present (after modification: Bhaskar Kanth J.V., e.a.: Selective Reduction of Carboxylic Acids into Alcohols Using NaBH, and If J. Org. Chem .56, 5964 (1991)). To transform the generated hydroxyls into poly 2-(2'methoxyethyloxy)ethyl ether, O-alkylation XVI-(g) is carried out in the form of "O-H insertion" of the in situ generated carbene (Fulton, J.R., e.a.: The Use of Tosylhydrazone Salts as a Safe

Alternative for Handling Diazo Compounds and Their Applications in Organic Synthesis Eur. J. Org. Chem. 1479 (2005)) according to scheme XVII (Fig. 19).

The order can advantageously be reversed without affecting the quality of the steric blocker. First, after the hydroxylation of XVI-(e), the alkane hemisphere is removed by extraction with hot cyclohexane, and the resulting intermediate XVI-(f) is reduced with the system sodium borohydride - iodine in triglyme at -15 °C in order to eliminate the carboxyls present, and then the hydroxylated hemisphere is treated by in situ generated carbene according to scheme (XVII).

An alternative route to the analogous, although not identical, unilaterally hydrophilized spheroidal polystyrene nanoparticles XVI-(i) is the direct alkylation of the α-position of the aliphatic chain of the skeleton of the polystyrene hemisphere XVI-(h) by the C-alkylation methodology in the form of "C-H insertion" of an in situ generated carbene (Fulton , J.R., e.a.: The Use of Tosylhydrazone Salts as a Safe

Alternatives. for Handling Diazo Compounds and Their Applications in Organic Synthesis Eur. J. Org. Chem. 1479(2005)) according to scheme XVIII (Fig. 20).

The basic advantage of performing hydrophilization using the "C-H insertion" method is the simplicity of the execution. 0.5 to 5% of CH groups functionalized in this way are sufficient on the polystyrene hemisphere, so the reaction is finished after 2 to 4 hours. Since the reactivity of the aliphatic CH of polystyrene (as well as aryl) and the CH of the alkane and the carbene chain is almost identical to the generated carbene, both the alkane hemisphere and the intramolecular carbene chain are functionalized. However, the alkylated alkane hemisphere is subsequently dissolved in hot cyclohexane during further processing, so the side reaction has no effect on the functionality of the thus prepared one-sided hydrophilized spheroidal polystyrene nanoparticle XVI-(i).

In both cases, after O-/C-alkylation, the hemisphere is removed from the alkane by extraction with hot cyclohexane.

Cross-linking is a key step in the production technology of spherical hollow nanoparticles with well-defined macropores. It serves to create a unitary layer of polysaccharide polycondensate, i.e. of a polymer with a polydispersity index equal to one. The degree and type of cross-linking is a controlling element of many application parameters of nanoparticles. Cross-linking is also used in the phase of building another isocentric layer, i.e. cross-linking is performed when creating the n -th isocentric layer and further cross-linking is performed when building (linking) the carriers of the n+1 isocentric layer and when cross-linking the own n+1 isocentric layer, as is shown in Scheme XIX (Fig. 21).

According to the production method used, either electrostatically sorbed or covalently bound saccharide units on the surface of the alkane nanosphere XIX-(a) are cross-linked by covalent bonding with bi- or tri-functional cross-linking units XIX-(b). Since carbohydrates are themselves polyfunctional substrates, in a preferred embodiment the cross-linking is carried out by bifunctional cross-linking units, with the fact that, depending on the cross-linking reaction conditions, the binding of three cross-linking units to one saccharide unit occurs in the range between 1 and 100% during the construction of the first cross-linked 3D isocentric layer, in in a preferred embodiment in the range between 10 to 30%, and in the range between 1 to 100% to bind four cross-linking units to one carbohydrate unit when building

- 18 CZ 309934 B6 the next cross-linked 3D isocentric layer, preferably in the range between 10 and 30%. The chain length of the cross-linking unit must correspond to the distance of the reactive groups of two adjacent saccharide units from the point of view of the future intermolecular bond. The result of the cross-linking performed under optimized conditions is a cross-linked isocentric monolayer of XIX-(c) carbohydrate polycondensate.

The method of production, i.e. the presented technology, makes it possible to use a different type of crosslinking unit at each stage or step of crosslinking and thus achieve specific effects of the arrangement of the skeleton of spherical hollow nanoparticles with precisely defined macroholes. This effect can best be used when building the linkers of the next isocentric layer XIX-(d) and XIX-(e). The very short cross-linking unit ensures that the two isocentric layers are very close to each other, which reduces the possible torsion angles during mechanical stress on the skeleton, which has a major impact on the mechanical sustainability of the skeleton geometry of the future therapeutic in a biological system. On the other hand, longer cross-linking units ensure greater flexibility between individual isocentric layers, which can be an advantage in other therapeutic applications, as the skeleton of nanotherapeutics will be more malleable, more adaptable and will also contain larger diffusion channels for ions and small molecules.

Another option for crosslinking is the use of reactive groups of the crosslinking units of the previous crosslinking: XIX-(e) and XIX-(g). In this way, it is possible to achieve a greater surface density of saccharide units in each higher isocentric layer XIX-(g), which subsequently XIX(i) has a critical impact on increasing the mechanical resistance of the nanospherical particle.

Cross-linking is carried out using four possible technological approaches. Each variant provides a different type of covalent bonds between the saccharide units and the crosslinking units, which has a major impact on the biodegradability and rate of metabolism of the framework of spherical hollow nanoparticles with precisely defined macroholes in the organism.

Crosslinking method "Polyester XX (Fig. 22). The cross-linking unit is a reactive polycarbonyl bis-acylating agent XXI (Fig. 23), where n is 0 to 8, where Gr is a leaving group from a hydroxyl group or Cl or Br or an active ester XXII (Fig. 24) or a ketene XXIII (Fig. 25), where the symbol "*" indicates a connection to the XXI skeleton.

If an acyl halide, carboxylic acid or active ester XXII is used in the polycondensation process, the index m in scheme XX is zero, if ketene XXIII is used, the index m in scheme XX is one.

The polyesterification reaction XX-(a) is carried out by adding the bis-acylation agent XXI to a suspension of nanoparticles containing free hydroxyls of saccharide units. The bis-acylation reagent XXI is used for reaction XX either prepared in advance or generated in situ.

The polyesterification reaction XX-(a) is carried out in the temperature range of -80°C to 95°C, preferably at temperatures lower than the transition temperature parameter and the freezing point of the alkane of the nanoparticle core.

The reaction with the bis-acylating agent XXI is carried out in solvents from the group of acetonitrile, N,Ndimethylformamide, NN-dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, dichloromethane, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, diethyl ether, 2-methoxy-2-methylpropane and their mixtures.

The reaction with the bis-acylating reagent XXI is carried out without the presence of other reagents and or for

- 19 CZ 309934 B6 of the presence of compounds from the group N,N'-dicyclohexylcarbodiimide, N-(3-dimethylaminopropyl)N-ethylcarbodiimide, N,N'-diispropylcarbodiimide, 2-(1H-1,2,3-benzotriazole-1- yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, 2-(1H-7-aza-1,2,3-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, 1-[(1- (cyano-2-ethoxy-2oxoethylideneaminooxy)dimethylaminomorpholinomethylene)]methanaminium hexafluorophosphate, 1,1'-(azodicarbonyl)dipiperidine, di-tert.-butylazodicarboxylate, diisopropylazodicarboxylate, diethylazodicarboxylate, N,N,N',N'-tetramethylazodicarboxamide, triethylamine, trimethylamine, N,N,N',N'-tetramethylethylenediamine, N-methylmorpholine, N,N-diisopropylethylamine, 1,8bis(dimethylamino)naphthalene, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1, 5-diazabicyclo[4.3.0]non-5ene, 1,4-diazabicyclo[2.2.2]octane, 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene, 4-( N,Ndimethylamino)pyridine, 4-(N,N-diethylamino)pyridine, 4-(4-pyridyl)morpholine, 4-(1pyrrolidinyl)pyridine, 2,6-lutidine, 2,4,6-collidine, 2,6 -di-tert-butylpyridine, 1H-1,2,3-benzotriazole, imidazole, triphenylphosphine, triethylphosphine, tributylphosphine, triethylphosphite or in the presence of combinations of the preceding.

After the polycondensation reaction XX-(a) is carried out, the product XX-(b) is reduced to polyol XX-(d) by the effect of a reducing agent from the group sodium borohydride, potassium borohydride, sodium cyanoborohydride, borane or agents that are prepared in advance from reagents of this group or in situ, preferably sodium tri(acetoxy)borohydride or borane-pyridine complex or zinc borohydride. The reduction of XX-(c) is carried out in solvents from the group tetrahydrofuran, dioxane, glyme, diglyme, triglyme, 2-methoxy-2-methylpropane, acetonitrile, dimethylformamide, water, methanol, ethanol, 2-propanol, 2-methyl-2- propanol and mixtures of these solvents at temperatures ranging from -90 °C to 80 °C.

2. Crosslinking method "Polyether" XXIV (Fig. 26). The cross-linking unit is the highly reactive polycarbonyl-α,ω-biscarbene XXV (Fig. 27), where the symbol n stands for 2 to 8, which must be generated in situ due to instability.

The XXIV “polyether” crosslinking method is performed by adding the polycarbonyl-α,ω biscarbene XXV precursor to a suspension of nanoparticles containing the free hydroxyls of XXIV-(a) saccharide units.

Reaction XXIV-(a) is carried out in the temperature range of -80°C to 95°C, preferably at temperatures lower than the transition temperature parameter and the freezing point of the alkane of the nanoparticle core.

Reaction XXIV-(a) is carried out in solvents from the group of acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, dichloromethane, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, diethyl ether, 2-methoxy- 2-methylpropane and their mixtures.

The precursor of polycarbonyl-α,ω-biscarbene XXV in reaction XXIV-(a) is bis-diazopolyketone XXVI (Fig. 28), where the symbol n means 2 to 8, which is prepared from precursors XXVII and used in reaction XXIV-(a) directly or is itself generated in situ in the reaction mixture from precursors XXVII (Fig. 29), where n is 2 to 8, where the symbol SS is H or phenyl or pentafluorophenyl or trifluoromethyl, where the symbol RR is methanesulfonyl, 4-toluenesulfonyl, 2 -toluenesulfonyl, 2,4,6-triisopropylbenzenesulfonyl, trifluoromethylsulfonyl, 4-nitrobenzenesulfonyl, 4-acetaminobenzenesulfonyl, 4carboxybenzenesulfonyl, where the symbol Me denotes a cation of sodium, lithium, potassium or a quaternary ammonium salt.

Bis-diazopolyketone XXVI is formed from precursors XXVII by the action of agents from the group N,N'-bis-4- 20 CZ 309934 B6 toluenesulfonylhydrazine, N,N'-bis-2-toluenesulfonylhydrazine, N,N'-bis-(2,4, 6triisopropylbenzenesulfonyl)hydrazine, N,N'-bis-methanesulfonylhydrazine, 4dodecylbenzenesulfonyl azide, 4-carboxybenzenesulfonyl azide, 4-acetamidobenzenesulfonyl azide, 4-nitrobenzenesulfonyl azide, 2,4,6-trinitrobenzenesulfonyl azide, 4-chlorobenzenesulfonyl azide, methanesulfonyl azide, 2,4,6-triisopropylbenzenesulfonyl azide, 2-naphthalenesulfonyl azide , diphenylazidophosphate, 2-azido-3-ethyl-1,3-benzothiazolium tetrafluoroborate, 2-azido-1-ethylpyridinium tetrafluoroborate, (azidochloromethylene)dimethylammonium chloride, azidotris(diethylamino)phosphonium bromide, triflylazide, imidazole-1-sulfonylazide, 2- azido-1,3dimethylimidazolium chloride, 1H-1,2,3-benzotriazol-1-yl-sulfonylazide, nonafluorobutanesulfonylazide, triethylamine, trimethylamine, N,N,N',N'-tetramethylethylenediamine, N-methylmorpholine, N,N-diisopropylethylamine , 1,8-bis(dimethylamino)naphthalene, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[2.2.2 ]octane, 7methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene, 4-(N,N-dimethylamino)pyridine, 4-(N,Ndiethylamino)pyridine, 4-(4-pyridyl) morpholine, 4-(1-pyrrolidinyl)pyridine, 2,6-lutidine, 2,4,6-collidine, 2,6-di-tert.-butylpyridine, 1H-1,2,3-benzotriazole, imidazole, triphenylphosphine, triethylphosphine, tributylphosphine, triethylphosphite and or from combinations of these agents.

Polycarbonyl-α,C')-biscarbene XXV is formed from bis-diazopolyketone XXVI by the action of reagents and catalysts from the group of copper chloride, copper acetylacetonate, copper trifluoromethanesulfonate, dirhodium tetraacetate, palladium chloride and their complexes with organic ligands. The reaction is carried out without or in the presence of stabilizers and carbene carriers from the group of organic sulfides, preferably in the presence of tetrahydrothiophene.

After carrying out the reaction XXIV-(a), the polyether XXIV-(b) is reduced to the polyol XXIV-(d) by the effect of a reducing agent from the group sodium borohydride, potassium borohydride, sodium cyanoborohydride, borane or agents that are prepared in advance from agents of this group or in situ, preferably by the effect of sodium borohydride. The reduction of XXIV-(c) is carried out in solvents from the group tetrahydrofuran, dioxane, glyme, diglyme, triglyme, 2-methoxy-2-methylpropane, acetonitrile, dimethylformamide, water, methanol, ethanol, 2-propanol, 2-methyl-2- propanol and mixtures of these solvents at temperatures ranging from -90°C to 80°C, preferably in methanol at room temperature.

3. "Polysaccharide" cross-linking method XXVIII (Fig. 30). The crosslinking unit is a highly reactive carbohydrate biscarbene XXVIII-(a) or triscarbene XXVIII-(c) or their mixture, which must be generated in situ due to instability and possible intramolecular reactions.

Reaction XXVIII is carried out by adding the carbohydrate carbene precursor XXIX (Fig. 31) to a suspension of nanoparticles containing free hydroxyls of carbohydrate units XXVIII, where n means 2 or 3, where Prot means methoxycarbonyl, ethoxycarbonyl, tert.-butyloxycarbonyl, acetoxyl groups, which form all hydroxyls that are protected carbohydrate.

Reaction XXVIII is carried out in the temperature range of -80 °C to 95 °C, preferably at temperatures lower than the transition temperature parameter and the freezing point of the alkane of the nanoparticle core.

Reaction XXVIII is carried out in solvents from the group of acetonitrile, N,N-dimethylformamide, N,Ndimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, dichloromethane, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, diethyl ether, 2-methoxy-2-methylpropane and their mixtures .

The precursor of the carbene of saccharide XXIX in reaction XXVIII are hydrazones XXX-(a) or Nnitrosoamides XXX-(b) of saccharide (Fig. 32), from which carbene XXIX is generated in situ in the reaction mixture,

- 21 CZ 309934 B6 where n means 2 or 3, where the symbol RR means methanesulfonyl, 4-toluenesulfonyl, 2-toluenesulfonyl, 2,4,6triisopropylbenzenesulfonyl, trifluoromethanesulfonyl, 4-nitrobenzenesulfonyl, 4acetaminobenzenesulfonyl, 4-carboxybenzenesulfonyl, benzoyl, 3,5 -dinitrobenzoyl, aminocarbonyl, methoxycarbonyl, ethoxycarbonyl, tert.-butyloxycarbonyl, acetyl, pivaloyl, where the symbol Me denotes a cation of sodium, lithium, potassium or a quaternary ammonium salt, where Prot denotes the groups acetoxyl, methoxycarbonyl, ethoxycarbonyl, tert.butyloxycarbonyl, which form the protected all hydroxyls of the saccharide.

The saccharide carbene XXIX is formed from the precursors XXX by the action of agents from the group of potassium carbonate, sodium carbonate, lithium carbonate, sodium bicarbonate, potassium bicarbonate, triethylamine, trimethylamine, N, N, N', N'-tetramethylethylenediamine, N-methylmorpholine, N, Ndiisopropylethylamine, 1,8-bis(dimethylamino)naphthalene, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[2.2.2] octane, 7-methyl-1,5,7triazabicyclo[4.4.0]dec-5-ene, 4-(N,N-dimethylamino)pyridine, 4-(N,N-diethylamino)pyridine, 4-(4pyridyl)morpholine , 4-(1-pyrrolidinyl)pyridine, 2,6-lutidine, 2,4,6-collidine, 2,6-di-tert.-butylpyridine, 1H-1,2,3-benzotriazole, imidazole, triphenylphosphine, triethylphosphine , tributylphosphine, triethylphosphite or by the action of combinations of these agents.

Carbene XXIX is generated and reacts with hydroxyls of saccharide units in reaction XXVIII without or under the influence of agents and catalysts from the group of copper chloride, copper acetylacetonate, copper trifluoromethanesulfonate and dirhodium tetraacetate and their complexes with organic ligands. The reaction is carried out without or in the presence of stabilizers and carbene carriers from the group of organic sulfides, preferably in the presence of tetrahydrothiophene.

After carrying out the reaction XXVIII-(a) and XXVIII-(c), the polysaccharide XXVIII-(b) and XXVIII-(d) are obtained by removing the protecting groups from the skeleton of the crosslinking units. The removal of protective supine is carried out by the effect of compounds from the group of potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium methanolate, sodium ethanolate, potassium methanolate. The removal of protective supines is carried out in solvents from the group of water, methanol, ethanol, 2-propanol, 2-methyl-2-propanol, 1,2-ethanediol and mixtures of these solvents at temperatures ranging from 0 °C to 80 °C, preferably in 90% wt. aqueous methanol at room temperature.

4. "glycidether" crosslinking method XXXI (Fig. 33). The crosslinking unit is the highly reactive glycidyl derivative XXXI-(a), where the symbol W stands for chlorine, bromine, methanesulfonoxyl, 4-toluenesulfonoxyl, 2-toluenesulfonoxyl, 2,4,6-triisopropylbenzenesulfonoxyl, trifluoromethanesulfonoxyl, 4-nitrobenzenesulfonoxyl, 4-acetaminobenzenesulfonoxyl, 4carboxybenzenesulfonoxyl.

The "glycidether" crosslinking method XXXI is performed by adding the glycidyl derivative XXXI-(a) to a suspension of nanoparticles containing free hydroxyls of saccharide units.

Reaction XXXI-(b) is carried out in the temperature range of -80°C to 95°C, preferably at temperatures lower than the transition temperature parameter and the freezing point of the alkane of the nanoparticle core.

Reaction XXXI-(b) is carried out in solvents from the group of acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, dichloromethane, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, diethyl ether, 2-methoxy- 2-methylpropane and their mixtures.

Reaction XXXI-(b) is carried out without or in the presence of reagents from the group potassium carbonate, sodium carbonate, lithium hydroxide, sodium hydroxide, potassium hydroxide, sodium methanolate,

- 22 CZ 309934 B6 lithium methanolate, potassium methanolate, sodium ethanolate, sodium hydride, potassium tert.-butanolate, sodium tert.butanolate, triethylamine, trimethylamine, N,N,N',N'-tetramethylethylenediamine, Nmethylmorpholine, N,N- diisopropylethylamine, 1,8-bis(dimethylamino)naphthalene, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[2.2. 2]octane, 7methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene, 4-(N,N-dimethylamino)pyridine, 4-(N,Ndiethylamino)pyridine, 4-(4-pyridyl )morpholine, 4-(1-pyrrolidinyl)pyridine, 2,6-lutidine, 2,4,6-collidine, 2,6-di-tert.-butylpyridine, lithium perchlorate, lithium chloride, lithium bromide, lithium iodide, nitrate lithium, lithium hexafluorophosphate, lithium tetrafluoroborate or in the presence of combinations of these agents.

The mentioned variants of the production methods make it possible to achieve the exact properties of spherical hollow nanoparticles with macroholes from the point of view of the following parameters:

1. Outer diameter of spherical particles. It is controlled by the diameter of the stabilized alkane-surfactant micelles (production variants 1 to 3) and the thickness of the nanoparticle's outer skeleton, which is controlled by the number of isocentric layers and the cross-linking rate of the skeleton. In accordance with Li. C., e.a.: "Formation and properties of paraffin wax submicron emulsions prepared by the emulsion inversion point method" Colloids and Surfaces A: Physicochem. Eng. Aspects 356, 71 (2010) and Hessien M., e.a.: Stability and Tunability of O/WNanoemulsions Prepared by Phase Inversion Composition Langmuir 27, 2299 (2011) it is a function of several variables: HLB of the surfactant or its mixture, stoichiometric ratio alkane - surfactant, concentration of surfactant in the medium (alkane concentration in the medium).

2. Diameter of macrohole and macrodiffusion hole. It is controlled by the type of steric blocker XVI used, the ratio of the diameters of the micelle nanoparticles: steric blocker and, to a limited extent, by the method of formation of the binary micelle - blocker particle. The type of steric blocker affects the range of embedding depth into the micelle, which is a function of the surface tension of the micelle and the entire blocker skeleton (hydrophobic and hydrophilic parts, or the part that is in a hydrophilic environment). Polymeric spherical nanoparticles XVI-(a) from polystyrene, possibly hydrophobized, do not contain native amphiphilic hemispheres and therefore the degree of embedding into the micelle is the most variable. Binary anisotropic polystyrene-alkane nanoparticles XVI-(c) provide significantly more defined macropore diameters, and unilaterally hydrophilized spheroidal polystyrene nanoparticles XVI-(i) provide macropores with high precision.

3. Diameter and areal density of diffusion channels. The diameter of the diffusion channels is controlled by the size of the carbohydrate structural units and the type and density of their cross-linking. Large structural units such as oligosaccharides with a low degree of cross-linking provide large diffusion channels, while small units with a high degree of cross-linking provide small diffusion channels. The area of the diffusion channel usually corresponds to the square of the length of the carbohydrate unit. Another factor controlling the diffusion channels is the degree of cross-linking, the type (especially the length of cross-linking units - linkers) of cross-linking XIX and the number of isocentric layers of the skeleton. Large diffusion channels are obtained if only carbohydrate units are cross-linked and the number of isocentric layers is low, small diffusion channels are obtained, on the contrary, if the subsequent cross-linking also includes the own cross-linking linker during the construction of isocentric layers XIX. A very important factor influencing the diameter and surface density of the diffusion holes is the surface density of the saccharide units allocated on the micelles. This is controlled (together with the micelle diameter) by the HLB of the surfactant or its mixture, the stoichiometric ratio of alkane - surfactant and the concentration of the surfactant in the medium (alkane concentration in the medium). The technology also allows the use of surfactant phantoms, i.e. in a mixture of surface-active substances with hydrophilic carbohydrate units (the second production approach) to have inert surfactants to adjust the HLB of the mixture or precisely so that part of the hydrophilic positions on the surface of the micelle are not occupied by carbohydrate units and thus greater distances between these units are achieved without micelle parameters were affected. Similarly (first and third production approach) part of the sulfonate surfactants can be replaced by neutral surfactants and thereby reduce the density of SO3H groups on the surface of the micelles, which results in a reduction of the surface density electrostatically

- 23 CZ 309934 B6 sorbed units of amino(deoxy)saccharides.

4. Elasticity and mechanical strength of particles. They are controlled by the degree and type of cross-linking, as well as the number of isocentric layers. A skeleton formed from disaccharide units with a low degree of cross-linking with long cross-linking linkers and a low number of isocentric layers will be very elastic and conformable in case of mechanical stress. The highly cross-linked skeleton with monosaccharide units with short cross-linking linkers will be very rigid and mechanically resistant.

5. Biodegradability and metabolizability. They are controlled primarily by the type of carbohydrate units used, the type and density of their cross-linking. The polyester XX crosslinking method is used to produce nanoparticles with the shortest half-life in the body, as biodegradation occurs as a result of the simple hydrolysis of ester groups derived from the secondary and primary hydroxyls of carbohydrates under the influence of pH (generally bases) in body fluids. Crosslinking methods polyether XXIV, polysaccharide XXVIII and glycidether XXXI are carriers of approximately identical biodegradability and metabolizability of the resulting nanoparticles and differ primarily in the type of metabolites. The arrangement of carbohydrate units, the type and density of their cross-linking form the so-called nano-time lock of the skeleton.

6. Density of antiviral active substances inside the nanoparticle. It is controlled by the surface density of functionalizable groups (amino groups: first and second production approaches), which is controlled by the surface density of (deoxy)saccharide units containing amino groups in the micelle formation process and by the surface density of functionalized saccharide units in the micelle formation process (third approach), i.e. fully analogously controlling the diameter and areal density of diffusion channels. The increasing density of antiviral active substances inside the nanoparticle has a fundamental impact on the application therapeutic effect.

Antiviral effective spherical hollow nanoparticles with macroholes prepared according to the present invention exhibit the following properties:

They have excellent chemical stability in aqueous solutions in the pH range of 3 to 11, especially in the physiological pH range, as they are composed only of covalent bonds of a hydrophilic polymer.

They have excellent physicochemical parameters, especially thermal and photochemical stability up to temperatures of 130 °C, and the nano-antiviral agent based on the concept of this invention can therefore be easily sterilized, easily lyophilized with steam sterilization or with a temperature curve above 100 °C, or easily vacuum dried without the mechanical parameters of the nanoparticle were disturbed.

They have a wide variability of the skeleton, the possibility of using different saccharide building units, and also the modifiability of mutual interconnection of saccharide building units in the wall polymer skeleton by choosing different crosslinking units and degree of crosslinking. The consequence of modifying the skeleton in this way is the unprecedented modifiability of biodegradability in the organism, controlled by the resistance of the skeleton polymer to the effect of extracellular enzymes and the effect of bases and nucleophiles in body fluids. In this way, the half-life of the nanoparticle in the organism can be controlled very effectively.

They have selective functionalizability and modifiability of the inner wall of the skeleton and thus create specific encapsulation cavities (cavities) without any effect on the external parameters (surface charge, hydrophilicity) of the nanoparticle skeleton. The outer wall of the skeleton has an extremely low affinity for peptides and lipids with respect to the building units and their degree of cross-linking.

They have metabolic products that are gentle on the human body. The product of biodegradation and metabolism of the nanoparticle skeleton are fragments of carbohydrates and carbohydrate metabolites, which are the body's own substances that serve either as cellular nutrition or as addresses for renal

- 24 CZ 309934 B6 transport from the organism (glucuronic acid), in all cases without any negative physiological functions and properties.

They have a suitable spherical shape for cyclic transport in the laminar flow of the vascular system, because the spherical shape concentrates the particles in the center of the capillary flow in the place with the maximum flow gradient, and conversely reduces the permeability through the cell walls (Doshi N., e.a.: Red blood cell-mimicking synthetic biomaterial particles Proc. Natl. Acad. Sci. USA 106, 21495 (2009); Decuzzi P., e.a.: Intravascular delivery of particulate systems: does geometry really matter? Pharm. Res. 26, 235 (2009)).

Antiviral-active spherical hollow nanoparticles with macroholes produced by the methods described above act in the manner shown in Scheme XXXII (Fig. 34).

The antiviral nanotherapeutic is placed in the body fluid, where it moves due to interactions with the moving fluid and its components (e.g. recirculation in the vascular system) or is carried away by free fluctuation. During the movement, stochastically controlled collisions occur with the contained fluid particles. Ions, molecules and supramolecules with an area up to the size of the effective area of the diffusion holes diffuse through the wall of the skeleton of the nanotherapeutic, or are guided by convection and, like larger supramolecular structures (typically: peptides) and fragments of vital particles, flow in the liquid through the macrohole or through the macrodiffusion holes present into the inner cavity of the nanotherapeutic . In the case of application of a nanotherapeutic agent in the vascular system, vital blood particles cannot enter the nanotherapeutic agent due to their size and are thus protected by the nanotherapeutic agent's skeleton against unwanted interaction with viricides located on the inner wall of the nanotherapeutic agent. However, pathogenic virus particles have a smaller diameter than the diameter of the macrohole, and due to statistical reasons, in case of effective collision, they enter the nanotherapeutics, where free movement limited by the space of the cavity inevitably hits the walls, which are modified by covalently bound viricides. At the surface of the inner wall, there is an extreme concentration of viricides, and at the point of contact between the virus and the inner wall, there is an interaction between the virion or capsid of the pathogen, and it is destructively irreversibly inactivated, i.e. it dies. Fragments of the destroyed pathogen are washed out through the macrohole or macrodiffusion holes present on the outside of the nanotherapeutic by continuous convection. The described principle of the function can best be described by the term - virus trap.

Depending on the setting of the so-called time nanolock of the skeleton, the skeleton of the nanotherapeutic is also gradually degraded. By metabolizing the skeleton, covalently bound viricide units are formed into molecules that act as a directive for renal excretion in the body, especially derivatives of glucuronic acid and its analogues. The skeleton of nanotherapeutics therefore already has a built-in directive (method) for the future elimination of xenobiotic metabolites of viricides from the organism. This significantly reduces the hemolytic activity (generally cytotoxicity) of the metabolites.

The designed antiviral effective nanotherapeutics brings a completely new pharmacokinetic and pharmacodynamic dimension of action and therapy XXXIII (Fig. 35). Existing molecular antivirals are limited by the so-called Selectivity Action Index (SAI), which is dependent on the activity (effective concentration) of the antiviral in tissue or fluid. Therefore, certain manifestations of cytotoxicity always occur when therapeutically effective activities are achieved, although often without acute pathological manifestations. Therapeutically effective activity, however, simultaneously means the achievement of a sufficient number of effective collisions of the molecular antiviral agent with pathogens. The frequency of these precipitations increases with the activity of the antiviral, and the low efficiency or ineffectiveness of a number of molecular antivirals is precisely due to the too low therapeutic activity imposed by SAI. Nevertheless, the basic advantage of the concept of molecular antivirals is precisely the high frequency of effective antiviral-pathogen collisions caused by the relatively high activity of antiviral molecules and the free movement of molecules.

On the other hand, antivirally effective nanotherapeutics are therapeutic means of macro dimensions, and their use cannot in principle be connected with the therapeutic activities of molecular antivirals. For example, a standard dosage of the antiviral oseltamivir 75 mg per day produces therapeutic activities in blood plasma of 0.33 μM (Brian E. Davies: Pharmacokinetics

- 25 CZ 309934 B6 of oseltamivir: an oral antiviral for the treatment and prophylaxis of influenza in diverse populations J. Antimicrob. Chemother. 65 (suppl 2), ii5-ii10 (2010)), which corresponds to roughly 2.10 ¹⁶ antiviral molecules per liter of plasma. In contrast, the intravenous therapeutic application of 1 ml of antivirally effective nanotherapeutics with a diameter of 2 microns means the presence of approximately 2.10 ¹⁰ nanoparticles in a liter of plasma, and an adequate volume of 75 mg would even be only 1.5.10 ⁹ nanoparticles. A decrease in therapeutic activity by seven orders of magnitude has an impact on the frequency of effective precipitation, which is why molecular antivirals have a higher probability of effective precipitation XXXIII than antiviral nanotherapeutics at the same mass therapeutic dose.

However, the fundamental advantage of the described concept of an antiviral effective nanotherapeutic compared to a molecular antiviral is the state after effective precipitation. Not every effective collision of a molecular antiviral with a pathogen leads to a therapeutic effect. For example, the H1N1 influenza virus shows a virus load in the blood plasma of up to 6.10 ⁵ clones/ml in patients with a severe course (Hung IFN, ea: Convalescent Plasma Treatment Reduced Mortality in Patients With Severe Pandemic Influenza A (H1N1) 2009 Virus Infection Clin. Infect. Dis .(2011); doi: 10.1093/cid/ciq106), which means that from the point of view of the pharmacokinetic collision model, more than 10 ⁷ antiviral molecules per unit of virus account for the above-mentioned therapeutic activity of the molecular antiviral. Nevertheless, the therapeutic effect of the molecular antiviral is not very convincing, or it does not appear at all.

Antivirally effective nanotherapeutics, on the other hand, use the synergism of higher therapeutic efficiency of viricides, from which vital plasma cells are protected by the wall of the nanotherapeutic, on the one hand, and high local concentrations of viricides at the surface of the inner wall of the nanotherapeutic, i.e. in the space of the cavity, on the other hand. While in plasma the marginal therapeutic activities of molecular antivirals are in the order of tenths, max. units of μΜ, at the surface of the inner wall of nanotherapeutics they can reach values of tenths to units of M, i.e. values 10 ⁶ to 10 ⁷ times higher. Therefore, from the point of view of collision coordinates (see XXXIII), the probability of achieving inactivation of the pathogen trapped in the cavity is orders of magnitude higher in the case of nanotherapeutics. The key pharmacokinetic factor of the application of an antiviral effective nanotherapeutic, i.e. the rate-determining event, therefore, is the phase of achieving effective precipitation, while the probability of effective precipitation in the plasma increases with the concentration of the nanotherapeutic.

Antiviral effective spherical hollow nanoparticles with macroholes and macrodiffusion holes in the arrangement produced by the methods described above have the following application advantages over existing solutions:

1. Minimal cytotoxicity and hemolytic activity against native (vital) cells in the organism due to the effect of the mechanical sieve of the macrohole, which is a selector (an element that makes a choice) between fluid particles that are allowed into the cavity and which are not. This principle of mechanical selection is not burdened by defects in receptor and membrane interactions and, in principle, viral pathogens cannot develop resistance to it. Antivirally effective nanotherapeutics XXXII are therefore the literal fulfillment of the concept of targeted medicine. Current molecular antivirals and their metabolites, on the other hand, act across the body and thus affect all cells (and tissues).

2. Full biodegradability of the nanotherapeutic skeleton after the end of the effect. This is controlled by the half-life of the skeleton, which is the result of the degree and type of cross-linking of the saccharide units, as well as the type of saccharide units of the skeleton, which forms the so-called time nanolock. The products of biodegradation and metabolism of the skeleton are native substances of zero toxicity corresponding to the breakdown of carbohydrates. Lactate is not produced by breaking down the skeleton, i.e. unwanted local anti-inflammatory processes are not triggered by the induction of cytokines (which is a fundamental difference from PLGA and similar hydrophilic polymers, see Dobrovolskaia M.A. e.a.: Immunological properties of engineered nanomaterials Nature Nanotechnology 2, 469 (2007)).

3. Internally built-in directive for the elimination of covalently bound viricide to the skeleton of the nanotherapeutic. The skeleton is therefore already native, recognizable and usable by the organism

- 26 CZ 309934 B6 function of future xenobiotic detoxification from the organism.

4. The exceptional biocompatibility of the skeleton resulting from the use of a polysaccharide skeleton (by analogy: Ficoll PM 70, Ficoll PM 400 Cell Separation 18-1158-27 AA, 2001-11 (Amersham Biosciences, 2011); Andrade F.K., e.a.: Studies on the biocompatibility of bacterial cellulose Journal of Bioactive and Compatible Polymers 28(1), 97 (2013); Dugan J.M., et.a.: Bacterial cellulose scaffolds and cellulose nanowhiskers for tissue engineering Nanomedicine 8(2), 287 (2013); Helenius G., et.a.: In vivo biocompatibility of bacterial cellulose DOI: 10.1002/jbm.a.30570)).

5. Minimal affinity for complement peptides and subsequently opsonins, which is a consequence of the highly hydrophilic surface of the skeleton without surface functional groups carrying a charge (zero electrokinetic, zeta potential). This suppresses the undesirable process of phagocytosis leading to the elimination of the nanotherapeutic from the organism as a whole (by analogy: Tabata Y., e.a.: Phagocytosis of Polymer Microspheres by Macrophages New Polymer Materials, DOI: 10.1007/BFb0043062). Of the important components of blood plasma, they show zero affinity to albumin, apolipoproteins, immunoglobulins, complement proteins, fibronectin, fibrinogen and C-reactive protein, they do not show any immunostimulatory or immunogenic properties (antigenicity), nor properties of antigenic adjuvants.

6. A biologically neutral highly hydrophilic surface containing a high surface density of aliphatic hydroxyls in the entire volume of the skeleton, which does not need to be specifically modified in any way. It is the surface containing a high surface density of aliphatic hydroxyls that is the carrier of immunity to complement proteins, and therefore opsonization (Reddy S.T., e.a.:

Exploiting lymphatic transport and complement activation in nanoparticle vaccines Nat. Biotechnol. 25, 1159 (2007)). Unlike hydrophobic nanoparticles, which require surface hydrophilization, most often by the introduction of PEG functional groups, which is not without risk, as mere modification of the PEG surface will never completely prevent the deposition of complement proteins and the subsequent triggering of the complement cascade (Hamad I., e.a.: Distinct polymer architecture mediates switching of complement activation pathways at the nanosphere-serum interface: implications for stealth nanoparticle engineering ACS Nano 11, 6629 (2010); Moghimi S.M., e.a.: Complement activation cascade triggered by PEG-PL engineered nanomedicines and carbon nanotubes: the challenges ahead J. Control. Release 146, 175 (2010)), possibly also through undesirable affinity to immunoglobulins (Chen H., e.a.: Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Forster resonance energy transfer imaging Proc. Natl. Acad. Sci. U.S.A. 105, 6596 (2008); Fast release of lipophilic agents from circulating PEGPDLLA micelles revealed by in vivo Forster resonance energy transfer imaging Langmuir 24, 5213 (2008)). Moreover, modification of the surface of the PEG nanoparticle causes serious pseudoallergenic reactions (Chahan-Khan A., e.a.: Complement activation following first exposure to pegylated liposomal doxorubicin (Doxil(R)): possible role in hypersensitivity reactions Ann. Oncol. 14, 1430 (2003)) .

7. Primarily a mechanically and interactively stable and robust skeleton, which is caused by the hydrophilic units of carbohydrates in the hydrophilic environment of the organism, which are cross-linked by hydrophilic linkers into a whole in which torsion angles can be eliminated. Stability is essential in flowing fluids due to collisions and possible mechanical stress. In the case of poorly mechanically resistant liposomes and micelles, an undesirable change in shape can occur and this can lead to easier opsonization, in the case of micelles made of hydrophilic PEG lactate esters, to interactions with immunoglobulins and cell walls (Chen H, e.a.: Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Forster resonance energy transfer imaging Proc. Natl. Acad. Sci. U. S. A. 105, 6596 (2008); Fast release of lipophilic agents from circulating PEG-PDLLA micelles revealed by in vivo Forster resonance energy transfer imaging Langmuir 24, 5213 (2008) ).

8. A suitable spherical shape equipped with at least one macro-hole, possibly i

- 27 CZ 309934 B6 macrodiffusion holes and many diffusion holes for optimal recirculation in the vascular system. The spherical shape ensures the aggregation of the nanotherapeutic in the place with the greatest flow gradient, i.e. in the middle of the blood vessels (Doshi N., e.a.: Red blood cell-mimicking synthetic biomaterial particles Proc. Natl. Acad. Sci. USA 106, 21495 (2009); Decuzzi P. , e.a.: Intravascular delivery of particulate systems: does geometry really matter? Pharm. Res. 26, 235 (2009)), the presence of multiple macroholes increases the probability of effective collision with the pathogen, and in the laminar flow of the vascular system, with the presence of each additional macrohole, a completely specific orientation of the nanotherapeutic in the flow gradient (the hydrodynamic properties of the shape change). The presence of macrodiffusion holes has a fundamental positive impact on the hydrodynamic resistance of the nanotherapeutic in both laminar and turbulent flow and the orientation of the nanoparticle in the flow.

9. A multi-antiviral effect, i.e. an effect independent of the type (class) and strain of the inactivated pathogen. They are therefore basically broad-spectrum antiviral therapeutics, which is a consequence of the effectiveness of strong covalently bound viricides. Any number of types of viricides with different accents on different types of viral pathogens can be covalently immobilized on the inner wall of the nanotherapeutic, thus further multiplying the broad-spectrum effect.

10. Selectivity between pathogens. If it is therapeutically desirable, the type of immobilized viricides and the diameter of the macrohole can achieve significant selectivity between classes and types of pathogens from the point of view of their inactivation, and the arrangement thus creates a selective antiviral nanotherapeutic.

11. Therapeutic effectiveness, because the half-life of antivirally effective XXXII nanotherapeutics can be in the order of tens of days, and during its life cycle each nanoparticle inactivates an order of magnitude higher number of viral pathogens, while with current molecular antivirals, the half-life of the medicinal substance is of the same order as the life cycle - replication. With existing molecular antivirals, either regular dosing is needed to maintain therapeutically active levels, or special depot forms for the same. In both cases, the organism is continuously burdened with xenobiotics, which brings with it significant secondary effects and risks. Therefore, with the antivirally effective nanotherapeutics XXXII, only a single therapeutic dose can be sufficient to inactivate all viral pathogens, a repeated dose can only serve to eliminate the risk of the presence of residual viruses and their transfer from other tissues or fluids.

Clarification of drawings

Giant. 1: Hollow spherical nano-particle with macroholes; Giant. 2: Hollow spherical nano-particle with macroholes; Giant. 3: The skeleton of a nanoparticle, which is formed by a covalently bound three-dimensionally cross-linked monodisperse polymer of A units; Giant. 4: The A unit of the nanoparticle skeleton, which is formed from the B units; Giant. 5: Unit B of the nanoparticle skeleton, which is formed from a saccharide; Giant. 6: Fragment of unit B; Giant. 7: Fragment of Unit B; Giant. 8: Fragment of Unit B; Giant. 9: Fragment of Unit B; Giant. 10: Linker; Giant. 11: Method of preparation of nanoparticles; Giant. 12: Immobilization of a biologically active substance (biocide) on the inner wall of the cavity; Giant. 13: Use of a steric blocker in the preparation of nanoparticles; Giant. 14: Lipophilic surfactant chain; Giant. 15: Lipophilic surfactant chain; Giant. 16: Building a nanoparticle skeleton; Giant. 17: Hydrophilic surfactant chain; Giant. 18: Steric blocker; Giant. 19: PEGylation of a steric blocker; Giant. 20: PEGylation of a steric blocker; Giant. 21: Cross-linking and skeleton building; Giant. 22: Cross-linking and skeleton building; Giant. 23: Cross-linking agent Fig. 24: Reactive forms of cross-linking agents; Giant. 25: Reactive form of cross-linking agent; Giant. 26: Cross-linking and skeleton building; Giant. 27: Reactive form of cross-linking agent; Giant. 28: Cross-linking agent; Giant. 29: Cross-linking agents; Giant. 30: Cross-linking and skeleton building; Giant. 31: Cross-linking agent; Giant. 32: Cross-linking agent; Giant. 33: Cross-linking and skeleton building; Giant. 34: Nanoparticle Effect on Pathogens; Giant. 35: Nanoparticle effect on pathogens compared to current antivirals.

- 28 CZ 309934 B6

Examples of implementation of the invention

Example 1

A mixture of 285 g of sodium hexadecylbenzene sulfonate, 215 g of sodium dodecane sulfonate (Alfa) and 2000 g of paraffin with a melting point of 52 °C is thoroughly mixed at 80 °C in a 20-liter container, and a total of 7500 ml of heated water is added to the mixture with intensive stirring during 15 minutes to 80 °C. The mixture is stirred for another 15 minutes at the same temperature, and polystyrene nanospherical particles with a diameter of 0.60 microns (PDI 1.03) are gradually added in approximately 5 g amounts, which have been hydrophobicized by previously standing in cyclohexane for 45 minutes and thoroughly dried under vacuum. The distribution of anisotropic particles in the system is monitored by optical microscopy and the addition is interrupted if the proportion of 2:1 systems exceeds 4% in the emulsion. A total of 320 g of polystyrene nanospherical particles are added within 40 minutes, which corresponds to an average stoichiometry of 1:2 of 103%. The temperature of the mixture is allowed to fall to 15°C during 15 minutes with very gentle stirring, and the resulting mixture is allowed to coagulate with stirring at the same temperature for at least another 180 minutes. Then the resulting dense suspension is vacuum filtered, the nanoparticles mixed with 5000 ml of water are carefully poured into a column with a frit, and a total of 1350 ml of 5% by weight is poured through the column within 30 minutes. hydrochloric acid and then deionized water until the acid reaction of the eluate is lost. The nanoparticles are washed on the column with dry methanol and then dried at laboratory temperature under vacuum. The yield is 99.1%, i.e. 2755 g of dry anisotropic precursor particles with an allocated steric blocker and sulfo groups in the form of free acid. All operations are performed without the use of mechanical aids, which can easily irreversibly deform soft nanoparticles.

2480 g of well-dried potassium hydrogenglucarate (prepared according to TN Smith, PhD Degree Thesis, The University of Montana (2008), p. 19 - 24; Smith TN, ea: Modifications in the nitric acid oxidation of D-glucose Carbohydrate Research 350, 6 (2012)) is added with constant stirring over 15 minutes to a 5 °C cooled solution prepared from 2000 ml of freshly distilled acetyl chloride and 13 liters of anhydrous methanol, which has been allowed to stand at 0 °C for 180 minutes. After all the potassium salt has been added, the temperature is allowed to rise to room temperature with constant stirring and then raised to boiling and the mixture is refluxed for 12 hours. After cooling to 0 °C, the precipitated potassium chloride is sucked off sharply, the filter is washed with dichloromethane and the combined filtrates are evaporated under vacuum to a yellowish, very viscous oil. 2320 g (96.2%) of dimethylglucarate is obtained. 1H NMR (DMSO-d6, 300 MHz) ď 3.63 (s, 6H, OCH3), 3.77 (m, 2H, OH), 4.30 (d, J = 8.1 Hz, 2H, CH- OH), 4.80 (m, 2H, OH), 4.91 (d, 2H, J = 8.1 Hz, CH-OH); ¹³ C NMR (DMSO-d 6 , 101 MHz) δ 51.43, 70.27, 71.20, 174.11.

240 g of dry dimethylglucarate are dissolved in 5 liters of dry 1,2-dichloroethane, 2230 g of well-ground dry N-bromosuccinimide are added and the mixture is kept at 90°C with constant stirring. After 11 hours, the reaction mixture begins to darken with the released bromine and is then stirred at 90 °C for at least 1 hour. The mixture is cooled to room temperature, allowed to stand for 2 hours, filtered sharply, concentrated on a vacuum evaporator to a volume of approximately 0.7 liters, cooled to 10 °C and 2 liters of dry, freshly redistilled diethyl ether are added. The resulting mixture is allowed to stand for 12 hours at room temperature, the residue is filtered off and the residue is evaporated under vacuum at 60°C to a yellow oily dimethyltetraoxoadipate, 193.5 g (84%), which is used in further reactions without purification. Ή NMR (CDQ 3 , 300 MHz) δ 3.94 (s, 6H, OCH 3 ). The crude ester is dissolved in 9 liters of methanol containing 2% by weight. of water and adds 2400 g of Amberlyst A-26 in OH ^- form. The suspension is vigorously stirred for 24 hours at room temperature, the annex is filtered off, thoroughly washed by pouring methanol over the column, and crude tetraoxoadipic acid is eluted from the column at a rate of 50 ml/min 20% by weight. with a solution of formic acid in methanol. Approximately 2 liters of eluate are taken from the detection of the acidic reaction of the eluate at the exit of the column. The eluate is evaporated to dryness under vacuum at 60°C and, after cooling, 153 g (76% for dimethylglucarate) of light brown 2,3,4,5-tetraoxoadipic acid are obtained. ¹³ C NMR (DMSO-d 6 , 101 MHz) δ 164.91, 182.38, 187.92.

153 g of 2,3,4,5-tetraoxoadipic acid are dissolved in 5800 ml of acetonitrile, cooled to 0 °C,

- 29 CZ 309934 B6 add 421 g of DMTMM tetrafluoroborate (prepared according to Raw S.A.: An improved process for the synthesis of DMTMM-based coupling reagents Tetr. Lett. 50, 946 (2009)), and with constant stirring and cooling during the hour a total of 157 g of N-methylmorpholine. The mixture is then stirred for another hour. Meanwhile, 1409 g of dry anisotropic precursor particles with an allocated steric blocker and sulfo groups in the form of free acid from the previous operation are slowly added with constant stirring at 5 °C to a solution of 286 g of 3-amino-3-deoxysucrose (prepared according to: Simiand C., e.a. : Synthesis of sucrose analogues modified at position 4” J. Carbohydr. Chem., 14, 977 (1995)) in 14.5 liters of water. The resulting mixture is stirred for another 30 minutes, filtered under vacuum and the nanoparticles are thoroughly washed with water, followed by methanol and dried at laboratory temperature under vacuum. The dried nanoparticles are mixed at laboratory temperature in 60 liters of dry acetonitrile (min. 99.9% by weight) and the reaction mixture of DMT active ester of 2,3,4,5-tetraoxoadipic acid is added to the mixture within twenty minutes. The mixture is then stirred for another 6 hours, 100 g of ethanol is added, stirred for another 5 hours, filtered and the solvent is recycled by distillation.

The content of free carboxyls in the nanoparticles is determined by back titration with 4-toluenesulfonic acid after the addition of aqueous ethanolamine with potentiometric indication. If the free carboxyl content of the batch is below 0.1% mol., they are used directly for further operations. If higher, then depending on the content (usually 0.4 to 3.3% mol.) carboxyls are reduced to carbinols either by the methodology described using the reduction of the active ester formed from 2-(1H-1,2,3-benzotriazol-1yl) -1,1,3,3-tetramethyluronium tetrafluoroborate by the action of sodium borohydride (MgGeary R.P.: Facile and chemoselective reduction of carboxylic acids to alcohols using BOP reagent and sodium borohydride Tetr. Lett. 39, 3319 (1998)), or by the iodine - borohydride system - glyme at -10°C (Bhaskar Kanth J.V., e.a.: Selective Reduction of Carboxylic Acids into Alcohols Using NaBH4, and If J. Org. Chem. 56,5964 (1991)). Crude nanoparticles are aspirated and reduced in the solvent system dioxane - methanol (5:1; vol./vol.) at -5 °C by the gradual addition of sodium borohydride according to Correa I.R., e.a.: Diastereoselective Reduction of E and Z Alkoxyimino-ketoesters by Sodium Borohydride Tetrahedron 55, 14221 (1999). The crude nanoparticles are aspirated and dried. After that, the action of four times the amount of DMT active ester of 2,3,4,5-tetraoxoadipic acid is repeated under the conditions described above, the reaction is carried out by stirring for 60 minutes at laboratory temperature in the presence of 10% mol. DMAP and after this time 330 g of sucrose is added over 3 hours and stirring is continued for another 24 hours. The active ester is applied in one dose. The resulting product is reduced from the free carboxyls as described above, and subsequently the polyketones are reduced to polyols. 2351 g of crude nanoparticles with two isocentric layers and polyester cross-linking of the skeleton are obtained.

The crude nanoparticles from the previous operation are suspended in 8 liters of methanol, the mixture is cooled to 20 °C and 4% by weight is added very slowly while stirring. solution of sodium methoxide in methanol under potentiometric monitoring of the basicity of the solution above the suspension. At the first excess of methanolate, the addition is stopped and the mixture is quickly filtered, washed thoroughly with methanol until complete loss of alkalinity, and dried under vacuum. The dried nanoparticles are suspended in 9.5 liters of cyclohexane, the mixture is heated to 80 °C and stirred at this temperature for 2 hours. The suspension visibly changes volume during this time and is filtered while hot through a microfilter with a pore size of 1 micron. Nanoparticles on the filter are extracted three more times in a similar manner with 5 liters of cyclohexane until the extract evaporates to zero. The combined extracts are cooled and after 2 hours the steric blocker is isolated by nanofiltration through a PTFE nanofilter and the cyclohexane is recycled. The steric blocker is then treated alternately at 35 °C with concentrated hydrochloric acid and then with 30% by weight. with methanolic sodium hydroxide at 40 °C, which removes the steric blocker from about 25 to 40% of the residues of the nanoparticle skeleton. After cooling, washing and drying, 291 g (92%) of the steric blocker is recovered.

The hollow spherical nanoparticles from the previous operation (864 g) are functionalized on the amino groups of the inner wall in the last stage:

N-trityl-tris(2-aminoethyl)amine is prepared from 58.4 g of tris(2-aminoethyl)amine according to Vandoorne F., e.a.: New approach to dextran derivatives containing primary amino functions Makromol. Chem. 192, 673 (1991) using dichloromethane as the solvent. 70.7 g is obtained

- 30 CZ 309934 B6 (46%) of the product as a colorless oil. 1H NMR (CDCl3, 300 MHz) ά 7.21 (m, 3H, Ar), 7.34 (m, 6H, Ar), 7.52 (m, 6H, Ar), 2.76 (m, J = 8.2 Hz, 6H, CH 2 ), 2.51 (m, 6H, CH 2 ). 17.6 ml of acetic acid is added to 38.8 g of N-trityl-tris(2-aminoethyl)amine dissolved in 450 ml of methanol and the mixture is cooled to 0 °C. 37.5 g of dodecanal and a total of 6.3 g of sodium cyanoborohydride are added during 20 minutes with vigorous stirring. Stirring is continued for another 5 hours, during which the temperature is allowed to rise to room temperature and 2 g of sodium cyanoborohydride are added. After an hour of stirring, 10 ml of 10% by weight is added to the mixture. of sodium hydroxide and stirred for another 30 minutes. Then the mixture is evaporated under vacuum, the residue is diluted with 30 ml of water and repeatedly extracted with 50 ml of dichloromethane, and the dichloromethane layer is collected. The combined extracts are evaporated to dryness under vacuum to constant weight at 45 °C, the brownish oily residue is dissolved in 400 ml of dichloromethane, 48 ml of DIPEA is added and at -20 °C the solution is added dropwise under a protective atmosphere of Ar during 2 hours with stirring 69 ml of methyl triflate in 250 ml of dichloromethane. The temperature is maintained at -20°C for a further 2 hours, then slowly allowed to rise to 0°C and held there for another hour. It is then kept under stirring at room temperature for another 10 hours, 10 ml of methanol is added and left to stand for 30 minutes. The reaction mixture is evaporated to dryness under vacuum, dissolved in 800 ml of anhydrous methanol, 5 g of Pd/C are added and under an overpressure of 0.4 atm. with stirring, the mixture is saturated with hydrogen for 12 hours at room temperature. The catalyst is filtered from the mixture, the mixture is concentrated to a volume of 200 ml, 4 ml of water are added and the mixture is left overnight in the refrigerator. The separated triphenylmethane is filtered off, thoroughly washed with aqueous methanol (90% by weight) and the combined filtrates are concentrated, extracted with hot (90 °C) 6 liters of water with 120 ml of concentrated hydrochloric acid and the aqueous extract is separated, alkalized with triethylamine to a weakly basic reaction , cooled and extracted with dichloromethane (6 x 50 mL). After evaporation of dichloromethane, 22.3 g (32%) of crude bis(N-dodecyl-NN-dimethyl-2-ammoniumethyl)-N-(2ammoniumethyl)methylammonium tetrachloride are obtained. 1H NMR (DMSO-d6, 300 MHz) ά 2.90 (d, J = 6.2 Hz, 2H, CH2), 2.46 (m, J = 14.9 Hz, 16H, CH2), 1.31 (m, J = 9.9 Hz, 40H, CH 2 ), 1.01 (t, 6H, J = 5.4 Hz, CH 3 ).

Triglycolic acid is prepared from triethylene glycol according to Lehn J.M., e.a.: Tetrahedron 29, 1629 (1973) in a yield of 61%. It is converted to an adequate active bis-ester by reaction with DMTMM tetrafluoroborate (prepared according to Raw S.A.: An improved process for the synthesis of DMTMM-based coupling reagents Tetr. Lett. 50, 946 (2009)). The active ester is used for the conjugation reaction immediately. The conjugation of bis(N-dodecyl-2-aminoethyl)(2aminoethyl)amine tetrahydrochloride with triglycolic acid is carried out in a stoichiometric ratio of 3:2 (amine:active ester) in methanol in the presence of 1.5 equiv. of N-methylmorpholine according to KunishimaM., e.a.: Formation of carboxamides by direct condensation of carboxylic acids and amines in alcohols using a new alcohol-and watersoluble condensing agent: DMT-MM Tetrahedron 57, 1551 (2001). The resulting mixture is used for conjugation with amino groups of nanospherical particles immediately and directly, without isolating the active ester.

18.8 g of hollow spherical nanoparticles with internal amino groups are mixed with an active ester solution prepared by conjugating 1.8 g of triglycolic acid and 10.4 g of bis(N-dodecyl-N,N-dimethyl2-ammoniumethyl)-N-(2-ammoniumethyl) )methylammonium tetrachloride in 130 ml of methanol at 0°C and stirred very vigorously for 30 minutes, then allowed to rise to room temperature and stirred vigorously for another 180 minutes. The nanoparticles are filtered off, thoroughly washed with methanol to remove dissolved residues, suspended in 500 ml of 10% by weight. of sodium chloride solution and stir vigorously in the solution for 20 minutes. Then thoroughly washed with water, followed by methanol and dried. 22.8 g of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macroholes: 2 diameter of macrohole: 0.5 micron (+ 0.01 / - 0.12 micron) number of macrodiffusion holes: 0 diameter of macrodiffusion holes: 0 number of isocentric layers: 2 diffusion holes (effective area): 2 to 4 nm ²

- 31 CZ 309934 B6 covalent binding of skeleton: type polysaccharide polyester antiviral effective box: lipophilic quaternary ammonium salt, polyethyleneimine derivative binding method of antiviral box: covalent, primary amide.

The number of surface-bound quaternary ammonium groups on the outer shell of the skeleton is determined spectrophotometrically by the formation of a surface associate with m-cresol purple. Detected: no presence of quaternary ammonium groups on the outer shell of the skeleton.

Example 2

The antiviral nanotherapeutic agent is prepared analogously to Example 1, with the difference that diglycolic acid is used as the linker of the conjugation unit. 22.4 g of antiviral nanotherapeutic is obtained.

Example 3

The antiviral effective nanotherapeutic is prepared analogously to example 1, with the difference that N-glycidyl-N, N, N-trimethylammonium chloride is used to modify the internal amino groups:

18.8 g of hollow spherical nanoparticles with free internal amino groups are suspended in 200 ml of tert.-butanol and a total of 6.6 ml of N-glycidyl-N,N,N-trimethylammonium chloride (94% by weight) is added at 0°C. and the mixture is stirred vigorously for 90 minutes. The nanoparticles are then filtered off, thoroughly washed repeatedly with methanol and dried. 20.4 g of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macroholes: 2 diameter of macrohole: 0.5 micron (+ 0.01 / - 0.12 micron) number of macrodiffusion holes: 0 diameter of macrodiffusion holes: 0 number of isocentric layers: 2 diffusion holes (effective area): 2 to 4 nm ² covalent binding skeleton: type polysaccharide polyester antiviral effective box: hydrophilic quaternary ammonium salt, aminoethanol derivative binding method of antiviral box: covalent, amine.

Example 4

The antiviral effective nanotherapeutic is prepared analogously to Example 1, with the difference that reductive amination of bis(N-dodecyl-N,N-dimethyl2-ammoniumethyl)(N-2-oxoethyl)-N-methyl-2- ammoniummethyl)methylammonia.

14.6 g of tris(2-aminoethyl)amine are dissolved in 350 ml of methanol, 17.6 ml of acetic acid are added and the mixture is cooled to 0 °C. With intensive stirring, 23.1 g of 45% by weight are added. of glyoxal dimethyl acetal and a total of 6.3 g of sodium cyanoborohydride in 20 minutes. Stirring is continued for another 5 hours, during which the temperature is allowed to rise to room temperature and 2 g of sodium cyanoborohydride are added. After an hour of stirring, 10 ml of 10% by weight is added to the mixture. of sodium hydroxide and stirred for another 30 minutes. Then the mixture is evaporated under vacuum, the residue is diluted with 30 ml of water and repeatedly extracted with 50 ml of dichloromethane, and the dichloromethane layer is collected. The combined extracts were evaporated to dryness under vacuum and the previous reductive amination was repeated with 37.5 g of dodecanal. By flash chromatography on silica gel in the ethyl acetate-triethylamine system (10:1, vol./vol.), 47.9 g (84%) of crude bis(N-dodecyl-2-aminoethyl)(N-(2bis( methoxy)ethyl)-2-aminoethyl)amine. 1H NMR (DMSO-d6, 300 MHz) δ 3.34 (s, 6H, OCH3), 4.52 (t, H, CH), 2.90 (d, J = 6.2 Hz, 2H, CH2) , 2.46 (m, J = 14.9 Hz, 16H, CH2), 1.31 (m, J = 9.9 Hz, 40H, CH2), 1.01 (t, 6H, J = 5.4 Hz, CH3); ¹³ C NMR (DMSO-d 6 , 101 MHz) δ 107.11 (CH).

- 32 CZ 309934 B6

The product is dissolved in 400 ml of dichloromethane, 48 ml of DIPEA is added, and a solution of 69 ml of methyl triflate in 250 ml of dichloromethane is added dropwise under a protective atmosphere of Ar at -20°C for 2 hours with stirring. The temperature is maintained at -20°C for a further 2 hours, then slowly allowed to rise to 0°C and held there for another hour. It is then kept under stirring at room temperature for another 10 hours, 10 ml of methanol is added and left to stand for 30 minutes. The reaction mixture is evaporated to dryness under vacuum, dissolved in 200 ml of methanol and stirred violently for 30 minutes with a mixture of 250 g of calcium chloride and 900 ml of water. The mixture is then extracted with 5 x 100 ml of dichloromethane and the combined extracts are evaporated under vacuum after drying with MgSO4. 48.5 g (71%) of the quaternary ammonium salt bis(N-dodecyl-N,N-dimethyl-2-ammoniumethyl)(N-(2-bis(methoxy)ethyl)-N-methyl-2ammoniumethyl)methylammonium tetrachloride are obtained in the form of a golden yellow viscous oil.

8.12 g of the quaternary ammonium salt from the previous embodiment is dissolved (after modification: Lipshutz B.H., e.a.: Hydrolysis of Acetals and Ketals Using LiBF4 Synthetic Communications 12(4), 267 (1982)) in a mixture of 2 ml of water and 100 ml of dry acetonitrile and add 10.2 g of lithium tetrafluoroborate. The mixture is vigorously stirred for 5 hours, a second portion of 5.1 g of lithium tetrafluoroborate and 1 ml of water is added and stirred for another 12 hours. The product is isolated by extraction with dichloromethane and evaporation of the extract at 0 °C to dryness. 9.12 g (94%) of bis(N-dodecyl-N,N-dimethyl-2ammoniumethyl)(N-2-oxoethyl)-N-methyl-2-ammoniumethyl)methylammonium tetrakis(tetrafluoroborate) are isolated, which low stability immediately used in the reaction of modification of internal amino groups of nanospherical particles.

9.1 g of bis(N-dodecyl-N,N-dimethyl-2-ammoniumethyl)(N-2-oxoethyl)-N-methyl-2ammoniumethyl)methylammonium tetrakistetrafluoroborate is dissolved in 250 ml of methanol and 18.8 g of hollow spherical of nanoparticles with loose internal amino groups and the mixture begins to mix intensively. 0.15 ml of acetic acid is added, the mixture is cooled to 0 °C and a total of 750 mg of sodium cyanoborohydride is added over 15 minutes. Then it is stirred for another 30 minutes at 0 °C, the temperature is allowed to rise to laboratory, 320 mg of sodium cyanoborohydride is added and again intensively stirred for another 90 minutes. The nanoparticles are filtered off, thoroughly washed with methanol to remove dissolved residues, suspended in 500 ml of 10% by weight. of sodium chloride solution and stir vigorously in the solution for 20 minutes. Then thoroughly washed with water, followed by methanol and dried. 22.6 g of antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macroholes: 2 diameter of macrohole: 0.5 micron (+ 0.01 / - 0.12 micron) number of macrodiffusion holes: 0 diameter of macrodiffusion holes: 0 number of isocentric layers: 2 diffusion holes (effective area): 2 to 4 nm ² covalent binding of skeleton: type polysaccharide polyester antiviral effective box: lipophilic quaternary ammonium salt, polyethyleneimine derivative binding method of antiviral box: covalent, polyamine.

Example 5

The antiviral effective nanotherapeutic is prepared analogously to Example 1, with the difference that anisotropic precursor particles are prepared from 348 g of sodium hexadecylbenzene sulfonate, 152 g of sodium dodecane sulfonate (Alfa) and 2000 g of paraffin with a melting point of 52 °C, and polystyrene nanospherical particles are used as a steric blocker with a diameter of 0.40 microns (PDI 1.04) in a stoichiometric ratio of 1:3. 22.4 g of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 0.85 micron (PDI 1.03) number of macroholes: 3 macrohole diameter: 0.5 micron (+ 0.01 / - 0.12 micron) number of macrodiffusion holes: 0

- 33 CZ 309934 B6 diameter of macrodiffusion holes: 0 number of isocentric layers: 2 diffusion holes (effective area): 2 to 4 nm ² covalent binding of the skeleton: type polysaccharide polyester antiviral effective box: lipophilic quaternary ammonium salt, polyethyleneimine derivative binding method of the antiviral box: covalent, amide.

Example 6

The antiviral effective nanotherapeutic agent is prepared analogously to example 1, with the difference that anisotropic precursor particles are prepared from 460 g of sodium hexadecane sulfonate and 2000 g of paraffin with a melting point of 52 °C and polystyrene nanospherical particles with a diameter of 0.90 microns are used as a steric blocker (PDI 1.04). 22.6 g of antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.8 micron (PDI 1.03) number of macroholes: 2 diameter of macrohole: 0.9 micron (+ 0.01 / - 0.21 micron) number of macrodiffusion holes: 0 diameter of macrodiffusion holes: 0 number of isocentric layers: 2 diffusion holes (effective area): 2 to 4 nm ² covalent binding skeleton: type polysaccharide polyester antiviral effective box: lipophilic quaternary ammonium salt, polyethyleneimine derivative binding method of antiviral box: covalent, amide.

Example 7

The antiviral effective nanotherapeutic is prepared analogously to example 1, with the difference that anisotropic precursor particles are prepared from 305 g of sodium hexadecylbenzene sulfonate, 195 g of sodium dodecane sulfonate (Alfa) and 2000 g of paraffin with a melting point of 52 °C, and polystyrene nanospherical particles are used as a steric blocker with a diameter of 0.36 microns (PDI 1.04). 23.1 g of antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 0.70 micron (PDI 1.03) number of macroholes: 2 macrohole diameter: 0.36 micron (+ 0.01 / - 0.11 micron) number of macrodiffusion holes: 0 diameter of macrodiffusion holes: 0 number of isocentric layers: 2 diffusion holes (effective area): 2 to 4 nm ² covalent binding skeleton: type polysaccharide polyester antiviral effective box: lipophilic quaternary ammonium salt, polyethyleneimine derivative binding method of antiviral box: covalent, amide.

Example 8

The antiviral effective nanotherapeutic is prepared analogously to Example 7, with the difference that D-glucosamine is used for sorption onto sulfo groups of dry anisotropic precursor particles with an allocated steric blocker and sulfo groups in the form of free acid. 23.9 g of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 0.70 micron (PDI 1.03) number of macroholes: 2 macrohole diameter: 0.36 micron (+ 0.01 / - 0.11 micron)

- 34 CZ 309934 B6 number of macrodiffusion holes: 0 diameter of macrodiffusion holes: 0 number of isocentric layers: 2 diffusion holes (effective area): 1 to 3 nm ² covalent binding of skeleton: polysaccharide type polyester antiviral effective box: lipophilic quaternary ammonium salt, polyethyleneimine derivative binding mode of antiviral box: covalent, amide.

Example 9

The antiviral effective nanotherapeutic is prepared analogously to example 7, with the difference that chitosan 3kDa is used for sorption onto sulfo groups of dry anisotropic precursor particles with an allocated steric blocker and sulfo groups in the form of free acid. 21.8 g of antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 0.70 micron (PDI 1.03) number of macroholes: 2 macrohole diameter: 0.36 micron (+ 0.01 / - 0.11 micron) number of macrodiffusion holes: 0 diameter of macrodiffusion holes: 0 number of isocentric layers: 2 diffusion holes (effective area): 2 to 16 nm ² covalent binding of skeleton: mixed type - polysaccharide polyester and polysaccharide antiviral effective box: lipophilic quaternary ammonium salt, polyethyleneimine derivative binding method of antiviral box: covalent, amide.

Example 10

An antiviral effective nanotherapeutic is prepared analogously to example 1, with the difference that N-glycidyl-N-dodecyl-N,N-dimethylammonium chloride is used to modify the internal amino groups:

18.8 g of hollow spherical nanoparticles with free internal amino groups are suspended in 200 ml of tert.-butanol and a total of 7.6 g of N-glycidyl-N-dodecyl-NNdimethylammonium chloride (prepared by standing 2.31 g of epichlorohydrin with 5.26 g of N,Ndimethylaminododecylamine in dioxane at room temperature for 48 hours) in 50 ml of dioxane and the mixture was stirred vigorously for 90 minutes. The nanoparticles are then filtered off, thoroughly washed repeatedly with methanol and dried. 20.1 g of antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macroholes: 2 diameter of macrohole: 0.5 micron (+ 0.01 / - 0.12 micron) number of macrodiffusion holes: 0 diameter of macrodiffusion holes: 0 number of isocentric layers: 2 diffusion holes (effective area): 2 to 4 nm ² covalent binding skeleton: type polysaccharide polyester antiviral effective box: lipophilic quaternary ammonium salt, aminoethanol derivative binding method of antiviral box: covalent, amine.

Example 11

A mixture of 195 g of sodium hexadecylbenzene sulfonate, 55 g of sodium dodecane sulfonate (Alfa) and 680 g of paraffin with a melting point of 61 °C is thoroughly mixed at 90 °C in a 10-liter vessel, and a total of 4000 ml of heated water is added to the mixture with intensive stirring during 15 minutes to 80 °C. The mixture is stirred for another 15 minutes at the same temperature and gradually added in approximately 3 g amounts

- 35 CZ 309934 B6 polystyrene nanospherical particles with a diameter of 0.20 microns (PDI 1.03), which were hydrophobized by previously standing in cyclohexane for 15 minutes and thoroughly dried under vacuum. The distribution of anisotropic particles in the system is monitored by optical microscopy and the addition is interrupted if the proportion of 2:1 systems exceeds 4% in the emulsion. A total of 92 g of polystyrene nanospherical particles are added within 30 minutes, which corresponds to an average stoichiometry of 1:1 of 104%. The mixture is stirred at the same temperature for another 15 minutes and polystyrene nanospherical particles with a diameter of 0.06 microns (PDI 1.03) are gradually added in approximately 1 g amounts. A total of 18 g of polystyrene nanospherical particles are added to the nanoemulsion, which corresponds to an average stoichiometry of 1:10 of 101%. The temperature of the mixture is then allowed to drop to 15°C during 15 minutes with very gentle stirring, and the resulting mixture is allowed to coagulate with stirring at the same temperature for at least another 180 minutes. Then the resulting dense suspension is vacuum filtered, the nanoparticles mixed with 3000 ml of water are carefully poured into a column with a frit, and a total of 850 ml of 5% by weight is poured through the column within 30 minutes. hydrochloric acid and then deionized water until the acid reaction of the eluate is lost. The nanoparticles are washed on the column with dry methanol and then dried at laboratory temperature under vacuum. The yield is 99.5%, i.e. 1021 g of dry anisotropic precursor particles with an allocated steric blocker and sulfo groups in the form of free acid. All operations are performed without the use of mechanical aids, which can easily irreversibly deform soft nanoparticles.

3-deoxysucrose-3-N,N,N-trimethylammonium hydroxide is prepared by modifying the methodology according to Rúnarsson OV, ea: N-selective One pot synthesis of highly N-substituted trimethyl chitosan (TMC) Carbohydrate Polymers 74, 740 (2008) of 3 -amino-3-deoxysucrose prepared according to Simiand C., ea: Synthesis of sucrose analogues modified at position 4 J. Carbohydr. Chem., 14, 977 (1995). 3-deoxysucrose-3-N,N,N-trimethylammonium hydroxide is isolated from the reaction mixture by elution of the reaction mixture through Amberlyst A-26 in OH ^- . The eluate contains sodium hydroxide and a quaternary ammonium salt. While cooling with crushed ice, the eluate is neutralized to neutral pH with dilute hydrochloric acid and the resulting solution of sodium chloride and 3-deoxysucrose-3-N,N,Ntrimethylammonium chloride is evaporated under vacuum at laboratory temperature to almost dryness. The dense semi-crystalline mass is triturated several times with hot 2-propanol, the triturates are combined, cooled to 0 °C, sharply filtered through a thin layer of celite and evaporated under vacuum to a viscous oil of 3-deoxysucrose 3-N,N,N-trimethylammonium chloride. Prior to reaction with anisotropic precursor particles with allocated steric blocker and sulfo groups, the chloride is converted to hydroxide by elution through an Amberlyst A-26 column in OH ^- .

157 g of anisotropic precursor particles with an allocated steric blocker and sulfo groups in the form of free acid from the previous operation are slowly added with constant stirring at 5 °C to a solution containing 44.5 g of 3-deoxysucrose-3-N,N,N-trimethylammonium hydroxide in 2 liters of water. The resulting mixture is stirred for another 30 minutes, filtered under vacuum and the nanoparticles are thoroughly washed with water, followed by methanol and dried at laboratory temperature under vacuum. 199.9 g (quant.) of nanoparticles are obtained.

The dried nanoparticles from the previous operation are stirred at laboratory temperature in 4 liters of dry dimethylformamide, 28 ml of DIPEA is added, the mixture is cooled to -55 °C and a solution of 30.9 g of glycidyl trifluoromethanesulfonate is added to the mixture in a protective Ar atmosphere during 2 hours with constant stirring. in 80 ml of dry freshly distilled diglyme. The temperature is allowed to rise to laboratory and stirred for another 48 hours. Without isolating the nanoparticles, 69 ml of DIPEA is added, a solution of 93 g of glycidyl-4-toluenesulfonate in 200 ml of dimethylformamide is added all at once, and the mixture is heated to 40 °C with gentle stirring and maintained at this temperature for 6 hours. After that, a saturated solution of 56 g of sucrose in dimethylformamide is added at 50°C over 4 hours and the mixture is stirred for another 16 hours. These two operations are repeated two more times, each time increasing the proportion of glycidyl-4-toluenesulfonate by 30% and sucrose by 25%, and before each operation the nanoparticles are filtered and the solvent is regenerated by vacuum distillation. Finally, the mixture is filtered, the solvent is recycled under vacuum and the crude nanoparticles are thoroughly washed with water and mixed at 30°C for 2 hours with 10% by weight. NaOH solution. They filter again, wash with water until the alkaline reaction is lost and dry under vacuum. 418 g (79.1%) of nanoparticles with a cross-linked skeleton are obtained.

- 36 CZ 309934 B6

The dried nanoparticles from the previous operation are suspended in 3 liters of cyclohexane, the mixture is heated to 80 °C and stirred at this temperature for 2 hours. The suspension is filtered through a microfilter with a pore size of 0.35 microns. The nanoparticles on the filter are extracted three more times in a similar manner with 2 liters of cyclohexane until the extract evaporates to zero. The combined extracts are cooled and after 2 hours the steric blocker is isolated by nanofiltration through a PTFE nanofilter and the cyclohexane is recycled. The steric blocker is then treated alternately at 35 °C with concentrated hydrochloric acid and then with 30% by weight. with methanolic sodium hydroxide at 40 °C, which removes the steric blocker from about 25 to 40% of the residues of the nanoparticle skeleton. After cooling, washing and drying, 13.2 g (92%) of the steric blocker mixture is recovered.

310 g of antiviral nanotherapeutics with the following parameters are obtained:

outer diameter: 0.45 micron (PDI 1.03) number of macroholes: 1 diameter of macrohole: 0.2 micron (+ 0.01 / - 0.08 micron) number of macrodiffusion holes: 10 diameter of macrodiffusion holes: 0.06 micron count of isocentric layers: 4 diffusion holes (effective area): 1 to 3 nm ² covalent binding of skeleton: type of polysaccharide glycide polycondensate antiviral effective box: hydrophilic quaternary trimethylammonium salt binding method of antiviral box: covalent, amine.

Example 12

An antiviral nanotherapeutic is prepared analogously to Example 11, with the difference that epibromohydrin is used for cross-linking. 220 g of antiviral nanotherapeutics with the following parameters are obtained:

outer diameter: 0.45 micron (PDI 1.03) number of macro holes: 1 macro hole diameter: 0.2 micron (+ 0.01 / - 0.08 micron) number of macro diffusion holes: 10 diameter of macro diffusion holes: 0.06 micron count of isocentric layers: 4 diffusion holes (effective area): 1 to 3 nm ² covalent binding of skeleton: type of polysaccharide glycide polycondensate antiviral effective box: hydrophilic quaternary trimethylammonium salt binding method of antiviral box: covalent, amine.

Example 13

The antiviral effective nanotherapeutic is prepared analogously to Example 11, with the difference that 6,6',1'-triglycidyl-2,3,4,3',4'-pentaethoxycarbonylsucrose prepared from 2,3,4,3' is used for crosslinking ,4'-pentaethoxycarbonylsucrose by treatment with three equivalents of glycidyl triflate in dichloromethane at room temperature in the presence of DBU in a yield of 91%. The cross-linking of each layer is carried out analogously to example 11 in dimethylformamide in the presence of 5% mol. DIPEA at 40 °C. The nanoparticles are then filtered off and the protective ethoxycarbonyls are hydrolyzed by the action of aqueous KOH (10% by weight) in the cold, and the nanoparticles treated in this way are cross-linked again by the further addition of 6,6',1'-triglycidyl-2,3,4,3',4' -pentaethoxycarbonylsucrose in dimethylformamide. The operation is repeated five more times, the amount of cross-linking component increases by 10% with each additional repetition. 314 g of antiviral nanotherapeutics with the following parameters are obtained:

outer diameter: 0.45 micron (PDI 1.03)

- 37 CZ 309934 B6 number of macroholes: 1 macrohole diameter: 0.2 micron (+ 0.01 / - 0.08 micron) number of macrodiffusion holes: 10 macrodiffusion hole diameter: 0.06 micron number of isocentric layers: 6 diffusion holes (effective area): 2 to 5 nm ² covalent binding of skeleton: type of polysaccharide glycide polycondensate antiviral effective box: hydrophilic quaternary trimethylammonium salt binding method of antiviral box: covalent, amine.

Example 14

An antiviral nanotherapeutic agent is prepared analogously to Example 11, with the difference that N,N,N-trimethyl-D-glucosamine hydroxide is used to react with anisotropic precursor particles with an allocated steric blocker and sulfo groups. Cross-linking is carried out analogously to example 13 by the action of 6,6',1'-triglycidyl-2,3,4,3',4'pentaethoxycarbonylsucrose in dimethylformamide. 274 g of antiviral nanotherapeutics with the following parameters are obtained:

outer diameter: 0.45 micron (PDI 1.03) number of macro holes: 1 macro hole diameter: 0.2 micron (+ 0.01 / - 0.08 micron) number of macro diffusion holes: 10 diameter of macro diffusion holes: 0.06 micron count of isocentric layers: 6 diffusion holes (effective area): 1 to 2 nm ² covalent binding of skeleton: type of polysaccharide glycide polycondensate antiviral effective box: hydrophilic quaternary trimethylammonium salt method of binding of antiviral box: covalent, amine.

Example 15

The antiviral effective nanotherapeutic is prepared analogously to example 11, with the difference that NNN-trimethyl-chitosan hydroxide 3 kDa prepared according to Rúnarsson O.V., e.a.: N-selective One poť synthesis of highly N-substituted trimethyl chitosan (TMC) Carbohydrate Polymers 74, 740 (2008). Cross-linking is carried out analogously to example 13 by the action of 6,6',1'-triglycidyl-2,3,4,3',4'-pentaethoxycarbonylsucrose in dimethylformamide, and only in three isocentric layers. 246 g of antiviral nanotherapeutics with the following parameters are obtained:

outer diameter: 0.45 micron (PDI 1.03) number of macro holes: 1 macro hole diameter: 0.2 micron (+ 0.01 / - 0.08 micron) number of macro diffusion holes: 10 diameter of macro diffusion holes: 0.06 micron count of isocentric layers: 3 diffusion holes (effective area): 2 to 10 nm ² covalent binding of skeleton: type of polysaccharide glycide polycondensate antiviral effective box: hydrophilic quaternary trimethylammonium salt method of binding of antiviral box: covalent, amine.

Example 16

Antiviral effective nanotherapeutic is prepared analogously to example 11, with the difference that

- 38 CZ 309934 B6 anisotropic precursor particles with an allocated steric blocker and sulfo groups are prepared as follows:

A mixture of 98 g of sodium hexadecylbenzene sulfonate, 28 g of sodium dodecane sulfonate (Alfa) and 680 g of paraffin with a melting point of 51 °C is thoroughly mixed at 80 °C in a 6-liter container, and a total of 4,000 ml of heated water is added to the mixture with intensive stirring during 15 minutes to 80 °C. The mixture is stirred for another 15 minutes at the same temperature, and polystyrene nanospherical particles with a diameter of 0.40 microns (PDI 1.03) are gradually added in approximately 3 g amounts, which were hydrophobicized by previously standing in cyclohexane for 15 minutes and thoroughly dried under vacuum. The distribution of anisotropic particles in the system is monitored by optical microscopy and the addition is interrupted if the proportion of 2:1 systems exceeds 4% in the emulsion. A total of 92 g of polystyrene nanospherical particles are thus added within 30 minutes, which corresponds to an average stoichiometry of 1:1 of 101%. The mixture is stirred at the same temperature for another 15 minutes and slightly hydrophobized polystyrene nanospherical particles with a diameter of 0.12 microns (PDI 1.02) are gradually added in approximately 2 g amounts. A total of 39 g of polystyrene nanospherical particles are added to the nanoemulsion, which corresponds to an average stoichiometry of 1:16 of 100%. The temperature of the mixture is then allowed to drop to 0°C during 15 minutes with very gentle stirring, and the resulting mixture is allowed to coagulate with stirring at the same temperature for at least another 180 minutes. Then the resulting dense suspension is vacuum filtered, the nanoparticles mixed with 3000 ml of water are carefully poured into a column with a frit, and a total of 650 ml of 5% by weight is poured through the column within 30 minutes. hydrochloric acid and then deionized water until the acid reaction of the eluate is lost. Nanoparticles are washed on the column with dry methanol and then dried at laboratory temperature under vacuum. The yield is 99.2%, i.e. 831 g of dry anisotropic precursor particles with an allocated steric blocker and sulfo groups in the form of free acid. All operations are performed without the use of mechanical aids, which can easily irreversibly deform soft nanoparticles.

The formation of the skeleton and cross-linking is carried out analogously to example 13 by the action of 6,6',1'triglycidyl-2,3,4,3',4'-pentaethoxycarbonylsucrose in dimethylformamide. 365 g of antiviral nanotherapeutics with the following parameters are obtained:

outer diameter: 0.8 micron (PDI 1.03) number of macro holes: 1 macro hole diameter: 0.4 micron (+ 0.01 / - 0.08 micron) number of macro diffusion holes: 16 diameter of macro diffusion holes: 0.12 micron count of isocentric layers: 6 diffusion holes (effective area): 1 to 3 nm ² covalent binding of skeleton: type of polysaccharide glycide polycondensate antiviral effective box: hydrophilic quaternary trimethylammonium salt binding method of antiviral box: covalent, amine.

Example 17

The antiviral nanotherapeutic agent is prepared analogously to Example 16, with the difference that the anisotropic precursor particles with an allocated steric blocker and sulfo groups are prepared as follows:

A mixture of 165 g of sodium hexadecylbenzenesulfonate, 35 g of sodium decanesulfonate (Alfa) and 880 g of paraffin with a melting point of 51 °C is thoroughly mixed at 80 °C in a 6-liter container, and a total of 3100 ml of heated water is added to the mixture with intensive stirring during 15 minutes to 80 °C. The mixture is stirred for another 15 minutes at the same temperature, and polystyrene nanospherical particles with a diameter of 0.40 microns (PDI 1.03) are gradually added in approximately 3 g amounts, which were hydrophobicized by previously standing in cyclohexane for 15 minutes and thoroughly dried under vacuum. The distribution of anisotropic particles in the system is monitored by optical microscopy and the addition is interrupted if the proportion of 2:1 systems exceeds 4% in the emulsion. It will be added within 30 minutes in total

- 39 CZ 309934 B6

122 g of polystyrene nanospherical particles, which corresponds to an average stoichiometry of 1:1 of 104%. The mixture is stirred at the same temperature for another 15 minutes and slightly hydrophobized polystyrene nanospherical particles with a diameter of 0.12 microns (PDI 1.02) are gradually added in approximately 2 g amounts. A total of 45 g of lightly hydrophobized polystyrene nanospherical particles is added to the nanoemulsion, which corresponds to an average stoichiometry of 1:20 of 101%. The temperature of the mixture is then allowed to drop to 0°C during 15 minutes with very gentle stirring, and the resulting mixture is allowed to coagulate with stirring at the same temperature for at least another 180 minutes. Then the resulting dense suspension is vacuum filtered, the nanoparticles mixed with 3000 ml of water are carefully poured into a column with a frit, and a total of 850 ml of 5% by weight is poured through the column within 30 minutes. hydrochloric acid and then deionized water until the acid reaction of the eluate is lost. The nanoparticles are washed on the column with dry methanol and then dried at laboratory temperature under vacuum. The yield is 99.1%, i.e. 1218 g of dry anisotropic precursor particles with an allocated steric blocker and sulfo groups in the form of free acid. All operations are performed without the use of mechanical aids, which can easily irreversibly deform soft nanoparticles.

To a solution of 6.64 g of 2-oxo-3,4,6,1',3',4',6'-heptaacetylsucrose (prepared by oxidation of 3,4,6,1',3',4',6'- of heptaacetylsucrose by NBS in dioxane in the presence of DIPEA) in 200 ml of dioxane at 0 °C, 1.30 ml of N,N,N'-trimethylethylenediamine is added, stirred for 10 minutes and then 3 g of sodium triacetoxyborohydride and 1.2 ml of acetic acid are added. The resulting mixture is stirred for 48 hours at laboratory temperature, then made alkaline by the addition of 12 g of potassium bicarbonate and, after stirring for 20 minutes, extracted with dichloromethane (5 x 100 ml). The combined extracts are evaporated to dryness under vacuum, dried in a desiccator over KOH, the residue is dissolved in 200 ml of dichloromethane, cooled to -20 °C and, at this temperature and intensively stirred, a solution of 22.6 ml is added during 10 minutes under a protective atmosphere of Ar of methyl triflate in 60 ml of dichloromethane. It is stirred for 30 minutes, the temperature is allowed to rise to laboratory and stirred for another 60 minutes. Then 5 ml of methanol and 3 g of sodium bicarbonate are added, stirring for another 10 minutes and the dichloromethane layer is separated, evaporated under vacuum, the residue is dissolved in 200 ml of methanol, 5 g of potassium hydroxide is added and stirred at room temperature for 6 hours. The resulting mixture is diluted by the addition of 300 ml of water and eluted through a column of Annex Amberlyst A-26 in OH ^- . The eluate is carefully acidified with HCl to a neutral pH while cooling with crushed ice and evaporated to dryness under vacuum at laboratory temperature. The residue is triturated with hot 2-propanol (3 x 200 ml), the combined triturates are cooled, sharply filtered through a layer of celite, and after evaporation 2.67 g (44%) of 2-(NNN'-trimethyl-2-ammonioethylammonium)-2deoxysucrose are obtained dichloride.

2.67 g (44%) of 2-(N,N,N'-trimethyl-2-ammonioethylammonium)-2-deoxysucrose dichloride is eluted through an Amberlyst Annex A-26 column in OH ^- . The eluate is diluted to a volume of 400 ml. 5.14 g of anisotropic precursor particles with an allocated steric blocker and sulfo groups in the form of free acid from the previous operation are slowly added to the eluate with constant stirring at 5 °C. The resulting mixture is stirred for another 60 minutes, filtered under vacuum and the nanoparticles are thoroughly washed with water, followed by methanol and dried at laboratory temperature under vacuum. 7.76 g (quant.) of nanoparticles are obtained.

The cross-linking of the skeleton and the formation of isocentric layers is carried out analogously to example 13 by the action of 6,6',1'-triglycidyl-2,3,4,3',4'-pentaethoxycarbonylsucrose in dimethylformamide. 10.6 g of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 0.9 micron (PDI 1.03) number of macroholes: 1 diameter of macrohole: 0.45 micron (+ 0.01 / - 0.12 micron) number of macrodiffusion holes: 20 diameter of macrodiffusion holes: 0.12 micron count isocentric layers: 6 diffusion holes (effective area): 1 to 3 nm ² covalent binding of skeleton: type of polysaccharide glycide polycondensate antiviral effective box: hydrophilic bis-trimethylammonium salt

- 40 CZ 309934 B6 binding method of antiviral box: covalent, amine.

Example 18

The antiviral effective nanotherapeutic is prepared analogously to Example 17, with the difference that the cross-linking is carried out analogously to Example 11, but the saccharide unit is dextrose. 251 g of antiviral nanotherapeutics with the following parameters are obtained:

outer diameter: 0.9 micron (PDI 1.03) number of macroholes: 1 diameter of macrohole: 0.45 micron (+ 0.01 / - 0.12 micron) number of macrodiffusion holes: 20 diameter of macrodiffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 1 to 3 nm ² covalent binding of the skeleton: type of polysaccharide glycide polycondensate antiviral effective box: hydrophilic quaternary trimethylammonium salt binding method of the antiviral box: covalent, amine.

Example 19

An antiviral nanotherapeutic is prepared analogously to Example 18, with the difference that the cross-linking is carried out with cellobiose as a carbohydrate unit. 280 g of antiviral nanotherapeutics with the following parameters are obtained:

outer diameter: 0.9 micron (PDI 1.03) number of macroholes: 1 diameter of macrohole: 0.45 micron (+ 0.01 / - 0.12 micron) number of macrodiffusion holes: 20 diameter of macrodiffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 1 to 3 nm ² covalent binding of the skeleton: type of polysaccharide glycide polycondensate antiviral effective box: hydrophilic quaternary trimethylammonium salt binding method of the antiviral box: covalent, amine.

Example 20

The antiviral nanotherapeutic agent is prepared analogously to Example 16, with the difference that the anisotropic precursor particles with an allocated steric blocker and sulfo groups are prepared as follows:

A mixture of 195.6 g of sodium hexadecylbenzene sulfonate, 104.4 g of sodium decanesulfonate (Alfa) and 1200 g of paraffin with a melting point of 58 °C is thoroughly mixed at 90 °C in a 10-liter vessel, and a total of 4500 ml of water heated to 90 °C. The mixture is stirred for another 15 minutes at the same temperature, and polystyrene nanospherical particles with a diameter of 0.70 microns (PDI 1.03) are gradually added in approximately 3 g amounts, which were hydrophobicized by previously standing in cyclohexane for 15 minutes and thoroughly dried under vacuum. The distribution of anisotropic particles in the system is monitored by optical microscopy and the addition is interrupted if the proportion of 2:1 systems exceeds 4% in the emulsion. A total of 259 g of polystyrene nanospherical particles are added within 30 minutes, which corresponds to an average stoichiometry of 1:1 of 102%. The mixture is stirred at the same temperature for another 15 minutes and slightly hydrophobized polystyrene nanospherical particles with a diameter of 0.12 microns (PDI 1.02) are gradually added in approximately 2 g amounts. A total of 26 g of slightly hydrophobized polystyrene nanospherical particles are added to the nanoemulsion, which corresponds to an average stoichiometry of 1:20 of 102%.

- 41 CZ 309934 B6

The temperature of the mixture is then allowed to drop to 0°C during 15 minutes with very gentle stirring, and the resulting mixture is allowed to coagulate with stirring at the same temperature for at least another 180 minutes. Then the resulting dense suspension is vacuum filtered, the nanoparticles mixed with 6000 ml of water are carefully poured into a column with a frit, and a total of 950 ml of 5% by weight is poured through the column within 30 minutes. hydrochloric acid and then deionized water until the acid reaction of the eluate is lost. The nanoparticles are washed on the column with dry methanol and then dried at laboratory temperature under vacuum. The yield is 99.0%, i.e. 1743 g of dry anisotropic precursor particles with an allocated steric blocker and sulfo groups in the form of free acid. All operations are performed without the use of mechanical aids, which can easily irreversibly deform soft nanoparticles.

13.0 ml of N,N, of N'-trimethylethylenediamine and 8 ml of water. The resulting mixture is heated to 75 °C for 12 hours with stirring, then evaporated at this temperature under vacuum to constant weight. The residue is dissolved in 200 ml of water and after sorption on a Dowex 50W cathexis column, 5% by weight is released. aqueous sodium hydroxide solution. After evaporating the eluate under vacuum at 60°C, 27.5 g (61%) of crude 2-(N,N,N'-trimethyl-2-aminoethylamino)-2-deoxysucrose are obtained.

27.5 g of crude 2-(N,NN'-trimethyl-2-aminoethylamino)-2-deoxysucrose from the previous embodiment are dissolved in 800 ml of water and 233 g of anisotropic precursor particles with an allocated steric blocker and sulfo groups in the form of free acid from the previous operation is added slowly with constant stirring at 5 °C to the solution. The resulting mixture is stirred for another 60 minutes, filtered under vacuum and the nanoparticles are thoroughly washed with water, followed by methanol and dried at laboratory temperature under vacuum. 260.1 g (quant.) of nanoparticles are obtained.

3,4-dioxoadipic acid is prepared according to Kozminikh V.O., e.a.: 1,3,4,6-tetrakabonilnye sistemy. Soobchenie 9 Vestnik OGU, No 5, 155 (2009) and subsequent ester hydrolysis. 26.1 g of 3,4-dioxoadipic acid is dissolved in 400 ml of dry, freshly distilled dimethylformamide and 63.3 g of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide is added. The mixture is stirred for 5 minutes and then 229.2 g of nanoparticles from the above operation in 4 liters of dry, freshly distilled dimethylformamide are added to the stirred suspension within 15 minutes. The mixture is stirred for another 15 minutes, the cross-linked nanoparticles are filtered and the solvent is recycled. The resulting nanoparticles are suspended in 5 liters of dry, freshly distilled dimethylformamide and a mixture of 52.2 g of 3,4-dioxoadipic acid and 63.3 g of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide in 500 ml of dimethylformamide is added. A saturated solution of 19.4 g of sucrose in dimethylformamide is then added over the course of an hour and stirred for another 2 hours. The crude nanoparticles are aspirated, washed thoroughly with water and dried, and then the carbonyls of the crosslinking linker present are reduced in the solvent system dioxane - methanol (5:1; vol./vol.) at -5 °C by the gradual addition of sodium borohydride according to Correa I.R., e.a.: Diastereoselective Reduction of E and Z Alkoxyimino-ketoesters by Sodium Borohydride Tetrahedron 55, 14221 (1999). The entire above operation is repeated two more times in order to create three isocentric layers. The alkane core and steric blocker are removed from the obtained nanoparticles as described in Example 16.

127 g of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macro holes: 1 macro hole diameter: 0.7 micron (+ 0.01 / - 0.21 micron) number of macro diffusion holes: 20 diameter of macro diffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 2 to 6 nm ² covalent binding of skeleton: type polysaccharide polyester antiviral box: hydrophilic bis-tertiary amine way of binding antiviral box: covalent, amine.

- 42 CZ 309934 B6

Example 21

The antiviral nanotherapeutic is prepared analogously to Example 20, with the difference that the antiviral nanotherapeutic from Example 20 is treated with methyl triflate in dichloromethane at -20°C and the resulting bis-quaternary ammonium salt is transformed into an adequate chloride by intensive mixing with 10% by weight. sodium chloride solution. 144 g of antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macro holes: 1 macro hole diameter: 0.7 micron (+ 0.01 / - 0.21 micron) number of macro diffusion holes: 20 diameter of macro diffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 2 to 6 nm ² covalent binding of skeleton: type of polysaccharide polyester antiviral effective box: hydrophilic bis-quaternary ammonium salt binding method of antiviral box: covalent, amine.

Example 22

The antiviral nanotherapeutic agent is prepared analogously to Example 20, with the difference that the antiviral nanotherapeutic agent from Example 20 is treated with two equivalents of dodecyl triflate in dichloromethane at 0°C and the resulting bis-quaternary ammonium salt is transformed into an adequate chloride by repeated vigorous mixing with 10% by weight. sodium chloride solution. 158 g of antiviral nanotherapeutics with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macro holes: 1 macro hole diameter: 0.7 micron (+ 0.01 / - 0.21 micron) number of macro diffusion holes: 20 diameter of macro diffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 2 to 6 nm ² covalent binding of skeleton: type of polysaccharide polyester antiviral effective box: lipophilic bis-quaternary ammonium salt binding method of antiviral box: covalent, amine.

Example 23

The antiviral nanotherapeutic agent is prepared analogously to Example 20, with the difference that the antiviral nanotherapeutic agent from Example 20 is treated with one equivalent of 1-hexyl triflate in dichloromethane at 0°C and the resulting quaternary ammonium salt is transformed into an adequate chloride by repeated violent mixing with 10% by weight. sodium chloride solution. 145 g of antiviral nanotherapeutics with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macro holes: 1 macro hole diameter: 0.7 micron (+ 0.01 / - 0.21 micron) number of macro diffusion holes: 20 diameter of macro diffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 2 to 6 nm ² covalent binding of the skeleton: polysaccharide polyester type

- 43 CZ 309934 B6 antiviral effective box: lipophilic quaternium ammonium salt way of binding antiviral box: covalent, amine.

Example 24

An antiviral nanotherapeutic is prepared analogously to Example 20, with the difference that the cross-linking and building of three isocentric layers is performed with glycidyl triflate under the conditions described in Example 11. 101 g of an antiviral nanotherapeutic with the following parameters is obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macro holes: 1 macro hole diameter: 0.7 micron (+ 0.01 / - 0.21 micron) number of macro diffusion holes: 20 diameter of macro diffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 2 to 6 nm ² covalent binding of skeleton: type of polysaccharide glycide polycondensate antiviral effective box: hydrophilic bi-tertiary amine type of binding of antiviral box: covalent, amine.

Example 25

An antiviral nanotherapeutic is prepared analogously to Example 24, with the difference that the product of Example 24 is treated with 1-dodecyl triflate in dichloromethane at 0°C. 104 g of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macro holes: 1 macro hole diameter: 0.7 micron (+ 0.01 / - 0.21 micron) number of macro diffusion holes: 20 diameter of macro diffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 2 to 6 nm ² covalent binding of skeleton: type of polysaccharide glycide polycondensate antiviral effective box: lipophilic bis-quaternary ammonium salt binding method of antiviral box: covalent, amine.

Example 26

The antiviral effective nanotherapeutic is prepared analogously to example 20, with the difference that 1-glucosylamine prepared by the reaction of N,N,N'-trimethylethylenediamine with Dglucose in methanol is used as an aminosaccharide according to Muhizi T., e.a.: Synthesis of N-Alkyl-0-D -glucosylamines and Their Antimicrobial Activity against Fusarium proliferatum, Salmonella typhimurium, and Listeria innocua J. Agric. Food Chem. 57, 11092 (2009).

It is subsequently quaternized by the action of methyl triflate in dichloromethane at 0 °C. The quaternary ammonium salt is converted to the hydroxide by elution through an Amberlyst A-26 annex column in OH ^- and the scaffold bases are formed by sorption onto anisotropic precursor particles with an allocated steric blocker and sulfo groups in the form of the free acid from Example 20. Cross-linking and scaffold building by formation of three isocentric layers is carried out analogously to example 20, with the exception that D-glucose is used as the saccharide unit. 102 g of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macroholes: 1 macrohole diameter: 0.7 micron (+ 0.01 / - 0.21 micron)

- 44 CZ 309934 B6 number of macrodiffusion holes: 20 diameter of macrodiffusion holes: 0.12 micron number of isocentric layers: 3 diffusion holes (effective area): 1 to 2 nm ² covalent bond of skeleton: type polysaccharide polyester antiviral effective box: hydrophilic bis-quaternary ammonium salt mode of attachment of antiviral box: covalent, amine.

Example 27

The antiviral effective nanotherapeutic is prepared analogously to example 26, with the difference that 1-glucosylamine prepared by the reaction of N,N,N'-trimethylethylenediamine with D-glucose in methanol according to Muhizi T., e.a.: Synthesis of N-Alkyl-f-D-glucosylamines and Their Antimicrobial Activity against Fusarium proliferatum, Salmonella typhimurium, and Listeria innocua J. Agric. Food Chem. 57, 11092 (2009) is subsequently quaternized by the action of one equivalent of dodecyl triflate in dichloromethane at 0 °C. 109 g of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macro holes: 1 macro hole diameter: 0.7 micron (+ 0.01 / - 0.21 micron) number of macro diffusion holes: 20 diameter of macro diffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 1 to 2 nm ² covalent binding of skeleton: type of polysaccharide polyester antiviral effective box: lipophilic quaternary ammonium salt binding method of antiviral box: covalent, amine.

Example 28

The antivirally effective nanotherapeutic is prepared analogously to example 26, with the difference that 3-glucosylamine prepared by reductive amination of 1,2,5,6di-O-isopropylidene-3-oxo-D-glucofuranose is used as the structural saccharide unit (Muhizi T., e.a. : Synthesis and Antibacterial

Activity of Aminodeoxyglucose Derivatives against Listeria innocua and Salmonella typhimurium J. Agric. Food Chem. 57, 8770 (2009)) with N,N,N'-trimethylethylenediamine by the effect of sodium triacetoxyborohydride in the presence of acetic acid (analogous to example 17). The resulting amine is quaternized with methyl triflate in dichloromethane at -20 °C and the acetal function is removed by acid hydrolysis (similar to Muhizi T., e.a.: Synthesis and Antibacterial Activity of

Aminodeoxyglucose Derivatives against Listeria innocua and Salmonella typhimurium J. Agric.

Food Chem. 57, 8770 (2009)). 103 g of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macro holes: 1 macro hole diameter: 0.7 micron (+ 0.01 / - 0.21 micron) number of macro diffusion holes: 20 diameter of macro diffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 1 to 2 nm ² covalent binding of skeleton: type of polysaccharide polyester antiviral effective box: hydrophilic bis-quaternary ammonium salt binding method of antiviral box: covalent, amine.

Example 29

Antiviral effective nanotherapeutic is prepared analogously to example 28, with the difference that

- 45 CZ 309934 B6 the resulting amine is quaternized with dodecyl triflate in dichloromethane at 0 °C. 108 g of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macro holes: 1 macro hole diameter: 0.7 micron (+ 0.01 / - 0.21 micron) number of macro diffusion holes: 20 diameter of macro diffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 1 to 2 nm ² covalent binding of skeleton: type polysaccharide polyester antiviral effective box: lipophilic quaternary ammonium salt way of binding antiviral box: covalent, amine.

Example 30

150 g of polystyrene nanoparticles with a diameter of 700 nm (+ 0.01 / - 0.03 microns) are suspended in a mixture of 100 g of paraffin with a melting point of 54 °C and 5 liters of 65% by weight. of aqueous 2-propanol and the mixture is heated to 72 °C with stirring and maintained at this temperature with constant stirring for 8 hours, during which the paraffin coagulates completely into polystyrene nanoparticles. The mixture is then allowed to cool to room temperature within 1 hour with slow stirring, the stirring is stopped and the solution above the nanoparticles is sucked off to a residual volume of approximately 1 liter. Anisotropic particles representing a steric blocker of type XVI-(c) are not separated due to their low mechanical resistance, but are used in suspension immediately in the next step.

A mixture of 195.6 g of sodium hexadecylbenzene sulfonate, 104.4 g of sodium decanesulfonate (Alfa) and 1200 g of paraffin with a melting point of 51 °C is thoroughly mixed at 80 °C in a 10-liter container, and a total of 4500 ml of water heated to 80 °C. The mixture is stirred for another 15 minutes at the same temperature, cooled to 65 °C and the steric blocker suspension prepared in the previous operation is gradually added in approximately 20 ml portions. The distribution of anisotropic particles in the system is monitored by optical microscopy and the addition is interrupted if the proportion of 2:1 systems exceeds 4% in the emulsion. The mixture is stirred at the same temperature for another 15 minutes and slightly hydrophobized polystyrene nanospherical particles with a diameter of 0.12 microns (PDI 1.02) are gradually added in approximately 3 g amounts. A total of 39 g of lightly hydrophobized polystyrene nanospherical particles are added to the nanoemulsion, which corresponds to an average stoichiometry of 1:30 of 101%. The temperature of the mixture is then allowed to drop to 0°C during 15 minutes with very gentle stirring, and the resulting mixture is allowed to coagulate with stirring at the same temperature for at least another 180 minutes. Then the resulting dense suspension is vacuum filtered, the nanoparticles mixed with 6000 ml of water are carefully poured into a column with a frit, and a total of 950 ml of 5% by weight is poured through the column within 30 minutes. hydrochloric acid and then deionized water until the acid reaction of the eluate is lost. The nanoparticles are washed on the column with dry methanol and then dried at laboratory temperature under vacuum. The yield is 99.0%, i.e. 1754 g of dry anisotropic precursor particles with an allocated steric blocker and sulfo groups in the form of free acid. All operations are performed without the use of mechanical aids, which can easily irreversibly deform soft nanoparticles.

13.0 ml of N,N, of N'-trimethylethylenediamine and 8 ml of water. The resulting mixture is heated to 75 °C for 12 hours with stirring, then evaporated at this temperature under vacuum to constant weight. The residue is dissolved in 200 ml of water and after sorption on a Dowex 50W cathexis column, 5% by weight is released. aqueous sodium hydroxide solution. After evaporating the eluate under vacuum at 60°C, 27.5 g (61%) of crude 2-(NNN'-trimethyl-2-aminoethylamino)-2-deoxysucrose are obtained.

27.5 g of crude 2-(N,N,N'-trimethyl-2-aminoethylamino)-2-deoxysucrose from the previous

- 46 CZ 309934 B6 version is dissolved in 250 ml of dimethylformamide, the mixture is cooled to -40 °C and a total of 13.8 ml of methyl triflate in 30 ml of dichloromethane is added during 15 minutes with constant stirring. The temperature is allowed to rise to room temperature and stirred for an additional 15 minutes. 4 ml of methanol are added and after 20 minutes the mixture is poured into 500 ml of water, the dichloromethane layer is separated and the aqueous layer is eluted through annex Amberlyst A-26 in OH ^- . A total of 235 g of anisotropic precursor particles with an allocated steric blocker and sulfo groups in the form of free acid from the previous operation are slowly added to the eluate with constant stirring at 5 °C. The resulting mixture is stirred for another 60 minutes, filtered under vacuum and the nanoparticles are thoroughly washed with water, followed by methanol and dried at laboratory temperature under vacuum. 260.5 g (quant.) of nanoparticles are obtained.

Dissolve 27.4 g of 2,3,4,5-tetraoxoadipic acid in 600 ml of acetonitrile, cool to 0 °C, add 72.8 g of DMTMM tetrafluoroborate (prepared according to Raw S.A.: An improved process for the synthesis of DMTMM-based coupling reagents Tetr. Lett. 50, 946 (2009)), and with constant stirring and cooling during the hour a total of 26.9 g of N-methylmorpholine. After another hour of mixing, the active DMT ester of 2,3,4,5-tetraoxoadipic acid prepared in this way is added to the suspension of 260.5 g of nanoparticles from the previous operation in 6 liters of dry dimethylformamide with constant stirring during twenty minutes. The mixture is then stirred for another 6 hours, 8 ml of ethanol is added, stirred for another 5 hours, filtered and the solvent is recycled by distillation. The carbonyls of the crosslinking linker present are reduced in the solvent system dioxane - methanol (5:1; vol./vol.) at -5 °C by the gradual addition of sodium borohydride according to Correa I.R., e.a.: Diastereoselective Reduction of E and Z Alkoxyimino-ketoesters by Sodium Borohydride Tetrahedron 55, 14221 (1999). The entire above operation is repeated two more times in order to create three isocentric layers. The stoichiometric ratio of the active ester to the nanoparticle skeleton increases by 100% in the second isocentric layer, and by 200% in the third. Sucrose is used as the building sugar unit. After removing the alkane core and the steric blocker analogously to the previous examples, 153.5 g of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macroholes: 1 diameter of macrohole: 0.7 micron (+ 0.01 / - 0.06 micron) number of macrodiffusion holes: 30 diameter of macrodiffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 2 to 6 nm ² covalent binding of skeleton: type of polysaccharide polyester antiviral effective box: hydrophilic bis-quaternary ammonium salt binding method of antiviral box: covalent, amine.

Example 31

The antiviral effective nanotherapeutic is prepared analogously to example 30, with the difference that 2,3,4,5-tetraoxoadipic acid dichloride prepared according to Chantarasriwong O., e.a.: Cl3CCONH2/PPh3: A Versatile Reagent for Synthesis of Esters is used as the reactive form of the cross-linker Synthetic Communications 38(16), 2845 (2008) with the difference that the reaction is carried out in dioxane at 40 °C. Polyesterification is carried out in the presence of DIPEA as a base. 169 g of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macroholes: 1 diameter of macrohole: 0.7 micron (+ 0.01 / - 0.06 micron) number of macrodiffusion holes: 30 diameter of macrodiffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 2 to 6 nm ² covalent binding of the skeleton: polysaccharide polyester type

- 47 CZ 309934 B6 antiviral effective box: hydrophilic bis-quaternary ammonium salt way of binding antiviral box: covalent, amine.

Example 32

An antiviral effective nanotherapeutic is prepared analogously to Example 30, with the difference that 2,3,4,5-hexanetetraone-1,6-biscarbene obtained from an adequate 1,6-bisdiazo-2,3,4, 5-Hexanetetraone:

Ethyl trifluoroacetoacetate is condensed with 2,3-dioxobutanoic acid dichloride in the presence of magnesium chloride and triethylamine in dichloromethane during 18 hours at laboratory temperature (according to Rathke M.W., e.a.: Procedures for the Acylation of Diethyl Malonate and Ethyl

Acetoacetate with Acid Chlorides Using Tertiary Amine Bases and Magnesium Chloride J. Org. Chem. 50, 2622 (1985)). The product is isolated by flash chromatography (ethyl acetate - cyclohexane; 2:1 vol./vol.) on silica gel. It is dissolved in dioxane in the presence of 5% by weight. of water and 1% 4-toluenesulfonic acid and heated with stirring for 4 hours at 85°C, when carbon dioxide escapes. After the end of gas evolution, it is stirred for another hour, cooled, diluted with water and 1,1,1,10,10,10hexafluoro-2,4,5,6,7,9-decanehexaone is isolated by extraction with dichloromethane and purified on a silica gel column by elution ethyl acetate. Ή NMR (CDCk 300 MHz) δ: 7.02 (s, 2H). Bis-diazoketone is generated by diazo transfer by the effect of mesylazide according to Danheiser R.L., e.a.: An Improved Method for the Synthesis of Diazo Ketones J. Org. Chem. 55, 1959 (1990). The reaction mixture containing bisdiazoketone is poured with stirring to 15% by weight. of potassium hydroxide solution at 0 °C and stirred intensively for 10 minutes. The organic layer is separated after the addition of an excess of sodium chloride, concentrated on a vacuum evaporator at 0 °C, dissolved in dry, freshly distilled dioxane, dried with magnesium sulfate and concentrated again to remove acetonitrile residues, and the solution of bis-diazoketone in dioxane thus prepared is used for crosslinking directly, without further cleaning. The active diazoketone content is determined gasometrically by the effect of copper chloride in water.

260 g of nanoparticles prepared by sorption of 2-(2-N, N, N-trimethylammonioethyl-NN-dimethyl ammonio)2-deoxysucrose to anisotropic particles according to Example 30 are suspended in 8 liters of acetonitrile, 650 ml of water, 840 mg of copper triflate and the resulting the mixture begins to stir. With constant stirring, the calculated amount of bis-diazoketone solution in dioxane corresponding to 2.2 equivalents of bis-diazoketone, i.e. 0.132 mol, is added. The addition is controlled so that the nitrogen is only released very slowly, so that nitrogen pockets do not form on the porous surface of the nanoparticles. This operation therefore takes about 1 hour. The reaction mixture is then stirred for another 2 hours. The nanoparticles are aspirated, the solvent is recycled and the linker polyketone is reduced to a polyol in the solvent system dioxane - methanol (5:1; vol./vol.) at -5 °C with the gradual addition of sodium borohydride according to Correa I.R., e.a.: Diastereoselective Reduction of E and Z alkoxyimino-ketoesters by Sodium Borohydride Tetrahedron 55, 14221 (1999) as described in the previous examples. Nanoparticles with a modified linker skeleton are suspended in 8 liters of aqueous acetonitrile and the O-H insertion process is repeated by the effect of 6 equivalents of bis-diazoketone during 30 minutes, followed by the gradual addition of 1.5 equivalents of sucrose in the form of a saturated solution in water. Further operations are repeated until 3 isocentric layers are formed. 238 g of antiviral nanotherapeutics with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macroholes: 1 diameter of macrohole: 0.7 micron (+ 0.01 / - 0.06 micron) number of macrodiffusion holes: 30 diameter of macrodiffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 2 to 6 nm ² covalent binding of the skeleton: type polyether - polysaccharide antivirally effective box: hydrophilic bis-quaternary ammonium salt binding method of the antiviral box: covalent, amine.

- 48 CZ 309934 B6

Example 33

The antiviral effective nanotherapeutic is prepared analogously to example 32, with the difference that 2% mol is used as an O-H insertion catalyst during cross-linking. dirhodium tetraacetate and work in acetonitrile with 3% wt. water. 158 g of antiviral nanotherapeutics with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macroholes: 1 diameter of macrohole: 0.7 micron (+ 0.01 / - 0.06 micron) number of macrodiffusion holes: 30 diameter of macrodiffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 2 to 6 nm ² covalent binding of the skeleton: type polyether - polysaccharide antivirally effective box: hydrophilic bis-quaternary ammonium salt binding method of the antiviral box: covalent, amine.

Example 34

An antiviral effective nanotherapeutic is prepared analogously to Example 32, with the difference that nanoparticles prepared as follows are used as a steric blocker:

150 g of polystyrene nanoparticles with a diameter of 700 nm (+ 0.01 / - 0.03 microns) are suspended in a mixture of 100 g of paraffin with a melting point of 54 °C and 5 liters of 65% by weight. of aqueous 2-propanol and the mixture is heated to 72 °C with stirring and maintained at this temperature with constant stirring for 8 hours, during which the paraffin coagulates completely into polystyrene nanoparticles. The mixture is then allowed to cool to room temperature within 1 hour with slow stirring, the stirring is stopped and the solution above the nanoparticles is suctioned off and washed with methanol suction. Subsequently, the nanoparticles are dried under vacuum. The dried nanoparticles are suspended at 0 °C in 150 ml of 70% by weight. of perchloric acid and, after cooling to -10 °C, a total of 180 mg of sodium chlorate is added in approximately 10 mg portions over 30 minutes and after each addition the container is slowly swirled to mix. The mixture is then allowed to stand for another 20 minutes and then transferred to a container with 400 ml of water and 0.3 kg of crushed ice. The nanoparticles are filtered off with suction, washed with water and extracted with hot cyclohexane (300 mL). After cooling, the nanoparticles are sucked off, thoroughly dried, and the hydroxylated polystyrene hemisphere generated in this way is reduced with the system sodium borohydride - iodine in triglyme at -15 °C in order to eliminate the carboxyls present (after modification: Bhaskar Kanth J.V., e.a.: Selective Reduction of Carboxylic Acids into Alcohols Using NaBH and I2 J. Org. Chem. 56, 5964 (1991)). To transform the generated hydroxyls into 2-(2'-methoxyethyloxy)ethyl ether, O-alkylation of XVI-(g) is carried out in the form of "O-H insertion" of the in situ generated carbene (Fulton, J.R., e.a.: The Use of Tosylhydrazone Salts as a Safe Alternative for Handling Diazo Compounds and Their Applications in Organic Synthesis Eur. J. Org. Chem. 1479 (2005)) according to Scheme XVII from the sodium salt of N-(2,4,6-triisopropylbenzenesulfo)hydrazone 3,6-dioxa-1-heptanal. 153 g of steric blocker type XVI-(i) is obtained.

172 g of antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macroholes: 1 diameter of macrohole: 0.65 micron (+ 0.01 / - 0.05 micron) number of macrodiffusion holes: 30 diameter of macrodiffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 2 to 6 nm ² covalent bond of skeleton: polyether - polysaccharide type

- 49 CZ 309934 B6 antiviral effective box: hydrophilic bis-quaternary ammonium salt binding method of antiviral box: covalent, amine.

Example 35

The antiviral effective nanotherapeutic is prepared analogously to example 32, with the difference that the bisdiazoketone is prepared by the action of 1,4-dibromo-2,3-butanedione (prepared according to Ruggli P., e.a.: 15. Ober die Addition von Benzol an symm. Dibromo-diacetyl Helv. Chim. Acta 29, 95 (1946)) to N,N'-ditosylhydrazine in the presence of DBU in dioxane according to Fukuyama (Toma T., e.a.: N,N'Ditosylhydrazine: A Convenient Reagent for Facile Synthesis of Diazoacetates Org. Letters 9(16), 3195 (2007)) and is used after elimination of sulfinane by aqueous hydroxide extraction in the cross-linking reaction directly, without separation.

177.5 g of antiviral nanotherapeutics with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macroholes: 1 diameter of macrohole: 0.7 micron (+ 0.01 / - 0.06 micron) number of macrodiffusion holes: 30 diameter of macrodiffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 2 to 6 nm ² covalent binding of the skeleton: type polyether - polysaccharide antivirally effective box: hydrophilic bis-quaternary ammonium salt method of binding of the antiviral box: covalent, amine.

Example 36

The antiviral effective nanotherapeutic is prepared analogously to example 32, with the difference that the bisdiazoketone is prepared by the action of 1,6-dibromo-2,3,4,5-hexanetetraone on N,N'-ditosylhydrazine in the presence of DBU in dioxane according to Fukuyama (Toma T. , e.a.: N,N-Ditosylhydrazine: A Convenient Reagent for Facile Synthesis of Diazoacetates Org. Letters 9(16), 3195 (2007)) and is used after elimination of sulfinane by aqueous hydroxide extraction in the cross-linking reaction directly, without separation. 1,6-dibromo-2,3,4,5-hexanetetraone is prepared by the reaction of diethyl malonate with 2,3-dioxobutanedioic acid chloride in the presence of magnesium ethanolate (Organic Syntheses, Coll. Vol. 4, p. 285 (1963); Vol. 37, p. 20 (1957)), by converting bis-acylmalonate to 2,3,4,5-hexanetetraone according to Astle M.J., e.a.: Cation-exchange resin catalyzed hydrolysis and decarboxylation of esters of acetoacetic and malonic acids J. Org. Chem. 26, 1713 (1961) and by bromination of NBS in dioxane.

251 g of antiviral nanotherapeutics with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macroholes: 1 diameter of macrohole: 0.7 micron (+ 0.01 / - 0.06 micron) number of macrodiffusion holes: 30 diameter of macrodiffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 2 to 6 nm ² covalent binding of the skeleton: type polyether - polysaccharide antivirally effective box: hydrophilic bis-quaternary ammonium salt binding method of the antiviral box: covalent, amine.

Example 37

Antiviral effective nanotherapeutic is prepared analogously to example 32, with the difference that

- 50 CZ 309934 B6 uses a bis-carbene generated from bis-N-(2,4,6triisopropylbenzenesulfo)hydrazone bisaldehyde obtained from 2,3,4,1',3',4'-hexaacetylsucrose (available by the gradual action of lipase) as a cross-linking component OF and chymotrypsin to octaacetylsucrose in a high yield of 94% according to Ong G.-T, e.a.: Selective deacylation on the glucosyl moiety of octa-Oacetylsucrose by enzymic hydrolysis: formation of 2,1',3',4',6'-penta -O-acetylsucrose Carbohydrate Research 241, 327 (1993)) by oxidation with the TCIA/TEMPO system in dichloromethane at 0 °C in a yield of 92% (according to De Luca L., e.a.: A Very Mild and Chemoselective Oxidation of Alcohols to Carbonyl Compounds Organic Letters 3(19), 3041 (2001)).

260 g of nanoparticles prepared by sorption of 2-(2-N, N, N-trimethylammonioethyl-NN-dimethyl ammonio)2-deoxysucrose on anisotropic particles according to Example 30 are suspended in 8 liters of acetonitrile, 6 g of TEBAC are added and finally a solution of 240 mg of dirhodium tetraacetate and 5.8 mL tetrahydrothiophene in 150 mL acetonitrile. With constant stirring, a saturated solution of 87.4 g of the disodium salt of bis-6,6'-N-(2,4,6-triisopropylbenzenesulfo)hydrazono-2,3,4,1',3',4'-hexaacetyl6 is added of ,6'-dideoxysucrose in acetonitrile. The addition is controlled so that the nitrogen is only released very slowly, so that nitrogen pockets do not form on the porous surface of the nanoparticles. This operation therefore takes approximately 2.5 hours. The reaction mixture is then stirred for another 2 hours. The nanoparticles are aspirated, the solvent is recycled, and the O-acetyl is removed from the cross-linker backbone by stirring the nanoparticles in methanolic potassium hydroxide at room temperature for 30 minutes. Nanoparticles with a modified linker skeleton are suspended in 8 liters of acetonitrile and the O-H insertion process is repeated with the effect of 1.5 times the disodium salt bis-6,6'-N-(2,4,6triisopropylbenzenesulfo)-2,3,4,1', of 3',4'-hexaacetyl-6,6'-dideoxysucrose for the third isocentric layer, 1.75 times for the fourth and 1.75 times for the fifth. 299 g of antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macroholes: 1 diameter of macrohole: 0.7 micron (+ 0.01 / - 0.06 micron) number of macrodiffusion holes: 30 diameter of macrodiffusion holes: 0.12 micron count of isocentric layers: 5 diffusion holes (effective area): 2 to 6 nm ² covalent binding of the skeleton: type of polysaccharide antiviral effective box: hydrophilic bis-quaternary ammonium salt binding method of the antiviral box: covalent, amine.

Example 38

182 g of D-glucose (99%, Lachema) is suspended in 800 g of (E)-2-octadecen-1ol (98%, Yieldpharma) in a 3-liter reaction kettle equipped with a powerful all-teflon mechanical stirrer and a thermometer and, with constant stirring, min. 300 rpm, a total of 90 grams of immobilized conc. of sulfuric acid on silica gel (20 wt%, 200 to 400 mesh, Lachema) and the mixture is placed in a thermostated ultrasonic bath (450 W). With constant stirring, the temperature rises to 55°C within 60 minutes and the mixture is maintained at this temperature for another 150 minutes. The crude mixture is cooled, 600 ml of dichloromethane is added, and 110 g of finely crushed potassium carbonate is gradually added to the resulting suspension. After the end of foaming, the mixture is filtered and the filtrate is slowly eluted through a silk gel (60 to 200 mesh, 3400 cm ³ , layer height 180 mm) with a mixture of cyclohexane dichloromethane (3:1, vol./vol.) and the product is released from the column by elution with the mixture ethyl acetate dichloromethane (2:1, vol./vol.). The product is a 12.2:1 α/β glycoside mixture. 376 g (84%) of the glycoside are obtained in the form of a brown-yellow oil. 1H NMR (CDCL, 300 MHz) δ 5.44 (t, 1H, J = 9.5Hz, H-3), 4.73 (t, 1H, J = 9.5Hz, H-4), 4 .76 (d, 1H, J = 8.7 Hz, H-1), 4.44 (dt, 1H, J = 9.5 Hz, 8.7 Hz, H-2), 4.40 (dd, 1H, J = 11.5 Hz, 9.9 Hz, H-6a), 4.06 (dd, 1H, J = 1.5 Hz, - 11.5 Hz, H6b), 4.34 (m, 1H , J = 11.0 Hz, 9.9 Hz, H-5), 4.06 (d, 2H, J = 7.4 Hz, O-CH2-), 5.41 (m, 1H, J = 15 .6 Hz, 6.9 Hz, -CH=), 0.93 (m, 3H, J = 7.0 Hz, -CH3), ¹³ C NMR (CDCk, 75 MHz) δ 100.3 (C-1 ), 79.8 (O-CH2-), 75.8 (-CH=), 62.6 (C-6), 53.9 (C-2), 69.2(C-4), 70, 4 (C-3), 71.1(C-5).

- 51 CZ 309934 B6

Reaction of N-dodecyl-N,N'-dimethylethylenediamine with D-glucose in methanol according to Muhizi T., e.a.: Synthesis of N-Alkyl-f-D-glucosylamines and Their Antimicrobial Activity against Fusarium proliferatum, Salmonella typhimurium, and Listeria innocua J. Agric . Food Chem. 57, 11092 (2009) the input aminodeoxysaccharide is prepared. 1-(NN'dimethyl-N-dodecyl-2-aminoethylamino)-1-deoxy-D-glucose is obtained in a total yield of 91%.

55.2 g of 1-O-(2-octadecen-1-yl)-α-D-glucopyranoside and 64.8 g of 1-(N,N'-dimethyl-N-dodecyl-2aminoethylamino)-1-deoxy-D-glucose is added to 1000 g of technical paraffin with a melting point of 62 °C heated to 75 °C and, while stirring, to a mixture of 10 liters of water preheated to 75 °C. The mixture is stirred for another 15 minutes at the same temperature, cooled to 70°C and the steric blocker prepared in Example 34 is added gradually in approximately 3 g portions. The distribution of anisotropic particles in the system is monitored by optical microscopy and the addition is stopped if the proportion of 2:1 systems exceeds 4% in the emulsion, which corresponds to 111 g of nanoparticles (106% of theory). The mixture is stirred at the same temperature for another 15 minutes and slightly hydrophobized polystyrene nanospherical particles with a diameter of 0.12 microns (PDI 1.02) are gradually added in approximately 3 g amounts. A total of 21 g of lightly hydrophobized polystyrene nanospherical particles is added to the nanoemulsion, which corresponds to an average stoichiometry of 1:20 of 99%. The temperature of the mixture is then allowed to drop to 0°C during 15 minutes with very gentle stirring, and the resulting mixture is allowed to coagulate with stirring at the same temperature for at least another 180 minutes. Then the resulting dense suspension is vacuum filtered, the nanoparticles are thoroughly washed with dry methanol and then dried at laboratory temperature under vacuum. 1235 g, i.e. 99.2% of crude nano-spherical particles, are obtained.

Dissolve 27.4 g of 2,3,4,5-tetraoxoadipic acid in 600 ml of acetonitrile, cool to 0 °C, add 72.8 g of DMTMM tetrafluoroborate (prepared according to Raw S.A.: An improved process for the synthesis of DMTMM-based coupling reagents Tetr. Lett. 50, 946 (2009)), and with constant stirring and cooling during the hour a total of 26.9 g of N-methylmorpholine. After another hour of mixing, the active DMT ester of 2,3,4,5-tetraoxoadipic acid prepared in this way is added to the suspension of 298 g of nanoparticles from the previous operation in 6 liters of dry dimethylformamide with constant stirring during twenty minutes. The mixture is then stirred for another 6 hours, 8 ml of ethanol is added, stirred for another 5 hours, filtered and the solvent is recycled by distillation. The carbonyls of the crosslinking linker present are reduced in the solvent system dioxane - methanol (5:1; vol./vol.) at -5 °C by the gradual addition of sodium borohydride according to Correa I.R., e.a.: Diastereoselective Reduction of E and Z Alkoxyimino-ketoesters by Sodium Borohydride Tetrahedron 55, 14221 (1999). The entire above operation is repeated two more times in order to create three isocentric layers. The stoichiometric ratio of the active ester to the nanoparticle skeleton increases by 100% in the second isocentric layer, and by 200% in the third. Sucrose is used as the building sugar unit. After removal of the alkane core and steric blocker analogously to the previous examples and hydrogenolysis of O-2-octadecen-1-yl, 194 g of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macro holes: 1 macro hole diameter: 0.7 micron (+ 0.01 / - 0.06 micron) number of macro diffusion holes: 20 diameter of macro diffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 1 to 2 nm ² covalent binding of skeleton: type of polysaccharide polyester antiviral effective box: lipophilic tertiary amine type of binding of antiviral box: covalent, amine.

Example 39

Antiviral effective nanotherapeutic is prepared analogously to example 38, with the difference that

- 52 CZ 309934 B6 the resulting antiviral nanotherapeutic from example 38 is quaternized by the effect of methyl triflate in dichloromethane at -20 °C and the resulting product is converted to dichloride by mixing with a saturated solution of sodium chloride in water. 199 g of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macroholes: 1 diameter of macrohole: 0.7 micron (+ 0.01 / - 0.06 micron) number of macrodiffusion holes: 30 diameter of macrodiffusion holes: 0.12 micron count of isocentric layers: 3 diffusion holes (effective area): 1 to 2 nm ² covalent binding of skeleton: type of polysaccharide polyester antiviral effective box: lipophilic quaternary ammonium salt binding method of antiviral box: covalent, amine.

Example 40

The antiviral effective nanotherapeutic is prepared analogously to example 1, with the difference that 311 mg of spherical nanoparticles with free amino groups on the inner wall of the skeleton are modified with the linker α-N-hydroxysuccinimidyl-o-maleimidyl-PEG (5 kDa) according to Gabriel M, e.a.: Preparation of LL- 37-Grafted Titanium Surfaces with Bactericidal Activity Bioconjugate Chem. 17, 548 (2006) and the resulting ω-maleimidyl-PEG nanoparticle derivative is conjugated without isolation with C-cys modified Magainin I according to Glinel K., e.a.: Antibacterial and antifoulingpolymer brushes incorporating antimicrobial peptide Bioconj. Chem. 20(1), 71 (2009). 342 mg of an antiviral nanotherapeutic with the following parameters are obtained:

outer diameter: 1.2 micron (PDI 1.03) number of macroholes: 2 diameter of macrohole: 0.5 micron (+ 0.01 / - 0.12 micron) number of macrodiffusion holes: 0 diameter of macrodiffusion holes: 0 number of isocentric layers: 2 Diffusion Holes (Effective Area): 2 to 4 nm ² Covalent Backbone Binding: Polysaccharide Polyester Antiviral Effective Box Type: A-Helix Cationic Antimicrobial Peptide Antiviral Box Binding Method: Covalent, Primary Amide.

Example 41

The antiviral effective nanotherapeutic is prepared analogously to example 17, with the difference that a mixture of 165 g of sodium hexadecylbenzenesulfonate, 35 g of sodium decanesulfonate (Alfa) and 880 g of paraffin with a melting point of 51 °C is thoroughly mixed at 80 °C in a 6-liter container and of the mixture, a total of 3100 ml of water heated to 80 °C is added during 15 minutes with intensive mixing. The mixture is stirred for another 15 minutes at the same temperature, and polystyrene nanospherical particles with a diameter of 0.40 microns (PDI 1.03) are gradually added in approximately 3 g amounts, which were hydrophobicized by previously standing in cyclohexane for 15 minutes and thoroughly dried under vacuum. The distribution of anisotropic particles in the system is monitored by optical microscopy and the addition is interrupted if the proportion of 3:1 systems exceeds 5% in the emulsion. A total of 247 g of polystyrene nanospherical particles are added within 30 minutes, which corresponds to an average stoichiometry of 1:2 of 105%. The mixture is stirred at the same temperature for another 15 minutes and slightly hydrophobized polystyrene nanospherical particles with a diameter of 0.10 microns (PDI 1.03) are gradually added in approximately 2 g amounts. A total of 18 g of lightly hydrophobized polystyrene nanospherical particles are added to the nanoemulsion, which corresponds to an average stoichiometry of 1:8 of 102%. The temperature of the mixture is then allowed to drop to 0°C over 15 minutes with very gentle stirring and the resulting mixture is left to stir

- 53 CZ 309934 B6 coagulate at the same temperature for at least another 180 minutes. Then the resulting dense suspension is vacuum filtered, the nanoparticles mixed with 3000 ml of water are carefully poured into a column with a frit, and a total of 850 ml of 5% by weight is poured through the column within 30 minutes. hydrochloric acid and then deionized water until the acid reaction of the eluate is lost. The nanoparticles are washed on the column with dry methanol and then dried at laboratory temperature under vacuum. The yield is 99.2%, i.e. 1315 g of dry anisotropic precursor particles with an allocated steric blocker and sulfo groups in the form of free acid. All operations are performed without the use of mechanical aids, which can easily irreversibly deform soft nanoparticles.

outer diameter: 0.9 micron (PDI 1.03) macro hole number: 2 macro hole diameter: 0.45 micron (+ 0.01 / - 0.12 micron) macro diffusion hole number: 8 macro diffusion hole diameter: 0.09 micron count of isocentric layers: 6.

Example 42

An antiviral active nanotherapeutic is prepared analogously to Example 41, with the difference that 11 g of slightly hydrophobized polystyrene nanospherical particles are added to the nanoemulsion, which corresponds to an average stoichiometry of 1:5 of 104%. A nanotherapeutic with the following parameters is obtained:

outer diameter: 0.9 micron (PDI 1.03) macro hole number: 2 macro hole diameter: 0.45 micron (+ 0.01 / - 0.12 micron) macro diffusion hole number: 5 macro diffusion hole diameter: 0.09 micron count of isocentric layers: 6.

Claims (50)

1. Hollow spherical nanoparticles with one or more macroholes, characterized in that the outer diameter of the nanoparticle is between 200 and 4000 nm, the diameter of the macrohole of the nanoparticle is between 150 and 900 nm, the skeleton of the nanoparticle contains 0 to 100 macrodiffusion holes with a diameter between 5 and 149 nm , diffusion channels with an effective area of 0.4 to 9 nm ² of each of the diffusion channels, the skeleton of the nanoparticle is formed by a covalently bound polymer of three dimensionally cross-linked saccharide units forming 1 to 12 isocentric layers of polysaccharide, and the inner wall of the nanoparticles is covalently modified with viricides or substances with antiviral properties or viruses with an inactivating effect, and the binding of these compounds to the inner wall of the nanoparticle skeleton is via linkers or spacers. 2. Hollow spherical nanoparticles with one or more macroholes according to claim 1, characterized in that the skeleton of the nanoparticles is formed by a covalently bound three-dimensionally cross-linked monodisperse polymer of the general formula I (I), where the index nn is 0 to 1000, where the index mm is 0 to (nn-1), where A/ is the structural unit of the polymer, where ξ is the index nn or mm, where any branch Α _ψ , where _ψ is the index a given branch between two A;, can be terminated independently of the other branches or can also continue without branching, where each structural unit A/ is formed independently of any other structural unit A; by the structure of the general formula II (Π), where ii to is are independently 0 to 100,000, where the symbol " * " denotes one or two or three bonds to any one or any two or three other structural units Β _φ , where _φ is an index from the range 1 up to 6, any structural unit A; and each structural unit Β _φ is formed by the skeleton of a saccharide or a modified saccharide of general formula III -55CZ 309934 B6 where w is 0 or 1, where one of the R 1 and R 2 substituents is H, where the other of the R 1 and R 2 substituents is either a “*” binding site for binding to another structural unit Β _φ , where _φ is an index from 1 to 6, 5 or is formed by a fragment of the general formula IV * * í * * * * . \ \ \ \ \ \ \ OH NH ₂ CH ₂ OH CH ₂ NH ₂ NHCOCHj ococh ₃ NHCONHj oconh ₂ och ₂ ch ₂ nh ₂ čh ₂ nhcoch ₃ ch ₂ och ₂ ch ₂ nh ₂ ch ₂ ococh ₃ cooh conh ₂ (IV) or is formed by a fragment of the general formula IN where the symbol " * " in both cases indicates a bond to the backbone unit B _φ , where g is 0 or 1, where Z is the crosslinking unit of formula VI 15 in which ki to ke are independently 0, 1, 2, 3, 4, 5 or 6, in which ki is 0 or 1, in which ks is 0 or 1, in which one of the substituents X® and Y®, where ® is an index in the range of 1 to 6, is H, and the other is formed by either a fragment of the general formula IV or the general formula V, where the symbol " * " in both cases indicates a bond to the backbone of the crosslinking unit of the formula VI in which Qi is carbonyl or methylene, in wherein Q 2 is carbonyl or methylene, or is formed by a fragment of general formula VII -56CZ 309934 B6 G------H Linker j------F- A nti vi r I \ 1 \ 1 J k (VII) where j is 0 or 1 where k is 0 or 1 where the symbol G means either O or NH, where Linker is a structural fragment of a linker or spacer, where Antivir is a structural fragment of a compound with a viricidal or antiviral or virus-inactivating effect, where the symbol "*" denotes a bond to the skeleton of the Β _φ unit, where one of the substituents FL and FL jc H and the second of the substituents FL and FL jc is either a binding site "*" for binding to another structural unit B _φ or is formed by a fragment of the general formula IV or is formed by a fragment of the general formula V or is formed by a fragment of the general formula VII, where one of the substituents L and R„ are H and the second of the substituents Rs and R„ is either a binding site "*" for binding to another structural unit B _φ or is formed by a fragment of the general formula IV or is formed by a fragment of the general formula V or is formed by a fragment of the general formula VII , where one of the substituents R? and Rs is H and the second of the substituents R? and Rs is either a binding site "*" for binding to another structural unit Β _φ or is formed by a fragment of the general formula IV or is formed by a fragment of the general formula V or is formed by a fragment of the general formula VII, where one of the substituents Rg and Rio is H and the other of the substituents Rg and Rio is either a binding site "*" for binding to another structural unit B _φ or is formed by a fragment of the general formula IV or is formed by a fragment of the general formula V or is formed by a fragment of the general formula VII, where the "Linker" in the formula V is formed by oligomers or polymers of general formula VIII type of polyethylene oxide, polyglycolate, polylactate, polyamide structures and their combinations, where ni is 0 to 3, where m is 0 to 500, where m is 0 to 40, where m is 0 to 500, where m is 0 to 40, where m is 0 to 500, where m is 0 to 3, where n 8 is 0 to 5, where n 9 is 0 or 1, where M 1 is either carbonyl (CO) or methylene (CLE) or ethylidene (CFLCH). where M2 forms a fragment from the group CO, CH2, CH3CH, NH or O, where M3 forms a fragment from the group CO, CH2, CH3CH, NH or O, where M4 forms a fragment from the group CO, CH2, CH3CH, NH or O, where M5 forms a fragment from the group CO, CH2, CH3CH, NH or O, where Me forms either O or NH, where Μγ forms a fragment from the group CO, CH2, CH3CH, NH or O, where Ms forms either CO or CH2 or CH3CH, where " Antivir" in the formula Vlije consists of an antiviral fragment of a compound with a viricidal or antiviral or virus-inactivating effect from the group of linear or branched polyethyleneimines with an Mr of less than 25 kDa or their N-alkylated or Λ'-polyalkylated derivatives or A-hctcropolyalkylation derived quaternary ammonium salts with aliphatic and alicyclic alkyls Cl to Cl8, or quaternary ammonium salts with aliphatic and alicyclic alkyls Cl to Cl8, or cationic and anionic antimicrobial peptides of the α-helix, β-sheet, cyclic β-sheet, β-loop, or free configuration, and their combination. 3. The method of preparation of spherical hollow nanoparticles mentioned in claim 2, characterized by the fact that in the first stage of preparation, a surfactant provided with a hydrophilic sulfo group is treated with an alkane in a polar solvent, the sulfo group is in the form of a free acid or an alkali metal salt, and due to the interaction of a steric blocker creates a macroscopic structure in which the steric blocker is partially embedded in the sphere skeleton formed by alkane micelles with surface -57CZ 309934 B6 sulfo groups, in the second stage of preparation, the nanoparticles prepared in the first stage are treated with a saccharide unit consisting of a monosaccharide, oligosaccharide, polysaccharide, their modified forms or their mixtures in polar protic or aprotic solvents under electrostatic sorption of the saccharide on the surface of the nanoparticle and defined orientation of the saccharide backbone such that the amino group of the saccharide converted to an ammonium cation is electrostatically bound to the sulfonate anion and points radially inward of the sphere, while the saccharide backbone is radially outside the sphere and where the steric blocker is present, no sulfo groups are present, and thus no sorption of the saccharide occurs , and in the third stage of preparation, the electrostatically bound carbohydrate units sorbed on the surface of the particles are cross-linked by the action of the cross-linking component. 4. Method for the preparation of spherical hollow nanoparticles according to claim 3, characterized in that the first phase of preparation includes the action of an alkane on a steric blocker and the subsequent formation of micelles by the action of a surfactant in a polar solvent or the addition of a steric blocker to the formed micelles from an alkane and a surfactant in a polar solvent , preferably including first the action of a solvent on a mixture of alkane and surfactant and the subsequent addition of a steric blocker. 5. Method for the preparation of spherical hollow nanoparticles according to claim 3, characterized in that in the first stage of preparation the surfactant is an aliphatic, alicyclic or aliphatic chain-substituted aromatic sulfonic acid or its salt with an alkali metal cation of a chain length corresponding to C10 to C45 and or a mixture of these surfactants, preferably the surfactant or their mixture has an HLB index in the range of 9.3 to 11.9 and is present in the reaction mixture in a concentration of 2 to 15 wt.%. 6. Method for preparing spherical hollow nanoparticles according to claim 3, characterized in that in the first stage of preparation, the alkane is an aliphatic or alicyclic hydrocarbon with a length corresponding to C16 to C60 or a mixture of these hydrocarbons, preferably technical paraffin, with a transition temperature parameter in the range of 35 °C to 80 °C and a melting temperature in the range of 35 °C to 80 °C, and the alkane concentration in the mixture is in the range of 5 to 40% by weight, preferably 20% by weight, and the solvent is water, methanol, N,N-dimethylformamide, NN-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, Nmethylmorpholine N-oxide and their mixtures, preferably the solvent is water and a mixture of methanol, ethanol, 2-propanol, 2-methyl-2-propanol, N,N-dimethylformamide, N,N- of dimethylacetamide, Nmethylpyrrolidone, dimethylsulfoxide with water, and if required by the design, the ionic strength of the solvent is increased by adding 0.01 to 36 wt.%. inorganic salts from the group sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium nitrate, potassium nitrate, ammonium sulfate, ammonium chloride, sodium iodide, potassium iodide, sodium bromide, potassium bromide, lithium chloride, lithium bromide, lithium iodide or salts organic acids from the group sodium acetate, potassium acetate or with the addition of tetramethylammonium chloride. 7. Method for the preparation of spherical hollow nanoparticles according to claim 3, characterized in that in the first stage of preparation the micelles are formed by effective mixing in the temperature range of 0 °C to 135 °C, preferably with technical paraffin with a melting point of 55 °C at a temperature in the range 85 to 95 °C. 8. Method for preparing spherical hollow nanoparticles according to claim 3, characterized in that the steric blocker used in the first stage of preparation is a polymer nanoparticle of spherical or anisotropic shape, size 5 to 1400 nm and surface arrangement of functional groups. 9. The method of preparation according to claim 8, characterized in that the steric blocker is a polymeric spherical nanoparticle made of polystyrene, which is optionally hydrophobized by the effect of non-polar solvents from the group of hexane, heptane, cyclohexane by standing together in the mixture for 10 minutes to 24 hours at room temperature . 10. The preparation method according to claim 8, characterized in that the steric blocker is a binary anisotropic nanoparticle from the polystyrene-alkane binary system. 11. The preparation method according to claim 8, characterized in that the steric blocker is a spheroidal polystyrene nanoparticle hydrophilized on one hemisphere or its part. - 58 CZ 309934 B6 12. The method of preparation according to claim 11, characterized in that the steric blocker is prepared by hydrophilizing the polystyrene part of the skeleton of the binary anisotropic nanoparticle with oligo-alkoxyalkyls in the form of C-insertion or O-H insertion after previous hydroxylation of the polystyrene skeleton, preferably by 2-methoxyethylation or 2-ethoxyethylation or 2-(2- methoxyethyloxy)ethylation. 13. The method of preparation of spherical hollow nanoparticles according to claim 3, characterized in that in the first stage of preparation, a steric blocker is added to the mixture in a ratio of particles in the range of 0.3 to 1.4 (blocker: micelle), preferably in a ratio in the range of 0 .8 to 0.9. 14. The method of preparing spherical hollow nanoparticles according to claim 3, characterized in that in the first stage of preparation, after the formation and stabilization of micelles with steric blockers, the mixture is cooled to a temperature lower than the melting point of the alkane, to form a suspension of spherical nanoparticles equipped with steric blockers, cooling is preferably carried out to a temperature 40°C lower than the melting point of the alkane. 15. Method for the preparation of spherical hollow nanoparticles according to claim 3, characterized in that the saccharide unit of general formula III used in the second stage of preparation contains in the skeleton at least one strongly basic amino group of general formula IV, the nanoparticles prepared in the first stage of preparation are preferably treated with a monosaccharide from group glucosamine, 1-glucosylamine, galactosamine, mannosamine, fructosamine, disaccharide from the chitobiose group, maltosamine, aminodeoxysucrose and lactosamine or the polysaccharide chitosan or a mixture of these carbohydrates. 16. Method for the preparation of spherical hollow nanoparticles according to claim 3, characterized in that the saccharide unit of general formula III used in the second stage of preparation contains a modified monosaccharide, or an oligosaccharide or a polysaccharide containing either quaternary ammonium groups or tertiary amino groups capable of quaternization, where w is 0 or 1, where one of the substituents R 1 to R 10 in formula III is formed by one of the fragments of general formula XV (XV) and where the remaining substituents from the group Ri to Rio in formula III form hydrogen or one of the substituents of formula IV or formula V, while in the fragments of general formula XV the index ai is between 0 and 3, the index a2 is between 0 and 2, the index as between 0 and 30, Z1, Z2 and Z3, independently of each other, aliphatic or alicyclic alkyl or alkyl substituted by an aromatic skeleton corresponding to the total chain length of Cl to Cl8, where the symbol "*" denotes a bond to the skeleton of general formula III, preferably in the case of monosaccharides such as -59CZ 309934 B6 saccharide units are worked with fragments of the general formula XV with indices ai and a2 values of 0 or 1, with a small ratio of the number of cations / molecular size. 17. Method for the preparation of spherical hollow nanoparticles according to claim 3, characterized in that in the third stage of the preparation the cross-linking of sorbed or allocated saccharide units is carried out by the cross-linking component after or without separation of the solvent used for sorption, while the degree of cross-linking is governed by the type and stoichiometric ratio between the nanoparticle and a cross-linking component. 18. Method for the preparation of spherical hollow nanoparticles according to claim 3, characterized in that the third phase of the preparation is carried out in a concentration of nanoparticles in the range of 0.1 to 20% by mass, in the mixture, preferably in a concentration of 1 to 3% by mass, of nanoparticles in the mixture, when the statistical average distance between the nanoparticles in the mixture is about 1 pm, to minimize the risk of intercorpuscular interaction. 19. The method of preparing spherical hollow nanoparticles according to claim 3, characterized in that the third phase of preparation is repeated by re-applying the same or different cross-linking component and subsequently by applying an excess of saccharide, and with each repetition of this procedure, the number of 3D isocentric layers of the nanosphere increases by one. 20. The preparation method according to claim 19, characterized in that the number of 3D isocentric layers of the nanosphere skeleton is in the range of 1 to 12. 21. The preparation method according to claim 19, characterized in that the last 3D isocentric layer of saccharides of the nanosphere skeleton is cross-linked or not, preferably biologically highly compatible saccharides are used for the last layer and they are not further cross-linked. 22. The method of preparing spherical hollow nanoparticles according to claim 3, characterized in that the cross-linking in the third stage of preparation is carried out by covalently linking carbohydrates on the surface of the particles with bi- or tri-functional cross-linking components, preferably the cross-linking is carried out by bi-functional cross-linking components, with that there is between 10 and 30% binding of three cross-linking units per one carbohydrate unit when building the first cross-linked 3D isocentric layer, preferably between 10 and 30 % binding of four cross-linking units per one carbohydrate unit when building the next cross-linked 3D isocentric layer layers. 23. Method for the preparation of spherical hollow nanoparticles according to claim 3, characterized in that in the third stage of preparation the crosslinking component is a reactive polycarbonyl bis-acylation agent of the formula XXI (xxi) where n is 0 to 8 where Gr is a leaving group from hydroxyl or Cl or Br or an active ester of formula XXII -60CZ 309934 B6 (XXII) or a ketene of formula XXIII •—c=c=o Η (XXIII), where the symbol " * " indicates the connection to the skeleton of formula XXI. 24. The preparation method according to claim 23, characterized in that the polyesterification reaction is carried out by adding a bis-acylating agent of the formula XXI to a suspension of nanoparticles containing free hydroxyls of saccharide units, while the bis-acylating agent of the formula XXI is used prepared in advance or is generated in situ in the temperature range of -80°C to 95°C, preferably at temperatures lower than the transition temperature parameter and the solidification temperature of the alkane of the nanoparticle core, in solvents from the acetonitrile, Λ'.Λ'-dimcthyltbramide group. Λ'.Λ'-Dimcthylacctamide. n-methylpyrrolidone, dimethylsulfoxide, dichloromethane, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, diethyl ether, 2-methoxy-2methylpropane and their mixtures. 25. The preparation method according to claim 24, characterized in that the polyesterification reaction is carried out with a bis-acylation agent of the formula XXI without the presence of other agents and or in the presence of compounds from the W-dicyclohexylcarbodiimide group. jN-(3-dimethylaminopropyl)-jN'ethylcarbodiimide, N,N'-diisopropylcarbodiimide, 2-(1H-1,2,3-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, 2-(ΙΗ- Ί-aza-1,2,3-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, l-[(l-(cyano-2-ethoxy-2oxoethylideneaminooxy)dimethylaminomorpholinomethylene)]methanaminium hexafluorophosphate, 1,1 'di(azodicarbonyl)dipiperidine, tert-butylazodicarboxylate, diisopropylazodicarboxylate, jVýVýVvyVtetramethylazodicarboxamide, diethylazodicarboxylate, triethylamine, trimethylamine, N,N,N',N'tetramethylethylenediamine, řV-methylmorpholine, diisopropylethylamine, 1,8bis(dimethylamino)naphthalene, 1,8- diazabicyclo [5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3,0]non-5-ene, 1,4-diazabicyclo[2.2.2]octane, 7-methyl-1,5,7- triazabicyclo[4.4.0]dec-5-ene, 4-(jVýV -61 CZ 309934 B6 dimethylamino)pyridine, 4-(Wdictylamino)pyridine. 4-(4-pyridyl)morpholine, 4-(1-pyrrolidinyl)pyridine, 2,6-lutidine, 2,4,6-collidine, 2,6-di-tert-butylpyridine, 1H-1,2,3-bcnztriazole. imidazole, triphenylphosphine, triethylphosphine, tributylphosphine, triethylphosphite or in the presence of combinations of the above and after performing the polycondensation reaction, the product is reduced to a polyol by the effect of a reducing agent from the group of sodium borohydride, potassium borohydride, sodium cyanoborohydride, borane and or agents that are prepared from agents of this group beforehand or in situ, preferably with sodium tri(acetoxy)borohydride or borane-pyridine complex or zinc borohydride. 26. Method for the preparation of spherical hollow nanoparticles according to claim 3, characterized in that in the third stage of preparation, the crosslinking component is a highly reactive polycarbonyl-a.m-biscarbene of the formula XXV (XXV), where the symbol n stands for 2 to 8, which is generated in situ, and the cross-linking is carried out by an ether polycondensation reaction or O-H insertion. 27. The preparation method according to claim 26, characterized in that the cross-linking is carried out by adding a precursor of the cross-linking component polycarbonyl-a.m-biscarbene of the formula XXV to a suspension of nanoparticles containing free hydroxyls of saccharide units in the temperature range of -80°C to 95°C, preferably at temperatures lower than the transition temperature parameter and the solidification temperature of the alkane of the nanoparticle core, in solvents from the group of acetonitrile, Wdimcthylformamide. N,Ndimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, dichloromethane, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, diethyl ether, 2-methoxy-2-methylpropane and their mixtures, wherein the precursor of polycarbonyl-α,co-biscarbene of formula XXV is bis-diazopolyketone formulas XXVI N=N=C θ ® H AD Θ (XXVI), where the symbol n stands for 2 to 8, which is prepared from precursors of general formula XXVII -62CZ 309934 B6 (and) (Φ (XXVII) and is used directly or is itself generated in situ in the reaction mixture from precursors of the formula XXVII, where n is 2 to 8, where the symbol SS is H or phenyl or pentafluorophenyl or trifluoromethyl, where the symbol RR is methanesulfonyl, 4-toluenesulfonyl, 2-Toluenesulfonyl, 2,4,6triisopropylbenzenesulfonyl, trifluoromethylsulfonyl, 4-nitrobenzenesulfonyl, 4-acetaminobenzenesulfonyl, 4-carboxybenzenesulfonyl, where the symbol Me denotes a cation of sodium, lithium, potassium or a quaternary ammonium salt. 28. The method of preparation according to claim 27, characterized in that the bis-diazopolyketone of formula XXVI is formed from precursors of formula XXVII by the action of reagents from the group W-bis-4toluenesulfonylhydrazine, W-bis-2-toluenesulfonylhydrazine. W-bis-(2.4.6triisopropylbenzenesulfonylhydrazine, N,N'-bismethanesulfonylhydrazine, 4dodecylbenzenesulfonyl azide, 4-carboxybenzenesulfonyl azide, 4-acetamidobenzenesulfonyl azide, 4-nitrobenzenesulfonyl azide, 2,4,6-trinitrobenzenesulfonyl azide, 4-chlorobenzenesulfonyl azide, methanesulfonyl azide, 2,4,6-triisopropylbenzenesulfonyl azide, 2-naphthalenesulfonylazide, diphenylazidophosphate, 2-azido-3-ethyl-1,3-benzothiazolium tetrafluoroborate, 2-azido-l-ethyl pyridinium tetrafluoroborate, (azidochloromethylene)dimethylammonium chloride, azidotris(diethylamino)phosphonium bromide, triflylazide, imidazole-l- sulfonylazide, 2-azido-1,3dimethylimidazolium chloride, 1H-1,2,3-benzotriazol-1-yl-sulfonylazide, nonafluorobutanesulfonylazide, trimethylamine, VVV.V-tctramctylcthylcndiamine. triethylamine, Nmethylmorpholine, Wdiisopropylcthylamine. l,8-bis(dimethylamino )naphthalene, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[2.2.2]octane, 7methyl-1,5 ,7-triazabicyclo[4.4.0]dec-5-ene, 4-(Wdimcthylaminojpyridine. 4-(W diethylamino)pyridine, 4-(4-pyridyl)morpholine, 4-(1-pyrrolidinyl)pyridine, 2,6- lutidine, 2,4,6-collidine, 2,6-di/cTC-biethylpyridine, 1H-1,2,3-bcnztriazole. imidazole, triphenylphosphine, triethylphosphine, tributylphosphine, triethylphosphite or from combinations of these reagents and polycarbonyl-a.m-biscarbcn XXV is formed from bis-diazopolyketone XXVI by the action of reagents and catalysts from the group of copper chloride, copper acetylacetonate, copper trifluoromethanesulfonate, dirhodium tetraacetate, palladium chloride and their complexes with organic ligands, without or in the presence of stabilizers and carbene carriers from the group of organic sulfides, preferably in the presence of tetrahydrothiophene. 29. The preparation method according to claim 26, characterized in that after the reaction is carried out, the polyether is reduced to a polyol by the effect of a reducing agent from the group of sodium borohydride, potassium borohydride, sodium cyanoborohydride, borane or agents that are prepared in advance or in situ from agents of this group , preferably by the effect of sodium borohydride, while the reduction is carried out in solvents from the group of tetrahydrofuran, dioxane, glyme, diglyme, triglyme, 2-methoxy-2-methylpropane, acetonitrile, dimethylformamide, water, methanol, ethanol, 2-propanol, 2-methyl- 2-propanol and mixtures of these solvents at temperatures ranging from -90°C to 80°C, preferably in methanol at room temperature. -63 CZ 309934 B6 30. The method of preparing spherical hollow nanoparticles according to claim 3, characterized in that in the third stage of preparation, the crosslinking component is a saccharide bis-carbene or a saccharide tris-carbene, or their mixtures, which are generated in situ, and the crosslinking is carried out by an ether polycondensation reaction or O-H insertion. 31. The preparation method according to claim 30, characterized in that the cross-linking is carried out by adding a saccharide carbene precursor of the formula XXIX (XXIX) to a suspension of nanoparticles containing free hydroxyls of saccharide units, where n denotes 2 or 3, where Prot denotes methoxycarbonyl, ethoxycarbonyl, tert -butyloxycarbonyl, acetoxyl which forms protected all hydroxyls of the saccharide in the temperature range of -80°C to 95°C, preferably at temperatures lower than the transition temperature parameter and the solidification temperature of the alkane of the nanoparticle core in solvents from the acetonitrile group, λ'.λ'- dimethylformamide. N,Ndimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, dichloromethane, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, diethyl ether, 2-methoxy-2-methylpropane and their mixtures. 32. The preparation method according to claim 31, characterized in that the carbene precursor of the saccharide of the formula XXIX is the hydrazones of the saccharide of the formula XXX-(a) or the JV-nitrosoamides of the saccharide of the formula XXX-(b) Prot Prot /\ (XXX), from which the carbene of formula XXIX is generated in situ in the reaction mixture, where n is 2 or 3, where the symbol RR is methanesulfonyl, 4-toluenesulfonyl, 2-toluenesulfonyl, 2,4,6triisopropylbenzenesulfonyl, trifluoromethanesulfonyl, 4-nitrobenzenesulfonyl, 4-acetaminobenzenesulfonyl, 4-carboxybenzenesulfonyl, benzoyl, 3,5-dinitrobenzoyl, aminocarbonyl, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, acetyl, pivaloyl, where the symbol Me denotes a sodium, lithium, potassium cation or quaternary ammonium salt, where Prot denotes groups acetoxyl, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, which form all protected hydroxyls of the saccharide, and where the saccharide carbene of the formula XXIX is formed from precursors of the general formula XXX by the action of agents from the group potassium carbonate, sodium carbonate, lithium carbonate, sodium bicarbonate, potassium bicarbonate, triethylamine , trimethylamine, N,N,N',N'tetramethylethylenediamine, JV-methylmorpholine, Λ'.Λ'-diisopropylcthylamine. 1,8-bis(dimethylamino)naphthalene, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3,0]non-5-ene, 1,4-diazabicyclo[2.2.2] octane, 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene, 4-(nVdimethylamino)pyridine, 4-(nV-diethylamino)pyridine, 4-(4-pyridyl)morpholine, 4 -(AND -64CZ 309934 B6 pyrrolidinyl)pyridine, 2,6-lutidine, 2,4,6-collidine, 2,6-di tert -butylpyridine, 1H-1,2,3-benzotriazole, imidazole, triphenylphosphine, triethylphosphine, tributylphosphine, triethylphosphite or by the action of combinations of these agents, without or under the action of agents and catalysts from the group of copper chloride, copper acetylacetonate, copper trifluoromethanesulfonate and dirhodium tetraacetate and their complexes with organic ligands, while the reaction is carried out without or in the presence of stabilizers and carbene carriers from the group of organic sulfides, preferably in the presence of tetrahydrothiophene. 33. The method of preparation according to claim 31, characterized in that after carrying out the reaction, the polysaccharide is obtained by removing protective groups from the skeleton of the cross-linking units, whereby the removal of protective groups is carried out by the effect of compounds from the group of potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, methanolate sodium, sodium ethanolate, potassium methanolate, while the removal of protective groups is carried out in solvents from the group of water, methanol, ethanol, 2-propanol, 2-methyl-2-propanol, 1,2-ethanediol and mixtures of these solvents at temperatures in the range of 0 °C to 80 °C, preferably in 90% by weight. aqueous methanol at room temperature. 34. Method for the preparation of spherical hollow nanoparticles according to claim 3, characterized in that in the third stage of preparation the crosslinking component is a highly reactive glycidyl derivative and the crosslinking is carried out by a glycide-ether polycondensation reaction, while the crosslinking units contain leaving groups chloride, bromide, methanesulfonoxyl, 4- toluenesulfonoxy, 2-toluenesulfonoxy, 2,4,6triisopropylbenzenesulfonoxy, trifluoromethanesulfonoxy, 4-nitrobenzenesulfonoxy, 4-acetaminobenzenesulfonoxy, 4-carboxybenzenesulfonoxy. 35. The preparation method according to claim 34, characterized in that the cross-linking is carried out by adding a glycidyl derivative to a suspension of nanoparticles containing free hydroxyls of saccharide units in the temperature range of -80°C to 95°C, preferably at temperatures lower than the temperature parameter of the transition and the temperature solidification of the alkane of the nanoparticle core in solvents from the group of acetonitrile, N,Ndimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, dichloromethane, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, diethyl ether, 2-methoxy-2-methylpropane and their mixtures without or in the presence of agents from the group potassium carbonate, sodium carbonate, lithium hydroxide, sodium hydroxide, potassium hydroxide, sodium methanolate, lithium methanolate, potassium methanolate, sodium ethanolate, sodium hydride, potassium tert-butanol, sodium tert-butanol, triethylamine, trimethylamine , N,N,N', N'-tetramethylethylenediamine, N-methylmorpholine, N,Ndiisopropylethylamine, 1,8-bis(dimethylamino)naphthalene, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5diazabicyclo [4.3.0]non-5-ene, 1,4-diazabicyclo[2.2.2]octane, 7-methyl-1,5,7triazabicyclo[4.4.0]dec-5-ene, 4-(N,N- dimethylamino)pyridine, 4-(N,N-diethylamino)pyridine, 4-(4pyridyl)morpholine, 4-(1-pyrrolidinyl)pyridine, 2,6-lutidine, 2,4,6-collidine, 2,6-diter -butylpyridine, lithium perchlorate, lithium chloride, lithium bromide, lithium iodide, lithium nitrate, lithium hexafluorophosphate, lithium tetrafluoroborate or in the presence of combinations of these agents. 36. The method of preparation of spherical hollow nanoparticles mentioned in claim 2, characterized by the fact that in the first stage of preparation, the surface-active substance formed by the structural unit of a saccharide, which is provided with a lipophilic group, is treated with an alkane in a polar solvent, while the interaction of a steric blocker creates a macroscopic a formation in which the steric blocker is partially embedded in the sphere skeleton formed by micelles of alkane with surface groups of saccharide, and the second and third stages of the preparation of nanoparticles are carried out by the method according to claims 15 to 35. 37. The method of preparing spherical hollow nanoparticles according to claim 36, characterized in that the first stage of preparation includes the action of an alkane on a steric blocker and the subsequent formation of micelles by the action of a surface-active substance in a polar solvent, or the addition of a steric blocker to the micelles formed from the alkane and a surface-active substance in polar solvent, preferably involves first the action of the solvent on the alkane-surfactant mixture and the subsequent addition of a steric blocker. 38. Method for preparing spherical hollow nanoparticles according to claim 36, characterized in that in the first stage of preparation the surface-active substance is a monosaccharide, disaccharide, oligosaccharide or polysaccharide with covalently bound one or more lipophilic aliphatic, alicyclic or aliphatic chains substituted by aromatic groups of a chain length corresponding to C10 to C45 and or - 65 CZ 309934 B6 a mixture of these saccharides, preferably the surfactant is a saccharide of general formula III, where w is 0 or 1, where either one of the substituents Ri to Rio in formula III is formed by one of the fragments of general formula XII j (ΧΠ), where the symbol "*" denotes a bond to the skeleton of general formula III, where K is O or NH, where e is 0 to 40, where Q is O or CH2, where J is H or NO2, where the remaining substituents from the group Ri to R 10 in formula III is hydrogen or any of the substituents of formula IV or formula V, or where any pair of substituents from the group Ri to R 10 outside of pairs of the same carbon atoms of the skeleton of formula III is formed by one of the fragments of general formula XIII (XIII), where the symbol "*" indicates a bond to the skeleton of the formula III, where e is 0 to 21, where Q is O or CH2, where J is H or NO2, where the remaining substituents from the group Ri to R10 in the formula III form hydrogen or any of the substituents having formula IV or formula V. 39. Method for preparing spherical hollow nanoparticles according to claim 36, characterized in that in the first stage of preparation, the lipophilic group of the surfactant is bound either through an ether bond or as a V-amino derivative, while the reaction mixture contains between 0 and 99% of saccharide with lipophilic ether-linked function and between 1 and 100% of a saccharide with a lipophilic N-alkylamine function, preferably in the reaction mixture there is between 1 and 100% of a saccharide with a lipophilic function according to general formula XII, where the symbol K is NH. 40. Method for preparing spherical hollow nanoparticles according to claim 36, characterized in that in the first stage of preparation, the alkane is an aliphatic or alicyclic hydrocarbon with a length corresponding to C16 to C60 or a mixture of these hydrocarbons, preferably technical paraffin, with a transition temperature parameter in the range of 35 °C to 80 °C and a melting temperature in the range of 35 °C to 80 °C, and the alkane concentration in the mixture is in the range of 5 to 40% by weight, preferably 20% by weight. and the solvent is water, methanol, N,N-dimethylformamide. -66CZ 309934 B6 N,N-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, N-methylmorpholine N-oxide and their mixtures, preferably the solvent is water and a mixture of methanol, ethanol, 2-propanol, 2-methyl-2propanol, N,N-dimethylformamide, N, of N-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide with water, and if required by the design, the ionic strength of the solvent is increased by the addition of 0.01 to 36% by weight. inorganic salts from the group sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium nitrate, potassium nitrate, ammonium sulfate, ammonium chloride, sodium iodide, potassium iodide, sodium bromide, potassium bromide, lithium chloride, lithium bromide, lithium iodide or salts organic acids from the group sodium acetate, potassium acetate or with the addition of tetramethylammonium chloride. 41. Method for the preparation of spherical hollow nanoparticles according to claim 36, characterized in that in the first stage of preparation the micelles are formed by effective mixing in the temperature range of 0 °C to 135 °C, preferably with technical paraffin with a melting temperature of 55 °C at a temperature in the range 85 to 95 °C. 42. Method for preparing spherical hollow nanoparticles according to claim 36, characterized in that the steric blocker used in the first stage of preparation is a polymeric nanoparticle of spherical or anisotropic shape, size 5 to 2000 nm and surface arrangement of functional groups. 43. The method of preparation according to claim 42, characterized in that the steric blocker is a polymeric spherical nanoparticle made of polystyrene, which is optionally hydrophobized by the effect of non-polar solvents from the group of hexane, heptane, cyclohexane by standing together in the mixture for 10 minutes to 24 hours at room temperature . 44. The preparation method according to claim 42, characterized in that the steric blocker is a binary anisotropic nanoparticle from the polystyrene-alkane binary system. 45. The preparation method according to claim 42, characterized in that the steric blocker is a spheroidal polystyrene nanoparticle hydrophilized on one hemisphere or its part. 46. The method of preparation according to claim 45, characterized in that the steric blocker is prepared by hydrophilizing the polystyrene part of the skeleton of a binary anisotropic nanoparticle with oligo-alkoxyalkyl in the form of C-insertion or O-H insertion after previous hydroxylation of the polystyrene skeleton, preferably by 2-methoxyethylation or 2-ethoxyethylation or 2-(2-methoxyethyloxy) ) by ethylation. 47. Method for preparing spherical hollow nanoparticles according to claim 36, characterized in that in the first stage of preparation, a steric blocker is added to the mixture in a ratio of particles in the range of 0.3 to 1.4 (blocker: micelle), preferably in a ratio in the range of 0 .8 to 0.9. 48. Method for preparing spherical hollow nanoparticles according to claim 36, characterized in that in the first stage of preparation after the formation and stabilization of micelles with steric blockers, the mixture is cooled to a temperature lower than the melting point of the alkane to form a suspension of spherical nanoparticles provided with steric blockers, preferably cooling is carried out to a temperature 40 °C lower than the melting point of the alkane. 49. The method of preparing spherical hollow nanoparticles according to claims 3 or 36, characterized in that by adding a steric blocker of a defined size in a defined amount to the reaction mixture in the first stage of preparation and its subsequent elimination after the third stage of preparation, a spherical hollow nanoparticle with a defined macropore size is formed the size of the steric blocker used and the number of macroholes defined by the amount of steric blockers in the reaction mixture. - 67 CZ 309934 B6 50. Use of hollow spherical nanoparticles with macroholes according to claim 1 as a biocidal agent of the virus trap type.