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WO2014141288A1 - The art, method, manner, process and system of a nano-biomineral for multi-modal contrast imaging and drug delivery - Google Patents

The art, method, manner, process and system of a nano-biomineral for multi-modal contrast imaging and drug delivery Download PDF

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Publication number
WO2014141288A1
WO2014141288A1 PCT/IN2013/000143 IN2013000143W WO2014141288A1 WO 2014141288 A1 WO2014141288 A1 WO 2014141288A1 IN 2013000143 W IN2013000143 W IN 2013000143W WO 2014141288 A1 WO2014141288 A1 WO 2014141288A1
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WIPO (PCT)
Prior art keywords
imaging
nanocontrast
multifunctional
calcium
nanoparticles
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PCT/IN2013/000143
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French (fr)
Inventor
Manzoor Koyakutty
Anusha ASHOKAN
Shantikumar V. Nair
Original Assignee
Amrita Vishwa Vidyapeetham University
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Application filed by Amrita Vishwa Vidyapeetham University filed Critical Amrita Vishwa Vidyapeetham University
Priority to PCT/IN2013/000143 priority Critical patent/WO2014141288A1/en
Publication of WO2014141288A1 publication Critical patent/WO2014141288A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4412—Non condensed pyridines; Hydrogenated derivatives thereof having oxo groups directly attached to the heterocyclic ring
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    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
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    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
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    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
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    • A61K49/183—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an inorganic material or being composed of an inorganic material entrapping the MRI-active nucleus, e.g. silica core doped with a MRI-active nucleus
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Definitions

  • the present invention relates to a nano-sized material that can provide contrast enhancement for multiple imaging methods and also deliver therapeutic molecules such as nucleic acids or chemo drugs to diseased sites such as cancer.
  • the present invention relates to nano-sized synthetic calcium phosphates and calcium apatite nanomaterials showing simultaneous contrast for at least any two of the medical imaging modalities including radio-, raman-, near-infrared fluorescence-, magnetic resonance- and X ray- imaging.
  • This invention relates to the preparation of a nanomaterial based on doped calcium phosphate or calcium apatite contrast agent for multimodal contrast imaging, drug-delivery and therapy applications.
  • Multimodal molecular imaging of disease using a combination of techniques such as single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), positron emission tomography (PET), X-ray computed tomography (CT) and near- infrared (NIR) fluorescence, raman spectroscopy is an emerging area of research [1,2].
  • SPECT single photon emission computed tomography
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • CT X-ray computed tomography
  • NIR near- infrared fluorescence
  • imaging systems and contrast agents are used independently to derive different anatomical and functional information such as location, size, angiogenesis, metastasis etc. It will be highly useful, if low-cost, but high resolution imaging modalities like optical (fluorescence) techniques can be combined with SPECT, MRI, CT Or raman spectroscopy to derive molecular information about the expression level of specific disease marker or biological process that influence the therapeutic outcome.
  • imaging modalities like optical (fluorescence) techniques can be combined with SPECT, MRI, CT Or raman spectroscopy to derive molecular information about the expression level of specific disease marker or biological process that influence the therapeutic outcome.
  • suitable contrast agents that can show different physical properties compatible with basic principles of imaging technique.
  • different molecular imaging techniques requires separate contrast agents because their working principles are distinctly different.
  • SPECT requires gamma radiation emitting nuclides
  • MRI requires paramagnetic
  • CT computed tomography
  • raman imaging requires raman active metals or dyes
  • in vivo fluorescence imaging requires near-infrared fluorescent contrast agents.
  • radionuclides such as Tc9 m is used for scintigraphy and 3D SPECT imaging
  • paramagnetic gadolinium complexes such as Gd-carboxylates or superparamagnetic iron oxide particles are used as MRI contrast agents
  • iodine or colloidal barium sulphate contrast agents are used in CT
  • Au Au
  • proteins Green fluorescent protein or Red fluorescent proteins
  • QDs quantum dots
  • contrast agents differ significantly.
  • An ideal solution is to provide a single contrast agent that can show all important contrast functionalities at the same time.
  • Luminescent quantum dots, superparamagnetic iron oxide nanoparticles, Ag nanoparticles and Au nanocrystals were studied for optical, magnetic, raman and photo- acoustic/two- photon imaging applications, respectively.
  • bi-functional nanosystems combining two separate agents such as luminescent QDs and magnetic iron oxide or fluorescent dyes and magnetic iron oxide were also reported for combinatorial (MRI and optical) imaging applications.
  • one of the major concerns about nanoparticle based contrast imaging is the efficient incorporation of multiple impurities and toxicity associated with each component within the nanomaterial.
  • luminescent semiconductor nanoparticles were found to be toxic to biological systems due to their heavy- metal composition (cadmium, selenium, tellurium, lead). Further, there exist no biocompatible single nanoparticle that can provide contrast enhancement for multiple imaging options such as SPECT, raman imaging, NI fluorescence, MRI and X-ray imaging.
  • HAp and calcium phosphates are extensively studied and used in clinical practice as implant material for in vivo bone tissue re-generation.
  • the present invention discloses, the preparation of improved calcium apatite and calcium phosphates (tricalcium phosphate, dicalcium phosphate, monocalcium phosphate) nanoparticles doped and labeled with multiple impurities that confer properties such as radioactivity, raman scattering, near-infrared fluorescence, X-ray absorption and super/para-magnetism.
  • the preparation of undoped hydroxyapatite and calcium phosphate nanoparticles is disclosed in several prior arts.
  • stoichiometric hydroxyapatite, Cai 0 (OH) 2 (P04)6 and calcium phosphate nanoparticles are prepared by adding ammonium phosphate or phosphoric acid and ammonium hydroxide mixture solution to a solution of Ca 2 ⁇ precursor.
  • Doped apatite/calcium phosphate nanoparticles is prepared by replacing calcium ions with fluorescent or paramagnetic metal ions.
  • Organic dye doped calcium phosphates can also be prepared by the incorporation of the dye within calcium phosphate matrix during its synthesis, wherein the dye can confer properties such NIR fluorescence or raman scattering.
  • Near infrared fluorescence based imaging is a recent advancement in biomedical research. Generally, in vivo fluorescence imaging using visible light is limited by low penetration of light photons into living tissue due to high absorption and scattering properties of tissue components. However, near- infrared region of the spectrum offers certain advantages because the hemoglobin and water absorb minimally in this spectral window (68o-90onm) so as to allow photons to penetrate for several centimeters deep inside the tissue. This allows mapping of molecular events in intact tissues using near-infrared fluorescence mediated molecular tomography (FMT), a new technique that can three- dimensionally image molecular events like gene expression by resolving fluorescence activation in deep tissues.
  • FMT near-infrared fluorescence mediated molecular tomography
  • an essential component of successful FMT imaging is the development of biocompatible NIR emitting fluorescent probes that can be targeted to the tissue.
  • various paramagnetic and superparamagnetic nanocontrast agents are being developed that has superior magnetic properties that their macro size counterparts.
  • Combining such nanocontrast agents with other imaging modalities such as NIR imaging provides added advantage of combining imaging of anatomic and functional characteristics of the diseased condition.
  • Research in combinatorial imaging approaches are exploring the advantages of combining anatomic imaging modalities such as MRI, 2D/3D CT imaging together with imaging modalities such as NIR, raman and radioactive imaging that give details on molecular characteristics of the pathological condition. It is therefore imperative to develop a biocompatible multimodal contrast agent that can combine the above mentioned modalities in combination to aid better and early stage disease diagnosis and treatment.
  • a novel biocompatible nanoparticle that can provide contrast enhancement for multiple molecular imaging modalities such as radioactive imaging, raman imaging, MRI, X-ray and/or near infrared (NIR) fluorescence together with targeted drug or gene delivery is disclosed.
  • the particle is based on nanoparticles of calcium phosphate (nCP) and calcium hydroxyapatite (nHAp), doped and/or conjugated with more than one impurity ion/molecule.
  • This multifunctional nano-composite particle enable combined molecular imaging using single photon emission computed tomography (SPECT), raman imaging, MRI, CT and/or optical near-infrared fluorescent imaging.
  • Another aspect of this invention relates to a method of making nanomedicine formulations based on the said nCP and nHAp particles, capable of delivering chemodrugs, small molecule inhibitors or nucleic acid drugs such as DNA, RNA, small interfering RNA (siRNA), micro RNA (miRNA) specifically to a targeted disease such as cancer.
  • nucleic acid drugs such as DNA, RNA, small interfering RNA (siRNA), micro RNA (miRNA) specifically to a targeted disease such as cancer.
  • nanoparticle refers to primary inventive nanoparticles formed by calcium phopshate or hydroxyapatite, measuring size about 1-500 nm, preferably l-ioonm, most preferably around 1- 50 nm in size, showing "multifunctional' properties such as radioactivity, raman scattering, NIR fluorescence, magnetism and X-ray absorption in different combinations with at least two properties together.
  • therapeutics refers to chemodrugs, nucleic acids such as DNA or RNA [small interfering RNA (siRNA) or micro RNA (miRNA)], bisphosphonates, antibodies, peptides, proteins, etc that have a therapeutic effect against a disease condition such as cancer, inflammatory disease or autoimmune disease.
  • nucleic acids such as DNA or RNA [small interfering RNA (siRNA) or micro RNA (miRNA)]
  • siRNA small interfering RNA
  • miRNA micro RNA
  • targeting ligand refers to biomolecules that can specifically identify and target another molecule like an antigen or receptor on the surface of cell-membrane of diseased cells such as that of cancer / tumor.
  • Targeting ligand include antibodies, peptides, aptamers, vitamins like folic acid, sugar molecules like mannose ligands, carbohydrates.
  • nanomedicine refers to a composite construct based on the said calcium phosphate or HAp nanoparticle which is connected or alternatively loaded with any of the above said therapeutics which will be delivered to the desired site using the said targeting ligands.
  • nanomedicine construct is formed by either : i) directly connecting the therapeutics and targeting ligands to the surface of said nanoparticles or alternatively, ii) the said nanoparticle will be taken as a core with another biodegradable polymer containing the therapeutics as shell with targeting ligands connected to its surface, or Hi) a biodegradable polymeric nanoparticle which embed nCP or nHAp together with the therapeutic agents and connected with the targeting ligands on its surface
  • the said nanomedicine construct preferably will have a size of 50- 20onm, more preferably 8o-i50nm and most preferably ⁇ ioo-i2onm.
  • the nanomedicine may be produced in the form of dry powders or liquid dispersions.
  • the nanoparticle may also be synthesized via colloidal synthesis or self- assembly of ions in saturated solutions like simulated body fluid (SBF) and may take the form of colloidal crystals with dopant ions or molecules in it.
  • SBF simulated body fluid
  • the term "doped” as used herein, refers to incorporation of small amount of (about 0.001-30 mole % or up to 30 weight %) another substance (inorganic elements, ions or organic molecules or ratioactive isotopes) within the said nanoparticles.
  • the dopant molecules include, elements, ions, dyes or molecular clusters that can provide radioactivity, raman scattering, NIR fluorescence, magnetic contrast by paramagnetic, superparamagnetic, ferromagnetic or ferrimagnetic properties or X-ray absorption.
  • the dopant ions may substitute either Ca or P ions, or may be present as molecular clusters within the interstitial space of nHAp or nCP lattice.
  • labelled refers to attaching the impurity ions, molecules or radioactive isotopes on the surface of the said nanoparticle with the help of a ligand such as bisphosphonates or functional groups such as carboxyl on the surface of the nanoparticle. This may involve electrostatic interactions , covalent-, ionic-, coordinate, or hydrogen bonding or hydrophobic, lipophilic or hydrophilic interactions
  • Figure 1 Schematic showing the method of preparation of multimodal and therapeutic nCP and nHAp
  • Figure 2 (a) XRD of MF-nHAp showing hexagonal HAp crystal structure (b) SEM image of MF-nHAp showing particle size ⁇ 100 nm. (c) DLS data of MF- nHAp giving a size distribution of 100 ⁇ 20nm (d) Digital photograph of MF- nHAp and nHAp solutions ( ⁇ 20 mg/mL) indicating the greenish body color due to ICG doping .
  • Figure 3 (A) Graph showing integrated fluorescence emission intensity at 800 nm with increase in MF-nHAp concentration. Inset: Fluorescence spectra of MF- nHAp showing excitation and emission peaks at 765 nm and 800 nm, respectively.
  • Figure 4 (a) Radioactive emission of 20mg/mL of MF-nCP samples conjugated to Tc-99m of varying concentrations (b) Fluorescence images of MF-nCP samples conjugated to varying concentrations of ICG (c) Graph showing the variation in radioactive emission of MF-nCP samples conjugated to varying concentrations of Tc-99m (d) Graph showing the fluorescence intensity variation of MF-nCP with varying doping % of ICG
  • FIG. 5 (A) Hemolysis data of nHAp and D-nHAp treated whole blood showing non-hemolytic action of the tested samples (B) Digital photograph of the whole blood treated with PBS, D-nHAp and triton showing no leakage of hemoglobin from PBS/D-nHAp treated samples compared to triton treated sample.
  • Figure 6 Scanning electron micrograph of RBC treated with (A) PBS (B) D-nHAp. Enlarged image (C))showing uniform distribution of D- nHAp particles although out the RBC without affecting its morphology (D) EDX spectrum of the D-nHAp that are distributed on the RBC.
  • Figure 7 Cytotoxicity data of D-nHAp treatment on peripheral blood derived mononuclear cells after 24 hours incubation showing no toxicity up to a concentration of 500 ug/mL.
  • Figure 8 Radioactive emission images (a-d) and fluorescence images (e-h) of MF-nCP sample imaged at varying depths in pork tissue.
  • the bottle containing the sample placed in pork tissue was rotated and images taken at varying angles. Depth at which the sample was imaged from, in tissue, is denoted as d and the angle at which the bottle is rotated is denoted as ⁇ .
  • Figure 9 In vivo multimodal imaging of MF-nCP after tail vein injection into mice.
  • Figure (a) shows the fluorescence image
  • (b) shows radioactive emission image
  • (c) shows radioactive emission image overlaid over X-ray images.
  • the method of preparation of said nanoparticle and nanomedicine formulation consists the steps of:
  • the precursor compound, part A is formed from a material selected from the group consisting of sulphate, phosphate, hydroxide, chloride, bromide, iodide, fluoride, nitrate, carbonate or oxide salts of calcium.
  • another precursor compound, Part B is formed from water soluble or miscible salt of phosphates including sodium (Na 3 P0 4 , Na 2 HP0 4 , NaH 2 P0 4 ), potassium (K 3 P0 4 , KsHPC-4, KH 2 P0 4 ) lithium (Li 3 P0 4 , Li 2 HP0 4 , LiH 2 P0 4 ), ammonium ((NH 4 ) 3 P0 4 , (NH 4 ) 2 HP0 4 , NH 4 H 2 P0 4 ) or phosphoric acid
  • the precursor compound containing hydroxyl anions (Part C) is formed by hydroxide salts of sodium, potassium, lithium, ammonium or calcium .
  • the dopant molecules (Part D) in the nanoparticle that gives raman scattering is formed by cyanine dyes (Cy3, Cy5, ICG), rhodamine (rhodamine 6G, tetramethyl rhodamine, rhodamine no), aniline blue, crystal violet, malachite green, basic fuchsin, variamine blue RT salt, triaminopyrimidine sulphate or porphyrins.
  • cyanine dyes Cy3, Cy5, ICG
  • rhodamine rhodamine 6G, tetramethyl rhodamine, rhodamine no
  • aniline blue crystal violet
  • malachite green malachite green
  • basic fuchsin basic fuchsin
  • variamine blue RT salt triaminopyrimidine sulphate or porphyrins.
  • the dopant molecules (Part D) that gives near-infrared fluorescence to the nanoparticle is NIR emitting dyes such as Indocyanine green (ICG).
  • the dopant ions (Part D) in the nanoparticle that gives magnetic contrast includes ions such as chromium(III), manganese(II), iron(II), iron (III), praseodymium (III), neodymium (III), samarium(III), ytterbium(III), gadolinium(lll), terbium(III), dysprosium(III), holmium(III), erbium(III).
  • the dopant ions (Part D) in the nanoparticle that gives X-ray contrast includes ions such as iodine or barium, bismuth, strontium tungsten, tantalum, hafnium, lanthanum, molybdenum, niobium, zirconium.
  • the doped nanoparticle is conjugated with a radioactive element that includes Sm-153, Tc- 99m, 12 3I, 18F, l 3 » I, '"In, l88 Re, l66 Ho, ⁇ ⁇ 0 r 82 Rb through ligands such as bisphOsphonates.
  • a radioactive element that includes Sm-153, Tc- 99m, 12 3I, 18F, l 3 » I, '"In, l88 Re, l66 Ho, ⁇ ⁇ 0 r 82 Rb through ligands such as bisphOsphonates.
  • the nanoparticle with dopant ions / molecules were formed by reacting precursor Part-A, B, C and D at a temperature range of 25-i50°C and pH range of 5 -12.
  • the dopant ions may be mixed preferably with either Part-A or Part B or added separately during the reaction between Part A, B and C.
  • the formed nanoparticles are grown to a preferred size scale, between 1-200 nm by aging of the reactants at time scales of any range between 0-24 Hrs, preferably, 0-6 Hrs and most preferably 1-4 hrs at a temperature range of 5-i50°C.
  • Figure 2b and 2c shows the SEM and DLS images of ICG and Gd3 + co-doped multifunctional hydroxyapatite nanoparticles (MF-nHAp). 90% of the particles synthesized were ⁇ 100 ⁇ 25 nm in size as seen in DLS and SEM data.
  • the XRD data of MF-nHAp gave characteristic peaks of hexagonal HAp crystal lattice as shown in Figure 2a.
  • inset of Figure 3A shows the fluorescence excitation and emission spectra of ICG and Gd3 + co-doped MF-nHAp, peaking at 760 nm and 830 nm proving the capability of the material for non invasive in vivo NIR imaging.
  • the total weight percentage is varied in the range of 0.01 - 1%.
  • the total concentration of ICG in the precursor solution is maintained less than 2xio _ s M to avoid agglomeration of dye molecules.
  • the said nanoparticle can be used for molecular imaging based NIR fluorescence for biomedical applications. This is displayed in phantom experiments as shown in Figure 3B that shows increase in fluorescence with increase in MF-nHAp concentration.
  • the nanoparticles show X-ray contrast properties (radio opacity) together with NIR imaging.
  • X-ray contrast imaging of powder nanoparticle samples of different dopant concentrations are displayed.
  • Undoped and doped samples were made into pellets and imaged using 12.87 KeV X-ray energy (0.4mm filter) for an exposure time of 30 seconds in a digital X-ray imaging station (Kodak in vivo multispectral imaging system, Carestream, USA).
  • the X-Ray density of the material which is a measure of the X-ray absorbed or attenuated, was calculated and plotted in Figure 3C.
  • the said NIR emitting nanoparticles show paramagnetism together with NIR fluorescence and X-ray contrast when co-doping ICG with paramagnetic Gd ions.
  • Figure 3E refers to magnetic studies carried out by keeping the sample under a varying magnetic field, in an instrument called vibrating sample magnetometer. When undoped sample show diamagnetic property the ICG and Gadolinium doped samples show paramagnetic property, which increases with the concentration of dopant ions, gadolinium in this case.
  • FIG. 4a shows the radioactive emission from MF- nCP samples. As seen in the figure tagging of Tc-99m increases with increase in Tc-99m concentration till a saturation value of 250 ⁇ ' (Figure 4c). Figure 4b shows increase in fluorescence with increase in % doping of ICG till a saturation value of 0.16 mole% ( Figure 4d).
  • FIG. 5A shows the hemocompatibility of the doped nHAp (D-nHAp) where D-nHAp and D- nHAp cause hemolytic effects upto a concentration of as shown in Figure 5A .
  • Figure 5B shows the blood samples treated with D-nHAp and triton where hemolysis is clearly visible in triton treated samples and D-nHAp treated sample is comparable to PBS treated blood.
  • Figure 6 shows SEM image of RBC treated with D-nHAp ( Figure 6B) that shows no membrane damage compared to PBS treated RBC ( Figure 6A).
  • Figure 6D shows EDAX data of D-nHAp distributed over RBC.
  • the said nanoparticles are highly bio-compatible to primary human cells, for eg; peripheral blood mononuclear cells, as shown in Figure 7 .
  • Phantom imaging using Tc tagged and ICG doped MF-nCP was done in pork tissue samples to check the efficiency of contrast when imaged in tissue samples.
  • Figure 8 shows that both NIR fluorescence and radioactivity is imageable at varying depths up to 2.5 cm.
  • Later imaging in mice was carried out using the same MF-nCP nanoparticles that proved the ability of the material for multimodal imaging under in vivo conditions.
  • Figure 9 shows the fluorescent and radioactive imaging of mice after tail vein injection of the MF-nCP sample. All these imaging experiment were carried out in an in vivo imaging station (Kodak in vivo multispectral imaging system, Carestream, USA).
  • the high resolution and blood penetration capability of near-infrared light can help doctors, during a surgery, to identify angiogenic blood vessels in the vicinity of a solid tumor, for its effective removal during a surgical procedure.
  • Angiogenesis is a process of formation of larger concentration of new blood vessels around a growing tumor and the removal of such blood vessels is critical in stopping recurrence of tumor.
  • the said nanoparticles conjugated with antibody targeted against angiogenic growth factors may illuminate the cancer related blood vessels together with providing contrast for the tumor.
  • the doped nanoparticles are cationic in nature, the same can be loaded with nucleic acid drugs such as DNA or RNA or its small fractions like siRNA, for therapeutic gene delivery applications.
  • nucleic acid drugs such as DNA or RNA or its small fractions like siRNA, for therapeutic gene delivery applications.
  • mAbs monoclonal antibodies
  • pep peptides
  • similar ligands ligands
  • the said nanoparticles can also be Coated with a biodegradable polymers containing anticancer drugs or nucleic acid drugs.
  • the biodegradable polymers can be formed by polymers such as poly-lactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyvinylpyrrolidon (PVP), polyvinyl alcohol (PVA), polyethyleneimine (PEI), polyethelene glycol (PEG), chitosan, carboxymethyl chitosan, cyclodextrin, thermosensitive polymers such as Poly(JV- isopropylacrylamide) and its derivative or proteins such as bovine or human serum albumin.
  • PLA poly-lactic acid
  • PLGA poly(lactic-co-glycolic acid)
  • PVP polyvinylpyrrolidon
  • PVA polyvinyl alcohol
  • PEI polyethyleneimine
  • PEG polyethelene glycol
  • chitosan carboxymethyl chitosan
  • cyclodextrin thermosensitive polymers
  • thermosensitive polymers such as Poly(JV- isopropylacrylamide) and its derivative or proteins such as
  • the said nanoparticle or drug loaded nanomedicine construct is connected, at the surface with cancer targeting ligands including antibodies, peptides or small molecular ligands such as folic acid or aptamers
  • the present invention provides an injectable composition or composition for oral administration comprising the said nanomedicine according to the present invention as described above, together with a pharmaceutically acceptable medium.
  • the present invention provides a method of image guided delivery of the drug, estimation of drug concentration at diseased site with respect to other regions, estimation of pharmacokinetics and pharmacodynamics, treatment planning and estimation of treatment efficacy after preferred treatment, all the above using any of the said contrast imaging property of the nanoparticle
  • essentially the present invention provides a method of simultaneously detecting and treating the disease like cancer with the help of more than one and upto 05 different molecular imaging techniques using a single nanoparticle system.
  • the inventors have discovered primarily a nanoparticle that provides novel concept for combining a number of important molecular imaging techniques such as radioactivity, raman scattering, near-infrared fluorescence imaging, which can register both spatial and temporal functional properties of a disease at microscopic level such as expression level of a cancer biomarker, with other properties such as super/para-magnetism and X-ray absorbance which gives micro/ macroscopic anatomical information.
  • the inventive step provide a nanomedicine formulation, containing the said nanoparticle as a mairi component together with therapeutic molecules such as chemo drugs or nucleic acids that can treat disease in a targeted manner by conjugation of a ligand specific to a receptor in the diseased tissue /cell.
  • Example 1 Multifunctional contrast agent based on calcium phosphate nanoparticles with 2 dopants for combined NIR and radioactive imaging.
  • the pH during the entire process was maintained at ⁇ 7.4 by the addition of 0.1M NH 4 OH solution.
  • the precipitate was left stirring for another 30 minutes, centrifuged and washed at least 2 times.
  • the entire process was carried out room temperature at ⁇ 25°C. - 3omg/mL of the washed product was treated with lmL of 250 ⁇ Tc-MDP (Tc- 99m conjugated to methylene diphosphate). The mixture is vortexed for 2 minutes and incubated for another 20 minutes.
  • the conjugate is washed at least twice and resuspended in PBS or distilled water for further applications.
  • ICG can be replaced with any NIR emitting dye or raman active dye molecules for the synthesis of other NIR emitting or raman active calcium phosphate nanoparticles.
  • Example 2 Multifunctional contrast agent based on calcium phosphate nanoparticles with more than 2 dopants for combined raman. magnetic. X-ray and radioactive imaging.
  • the entire process was carried out room temperature at ⁇ 25 C. ⁇ 3omg/mL of the washed product was treated with lmL of 250 ⁇ Tc-MDP(Tc-99m conjugated to methylene diphosphate). The mixture was vortexed for 2 minutes and incubated for another 20 minutes. The conjugate was washed at least twice and resuspended in PBS or distilled water for further applications.
  • Aniline blue can be replaced by any other raman active or NIR emitting dye for the synthesis of other raman active or NIR emitting calcium phosphate nanoparticles.
  • Gadolinium can be replaced by any other impurity ion to give magnetic or X-ray contrast properties.
  • Tc-99m can be replaced with any other radioactive element to synthesize other radioactive calcium phosphate nanoparticles.
  • Example 3 Multifunctional contrast agent based on hydroxyapatite nanoparticles with more than 2 dopants for combined raman. magnetic. X-rav and radioactive imaging,
  • Aniline blue can be replaced by any other raman active or NIR emitting dye for the synthesis of other raman active or NIR emitting hydroxyapatite nanoparticles.
  • Gadolinium can be replaced by any other impurity ion to give magnetic or X-ray contrast properties.
  • Tc-99m can be replaced with any other radioactive element to synthesize other radioactive hydroxyapatite nanoparticles.
  • Example 4 Preparation of siRNA incorporated calcium phosphate nanoparticles based nanomedicines capable of image guided delivery.
  • siRNA therapeutic small interfering RNA
  • Antisense strand 3 1 -dTdTCGGCGAGCAACCUUGAGGU— 5 1
  • lmg/ml bare nanoparticles prepared as said in any of the above examples 1-3 is prepared in phosphate buffer saline (PBS, Sigma, USA)) and sonicated for 10 minutes to get a fine dispersion.
  • PBS phosphate buffer saline
  • the nCP-siRNA conjugates were further treated with o.oimg/ml BSA (Bovine Serum Albumin) and o.img/ml EDC (i-ethyl-3-(3- dimethylaminopropyl) carbodiimi.de) for 3omin at 37°C. After 30min, the nanoconjugates are removed by centrifugation and re-suspended in PBS. This forms a protective shell of albumin protein that can be derivatized using targeting ligands.
  • BSA Bovine Serum Albumin
  • EDC i-ethyl-3-(3- dimethylaminopropyl) carbodiimi.de
  • this example provides preparation of a representative nanomedicine formed by loading the said multifunctional calcium phosphate nanoparticles with a representative gene.
  • a gene preferably a marker gene that can be readily detected by simple laboratory tools
  • An appropriate marker gene selected for this example is beta.- galactosidase ( ⁇ -gal) since its expression can be readily detected by addition of X- gal, a substrate which yields a blue color when the active enzyme is present.
  • ⁇ -gal beta.- galactosidase
  • this example is not limited to the said marker gene, but any gene intended for a desired function such as inhibition of tumor growth.
  • Part B Another two solutions containing phosphate group (Part B), ammonium dihydrogen phosphate, (5 mL, 0.3M, 98%, Qualigens, India) and 1 ml NH 4 OH (0.1M, 25% NH 3 , Qualigens, India) are prepared. Both these solutions are added drop wise to the precursor mixture of Part A+D with continuous stirring over a period of 15 minutes. The rate of addition of ammonium hydroxide solution is adjusted to maintain the pH of the reaction medium at ⁇ 7.4 throughout the reaction. After completion of precipitation, the mixture is kept stirring for 1 hour followed by separation of the precipitate by centrifugation at 3000 rpm for lominutes and washing with ice cold water for 3-5 times.
  • the DNA embedded within the doped calcium phosphate matrix is optionally protected from enzymatic degradation using a protective coat of BSA, by treating the nCP-DNA conjugates with o.oimg of BSA and o.img of EDC in 5ml PBS for 30mm at 37°C, followed by washing with water.
  • Example 6 Preparation of nCP-polymer nanomedicine containing chemo drugs capable of image guided delivery
  • a representative nanomedicine formed by the said multifunctional calcium phosphate nanoparticles embedded within polymeric nanoparticle containing chemical drugs, for example a small molecule inhibitor, Sorafenib is presented, l mg/ml bare nanoparticles prepared as said in any of the above examples 1-3 is prepared in DMSO (dimethyl sulfoxide) medium and sonicated for 10 minutes to get a fine dispersion.
  • DMSO dimethyl sulfoxide
  • ⁇ ⁇ Sorafenib in lmg/ml PLGA poly(lactic-co-glycolic acid)
  • DMSO dimethyl sulfoxide
  • ⁇ 500 ⁇ , of H 2 0 is added to the CP-Sorafenib-PLGA mixture to precipitate the polymeric nanoparticles that contain CP and Sorafenib embedded in it.
  • This nanomedicine formulation can be used to deliver drug actively or passively to tumor sites together with imaging using multiple imaging modalities.
  • PLGA other biodegradable polymers such as PEI, PLA, PCL, PVA, PPV etc also can be used for embedding the nCP and chemical drug.
  • Example 7 Bisphosphonate tagged nCP for imaging and therapeutic applications
  • synthesis of bisphosphonate drug, alendronate, tagged Tc99m labeled doped calcium phosphate nanoparticles for imaging and therapy of diseases such as bone metastasis or other bone metabolic disorders, osteoporosis etc is presented.
  • the precipitate was left stirring for another 30 minutes, centrifuged and washed at least 2 times. The entire process was carried out room temperature at ⁇ 25°C. ⁇ 30mg/mL of the washed product was treated with lmL of 250 ⁇ Tc99m-alendronate (Tc-99m conjugated to alendronate drug). The mixture is vortexed for 2 minutes and incubated for another 20 minutes. The conjugate is washed at least twice and resuspended in PBS or distilled water for further applications.

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Abstract

The present invention relates to a nano-sized material that can provide contrast enhancement for multple imaging methods and also deliver therapeutic molecules such as nucleic acids or chemo drugs to diseased sites such as cancer. In particular, the present invention relates to nano-sized synthetic calcium phosphates and calcium apatite nanomaterials showing simultaneous contrast for at least any two of the medical imaging modalities including radio, raman-, near-infrared fluroscence-, magnetic resonance and x-ray-imaging.

Description

FORM-2
THE PATENTS ACT.1970
(39 of 1970) AND
THE PATENT RULES 2003
COMPLETE SPECIFICATION
(See Sections 10 rule 13)
1. TITLE OF THE INVENTION
The Art. Method. Manner. Process and System of nano-biomineral for multimodal contrast imaging and drug delivery
2. APPLICANT(S)
(a) Name: AMRITA VISHWAVIDYAPEETHAM UNIVERSITY represented by its Director, Amrita Centre of Nano Sciences, Dr. Shantikumar Nair
(b) Nationality : Indian.
(c) Address ; "Elamakkara P.O., Cochin 682 026, Kerala
2. PREAMBLE TO THE DESCRIPTION COMPLETE SPECIFICATION
The following specification describes the invention
FIELD OF THE INVENTION
The present invention relates to a nano-sized material that can provide contrast enhancement for multiple imaging methods and also deliver therapeutic molecules such as nucleic acids or chemo drugs to diseased sites such as cancer. In particular, the present invention relates to nano-sized synthetic calcium phosphates and calcium apatite nanomaterials showing simultaneous contrast for at least any two of the medical imaging modalities including radio-, raman-, near-infrared fluorescence-, magnetic resonance- and X ray- imaging.
BACKGROUND OF THE INVENTION
This invention relates to the preparation of a nanomaterial based on doped calcium phosphate or calcium apatite contrast agent for multimodal contrast imaging, drug-delivery and therapy applications. Multimodal molecular imaging of disease using a combination of techniques such as single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), positron emission tomography (PET), X-ray computed tomography (CT) and near- infrared (NIR) fluorescence, raman spectroscopy is an emerging area of research [1,2]. Combinatorial imaging can provide simultaneous high definition anatomical, physiological and functional information about the diseases like cancer leading to early stage diagnostics, image guided drug deliver}' and therapeutics, and efficacy analysis of the treatment [3,4]. At present, separate imaging systems and contrast agents are used independently to derive different anatomical and functional information such as location, size, angiogenesis, metastasis etc. It will be highly useful, if low-cost, but high resolution imaging modalities like optical (fluorescence) techniques can be combined with SPECT, MRI, CT Or raman spectroscopy to derive molecular information about the expression level of specific disease marker or biological process that influence the therapeutic outcome. However, one of the major challenges in multi-modal molecular imaging is the requirement of suitable contrast agents that can show different physical properties compatible with basic principles of imaging technique. At present, different molecular imaging techniques requires separate contrast agents because their working principles are distinctly different. For example, SPECT requires gamma radiation emitting nuclides, MRI requires paramagnetic, super paramagnetic contrast agents, computed tomography (CT) requires X-ray absorbing contrast agent, raman imaging requires raman active metals or dyes and in vivo fluorescence imaging requires near-infrared fluorescent contrast agents. Accordingly, radionuclides such as Tc9 m is used for scintigraphy and 3D SPECT imaging, paramagnetic gadolinium complexes such as Gd-carboxylates or superparamagnetic iron oxide particles are used as MRI contrast agents, iodine or colloidal barium sulphate contrast agents are used in CT, Au, Ag nanoparticles for raman imaging and fluorescent molecules, proteins (Green fluorescent protein or Red fluorescent proteins) or quantum dots (QDs) are used for optical imaging [5-9]. For combining all these different imaging techniques to obtain both functional as well as anatomical information, separate contrast agents need to be injected in to the human body. Further, these contrast agents should reach to the disease site at the same time, at sufficient concentrations. This invite
r
formidable challenges because the pharmacokinetics of all the above mentioned i
contrast agents differ significantly. An ideal solution is to provide a single contrast agent that can show all important contrast functionalities at the same time.
With the emergence of nanotechnology, a number of new nanocontrast agents were proposed for molecular imaging applications. Luminescent quantum dots, superparamagnetic iron oxide nanoparticles, Ag nanoparticles and Au nanocrystals were studied for optical, magnetic, raman and photo- acoustic/two- photon imaging applications, respectively. In addition, bi-functional nanosystems combining two separate agents such as luminescent QDs and magnetic iron oxide or fluorescent dyes and magnetic iron oxide were also reported for combinatorial (MRI and optical) imaging applications. However, one of the major concerns about nanoparticle based contrast imaging is the efficient incorporation of multiple impurities and toxicity associated with each component within the nanomaterial. For example, luminescent semiconductor nanoparticles (quantum dots) were found to be toxic to biological systems due to their heavy- metal composition (cadmium, selenium, tellurium, lead). Further, there exist no biocompatible single nanoparticle that can provide contrast enhancement for multiple imaging options such as SPECT, raman imaging, NI fluorescence, MRI and X-ray imaging.
In the present invention, we disclose a method of preparation of nanocontrast agent based on calcium phosphates or calcium apatite doped and labeled with multiple impurities, including inorganic and organic, for deriving different physical properties suitable for simultaneous multi-modal contrast imaging. Calcium , phosphate [Ca3(P04)2] and calcium hydroxyapatite [Caio(P04)6(OH)2] forms the biomineral constituent of human bone and teeth and hence offer excellent biocompatibility. HAp and calcium phosphates are extensively studied and used in clinical practice as implant material for in vivo bone tissue re-generation.
In the prior art, the preparation of calcium phosphate and apatite particles doped with either NIR, paramagnetic, X-ray absorbing impurities or ultrasound contrast were reported [US patent no: 5342609, 5690908, 5468465, 5344640, US patent Application number: 12/941,719, Indian patent application no. 2633/CHE/2009] as listed in the reference. However, there exist no prior art on the preparation of calcium apatite or calcium phosphate nanocrystals doped and labeled with multiple impurities showing radioactivity, raman scattering. NIR contrast, magnetism and X-rav contrast properties in combination suitable for multimodal medical imaging.
Accordingly, the present invention discloses, the preparation of improved calcium apatite and calcium phosphates (tricalcium phosphate, dicalcium phosphate, monocalcium phosphate) nanoparticles doped and labeled with multiple impurities that confer properties such as radioactivity, raman scattering, near-infrared fluorescence, X-ray absorption and super/para-magnetism. In general, the preparation of undoped hydroxyapatite and calcium phosphate nanoparticles is disclosed in several prior arts. For example, stoichiometric hydroxyapatite, Cai0(OH)2(P04)6 and calcium phosphate nanoparticles are prepared by adding ammonium phosphate or phosphoric acid and ammonium hydroxide mixture solution to a solution of Ca2† precursor. Doped apatite/calcium phosphate nanoparticles is prepared by replacing calcium ions with fluorescent or paramagnetic metal ions. Organic dye doped calcium phosphates can also be prepared by the incorporation of the dye within calcium phosphate matrix during its synthesis, wherein the dye can confer properties such NIR fluorescence or raman scattering.
Various techniques for controlling the particle size for calcium apatite or calcium phosphates are also disclosed in the prior arts. For example, basic pH, slower addition of reactants, faster stirring, higher reaction temperatures, and lower concentrations generally result in smaller particles. In addition, sonication during precipitation, turbulent flow or impingement mixers and homogenization are also used to control particle size. Other means, such as computer controlled auto-burettes, peristaltic pumps, and syringes, may be used to control the release of precipitating ions to produce smaller particles.
Near infrared fluorescence based imaging is a recent advancement in biomedical research. Generally, in vivo fluorescence imaging using visible light is limited by low penetration of light photons into living tissue due to high absorption and scattering properties of tissue components. However, near- infrared region of the spectrum offers certain advantages because the hemoglobin and water absorb minimally in this spectral window (68o-90onm) so as to allow photons to penetrate for several centimeters deep inside the tissue. This allows mapping of molecular events in intact tissues using near-infrared fluorescence mediated molecular tomography (FMT), a new technique that can three- dimensionally image molecular events like gene expression by resolving fluorescence activation in deep tissues. Obviously, an essential component of successful FMT imaging is the development of biocompatible NIR emitting fluorescent probes that can be targeted to the tissue. In MRI, various paramagnetic and superparamagnetic nanocontrast agents are being developed that has superior magnetic properties that their macro size counterparts. Combining such nanocontrast agents with other imaging modalities such as NIR imaging, provides added advantage of combining imaging of anatomic and functional characteristics of the diseased condition. Research in combinatorial imaging approaches are exploring the advantages of combining anatomic imaging modalities such as MRI, 2D/3D CT imaging together with imaging modalities such as NIR, raman and radioactive imaging that give details on molecular characteristics of the pathological condition. It is therefore imperative to develop a biocompatible multimodal contrast agent that can combine the above mentioned modalities in combination to aid better and early stage disease diagnosis and treatment.
This is achieved, in the present invention, by doping calcium phosphates or apatite with multiple impurities and impurity clusters, while maintaining the particle size range ~ 30-ioonm. In the prior art, while the doping of apatite with individual atoms, that replaces either calcium or hydroxyl ions were disclosed, here we disclose, an improved novel method of doping of calcium phosphates or calcium apatite with multiple impurity ions/molecules. These doped impurities show its own new properties of practical significance such as radioactivity, raman scattering, near-infrared fluorescence, magnetism and/or X-ray contrast. In addition, we also disclose, a method of utilising such new materials for producing nanomedical formulation for its use in human health care, more specifically in image guided drug delivery and therapy.
SUMMARY OF THE INVENTION
In the present invention, a novel biocompatible nanoparticle that can provide contrast enhancement for multiple molecular imaging modalities such as radioactive imaging, raman imaging, MRI, X-ray and/or near infrared (NIR) fluorescence together with targeted drug or gene delivery is disclosed. Specifically, the particle is based on nanoparticles of calcium phosphate (nCP) and calcium hydroxyapatite (nHAp), doped and/or conjugated with more than one impurity ion/molecule. This multifunctional nano-composite particle enable combined molecular imaging using single photon emission computed tomography (SPECT), raman imaging, MRI, CT and/or optical near-infrared fluorescent imaging. Another aspect of this invention relates to a method of making nanomedicine formulations based on the said nCP and nHAp particles, capable of delivering chemodrugs, small molecule inhibitors or nucleic acid drugs such as DNA, RNA, small interfering RNA (siRNA), micro RNA (miRNA) specifically to a targeted disease such as cancer.
DETAILED DESCRIPTION OF THE INVENTION
Definitions : The term "nanoparticle " as used herein refers to primary inventive nanoparticles formed by calcium phopshate or hydroxyapatite, measuring size about 1-500 nm, preferably l-ioonm, most preferably around 1- 50 nm in size, showing "multifunctional' properties such as radioactivity, raman scattering, NIR fluorescence, magnetism and X-ray absorption in different combinations with at least two properties together.
The term " therapeutics" as used herein refers to chemodrugs, nucleic acids such as DNA or RNA [small interfering RNA (siRNA) or micro RNA (miRNA)], bisphosphonates, antibodies, peptides, proteins, etc that have a therapeutic effect against a disease condition such as cancer, inflammatory disease or autoimmune disease.
The term "targeting ligand" as used herein refers to biomolecules that can specifically identify and target another molecule like an antigen or receptor on the surface of cell-membrane of diseased cells such as that of cancer / tumor. Targeting ligand include antibodies, peptides, aptamers, vitamins like folic acid, sugar molecules like mannose ligands, carbohydrates.
The term "nanomedicine' as used herein refers to a composite construct based on the said calcium phosphate or HAp nanoparticle which is connected or alternatively loaded with any of the above said therapeutics which will be delivered to the desired site using the said targeting ligands.
The above said "nanomedicine " construct is formed by either : i) directly connecting the therapeutics and targeting ligands to the surface of said nanoparticles or alternatively, ii) the said nanoparticle will be taken as a core with another biodegradable polymer containing the therapeutics as shell with targeting ligands connected to its surface, or Hi) a biodegradable polymeric nanoparticle which embed nCP or nHAp together with the therapeutic agents and connected with the targeting ligands on its surface
The said nanomedicine construct preferably will have a size of 50- 20onm, more preferably 8o-i50nm and most preferably ~ioo-i2onm. The nanomedicine may be produced in the form of dry powders or liquid dispersions. The nanoparticle may also be synthesized via colloidal synthesis or self- assembly of ions in saturated solutions like simulated body fluid (SBF) and may take the form of colloidal crystals with dopant ions or molecules in it.
The term "doped" as used herein, refers to incorporation of small amount of (about 0.001-30 mole % or up to 30 weight %) another substance (inorganic elements, ions or organic molecules or ratioactive isotopes) within the said nanoparticles. In the present case, the dopant molecules include, elements, ions, dyes or molecular clusters that can provide radioactivity, raman scattering, NIR fluorescence, magnetic contrast by paramagnetic, superparamagnetic, ferromagnetic or ferrimagnetic properties or X-ray absorption. The dopant ions may substitute either Ca or P ions, or may be present as molecular clusters within the interstitial space of nHAp or nCP lattice.
The term "labelled" as used herein refers to attaching the impurity ions, molecules or radioactive isotopes on the surface of the said nanoparticle with the help of a ligand such as bisphosphonates or functional groups such as carboxyl on the surface of the nanoparticle. This may involve electrostatic interactions , covalent-, ionic-, coordinate, or hydrogen bonding or hydrophobic, lipophilic or hydrophilic interactions
Figure captions:
Figure 1: Schematic showing the method of preparation of multimodal and therapeutic nCP and nHAp
Figure 2: (a) XRD of MF-nHAp showing hexagonal HAp crystal structure (b) SEM image of MF-nHAp showing particle size ~ 100 nm. (c) DLS data of MF- nHAp giving a size distribution of 100 ± 20nm (d) Digital photograph of MF- nHAp and nHAp solutions (~ 20 mg/mL) indicating the greenish body color due to ICG doping .
Figure 3: (A) Graph showing integrated fluorescence emission intensity at 800 nm with increase in MF-nHAp concentration. Inset: Fluorescence spectra of MF- nHAp showing excitation and emission peaks at 765 nm and 800 nm, respectively. (B) NIR contrast images of MF-nHAp samples of varying concentration at ~ 800 ± 15 nm NIR band (C) Graph showing increase in X-ray attenuation with increase in doping % of Gd3+ within MF-nHAp (D) X-ray contrast images of undoped nHAp and MF-nHAp showing an increase in contrast with increase in percentage doping of Gd3+ (E) Vibration sample magnetometer data showing the paramagnetic behavior of MF-nHAp versus diamagnetic property of nHAp (F) Phantom NMR images of different concentrations of MF-nHAp measured using 1.5T MR system giving a bright Ti contrast that increases with MF-nHAp concentration.
Figure 4: (a) Radioactive emission of 20mg/mL of MF-nCP samples conjugated to Tc-99m of varying concentrations (b) Fluorescence images of MF-nCP samples conjugated to varying concentrations of ICG (c) Graph showing the variation in radioactive emission of MF-nCP samples conjugated to varying concentrations of Tc-99m (d) Graph showing the fluorescence intensity variation of MF-nCP with varying doping % of ICG
Figure 5 . (A) Hemolysis data of nHAp and D-nHAp treated whole blood showing non-hemolytic action of the tested samples (B) Digital photograph of the whole blood treated with PBS,
Figure imgf000013_0001
D-nHAp and triton showing no leakage of hemoglobin from PBS/D-nHAp treated samples compared to triton treated sample.
Figure 6: Scanning electron micrograph of RBC treated with (A) PBS (B)
Figure imgf000013_0002
D-nHAp. Enlarged image (C))showing uniform distribution of D- nHAp particles although out the RBC without affecting its morphology (D) EDX spectrum of the D-nHAp that are distributed on the RBC.
Figure 7: Cytotoxicity data of D-nHAp treatment on peripheral blood derived mononuclear cells after 24 hours incubation showing no toxicity up to a concentration of 500 ug/mL.
Figure 8: Radioactive emission images (a-d) and fluorescence images (e-h) of MF-nCP sample imaged at varying depths in pork tissue. The bottle containing the sample placed in pork tissue was rotated and images taken at varying angles. Depth at which the sample was imaged from, in tissue, is denoted as d and the angle at which the bottle is rotated is denoted as Θ.
Figure 9: In vivo multimodal imaging of MF-nCP after tail vein injection into mice. Figure (a) shows the fluorescence image (b) shows radioactive emission image (c) shows radioactive emission image overlaid over X-ray images.
Detailed Description:
Referring the flowchart as shown in Fig.i, the method of preparation of said nanoparticle and nanomedicine formulation, consists the steps of:
• Providing the nanoparticle precursor solutions containing Ca2+ (Part A), PO- (Part B) and OH- (Part C) ions
• Providing the precursor solutions containing multiple dopant ions, Part D, that provide radioactivity, raman scattering, near infrared fluorescence, magnetism and/or X-ray absorption in specific combinations.
• Forming the impurity doped nanoparticle by reacting the Part A, B, C and D by drop wise mixing each reactants at appropriate sequences, rates, pH and temperature conditions.
• Aging the colloidal precipitate at 25-40^ for nCP and 8o-i20°C for nHAp for a time period 1-24 Hrs.
• Washing the precipitated nanoparticles to remove unwanted byproducts.
• Conjugation of the nanoparticles with radioactive elements through bisphosphonates.
• Coupling the said nanoparticles with anticancer chemodrugs or nucleic acid drugs or making the nanomedicine formulation containing said nanoparticle in biodegradable polymeric shell containing the drugs, or biodegradable polymer containing both said nanoparticles and drugs.
• Connecting the said nanoparticles or associated nanomedicines with a suitable targeting ligands. In a preferred embodiment of said method of making nanoparticles, the precursor compound, part A, is formed from a material selected from the group consisting of sulphate, phosphate, hydroxide, chloride, bromide, iodide, fluoride, nitrate, carbonate or oxide salts of calcium.
In another preferred embodiment of said method of making nanoparticles, another precursor compound, Part B, is formed from water soluble or miscible salt of phosphates including sodium (Na3P04, Na2HP04, NaH2P04), potassium (K3P04, KsHPC-4, KH2P04) lithium (Li3P04, Li2HP04, LiH2P04), ammonium ((NH4)3P04, (NH4)2HP04, NH4H2P04) or phosphoric acid
In another preferred embodiment of said method, the precursor compound containing hydroxyl anions (Part C) is formed by hydroxide salts of sodium, potassium, lithium, ammonium or calcium .
In another preferred embodiment of said method, the dopant molecules (Part D) in the nanoparticle that gives raman scattering is formed by cyanine dyes (Cy3, Cy5, ICG), rhodamine (rhodamine 6G, tetramethyl rhodamine, rhodamine no), aniline blue, crystal violet, malachite green, basic fuchsin, variamine blue RT salt, triaminopyrimidine sulphate or porphyrins.
In another preferred embodiment of said method, the dopant molecules (Part D) that gives near-infrared fluorescence to the nanoparticle, is NIR emitting dyes such as Indocyanine green (ICG).
In yet another preferred embodiment of said method, the dopant ions (Part D) in the nanoparticle that gives magnetic contrast includes ions such as chromium(III), manganese(II), iron(II), iron (III), praseodymium (III), neodymium (III), samarium(III), ytterbium(III), gadolinium(lll), terbium(III), dysprosium(III), holmium(III), erbium(III). In yet another preferred embodiment of said method, the dopant ions (Part D) in the nanoparticle that gives X-ray contrast includes ions such as iodine or barium, bismuth, strontium tungsten, tantalum, hafnium, lanthanum, molybdenum, niobium, zirconium.
In yet another preferred embodiment of said method, the doped nanoparticle is conjugated with a radioactive element that includes Sm-153, Tc- 99m, 123I, 18F, l3»I, '"In, l88Re, l66Ho, < ογ 0r 82Rb through ligands such as bisphOsphonates.
In yet another preferred embodiment of said method, the nanoparticle with dopant ions / molecules (Part D) were formed by reacting precursor Part-A, B, C and D at a temperature range of 25-i50°C and pH range of 5 -12. The dopant ions may be mixed preferably with either Part-A or Part B or added separately during the reaction between Part A, B and C.
In yet another preferred embodiment of said method, the formed nanoparticles are grown to a preferred size scale, between 1-200 nm by aging of the reactants at time scales of any range between 0-24 Hrs, preferably, 0-6 Hrs and most preferably 1-4 hrs at a temperature range of 5-i50°C. Figure 2b and 2c shows the SEM and DLS images of ICG and Gd3+ co-doped multifunctional hydroxyapatite nanoparticles (MF-nHAp). 90% of the particles synthesized were ~100±25 nm in size as seen in DLS and SEM data. The XRD data of MF-nHAp gave characteristic peaks of hexagonal HAp crystal lattice as shown in Figure 2a. Lattice constant values for doped MF-nHAp and undoped nHAp samples showed slight deviations, suggesting lattice distortions caused by the incorporation of dopant ions/molecules. Figure 2d shows undoped nHAp and MF-nHAp at a concentration of 20mg/mL, where the doped MF-nHAp shows the green body color due to the presence of ICG.
In relation to the above aspect of doping nCP and nHAp with NIR dyes such as ICG, inset of Figure 3A shows the fluorescence excitation and emission spectra of ICG and Gd3+ co-doped MF-nHAp, peaking at 760 nm and 830 nm proving the capability of the material for non invasive in vivo NIR imaging. The total weight percentage is varied in the range of 0.01 - 1%. The total concentration of ICG in the precursor solution is maintained less than 2xio_s M to avoid agglomeration of dye molecules. In relation to the above said novel aspect of NIR fluorescence, the said nanoparticle can be used for molecular imaging based NIR fluorescence for biomedical applications. This is displayed in phantom experiments as shown in Figure 3B that shows increase in fluorescence with increase in MF-nHAp concentration.
1 In yet another aspect of this present invention, the nanoparticles show X-ray contrast properties (radio opacity) together with NIR imaging. This is demonstrated in Figure 3D where the X-ray contrast imaging of powder nanoparticle samples of different dopant concentrations are displayed. Undoped and doped samples were made into pellets and imaged using 12.87 KeV X-ray energy (0.4mm filter) for an exposure time of 30 seconds in a digital X-ray imaging station (Kodak in vivo multispectral imaging system, Carestream, USA). The X-Ray density of the material, which is a measure of the X-ray absorbed or attenuated, was calculated and plotted in Figure 3C. The results discloses that, while undoped nanoparticle of HAp attenuated only -65% of incident X-ray, MF- nHAp samples attenuated ~ 75% of incident X-ray energy, clearly suggesting that the said nanoparticles can provide very good X-ray contrast together with both NIR fluorescence contrast imaging.
In another aspect of the present invention, the said NIR emitting nanoparticles show paramagnetism together with NIR fluorescence and X-ray contrast when co-doping ICG with paramagnetic Gd ions. Figure 3E refers to magnetic studies carried out by keeping the sample under a varying magnetic field, in an instrument called vibrating sample magnetometer. When undoped sample show diamagnetic property the ICG and Gadolinium doped samples show paramagnetic property, which increases with the concentration of dopant ions, gadolinium in this case. This property makes it possible to use the said nanoparticle as a contrast agent for magnetic resonance imaging, as demonstrated in Figure 3F wherein the said nanoparticles taken in low (0.1 mg/ml) to high concentration (5 mg/ml) and imaged under a clinical MRI system show excellent Ti weighted contrast properties, which increases with increase in concentration.
Citing another combination where NIR fluorescence and radioimaging is used together, ICG doped and Tc-99m tagged calcium phosphate nanoparticles (MF-nCP) were synthesized. Figure 4a shows the radioactive emission from MF- nCP samples. As seen in the figure tagging of Tc-99m increases with increase in Tc-99m concentration till a saturation value of 250 μθι' (Figure 4c). Figure 4b shows increase in fluorescence with increase in % doping of ICG till a saturation value of 0.16 mole% (Figure 4d).
In another aspect of the present invention the hemocompatibility of the doped nHAp (D-nHAp) was studied, and it was found that neither nHAp nor D- nHAp cause hemolytic effects upto a concentration of
Figure imgf000018_0001
as shown in Figure 5A . Figure 5B shows the blood samples treated with D-nHAp and triton where hemolysis is clearly visible in triton treated samples and D-nHAp treated sample is comparable to PBS treated blood. Figure 6 shows SEM image of RBC treated with D-nHAp (Figure 6B) that shows no membrane damage compared to PBS treated RBC (Figure 6A). Figure 6D shows EDAX data of D-nHAp distributed over RBC.
In another important aspect of the present inventions, the said nanoparticles are highly bio-compatible to primary human cells, for eg; peripheral blood mononuclear cells, as shown in Figure 7 . Phantom imaging using Tc tagged and ICG doped MF-nCP was done in pork tissue samples to check the efficiency of contrast when imaged in tissue samples. Figure 8 shows that both NIR fluorescence and radioactivity is imageable at varying depths up to 2.5 cm. Later imaging in mice was carried out using the same MF-nCP nanoparticles that proved the ability of the material for multimodal imaging under in vivo conditions. Figure 9 shows the fluorescent and radioactive imaging of mice after tail vein injection of the MF-nCP sample. All these imaging experiment were carried out in an in vivo imaging station (Kodak in vivo multispectral imaging system, Carestream, USA).
In yet another novel aspect of the present invention, the high resolution and blood penetration capability of near-infrared light can help doctors, during a surgery, to identify angiogenic blood vessels in the vicinity of a solid tumor, for its effective removal during a surgical procedure. Angiogenesis is a process of formation of larger concentration of new blood vessels around a growing tumor and the removal of such blood vessels is critical in stopping recurrence of tumor. The said nanoparticles conjugated with antibody targeted against angiogenic growth factors may illuminate the cancer related blood vessels together with providing contrast for the tumor.
In another important aspect of the present invention, since the doped nanoparticles are cationic in nature, the same can be loaded with nucleic acid drugs such as DNA or RNA or its small fractions like siRNA, for therapeutic gene delivery applications. These nanomedicine constructs can be targeted using monoclonal antibodies (mAbs), peptides (pep) or similar ligands
In another aspect of the present invention, the said nanoparticles can also be Coated with a biodegradable polymers containing anticancer drugs or nucleic acid drugs.
The biodegradable polymers can be formed by polymers such as poly-lactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyvinylpyrrolidon (PVP), polyvinyl alcohol (PVA), polyethyleneimine (PEI), polyethelene glycol (PEG), chitosan, carboxymethyl chitosan, cyclodextrin, thermosensitive polymers such as Poly(JV- isopropylacrylamide) and its derivative or proteins such as bovine or human serum albumin.
In another aspect of the present invention the said nanoparticle or drug loaded nanomedicine construct is connected, at the surface with cancer targeting ligands including antibodies, peptides or small molecular ligands such as folic acid or aptamers
In another aspect, the present invention provides an injectable composition or composition for oral administration comprising the said nanomedicine according to the present invention as described above, together with a pharmaceutically acceptable medium.
In another aspect, the present invention provides a method of image guided delivery of the drug, estimation of drug concentration at diseased site with respect to other regions, estimation of pharmacokinetics and pharmacodynamics, treatment planning and estimation of treatment efficacy after preferred treatment, all the above using any of the said contrast imaging property of the nanoparticle
In another aspect, essentially the present invention provides a method of simultaneously detecting and treating the disease like cancer with the help of more than one and upto 05 different molecular imaging techniques using a single nanoparticle system.
The inventors have discovered primarily a nanoparticle that provides novel concept for combining a number of important molecular imaging techniques such as radioactivity, raman scattering, near-infrared fluorescence imaging, which can register both spatial and temporal functional properties of a disease at microscopic level such as expression level of a cancer biomarker, with other properties such as super/para-magnetism and X-ray absorbance which gives micro/ macroscopic anatomical information. In addition, the inventive step provide a nanomedicine formulation, containing the said nanoparticle as a mairi component together with therapeutic molecules such as chemo drugs or nucleic acids that can treat disease in a targeted manner by conjugation of a ligand specific to a receptor in the diseased tissue /cell.
Method of preparation of the multimodal nanocontrast agent
Different methods of preparation of nanocontrast agents are discussed below: Example 1: Multifunctional contrast agent based on calcium phosphate nanoparticles with 2 dopants for combined NIR and radioactive imaging.
In this synthesis procedure the preparation of 0.04 weight % ICG doped and 250 μθΐ Tc-99m labeled calcium phosphate nanoparticles for combined NIR arid radioactive imaging is presented. In a typical reaction procedure, smL of 0.5M calcium chloride dihydrate (CaCl2.2H20, Merck, India) was mixed with ιοομΐ-, imM ICG (molecular weight 774.98, Sigma Aldrich, USA) and left for 30 minutes stirring. 5mL of 0.3 M diammonium hydrogen phosphate ((NH4)2HP04), S.D Fine Chemicals, India) was added drop wise to the mixture. The pH during the entire process was maintained at ~ 7.4 by the addition of 0.1M NH4OH solution. The precipitate was left stirring for another 30 minutes, centrifuged and washed at least 2 times. The entire process was carried out room temperature at ~ 25°C. - 3omg/mL of the washed product was treated with lmL of 250 μθϊ Tc-MDP (Tc- 99m conjugated to methylene diphosphate). The mixture is vortexed for 2 minutes and incubated for another 20 minutes. The conjugate is washed at least twice and resuspended in PBS or distilled water for further applications.
ICG can be replaced with any NIR emitting dye or raman active dye molecules for the synthesis of other NIR emitting or raman active calcium phosphate nanoparticles.
Example 2: Multifunctional contrast agent based on calcium phosphate nanoparticles with more than 2 dopants for combined raman. magnetic. X-ray and radioactive imaging.
In this synthesis procedure the preparation of 0.4 weight % aniline blue and 10 atomic % (wrt Ca2+) Gadolinium doped and 250 μθϊ Tc-99m labeled calcium phosphate nanoparticles for combined raman, magnetic and radioactive imaging is presented. In a typical reaction procedure, 5 mL of 0.5 M calcium chloride dihydrate (CaCl2.2H20, Merck, India) was mixed with 135 μΐ., o.oi M Aniline blue (molecular weight 737-73» Spectrochem, India) and 2.5mL 0.1M gadolinium nitrate and left for 3.0 minutes stirring. 5mL of 0.3 M diammonium hydrogen phosphate ((NH4)2HP04), S.D Fine Chemicals, India) was added drop wise to the mixture. The pH during the entire process was maintained at ~ 7.4 by the addition of 0.1M NH4OH solution. The precipitate was left stirring for another 30 minutes, centrifuged and washed at least 2 times.
The entire process was carried out room temperature at ~ 25 C. ~ 3omg/mL of the washed product was treated with lmL of 250 μθι Tc-MDP(Tc-99m conjugated to methylene diphosphate). The mixture was vortexed for 2 minutes and incubated for another 20 minutes. The conjugate was washed at least twice and resuspended in PBS or distilled water for further applications.
Aniline blue can be replaced by any other raman active or NIR emitting dye for the synthesis of other raman active or NIR emitting calcium phosphate nanoparticles. Gadolinium can be replaced by any other impurity ion to give magnetic or X-ray contrast properties. Tc-99m can be replaced with any other radioactive element to synthesize other radioactive calcium phosphate nanoparticles.
Example 3: Multifunctional contrast agent based on hydroxyapatite nanoparticles with more than 2 dopants for combined raman. magnetic. X-rav and radioactive imaging,
In this synthesis procedure the preparation of 0.4 weight % aniline blue and 10 atomic % (wrt Ca2+) Gadolinium doped and 250 μΟΊ Tc-99m labeled hydroxyapatite nanoparticles for combined raman, magnetic and radioactive imaging is presented. In a typical reaction procedure, 5 mL of 0.5 M calcium chloride dihydrate (CaCl2.2H20, Merck, India) was mixed with 135 μΐ-, o.oi M Aniline blue (molecular weight 737.73, Spectrochem, India) and 2.5mL 0.1M gadolinium nitrate and heated to 90°C. The: mixture was left for 30 minutes stirring. 5mL of 0.3 M diammonium hydrogen phosphate ((NH4)2HP04), S. D Fine Chemicals, India) was added drop wise to the mixture. The pH during the entire process was maintained at ~ 7.4 by the addition of 0.1M NH4OH solution. The precipitate was left stirring for another 30 minutes, centrifuged and washed at least 2 times. The entire process was carried out room temperature at ~ 90°C. ~ 30mg/mL of the washed product was treated with lmL of 250 μθϊ Tc-MDP(Tc- 99m conjugated to methylene diphosphate). The mixture was vortexed for 2 minutes and incubated for another 20 minutes. The conjugate was washed at least twice and resuspended in PBS or distilled water for further applications.
Aniline blue can be replaced by any other raman active or NIR emitting dye for the synthesis of other raman active or NIR emitting hydroxyapatite nanoparticles. Gadolinium can be replaced by any other impurity ion to give magnetic or X-ray contrast properties. Tc-99m can be replaced with any other radioactive element to synthesize other radioactive hydroxyapatite nanoparticles.
Example 4: Preparation of siRNA incorporated calcium phosphate nanoparticles based nanomedicines capable of image guided delivery.
In this example, preparation of a representative nanomedicine formulation formed by loading the said multifunctional calcium phosphate nanoparticles (MF-nCP) with small length nucleic acid drugs, for example therapeutic small interfering RNA (siRNA) is presented. The sequence of siRNA selected for this example is the one targeted against 315T mutations in the kinase domain (KD) of BCR-ABL that is prevalent mechanism of acquired drug resistance in patients with chronic myeloid leukaemia (CML).
Sequence details are:
Sense strand: 51 -GCCGCUCGUUGGAACUCCAdTdT— 31
Antisense strand: 31 -dTdTCGGCGAGCAACCUUGAGGU— 51
Length: 21 base pair For the loading reaction, lmg/ml bare nanoparticles prepared as said in any of the above examples 1-3 , is prepared in phosphate buffer saline (PBS, Sigma, USA)) and sonicated for 10 minutes to get a fine dispersion. To this, ~ Ο.ΐμΜ siRNA
(Sense strand: 51 -GCCGCUCGUUGGAACUCCAdTdT- 3' Antisense strand: 31 -dTdTCGGCGAGCAACCUUGAGGU— 51) solution prepared in PBS is mixed and reacted for 3 Hrs at 37°C in a water bath, while stirring ultrasonically. The sequence of the siRNA is only representative and any other sequence of therapeutic importance shall be used. After 03 hrs, the rianoparticle-siRNA conjugates were removed from the medium by centrifugation and unbound siRNA from the supernatant is discarded. To protect the siRNA bound to the surface of nanoparticles from enzymatic degradation during the transfection, the nCP-siRNA conjugates were further treated with o.oimg/ml BSA (Bovine Serum Albumin) and o.img/ml EDC (i-ethyl-3-(3- dimethylaminopropyl) carbodiimi.de) for 3omin at 37°C. After 30min, the nanoconjugates are removed by centrifugation and re-suspended in PBS. This forms a protective shell of albumin protein that can be derivatized using targeting ligands.
Example : Preparation of nCP-DNA nanomedicines capable of image guided delivery
In an alternative method, this example provides preparation of a representative nanomedicine formed by loading the said multifunctional calcium phosphate nanoparticles with a representative gene. Here we show encapsulating a gene, preferably a marker gene that can be readily detected by simple laboratory tools, into the nanoparticle during the synthesis of nanoparticles. An appropriate marker gene selected for this example is beta.- galactosidase (β-gal) since its expression can be readily detected by addition of X- gal, a substrate which yields a blue color when the active enzyme is present. However, this example is not limited to the said marker gene, but any gene intended for a desired function such as inhibition of tumor growth. For the encapsulation of linear and super coiled β-gal coding DNA into nHAp, img/ml DNA is dissolved in PBS and mixed with 5mL CaCl2, 0.5M, 98%, Sigma, USA (Part A) and stirred for ~ 3omin at 37°C. To this, 2.5 ml each of 0.1 M Gd(N03)3 and 100 μΐ. ICG, dopant ion precursors (Part D), is added and mixed for 30 minutes. Another two solutions containing phosphate group (Part B), ammonium dihydrogen phosphate, (5 mL, 0.3M, 98%, Qualigens, India) and 1 ml NH4OH (0.1M, 25% NH3, Qualigens, India) are prepared. Both these solutions are added drop wise to the precursor mixture of Part A+D with continuous stirring over a period of 15 minutes. The rate of addition of ammonium hydroxide solution is adjusted to maintain the pH of the reaction medium at ~ 7.4 throughout the reaction. After completion of precipitation, the mixture is kept stirring for 1 hour followed by separation of the precipitate by centrifugation at 3000 rpm for lominutes and washing with ice cold water for 3-5 times. The DNA embedded within the doped calcium phosphate matrix is optionally protected from enzymatic degradation using a protective coat of BSA, by treating the nCP-DNA conjugates with o.oimg of BSA and o.img of EDC in 5ml PBS for 30mm at 37°C, followed by washing with water.
Example 6: Preparation of nCP-polymer nanomedicine containing chemo drugs capable of image guided delivery
In this example, preparation of a representative nanomedicine formed by the said multifunctional calcium phosphate nanoparticles embedded within polymeric nanoparticle containing chemical drugs, for example a small molecule inhibitor, Sorafenib, is presented, l mg/ml bare nanoparticles prepared as said in any of the above examples 1-3 is prepared in DMSO (dimethyl sulfoxide) medium and sonicated for 10 minutes to get a fine dispersion. To this solution, ~ ιμΜ Sorafenib in lmg/ml PLGA (poly(lactic-co-glycolic acid)) , dissolved in DMSO is added and reacted for ~ 2 Hrs with continuous stirring. After 02 hrs, ~ 500 μΐ, of H20 is added to the CP-Sorafenib-PLGA mixture to precipitate the polymeric nanoparticles that contain CP and Sorafenib embedded in it. This nanomedicine formulation can be used to deliver drug actively or passively to tumor sites together with imaging using multiple imaging modalities. Alternative to PLGA, other biodegradable polymers such as PEI, PLA, PCL, PVA, PPV etc also can be used for embedding the nCP and chemical drug.
Example 7: Bisphosphonate tagged nCP for imaging and therapeutic applications In this example synthesis of bisphosphonate drug, alendronate, tagged Tc99m labeled doped calcium phosphate nanoparticles for imaging and therapy of diseases such as bone metastasis or other bone metabolic disorders, osteoporosis etc is presented.
In this synthesis procedure 0.04 weight % ICG doped and 250 μΟΊ Tc- 99m-alendronate labeled calcium phosphate nanoparticles is prepared. In a typical reaction procedure, smL of 0.5M calcium chloride dihydrate (CaCl2.2H20, Merck, India) was mixed with ιοομί,, imM ICG (molecular weight 774.98, Sigma Aldrich, USA) and left for 30 minutes stirring. 5mL of 0.3 M diammonium hydrogen phosphate ((NH4)2HP04), S.D Fine Chemicals, India) was added drop wise to the mixture. The pH during the entire process was maintained at ~ 7.4 by the addition of 0.1M NH4OH solution. The precipitate was left stirring for another 30 minutes, centrifuged and washed at least 2 times. The entire process was carried out room temperature at ~ 25°C. ~ 30mg/mL of the washed product was treated with lmL of 250 μϋϊ Tc99m-alendronate (Tc-99m conjugated to alendronate drug). The mixture is vortexed for 2 minutes and incubated for another 20 minutes. The conjugate is washed at least twice and resuspended in PBS or distilled water for further applications.

Claims

Claims
l. A nanomaterial showing multi-functional properties such as radioactivity, raman scattering, near-infrared (NIR) fluorescence, para- or superpara- magnetism and X-ray absorption, at least any two properties simultaneously, making it suitable for contrast enhancer for multi-modal medical imaging using uses, radiation, raman scattering, near-infrared fluorescence, para- or superpara- magnetism and X-ray absorption, in combination or separately.
Ί. The multifunctional nanocontrast agent as in claim l, shows radioactive emission, raman scattering, fluorescence emission in the NIR region, para-/super para-magnetism and X-ray absorption, all or at least two properties in combination making it suitable for multi-modal medical imaging using 2D scintigraphy, positron emission tomography (PET), single-photon emission computed tomography (SPECT), raman-spectroscopy, near-infrared optical imaging, magnetic resonance imaging (MRI), 2D/3D X-ray computed tomography.
3. The multifunctional nanocontrast agent as in claim 1 is hydroxyapatite ((Caio(P04)6(OH)2) or calcium phosphate ((Ca3(P04)2) or calcium dihydrogen phosphate (Ca(H2P04)2) or calcium hydrogen phosphate (CaHP04) nanoparticles doped and labeled with organic dyes and/or inorganic impurities.
4. The impurities doped and/or labeled/conjugated to the nanocontrast agent as in claim 3 includes: Raman dyes, magnetic impurities, X-ray contrast impurities, near-infrared emitting dyes and radio isotopes, all in combinations or at least two in the same nanomaterial.
5. The different organic dyes used in claim 3 comprises cyanine dyes (Cy3, Cys, ICG), rhodamine (rhodamine 6G, tetramethyl rhodamine, rhodamine 110), aniline blue, crystal violet, malachite green, basic fuchsin, variamine blue RT salt, triaminopyrimidine sulphate and porphyrins to obtain near infrared fluorescence and / or raman contrast.
6. The different inorganic impurities as in claim 3 comprises ^S , 9 Tc, 123l, ^F, 1311, inln, l88Re, l66Ho, 9°Y or 82Rb for radioactive imaging, Tb3+, Er3-. Dy3+, Ho3+, Gd3+, Tm3+, Mn2+, Fe2+, Fe3+ for paramagnetic or super paramagnetic imaging and iodine, barium, gadolinium, bismuth, strontium tungsten, tantalum, hafnium, lanthanum, molybdenum, niobium, zirconium for X-ray contrast imaging
7. The impurities mentioned in claim 3 can either substitute calcium or hydroxyl or phosphorus ion site depending on the charge/composition or be incorporated within the nanoparticles as individual atoms/molecule or cluster of atoms/molecules or be labeled/conjugated on the surface of the nanoparticles directly or using a conjugating ligand.
8. The multifunctional nanocontrast agent as in claim 1 has spherical or non spherical shape and size ranging from 1 to 200 nm and can be delivered intravenously, intramuscularly or orally.
9. The multifunctional nanocontrast agent as in claim 1, can be conjugated or loaded with drug molecules such as bisphosphonates, chemodrugs, anticancer gene therapy agents, RNA fragments (siRNA, miRNA), photosensitive drugs, small molecule inhibitors, antibiotics.
10. A method of making the multifunctional nanocontrast agent as in claim ι, consist the steps of (a) preparing an aqueous or Simulated Body Fluid (SBF) based solution containing calcium (Ca2+) ions as Part-A (b) preparing an aqueous or SBF based solution containing phosphate P043- ions as Part-B c) preparing an aqueous or SBF based solution containing hydroxyl, OH-, ions as Part-C d) preparing an aqueous or SBF based solution containing dopant ions or dyes separately or in combination as Part-D e) reacting Part-A, Part-B, and Part-C and Part-D at a pH range and temperature range for a stipulated period of time in open air on a hot plate, e) Continuously stirring the reaction medium to precipitate inventive particles while maintaining the pH at a particular range by adding Part C intermittently f) aging the precipitated particles for a stipulated period at a stipulated temperature range g) separating the precipitated particles from the growth medium by centrifugation h) washing the precipitated particles with distilled water i) re-dispersing the precipitate in water or phosphate buffer saline j) conjugating the precipitate with capping agents and other moieties such as radiolabels or targeting ligands.
11. A method of multifunctional nanocontrast as in claim 10, the part A is formed from water soluble, miscible or dispersible hydroxide, chloride, bromide, iodide, fluoride, nitrate, sulphate, carbonate or oxide salts of calcium.
12. A method multifunctional nanocontrast as in claim io, the part B is formed from water soluble or miscible salt of phosphate including sodium (Na3P04, Na2HP04, NaH2P04), potassium (K3P04, K2HP04, KH2P04) lithium (Li3P04, Li2HP04, LiH2P04), ammonium ((NH4)3P04, (NH4)2HP04, NH4H2P04) and phosphoric acid (H3P04).
13. A method of making multifunctional nanocontrast as in claim 10, Part C is formed by sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonium hydroxides, calcium hydroxide or all possible water soluble hydroxides.
14. A method of making multifunctional nanocontrast as in claim 10, the Part D is formed by water soluble or miscible chloride, bromide, iodide, fluoride, nitrate, sulphate, carbonate or oxide salts of the inorganic impurity ions and/or organic dyes.
15. A method of making multifunctional nanocontrast as in claim 10, all reactions are carried out within a pH range of 5-12 and a temperature range of 25-i20°C.
16. A method of making multifunctional nanocontrast as in claim 10, the water or PBS dispersed particles are preferentially conjugated at its surface with targeting ligarids including folic acid, antibodies, peptides, aptamers or carbohydrates.
17. The capping agents mentioned in claim 10 include citrate, polymers including polyethylene glycol (PEG), Polyethylene imine (PEI), bisphosphonates that can be added after or during the synthesis of the nanocontrast agent mixed with either Part A, B, C or D.
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