KR100809046B1 - Self assembled nanoparticle having pullulan and methods of using thereof - Google Patents

Abstract

The present invention relates to a nano self-aggregate having a pullulan and a method of using the same, and provides a conjugate of a pullulan and a hydrophobized monomer containing a carboxyl group. More specifically, the present invention relates to a method for preparing nano-aggregates by esterification bonds of pullulan and the hydrophobization monomer. In addition, the present invention relates to a method of using the nano-self-aggregates, the nano- self-aggregates can be used as a biomaterial because of the degradability in vivo, and in particular, as a drug carrier, it can be used for delivery of anticancer agents.

Pullulan, Nanoparticles, Drug Delivery, Folic Acid, Biotin, Bile Acid

Description

Self assembled nanoparticle having pullulan and methods of using}

1 shows the biosynthesis process of folic acid- pullulan conjugate (FA / Pul). DCC: N, N'-dicyclohexyl carbodiimide; DMAP: dimethylaminopyridine; DMSO: Dimethyl sulfoxide.

Figure 2 shows the biosynthesis process of pullulan / biotin (PU / Bio). (a) pullulan, (b) biotin, (C) pullulan / biotin (PU / Bio) polymer.

Figure 3 shows the biosynthesis process of pullulan / bile acid polymer. (a) pullulan, (b) bile acid, (C) pullulan / bile acid polymer.

Figure 4 shows the size distribution of folic acid / pullulan (FA / Pul) nanoparticles measured by dynamic light scattering (DLS). Two main groups were observed in the range of 60-80 and 230-300 nm. At around 1000 nm, small peaks disappeared completely as the degree (DS) of the substituted monomers of folic acid increased, while at the same time the percentage of small particles near 70 nm was lowered. (a) FA4 / Pul: 4 folates per 100 units of glucose of pullulan; (b) FA6 / Pul: 6 folates per 100 units of pullulan glucose; ( 8 ) FA8 / Pul: 8 folates per 100 units of pullulan glucose.

5 shows the transmission electron micrograph of the folic acid / pullulan conjugate in the dry state. Many small aggregates (<20 nm) were observed in FA4 / Pul, but fused into larger particles (ca 50 nm) with increasing degree of substituted monomer of folic acid. The scale bar below each image represents 500 nm (a), (b) or 100 nm (c). (a) FA4 / Pul: Photograph with four folates per 100 units of glucose of pullulan; (b) FA6 / Pul: photograph of six folates per 100 units of pullulan glucose; (c) FA8 / Pul: Picture of 8 folates per 100 units of pullulan glucose.

6 shows particle size (a) and transmission electron microscopy (TEM) images (b) measured by dynamic light scattering (DLS) method of PU / Bio2 nanogels.

7 shows the polymer concentration excitation spectrum. Pyrene (6.0 × 10 ⁻² M) was used as a fluorescence probe to measure the self-aggregation results of PU / Bio. As the self-aggregation-forming nanogel is generated from the PU / Bio conjugate, pyrene is released to the inside of the hydrophobic nanogel, and the wavelength of the fluorescence band in the fluorescence analyzer is shifted from 334 nm to 337 nm. This result suggests that the conjugates form self-aggregates above the critical aggregation concentration (CAC).

FIG. 8 shows a relative comparison of excitation spectra and concentration log values of PU / Bio 1,2,3 self-organized nanogels. It is the result of investigating critical aggregation concentration (CAC) by comparing each concentration of PU / Bio polymer and I ₃₃₇ / I ₃₃₄ intensity value. The CAC value for each PU / Bio represents a minimum concentration to be formed in a PU / Bio form.

9 shows doxorubicin (DOX) release from FA / Pul nanoparticles (circle ●, FA4 / Pul; triangle ▲, FA6 / Pul; and rectangle ■, FA8 / Pul). The more folate-substituted pullulan, the later DOX was released. Data is expressed as mean standard deviation (n = 3).

FIG. 10 is a fluorescence microscope image to show the effect of the degree of substituted monomer of folic acid on cell binding and uptake of DOX-filled FA / Pul nanoparticles. The left (a) and right (b) panels show KB and CHO-K1 cells, respectively. FITC channels for the polymer (green) and RITC channels for the DOX (red) were provided simultaneously (FA4 / Pul, FA6 / Pul, and FA8 / Pul from above). No significant difference was observed depending on the degree of substituted folic acid.

FIG. 11 shows competition between free FA and DOX charged FA8 / Pul nanoparticles. The left (a) and right (b) panels show KB and CHO-K1 cells, respectively. FITC channel for polymer (green) and RITC channel for DOX (red) were provided simultaneously (0.5, 0.1, and 0.05 mg / mL of folic acid from above). The higher the free folic acid, the less the binding and absorption of the nanoparticles.

12 shows the change in fluorescence intensity due to non-interaction between cells and nanogels. In order to examine the interaction with cancer cells, a microplate-fluorescence reader was prepared by reacting a hepatocellular carcinoma cell line (HepG2) with PU / Bio nanogels conjugated with rhodamine B isothio-cyanate (RITC). Analyze The result was a higher specific interaction at PU / Bio than at RITC labeled pullulan and at 2, 3 than at PU / Bio1. Biotin forms a hydrophobic center inside the PU / Bio nanogel and also comes out to the outside, meaning that it can act as a ligand when interacting with cancer cells. Thus, biotin has the property of targeting cancer cells as a nonspecific interaction with cancer cells.

In FIG. 13 , specific interaction between cells and nanogels is shown as RITC fluorescence. The results of observing PU / Bio nanogel acceptance in HepG2 cancer cell line by fluorescence microscopy (Confocal Laser microscopy). PU / Bio 2 and 3 prepared in this study showed strong fluorescence by penetrating into HepG2 cells and relatively weak internal penetration of PU / Bio1. The different results of PU / Bio 1,2,3 suggest that biotin content contained in PU / Bio at different concentrations is a significant factor for cancer cells. These PU / Bio nanogels are sufficiently applicable even if they are applied to the correlation system of other similar cells and nanogel particles, and biotin content is considered to be the most important factor.

Common magnetic aggregates are obtained by conjugating hydrophobic moieties such as deoxycholic acid or 5-cholic acid to a water soluble polymer. Hydrophobic compounds can be used for the backbone chain with natural polymers including bile acids containing cholesterol structures and heparin or chitosan derivatives. Biosynthetic amphiphilic polymers spontaneously produce nano-sized aggregates in aqueous solutions and load hydrophobic drugs in aqueous environments. Nanoparticles prepared in this way are very stable in aqueous solution. In addition, in terms of the principle that "similar ones dissolve similar" drugs present in the center of the particle with a hydrophobic moiety are more stable to self-aggregates.

Medicine-encapsulated nanoparticles without any target moiety can be used by themselves for cancer treatment. In this case, the therapeutic effect is obtained by the specific pathophysiological characteristics of cancer cells called enhanced permeability and retention (EPR) effects. Because in the body, malignant neoplasms grow faster than normal tissues and require a lot of nutrients and oxygen. This leads to the creation of new blood vessels around cancer cells. Because of their rapid growth, these neovascularized vessels have large outflow openings that are large enough for the nanoparticles to escape from the vessels, thus allowing the nanoparticles to accumulate in cancerous tissue.

On the other hand, introducing a target moiety into the nanoparticle makes the nanoparticle more specific to the target tissue. In order to have such targets, compounds containing folic acid, transferrin or monoclonal antibodies have been conjugated to amphiphilic polymers (Chung, JE, M. Yokoyama, and T. Okano. 2000. Inner core segment design for delivery control of thermo-responsive polymeric micelles J. Control Release 65:.. 93-103; Gref, R., Y. Minamitake, MT Peracchia, V. Trubetskoy, V. Torchilin, and R. Langer, 1994. biodegradable long-circulating polymeric nanospheres, Science 263: 1600-1603 .; Hashida, M., H. Hirabayashi, M. Nishikawa, and Y. Takakura, 1997. Targeted delivery of drugs and proteins to the liver via receptor-mediated endocytosis, J. Control. Release 46: 129-137.) Moieties leading to cancer cells make the therapeutic effect more specific, but require one or more chemical steps. One consideration is that there has been no report on whether the conjugation of the target compound affects the physicochemical properties of the polymer or nanoparticles, although most such molecules have excessive hydrophobicity or large molecular weight.

Pullulan is a homopolysaccharide that is of interest in the pharmaceutical field for its low immunogenicity and biodegradability. In addition, the natural polymer is well soluble in organic solvent dimethylsulfoxide (DMSO). Since anhydrous conditions are very important for obtaining high yields in the linking reaction, solubility in organic solvents makes chemical modifications easier. For this reason, new pullulans have been synthesized, and materials with various physicochemical properties have been synthesized in the field of biomedical research.

Since folic acid, biotin, and bile acids have carboxyl groups and low solubility in polar substances or neutral aqueous solutions, the conjugation of hydrophobic substances with pullulan in a single step of chemical manipulation is expected to produce new kinds of compounds. .

The inventors have found that monomers having carboxyl groups such as folic acid, biotin, and bile acids can be hydrophobized by ester bonding in a simple reaction such as one-step reaction with pullulan to form nano-size self-aggregates. Has been found to have specificity for binding to target cells. In addition, the present inventors have found that the degree of the monomer to be substituted is closely related to the release of the drug, and completed the present invention.

The present invention finds a conjugate formation reaction between a pullulan and a hydrophobized monomer having a carboxyl group such as folic acid, biotin, and bile acid, and a drug release control mechanism in a drug carrier, and has a cell-specific reaction, a nanoconjugate, a drug carrier, Provided are biomaterials, methods for their preparation, and methods for drug delivery.

The present invention is a self-assembled nano-aggregates prepared by conjugation of a plurality of hydrophobized monomers (folic acid, biotin, bile acids, etc.) having a carboxyl group to pullulan, a polysaccharide, through an ester bond. nanoparticles). Hereinafter, the term 'hydrophobization monomer containing carboxyl group' refers to a carboxyl group-containing cyclic compound, which shows hydrophilicity before bonding, but is water soluble in the monomer after forming a conjugate by ester bond with a hydroxyl group of pullulan. It is a substance that decreases hydrophilicity and adds hydrophobicity due to the disappearance of carboxylic acid. Examples of such hydrophobized monomers include retinoic acid, folic acid, vitamins such as biotin, and bile acid.

A first aspect of the present invention provides ester conjugates of hydrophobized monomers containing pullulan and carboxyl groups, nano self-aggregates thereof, and drug delivery agents using them. Preferably, the hydrophobization monomer is selected from the group consisting of cyclic compounds such as retinol acid, folic acid, biotin, bile acid and the like.

A second aspect of the present invention provides an ester conjugate of a hydrophobized monomer containing pullulan and a carboxyl group, nano self-aggregates thereof, and a method for producing a drug carrier using the same.

The third aspect of the present invention provides a drug delivery method using the nano-self aggregates. Drugs include chemotherapy, antitumor, anti-inflammatory, and natural substances. The form of the drug may also be used compounds, compositions, proteins, nucleic acids and the like.

A fourth aspect of the present invention relates to a method for controlling drug delivery, characterized in that for controlling the number of bonds to which hydrophobized monomers bind to pullulan in the conjugate. The number of bonds of the hydrophobization monomer of the conjugate is determined in comparison with the glucose units constituting pullulan, and refers to the number of hydrophobization monomers bound per 100 units of glucose. It provides a method of reducing the drug delivery rate by binding more than 6 hydrophobization monomer per 100 units of glucose and a method of increasing the drug delivery rate by combining less than 6 monomers per 100 units of glucose.

A fifth aspect of the present invention provides a biomaterial comprising a conjugate of a pullulan and a hydrophobization monomer containing a carboxyl group and nano self-aggregates thereof. Biomaterial refers to a substance used to replace or replace a function of a living body as an artificial substance which is intermittently or continuously in direct contact with surrounding tissues and exposed to body fluid in order to replace a function of a tissue. Its use is widely used from disposable syringes to artificial heart materials. These materials are currently used in a variety of dental and medical fields. All of these materials refer to materials having excellent affinity or biocompatibility for the human body. Initially, the types and use of biomaterials were very limited, but nowadays, a wide variety of biomaterials have been developed and used. The biomaterial including the conjugate of the pullulan and the hydrophobization monomer containing a carboxyl group and nano self-aggregates thereof in the present invention is excellent in biocompatibility, and can be usefully used for drug delivery since it is degraded in vivo. The material may be used as an MRI contrast agent or an anti-adhesion film that inhibits the prevention of adhesion after surgery.

Hereinafter, the present invention will be described in detail through the following examples. However, the present invention is not limited to the following examples.

<Example 1> Preparation of conjugate of hydrophobization monomer containing pullulan / carboxyl group

(1) Preparation of conjugates of pullulan / folic acid

1) Materials and Methods

Fetal boval serum, antibiotics (penicillin / streptomycin), and Dulbecco's phosphate buffer (DPBS) were purchased from Gibcovial (Invitrogen, Carlsbed, Calif.). All reagents were at least cell culture or spectroscopic quality.

Since folic acid contained 8.1% (w / w) moisture, it refine | purified as follows. After dissolving in 200 mL DMSO (1%, w / v) continuously stirred at 40 ° C., folic acid was precipitated against 800 mL of deionized water. The solution was further acidified to 4.0 by addition of 2N HCl solution, which became a yellow slurry. The solid component was obtained by filtration and dissolved in 2N NaOH solution. After removing the insoluble particles, the solution was again acidified (pH ≒ 4.5). The precipitate was collected by filter and dried at 40 ° C. for 2 days with P ₂ O ₅ (yield 88%).

Pullulan (Pul) was purchased from Hayashibara (MW 100,000 Da, Okayama, Japan) and purified by the following method. First, pullulan was dissolved in 300 ml of double distilled water (2% w / v) and the solution was dropped into 700 ml ethyl alcohol. A white precipitate was obtained and dried under vacuum conditions for one day. The dried pullulan was dissolved again in 300 ml of tertiary distilled water and filtered continuously with 5, 0.8, and 0.2 μm pore sizes (Millipore Corp., Billerica, Mass.). The final product was dried under vacuum conditions with condensation of the filtrate and P ₂ O ₅ at room temperature (yield 96%).

 2) conjugation of folic acid with pullulan.

Degree of substitution (degree of substitution; DS) that the other three folate / pullulan (FA / Pul) was bonded the composite, are shown in Fig. Purified pullulan (1 g. 6.173 mmole of repeated glucose units) was dissolved in 30 ml of anhydrous DMSO at 80 ° C. for 20 minutes, and the amount of folic acid and DMAP (4, 6, 8% degree of substitution) was varied. For each 247, 370 and 474 μmole, and the initial rate of folic acid, FA / Pul was named FA4 / Pul, FA6 / Pul and FA8 / Pul, respectively.

To dissolve folic acid cleanly, the mixture was stirred at room temperature overnight. After adding a slight excess of DCC to each flask, the reaction was carried out at 25 ° C. for two days. The solution was placed directly on the dialysis membrane (MWCO 12,000-14,000 DA, Spectra / Por RC dialysis membrane, Spectrum Laboratories, Rancho Dominguez, Calif.) And dialyzed with excess tertiary distilled water at room temperature for one day and for another four days at 4 ° C. It was.

FA / Pul end products were obtained by freezing and vacuum drying. All procedures were performed under conditions protected from light, and each FA / Pul was stored at -20 ° C for further testing (yield 89-93%).

Chemical conjugation was confirmed following ¹ H-NMR spectroscopy, and the degree of substitution (DS) of the monomers to be substituted was determined by UV-VIS spectroscopy (Model Lamda 18, PerkinElmer Inc., Wellesley, Mass.). After dissolving FA / Pul and folic acid in DMSO at various concentrations, the absorption spectra were scanned from 190 to 450 nm, showing three peaks at 320, 360 and 380 nm, of which 380 nm. The absorbance values of folic acid solutions at different concentrations at gave a good plot of first regression. Using a standard sample curve, the molar extinction coefficient (ε) was given.

Fluorophore labeling of FA / Puls was performed by mixing 100 mg FA / Pul with 0.5 mg FITC in 10 ml of DMSO containing anhydrous 1 L dibutylin dilaurate. After 18 hours of reaction at room temperature, the solution was dialyzed (MWCO 12,000 -14,000 Da, Spectra / Por membrane RC) with excess tertiary distilled water at 4 ° C. for 5 days and frozen. Since the amount of FITC was too small to modify the physicochemical properties of FA / Pul, each FITC-labeled FA / Pul was used for further experimentation without further characterization.

 3) results

Chemical conjugation was carried out by a general carbodiimide reaction. It is well known that the carbonyl compound preferentially binds to the hydroxyl group of C (6) of the polysaccharide. Pullulan is one of the few polysaccharides that can be easily dissolved in organic solvents. This solubility facilitates chemical modification. ¹ H-NMR spectra showed that folic acid was successfully conjugated to pullulan. ¹ H-NMR (300 MHz, DMSO- d ₆ ) is 2.29 (t, 2H, COCH ₂ ), 4.27 (q, 2H, C H ₂ CH), 6.9 (t, 1H, CHCO), 7.64 (for folic acid). A plurality of peaks ranging from 3 to 6 ppm were shown for s, 2H, COC H = CH), 6.64 (s, 2H, COCH = C H ), 8.10 (s, 2H, CH ₂ C) and pullulan.

(2) Preparation of conjugate of pullulan / biotin

 1) Materials and Methods

  5 g of hydrophobized pullulan was dissolved in 50 ml of dimethyl sulfoxide (DMSO) completely dehydrated, and various concentrations of biotin were added to the DMSO solution, followed by N, N'-dicyclohexyl carbodiimide ( DCC) and dimethylaminopyridine (DMAP) were added and reacted at room temperature for one day. The dicyclohexyluria (DCU) produced during the reaction was filtered off, and the solution was dialyzed in distilled water and then lyophilized to prepare a biotin / pullulan conjugate.

Biotin was linked to pullulan by DCC and DMAP-mediated ester formation. The carboxyl group of biotin (1 g) in dry DMSO was activated by the addition of DCC (600 mg) and DMAP (400 mg) ( FIG. 2 ). Various amounts of activated biotin (100-1000 mg) were added to dry 50 mL DMSO containing 1 g pullulan and reacted for 2 hours at room temperature. After 24 hours, the mixture of reactants was filtered and dialyzed against distilled water for 3 days using a dialysis tube of molecular cut-off 12,000. Distilled water was changed every 3-6 hours. The precipitated pullulan / biotin conjugate was filtered off, washed sufficiently with diethyl ether and distilled water, recovered and freeze-dried. To completely remove the unreacted materials, the freeze-dried sample was dissolved in DMSO again and dialyzed with distilled water. This process was repeated three times. The degree of substitution (DS), defined as the number of biotin groups per 100 anhydroglucose units of pullulan, was determined by ¹ H-NMR. In NMR measurements, all samples were dissolved in DMSO-d ₆ (Aldrich) and tetramethylsilane (TMS; Aldrich) as reference materials.

2) results

Conjugation of pullulan and biotin through the formation of ester bonds was carried out in a traditional carbodiimide reaction. This method is known to adhere the carbonyl compound to the hydroxyl group on carbon 6 of the polysaccharide. ¹ H-NMR spectra showed that biotin successfully combined with pullulan. ¹ H-NMR (300 MHz, DMSO- d ₆ ) gives δ 1.89-2.11 (2H, CH _{2 in} methylene; -C, -SC), 2.51 (2H, CH _{2 in} methylene; -C, -O for biotin , -OC) and 3.37 (H, CH in tetrahydrothiophene) and broad complex peaks in the range of 3.7 to 5.5 ppm for pullulan. The peak of OH in the carboxyl group of biotin disappeared at δ11.5. The calculations for each pullulan substitution with biotin (number of biotin per 100 glucose units of pullulan) were 11.34, 18.97 and 23.56.

(3) Preparation of conjugate of pullulan and bile acid

In order to introduce amphiphility into pullulan, a hydrophobized monomer, Deoxycholic acid (DOCA), was used. The reaction mechanism was to generate an ester bond by reacting the hydroxyl group of the polysaccharide with the carboxyl group of DOCA. First, 5 g of pullulan was added to 50 ml of dimethyl sulfoxide (DMSO) from which water was completely removed, and the temperature was raised to 60 ° C. to dissolve polysaccharides, and the mixture was left at room temperature. DOCA of various concentrations was added to the dissolved DMSO solution at room temperature, and then N, N'-dicyclohexyl carbodiimide (DCC) and dimethylaminopyridine (DMAP) were added according to the molar ratio. The reaction was carried out at room temperature for one day. The dicyclohexyluria (DCU) produced during the reaction was filtered off, and the solution was dialyzed in distilled water and then lyophilized to obtain pullulan-DOCA conjugate. Ester bond reaction between flulan and bile acid and the chemical structure of the product are shown in FIG.

Example 2 Preparation and Characterization of Nano-Agglomerates

(1) Folic Acid / Pululan Nanoparticles

1) Materials and Methods

 Nanoparticles were prepared by dialysis and solvent evaporation. 100 mg of the conjugate prepared in Example 1 was completely dissolved in 30 ml of DMSO, and then put in a dialysis membrane (cutoff: 1000) and dialyzed in aqueous solution. Replacement of the aqueous solution is carried out every 3 hours, and this process is continued for 2 days and then lyophilized.

Further, 100 mg of the conjugate synthesized in Example 1 was completely dissolved in 30 ml of dichloromethane (MC), respectively, poured into 50 ml of aqueous solution, and then vigorously stirred at 1,00-10,000 at 30 ° C. for 2 hours. Remove dichloromethane. Nanoparticles are made in the process of dichloromethane removal.

To make self-aggregated nanoparticles, each FA / Pul (100 mg) was dissolved in 10 mL DMSO and dialyzed against excess tertiary distilled water in a dark cold room (MWCO 3,500 Da, Spectra / Por membrane RC) ). For 3 days, the dialysates were collected and their volume adjusted to 100 mL (final concentration 1 mg / mL). After each sample was filtered with a 0.8 μm syringe filter, the particle size and distribution were measured by dynamic light scattering (Argon ion laser system, Lacsel laser model 95, Brookhaven Instrument Corp., Holtsville, NY). Acceleration voltages of transmission electron microscopes (TEM, Philips CM 30, Philips Electron Optics, Eindhoven, the Netherlands) were carried out at 200 keV to observe the particles in the dry state.

 2) Results-Generation of Nanoparticles

Figure 4 shows the size distribution of folic acid / pullulan (FA / Pul) nanoparticles measured by dynamic light scattering (DLS). ( FIG. 4 (a) FA4 / Pul: FIG. 4 (b) FA6 / Pul: and FIG. 4 (c) FA8 / Pul). Two main peaks and one small peak are observed in the FA / Pul sample. The predominant particle size was about 230-300 nm (mean values 261, 276 and 272 nm for FA4 / Pul, FA6 / Pul, and FA8 / Pul respectively) and other predominant sizes distributed in the 60-80 nm range (the same 71, 75, and 74 nm in order). The size and distribution of the particles within these ranges did not vary with the degree of substitution by folic acid. On the other hand, particles appearing around 1,000 nm became apparent as the degree of substitution of monomers increased ( 6, 2, and 0% intensity in each of FIGS . 4 (a), 4 (b) , and 4 (c) ). .

Nanoparticles from FA / Pul are very stable in the dry state, which was confirmed by TEM images ( Fig. 5 (a) FA4 / Pul; Fig. 5 (b) FA6 / Pul; and Fig. 5 (c) FA8 / Pul). However, the image of FA4 / Pul showed a number of small aggregates, which would result in additional aggregation of each nanoparticle. The population of these small particles decreased with increasing hydrophobicity or DS value of the polymer. The overall size of the nanoparticles was less than 50 nm because water was removed from the sample. In combination with the light scattering results, their particle size shrinkage shows that the particles have a huge amount of hydrodynamics.

(2) Biotin / Pululan Nanoparticles

1) Materials and Methods

 Nanoparticles of nanogel self-organized from the conjugate of pullulan and biotin were prepared by diafiltration method with a fractional molecular weight of 2,000. Each pullulan / biotin conjugate with different degrees of substitution (DS) was dissolved in 20 mL of DMSO. Each solution was dialyzed at room temperature and dialyzed with distilled water for 3 days. The solution was filtered through a 0.45 μm filter to remove precipitates.

Pyrene stock solution (6.0 × 10 ⁻² M) was prepared in acetone for measurement of fluorescence spectroscopy and stored at 5 ° C. until use. Pyrene solution under acetone was added to distilled water to adjust pyrene concentration 12.0 × 10 ⁻⁷ M for measurement of steady state fluorescence, and then the solution was distilled at 60 ° C. for 1 hour to remove acetone from the solution. Acetone-free pyrene solutions were mixed with self-aggregating nanoparticles in concentrations ranging from 1 × 10 ⁻⁴ to 1.0 mg / mL. The final concentration of pyrene in each sample was 6.0 × 10 ^-7 M, which is about the same as the solubility in water at 25 ° C.

The measurement of anisotropy value for 1,6-diphenyl-1,3,5, -hexatriene (DPH) in the PU / Bio nanoparticles, the anisotropic value of DPH (2.1 × 10 ^-6 M) in the nanoparticles was determined by the L-shape array of the sense. Fluorescence anisotropy (r) was measured from the following relationship.

(수학식 1)

(Equation 1)

I ^s represents the contribution of light scattered from the sample solution in the absence of DPH. G (I _VH / I _HH ) is a modifier and I _VV , I _VH , I _HV and I _HH are vertical or horizontal detection when excited with vertically or horizontally polarized first subindex The resulting emission intensity is polarized in the second subindex. The excited wavelength was 360 nm and emission was measured at 430 nm.

2) results

2 shows the chemical structures of pullulan (a), biotin (b) and pullulan / biotin (Pu / Bio) polymer (C). The degree of biotin substitution was 11, 19 and 24 biotin groups in 100 anhydrous glucose units of pullulan. Physicochemical properties of nanogels (Pu / Bio1, 2, and 3) in aqueous medium were determined by dynamic light scattering, transmission electron microscopy, and fluorescence spectroscopy. The average diameter of all samples was unimodal, less than 300 nm. Critical aggregation concentration (CAC) in distilled water was 2.8x10 ^-2 , 1.6x10 ^-2 and 0.7x10 ^-2 mg / ml for Pu / Bio1, 2, 3 respectively. The aggregation behavior of the nanogels has shown that biotin can act as a hydrophobic moiety. To see specific interactions with the hepatic cancer cell line (HepG2), rhodamine B isothiosayanate (RITC) was labeled on the conjugate and their extent was specified using a fluorescent microplate reader. HepG2 treated with fluorescently labeled PU / Bio nanoparticles emitted more strongly than the control (pullulan). Confocal laser microscopy showed the cancer cells entering the Pu / bio nanogel. This shows that in the combination of biotin and pullulan, this biotin acts as a moiety for both hydrophobization for self-aggregation and tumor targeting for specific interactions with tumor cells. It can be seen that it is useful as a drug carrier for treatment.

The pullulan / biotin nanoparticles synthesized three conjugates replacing 11 (PU / Bio1), 19 (PU / Bio2), and 24 (PU / Bio3) biotin groups per 100 pullulan glucose units. Self-organized nanogels were prepared from these conjugates using the dialysis method. This method has several advantages, such as small particle formation, rapid precipitation prevention and narrow size dispersion (Yasugi, K., Y. Nagasaki, M. Kato, and K. Kataoka, 1999. Preparation and characterization of polymer micelles from poly (ethylene glycol) -poly (d, l-lactide) block copolymers as potential drug carrier, J. Control.Release 62: 89-100.). After dialysis of the PU / Bio solution, a significant amount of light scattering intensity change was observed. This indicates that the nanogel was formed from the conjugate.

The size and size distribution of self-organized nanogels in distilled water were measured by DLS, and morphological structures were observed using TEM.

Figure 6 (a) shows the particle size and particle distribution of PU / Bio 2 in the solution medium. The swollen PU / Bio2 nanogels at pH 7.4 have an average diameter of 217 nm with a unimodal size distribution (± 182 nm). The morphological structure of pullulan / biotin 2 nanogels observed by TEM is shown in FIG. 6 (b) . PU / Bio2 nanogels are spherical with a radius of less than 100 nm in dry size.

Self-organized nanogel formation of pullulan and biotin conjugates in solution medium was investigated using a fluorescence probe.

Fluorescence excitation spectra of PU / Bio3 nanogels at various concentrations of polymer in the presence of 6.0 × 10 ⁻⁷ M pyrene are shown in FIG. 7 . At low concentrations of PU / Bio3, the change in total fluorescence intensity and the shift of the (0,0) band at 334 nm were negligible. However, as the PU / Bio3 concentration increased, the increase in total fluorescence intensity and red shift of the (0,0) band were clearly observed. The increase in total fluorescence intensity with the addition of the PU / Bio3 conjugate shows that the probe has migrated from the aqueous medium to the hydrophobic microdomains inside the nanogels. The (0,0) band for pyrene at 334 nm varied to 337 nm with the addition of PU / Bio3. Critical aggregation concentration (CAC), the threshold concentration of self-assembling nanoparticle formation by internal and / or mutual molecular association, is due to the change in the intensity ratio of pyrene (I ₃₃₇ / I ₃₃₄ ) in the presence of PU / Bio. Decided. 8 shows the intensity ratio (I ₃₃₇ / I ₃₃₄ ) of PU / Bio nanogels as a function of polymer concentration. CAC values were calculated from the intersection at low concentrations.

The physicochemical properties of the self-organized nanogels are shown in Table 1 .

Table 1. Properties of self-organized nanogels in aqueous media

sample DSa Particle size b ± standard deviation (nm) CACc X 102 (mg / mL)  rd (anisotropic value) PU / Bio1  11 130 ± 114 2.8 0.182 PU / Bio2 19 217 ± 182 1.6 0.196 PU / Bio3 24 275 ± 223 0.7 0.201

^a degree of substitution of biotin per 100 anhydroglucose units of pullulan (using NMR)

^b Average diameter (intensity mean) measured by dynamic light scattering

Critical Condensation Concentration Determined from ^C I ₃₃₇ / I ₃₃₄

^{d The} value calculated from Equation 1

The average radii of Pu / Bio 1, 2 and 3 nanogels are 130 ± 114, 217 ± 182 and 275 ± 223 nm, respectively. The particle size of self-organized nanogels increased with increasing biotin content due to increased aggregation between biotin. In general, carriers with a radius larger than about 300 nm are known to cause nonspecific clearance through monocytes and the reticuloendothelial system (RES) (Na, K., ES Lee, and YH Bae. 2003. Adriamycin loaded pullulan acetate / sulfonamide conjugate nanoparticles responding to tumor pH: pH-dependent cell interaction, internalization and cytotoxicity in vitro.J. Control.Release 87: 3-13.Yokoyama, M., and T. Okano, 1996. Targetable drug carriers: present status and a future perspective.Adv.Drug Del. Rev. 21: 77-80.). By this method nanogels will be able to avoid entrapment by RES.

PU / Bio1, 2, and 3 produced in the present invention exhibited characteristics such as different DS (units of biotin per 100 anhydrous grapes, unit of pullulan), particle size, and CAC (critical concentration concentration). As the particle size increased, the amount of biotin aggregated increased. Since the feeding by leukocytes macrophages above 300nm particle size, PU / Bio nanogel has a characteristic that is not captured by the reticuloendothelial system (RES). Biotin contained in PU / Bio nanogels imparts hydrophobicity to the inner core, affects the reduction of CAC values in PU / Bio polymers, and has a critical effect on the physicochemical properties of nanogels.

CAC shows the dependence on biotin content in pullulan / biotin conjugates ( Table 1 ). This means that the increase in hydrophobicity resulting from the introduction of biotin decreases the CAC value. For detailed evidence of the function of biotin as a hydrophobic moiety, the internal structure of pullulan / biotin nanogels was investigated by measuring microviscosity by DPH.

The microviscosity of the inner nucleus of the particles was determined by measuring molecular anisotropy as a result of molecular rotational diffusion. The value of anisotropy for DPH increases with central microviscosity due to limited rotational motion. The r value of Pu / Bio3 nanogels is shown to be the highest among the samples tested ( Table 1 ). Higher r values mean increased strength of the inner center of the particle, which means that Pu / Bio3 nanogels have the hardest inner center. The results support that the hydrophobicity of nanogels increases with increasing biotin content of the polymer. The results indicate that biotin can act as a hydrophobic moiety to make self-organized nanogels, and that the degree of substitution of biotin in the conjugate can control the physicochemical properties of the nanogels.

Example 3 Encapsulation and Release of Anticancer Agents in Nanoparticles

(1) materials and methods

DOX (doxorubicin.HCl, 20 mg, 34.484 μmol) was dissolved in anhydrous DMSO containing 6.2 μl TEA (44.829 mol). After each FA / Pul conjugate (50 mg) was added to the solution, the mixture was stirred in a dark cold room. To make DOX-encapsulated FA / Pul nanoparticles, the solution was transferred to a wet dialysis membrane (Spectra / Por RC dialysis membrane, MWCO 1,000 Da) and dialyzed at room temperature for excess deionized water. Water was changed every 3 hours for 24 hours, and the dialysate was filtered (0.45 membrane filter, Millipore) to remove aggregated precipitate. Finally, the filtrate was frozen for 3 days and the product was stored at -20 ° C for further testing.

To determine the drug-loading amount, the dry sample was suspended in DMAC and vigorously stirred for 2 hours. After a short sonication (3 min), the solution was centrifuged at 20,000 g for 30 min. The UV absorbance of the supernatant containing DOX extracted from the nanoparticles was measured at 490 nm. The linear standard curve from free DOX in DMAC was used to calculate the encapsulation efficiency, which was 12 ± 4.4, 18 ± 6.1, and 22 ± 6.9% (w / F) for FA4 / Pul, FA6 / Pul, and FA8 / Pul, respectively. w) was. In vitro drug release testing was performed using a dialysis tube. After holding 2 mL DOX-encapsulated FA / Pul nanoparticles (0.1 mg / mL DOX amount), the dialysis tube is placed in a vial containing 10 mL sterile PBS (pH 7.4) and lightly at 100 ° C. (100 rpm). Stir it. In some cases, the entire exclusion was taken and stored at 4 ° C. and fresh PBS was added without breaking the precipitate. All sample media containing the released DOX residues were analyzed by UV spectroscopy as described above, which showed an emission plot as a function of time.

(2) results

In vitro release profiles of DOX from FA / Pul nanoparticles were examined and the results are shown in FIG. 9 . The regression equations for each group of FA4 / Pul, FA6 / Pul, and FA8 / Pul are y = 3.133 + 92.9794 (1-e ^-0.0875x ), y = 0.8497 + 93.7875 (1-e ^-0.0584x ), and y = 2.4669 +93.2957 (1-e ^-0.0367x ). Each equation shows a good correlation factor, so that the R ² values are 0.9987, 0.9985, and 0.9987 in the same order. Half-maximal release time (t _1/2 ), ie 50% release of encapsulated DOX from nanoparticles, was dependent on the folate's DS, which was applied to FA4 / Pul, FA6 / Pul, and FA8 / Pul respectively. For 8.0, 12.7, and 19.4 hours. The more folic acid was substituted, the longer t _1/2 was. In other words, DOX release from nanoparticles was promoted as DS decreased. FA4 / Pul produced about 0.13 mg DOX (63% of the total 0.2 mg) within 12 hours, while FA8 / Pul made only 37%. Over three days, the amount of released DOX was 96, 93, and 89 percent of each nanoparticle in the same order. The saturation point of each nanoparticle was about 95-96 percent. This shows that most drugs are separated from nanoparticles within 3-4 days.

Example 4 Confirmation of Specific Interaction with Cells

(1) Cell specific interaction of FA / Pul

1) Materials and Methods

Absorption of free or DOX-enclosed nanoparticles was investigated using fluorescence microscopy (Karl Zeiss, Oberkochen, Germany). Initially, KB and CHO-K1 cells (10 ⁵ / well) were planted in 6-well cell culture plates and incubated in 2 mL FBS- and antibiotic-supplemented media for one day. Cells were washed twice with warm PBS and placed in 1.8 mL KRH containing 0.1% (w / v) BSA. To test whether the degree of substitution by folic acid (DS) affects the binding properties of nanoparticles to cells, the same amount of FITC-labeled FA4 / Pul, FA6 / Pul, and FA8 / Pul were treated in each well. It became. In this experiment, the amount of FITC-labeled FA / Pul was 1 mg / mL regardless of DOX encapsulation efficiency. Before adding the nanoparticles, each well was washed again with warm PBS and fixed with 2% glutaraldehyde and 2% formaldehyde (fixed solution) in PBS for 5 minutes at room temperature. Each sample was stored at 4 ° C. prior to performing fluorescence microscopy. To observe the fluorescence image, 2 mL PBS was added to each well, each sample was repeated three times, and five different images per well were obtained. Using the underwater-precipitating lens (x400), the sample was photographed with an exposure time of 200 ms. The obtained image was not further adjusted except to montage or resize by NIH image J, version 1.33u.

Specific interactions with cells expressing FA / Pul's folate receptors (FARs) were studied in a competitive study. 1 mg / mL FA8 / Pul was added to a 6-well culture plate containing KB or CHO-K1 cells in 1 mL KRH (w / 0.1% BSA, pH 7.4), followed by different concentrations (0.05, 0.1, and 0.5). mg / mL) and folic acid solution in PBS were treated simultaneously. Within two hours, cells were washed twice with PBS and 1 mL fixed solution was applied. Fluorescence microscopy was performed by the method as mentioned above.

All experiments were repeated at least three times, and the data obtained were expressed as mean standard deviation. In the UV absorbance test, a simple model of the first cycle (y = ax + b) was used and the a value when the b value was close to zero was chosen as the molar extinction coefficient. Graphs from the release test were analyzed with the exponential increase model y = y ₀ + a (1-e ^-bx ). Survival from MTT was analyzed by Student's unpaired t-test, and the statistical significance of each value by comparison with the control at a given time was determined by both Estrike (*, P <0.01) and cross (+, P <0.05 ).

2) results

Since there was no difference in fluorescence between each group, FIG. 10 shows that the effect of DS on intracellular uptake of nanoparticles is negligible. However, KB cells appear to attract more nanoparticles inwards than CHO-K1 cells do. Within an hour after administration of the DOX-filled nanoparticles, most of the nanoparticles are observed in the cytoplasm of both KB and CHO-K1 cells. FITC channels, FITC-labeled FA / Pul and RITC (rhodamine B isothiocyanate) channels showed DOX fluorescence.

In order to ensure that the differences are caused by the over-expression of folic acid receptors, KB cells, the same, by performing the analysis of the competition (competition), are shown in Fig. This shows that free folic acid competes effectively with FA / Pul nanoparticles. The fluorescence emission observed in the FITC and RITC channels gradually decreased with increasing free folic acid. When treated with 0.5 mg / mL folic acid, most all nanoparticles could not enter KB cells. Since CHO-K1 cells express relatively small amounts of folic acid, uptake of nanoparticles was also reduced, although no significant difference was observed.

(2) Pu-Bio Cell Specific Interactions

Another role for biotin in Pu / Bio nanogels is the introduction of specific interactions with malignant tumor cells. Pu / Bio conjugates were labeled with RITC with a fluorescence probe to observe the interaction. HepG2 cells (liver cancer cell line) were used as another malignant tumor cell. The interaction between Pu / Bio nanogels and HepG2 was quantified with a microplate-fluorescence-reader. Pu / Bio nanogels showed similar dependence on biotin content as in the interaction between nanogels and HepG 2. The degree of interaction between Pu / Bio nanogels and cells was higher than that of RITC labeled pullulan ( FIG. 12 ). In part, Pu / Bio2 and 3 interacted more strongly with HepG2 cells than Pu / Bio1. Specifically, biotin acts first as a hydrophobic moiety to form a self-aggregate, and biotin exposed to the outer shell of the nanogel acts as a target moiety for specific interaction with malignant tumor cells.

Internalization of drug carriers, including anticancer drugs, is one of the effective methods to improve the cytotoxicity of drugs. As the intracellular content of the drug through endocytosis of the carrier increases, the cytotoxic effect on the target cells will increase significantly. Thus, the effect of biotin as a target moiety on the internalization of nanogels was observed using HepG 2 treated with 10 μg / mL nanogels for two hours. Confocal laser microscopy was used to observe the internalization of Pu / Bio nanogels in HepG2 cells. Pu / Bio2 and 3 nanogels showed intracellular localization in HepG 2 cells, whereas pullulan / biotin 1 nanogels were only slightly localized. Confocal microscopy also showed that the degree of interaction of HepG2 cells depends on the biotin content ( FIG. 13 ). As a result, Pu / Bio nanogels can interact with cancer cells through any mechanism of internalization and biotin-cell interaction of nanogels with biotin that depends on biotin concentration.

The present invention relates to an ester conjugate of a pullulan and a hydrophobized monomer having a carboxyl group, a method for producing a self-aggregate by esterification of a pullulan and a hydrophobized monomer, and a method of using the nano-aggregate. It has been shown that hydrophobized monomers with carboxyl groups such as biotin and bile acids and pullulan can form nano-size self-aggregates in a simple reaction such as one-step reaction.

The nanoparticles may be used as drug delivery materials, and may have specificity to bind to target cells such as malignant tumor cells, and thus anticancer agents may be used as delivery agents.

In addition, in the present invention, it was confirmed that there is a close relationship between the degree of the monomer to be substituted and the release of the drug, by combining 6 or more hydrophobization monomer per 100 units of glucose to reduce drug delivery rate, or less than 6 monomers per 100 units of glucose The combination provides a method of speeding up drug delivery. This can be used as a drug delivery method and a drug delivery rate control method.

In addition, the nano-self-agglomerate has excellent affinity and biocompatibility for the human body and can be used as a biomaterial because it has biodegradability.

Claims (18)

In the method for producing an ester-bonded conjugate of any one cyclic compound and pullulan selected from a hydrophobized monomer cyclic compound containing a carboxyl group consisting of retinolic acid, folic acid, biotin and bile acid, (a) dissolving pullulan in dimethyl sulfoxide (DMSO) to prepare a dimethyl sulfoxide (DMSO) solution in which pullulan is dissolved; (b) hydrophobization monomer containing carboxyl group adding the cyclic compound, N, N'-dicyclohexyl carbodiimide (DCC) and dimethylaminopyridine (DMAP) to the solution prepared in step (a) ; (c) filtering out dicyclohexyluria (DCU) from the solution produced in step (b); And (D) a method for producing an ester bond conjugate of a cyclic compound and pullulan, comprising dialysis and lyophilizing the solution from which dicyclohexyluria (DCU) is filtered out in step (c). An ester bond conjugate of a hydrophobized monomer cyclic compound containing a carboxyl group prepared by the method of claim 1 and pullulan. The conjugate according to claim 2, wherein the hydrophobized monomer cyclic compound containing a carboxyl group is folic acid. The conjugate according to claim 2, wherein the hydrophobized monomer cyclic compound containing a carboxyl group is biotin. The conjugate according to claim 2, wherein the hydrophobized monomer cyclic compound containing a carboxyl group is bile acid. The nanoaggregate obtained by self-aggregation of the conjugate of claim 2. The nanoaggregate obtained by self-aggregation of the conjugate of claim 3. The nanoaggregate obtained by self-aggregation of the conjugate of claim 4. A nanoaggregate obtained by self-aggregation of the conjugate of claim 5. A drug carrier in which a drug is bound to any one or a combination of the nanoaggregates of claims 6 to 9. delete delete A drug delivery method for animals other than humans using any one or a combination of the nanoaggregates of claims 6 to 9. The method of claim 13, wherein the drug is selected from the group consisting of proteins, nucleic acids, chemicals, and natural substances. delete 15. The method of claim 13, wherein in the conjugate forming the nano-aggregate, the number of the hydrophobized monomers per glucose unit of pullulan is increased to 6 units or more and 20 units or less to reduce the drug delivery rate. Drug delivery method.  14. The drug delivery to animals other than humans according to claim 13, wherein the rate of drug delivery is increased by reducing the number of bonds of hydrophobization monomer per glucose unit of pullulan to less than 6 units in the conjugate forming the nanoaggregate. Way. A biomaterial comprising any one or a combination of the conjugates of claim 2.

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