WO2013115559A1 - Method for preparing albumin nanoparticles containing poorly water soluble drug therein - Google Patents

Abstract

The present invention relates to a method for preparing non-peptidic polymer and albumin assembly nanoparticles containing a poorly water soluble drug therein and capable of mediating in vivo delivery through solubilization and stabilization in an aqueous solution. Particularly, a non-peptidic polymer and albumin assembly and a poorly water soluble drug can be simultaneously dissolved even if using only an organic solvent, and thus the two components are mixed well even if a separate chemical or physical action is not applied. In addition, a method for preparing polyethylene glycol and albumin assembly nanoparticles according to the present invention does not contain a surfactant and a solubilizing agent, and thus does not cause a hypersensitive reaction or a toxic reaction due to the formulation of a poorly water soluble drug and can increase the compliance of a patient. Further, the present invention can also be useful for the stabilization and the like of inorganic nanoparticles for image/diagnosis and of various poorly water soluble drugs.

Description

Method for producing albumin nanoparticles containing a poorly water-soluble drug therein

The present invention relates to a method for producing polyethylene glycol and albumin aggregate nanoparticles containing a poorly water-soluble drug therein that can mediate in vivo delivery through solubilization and stabilization in aqueous solution.

Many of the pharmacologically active substances used for the treatment of diseases are poorly water-soluble and are formulated with solubilizers or organic solvents, including various surfactants that can cause irritation or toxicity when administered in vivo to increase the solubility of the drug. Formulated with emulsifiers. For example, paclitaxel, a plant-derived anticancer agent, is a highly hydrophobic, poorly water-soluble drug that is made into an injection by solubilizing in a co-solvent of Cremophor EL and ethanol. However, cremophore can cause severe hypersensitivity reactions, including allergic reactions, which requires preparation before treatment through administration of steroids, H2-antagonists, and antihistamines, and 3 to 24 hours of administration time to avoid severe acute allergic reactions. This is necessary. Therefore, patient discomfort due to a long infusion time, patient monitoring during the administration time, preparation and injection process requires a lot of additional costs for hospitalization.

In order to reduce such serious side effects, a method of using a polymer micelle or microsphere as a carrier of a poorly water-soluble anticancer agent and a method of combining an anticancer agent with a hydrophilic polymer have been studied. In the case of using double micelles or microspheres as carriers, the amphiphilic copolymer having hydrophilicity and hydrophobic blocks in one chain is mainly used to form colloidal particles having a core-shell structure which is thermodynamically stable in aqueous solution. Techniques have been developed to encapsulate and deliver poorly water-soluble drugs in a hydrophobic core inside micelles or autoaggregates.

Microspheres using proteins in vivo have been reported to be used as carriers for drugs or diagnostic agents. Among various biological proteins, albumin has a well-established separation technology and accounts for about 70% of plasma proteins in the blood, which is more economical than other proteins in terms of isolation and use, is easy to acquire, and is highly biocompatible. It is a carrier that is readily available in vivo and can provide a variety of dosage forms, including injections. For example, a method for preparing albumin microspheres by chemical crosslinking using glutaraldehyde as an emulsifying and crosslinking agent (US Pat. No. 4,671,954) and a heat-modifying method for producing microspheres by heating an emulsion mixture at 100 ° C to 150 ° C (Leucuta et al. , International Journal of Pharmaceutics 41: 213-217 (1988)).

However, such a method for producing microspheres is suitable for inclusion of water-soluble materials due to technical limitations in forming water-modified or cross-linked particles in the aqueous phase of water-in-oil emulsions, but not suitable as a carrier for poorly water-soluble materials.

In addition, a copolymer of polylactide and the like is dissolved in a poorly water-soluble drug and an organic phase and introduced into an aqueous phase to emulsify with a water-in-oil emulsion, whereby nanoparticles are prepared, and a preparation method using albumin as a surfactant for surface stabilization is presented in the literature. (Bazile et al., Biomaterials, 13: 1093 (1992)).

Havre developed a lock System Biosciences (Abraxis, USA) is not chemically bonded to the poorly water-soluble anticancer agent is paclitaxel to albumin using the problem of existing crushers all pores of Taxol ^® alternative protein nano-formulation stability Havre Leshan ^®.

In addition, Vivorex (VivoRX, USA) developed nanoparticle capsol ^® containing a poorly water-soluble drug without the use of cremophors or surfactants using high pressure, high shear forces under certain oil / water emulsion conditions (US patent). 5,439,686, Korean Patent 10-0923172).

However, the above-described method for preparing ABRAXANE ^® and CAPSOL ^® requires the use of chemical bonding of poorly water-soluble drugs and high pressure and high shear forces in the emulsion phase. When the nanoparticles are manufactured using high pressure and high shear force, there is a problem that the stability of the drug is lowered and the time and cost increase.

In addition, according to Chinese Patent Application Publication No. 101543630, a technique of imparting amphiphilicity by introducing a hydrophobic block and a hydrophilic block into a specific copolymer or protein at the same time has been known. Therefore, there is a need for a technique for stabilizing poorly water-soluble drugs without the introduction of additional hydrophobic groups.

Therefore, the present inventors have high solubility in organic solvents without using a surfactant or using chemical bonding method and high pressure and high shear force in an emulsion phase when using a combination of a non-peptidyl polymer and albumin. It was confirmed that coalescing could be formed.

In addition, after preparing the association of non-peptidyl polymer and albumin, the organic solvent is removed, and then, a hydrophilic solvent is added, and the nanoparticles containing poorly water-soluble drugs by self-assembly by simple nanoparticle manufacturing methods such as solvent diffusion and film formation. It was confirmed that the particles can be formed, the nanoparticles containing the poorly water-soluble drug prepared by the above method compared with the albumin nanoparticles containing the known anticancer agent, the anticancer agent is well delivered to confirm that the anticancer effect is excellent The present invention has been completed.

The present invention solves the problem of using a surfactant or a high pressure, high shear force in the chemical bonding method and emulsion as a method for producing nanoparticles for in vivo delivery of conventional poorly water-soluble drugs or inorganic nanoparticles for bioimaging. It is to.

The present invention provides a method for preparing a non-peptidyl polymer and albumin aggregate and dissolving it in an organic solvent in a fixed molar ratio with a poorly water-soluble drug to remove the organic solvent and to form nanoparticles by self-assembly in a hydrophilic solvent. It is to.

In addition, since the non-peptidyl polymer and albumin aggregate nanoparticles prepared by the present invention can mediate in vivo delivery through solubilization and stabilization in aqueous solution, they can be used as carriers of poorly water-soluble drugs or inorganic nanoparticles for bioimaging. The present invention is to provide an association nanoparticle of the non-peptidyl polymer and albumin prepared by the above production method.

In order to solve the above problems, the present invention is a first step of forming an association of the non-peptidyl polymer and albumin by mixing the non-peptidyl polymer with albumin;

Preparing a mixture by dissolving a poorly water-soluble drug or inorganic nanoparticles for bioimaging, and an association of the non-peptidyl polymer and albumin on an organic solvent; And

Removing the organic solvent from the mixture and adding a hydrophilic solvent to prepare nanoparticles by self-assembly,

The present invention provides a method for producing a conjugated nanoparticles of albumin and a non-peptidyl polymer containing a poorly water-soluble drug or inorganic nanoparticles for bioimaging.

Step 1 is a step of mixing the non-peptidyl polymer with albumin to form a combination of the non-peptidyl polymer and albumin, the non-peptidyl polymer can react with the amine group of the albumin to mix the non-peptidyl polymer with albumin It may be substituted with a substituent. At this time, the reactor capable of reacting with the amine of albumin is composed of N-hydroxysuccinimide, succinimidyl succinate, succinimide propionate, succinimidyl butanoate, benzotriazole carbonate, aldehyde and catechol It may be selected from the group, but is not limited thereto.

In the present invention, the non-peptidyl polymer is polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylene, polyoxazoline, polyurethane, polyphosphazene, polysaccharide, dextran, polyvinyl alcohol, poly Vinyl pyrrolidone, polyvinyl ethyl ether, polyacryl amide, polyacrylate, polycyanoacrylate, lipid polymers, chitin, hyaluronic acid, heparin and combinations thereof. In the present invention, it is preferable to use polyethylene glycol, and derivatives of such polyethylene glycol can be used. The derivatives of polyethylene glycol are straight or branched and the branched may have two or more multiple groups.

Albumin used in the present invention is preferably human albumin (human albumin, HSA). However, the present invention is not limited thereto.

In the present invention, the mole ratio of the non-peptidyl polymer and albumin is preferably 5: 1 to 50: 1.

In the present invention, the molecular weight of the non-peptidyl polymer is preferably selected from the range of 750 Da to 300,000 Da.

In step 1, the non-peptidyl polymer may react with an amine group located in the hydrophilic domain of the albumin protein to form a covalent bond. Although non-peptidyl polymers are hydrophilic polymers, they can exhibit very high solubility not only in aqueous solvents but also in various kinds of organic solvents. Furthermore, the non-peptidyl polymer prepared by step 1 and the albumin association are non-peptidyl polymers bound to the hydrophilic domain of albumin protein, so the hydrophilic region of albumin protein is reduced and the hydrophobic region remains. Compared with the case where only the polymer is used, the solubility in the organic solvent can be further increased by forming the association.

Step 2 is a step of preparing a mixture by dissolving a poorly water-soluble drug or inorganic nanoparticles for bioimaging, and the association of the non-peptidyl polymer and albumin on an organic solvent.

Albumin molecules act as carriers of hydrophobic substances such as cholesterol and lipid molecules in vivo due to amphipathic chains, and may play an important role in binding to hydrophobic drugs administered in the blood. This property is due to the hydrophobic domain formed inside the albumin molecule.

The association of the non-peptidyl polymer and albumin on the organic solvent exposes the albumin hydrophobic domain to the organic solvent through chain rearrangement of the peptide chain.

In addition, non-peptidyl polymer chains of associations of non-peptidyl polymers with albumin may also be exposed in the organic solvent to help stabilize albumin proteins in organic solvents.

Therefore, phase separation does not occur in the state where the poorly water-soluble drug is dissolved in the association of the non-peptidyl polymer and albumin and the organic solvent, and it is possible to form a film in which the association and the drug are uniformly present when the solvent is removed. .

The organic solvent used in the present invention is ethanol, methanol, isopropyl alcohol, butanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), acetonitrile, dichloromethane, ethyl acetate, hexane, Diethyl ether, benzene, chloroform, acetone and combinations thereof may be used. In addition, a water-soluble solvent may be mixed with the organic solvent. However, it is not limited thereto.

It is preferable to use the organic solvent which does not form an emulsion by mixing with an aqueous phase. Examples include, but are not limited to, ethyl alcohol, methyl alcohol, isopropyl alcohol, butyl alcohol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF) or acetonitrile.

In the present invention, the molar ratio of the poorly water-soluble drug or the inorganic nanoparticles for bioimaging, the association of the non-peptidyl polymer and albumin is 1: 5 to 1:50. The optimal molar ratio can be set differently depending on the water soluble drug used or the application (eg the long-circulation formulation of SPION).

Step 2 takes advantage of the fact that the poorly water-soluble drug can be stabilized in a state in which the non-peptidyl polymer and the association of albumin and the organic solvent are dissolved together without introducing an additional hydrophobic group such as hydrophobic alkyl (acyl group) or bile acid.

Therefore, in the present invention, albumin may not only serve as a skeleton for introducing hydrophilic groups and hydrophobic groups, but may dissolve poorly water-soluble drugs using hydrophobic domains in the non-peptidyl polymer and albumin itself without introducing additional hydrophobic groups.

In the present invention, the poorly water-soluble drug may be an anticancer agent, an antibiotic, an antifungal agent, an anti-inflammatory agent, an immunosuppressive agent or a nutrient, and the inorganic nanoparticles for biological imaging may be a diagnostic agent or a contrast agent.

As anticancer agent, paclitaxel, docetaxel or ortataxel can be used as taxane or its derivative (s).

In addition, as an anticancer agent, adriamycin, colchicine, cyclophosphamide, actinomycin, bleomycin, duanorubicin, epirubicin, mitomycin, methotrexate, mitoxantrone, fluorouracil, carboplatin, carmustine (BCNU) ), Methyl CCNU, cisplatin, etoposide, interferon, camptothecin and derivatives thereof, penesterin, tofetecan, vinblastine, vincristine, tamoxifen, piposulfan, irinotecan, gemcitabine, herceptin, vinorelbine, Capecitabine, Alumta, Avastin, Velcade, Tarceva, Neurastar, Lapatinib and Sorafenib can be used. The anticancer agent which can be used is more preferably paclitaxel or camptothecin. However, the present invention is not limited thereto.

Antibiotics include neomycin, amikacin, aztreonam, chloramphenicol, palmitic acid chloramphenicol, chloramphenicol sodium succinate, ciprofloxacin, clindamycin, metronidazole, gentamicin, lincomycin, tobramycin, vancomycin, polymyxin B, colistin Metate sodium and colistin can be used. However, the present invention is not limited thereto.

Antifungal agents may include griseofulvin, keloconazole, amphotericin B, nystatin or candicidine. However, the present invention is not limited thereto.

As anti-inflammatory agents, nonsteroidal anti-inflammatory agents such as indomethacin, naproxen, ibuprofen, ramipenazone, pyroxicam and steroidal anti-inflammatory agents such as cortisone, dexamethasone, fluazacort, hydrocortisone, prednisolone, prednisone, etc. can be used. Can be. However, the present invention is not limited thereto.

As immunosuppressive agents, cyclosporin, azathioprine, myzoribin or tacrolimus can be used. However, the present invention is not limited thereto.

As a nutrient, fat-soluble vitamins such as vitamins A, D, E, and K can be used. However, the present invention is not limited thereto.

Diagnostic or contrast agents include MR magnetic contrast agents such as iron oxide nanoparticles, fluorocarbons, fat-soluble paramagnetic compounds, and the like; Ultrasound contrast agent; Radiographic agents such as iodo-octane, halocarbon, graphene and the like; And other diagnostics that cannot be delivered without physical or physicochemical modifications of substantially water insoluble properties. The diagnostic or contrast agent that can be used is more preferably iron oxide nanoparticles. However, the present invention is not limited thereto.

According to one embodiment of the present invention it is possible to dissolve the association of polyethylene glycol and albumin prepared in step 2 using dimethyl sulfoxide (DMSO). In addition, in the case of poorly water-soluble drugs or inorganic nanoparticles for bioimaging, the organic solvent can be dissolved in both the association of polyethylene glycol and albumin and the poorly water-soluble drug or bioimaging inorganic nanoparticles in the organic solvent.

Step 3 is a step of preparing nanoparticles by self-assembly by removing the organic solvent and adding a hydrophilic solvent. Removing the organic solvent and obtaining a mixture in which poorly water-soluble drugs or inorganic nanoparticles for biological imaging are uniformly mixed with a non-peptide polymer and an association of albumin, and having hydrophobicity by self-assembly by adding a hydrophilic solvent to the mixture. The portion of the protein is located inside the particle and the hydrophilic part is located toward the aqueous solution, the poorly soluble drugs or inorganic nanoparticles for bioimaging are collected in the hydrophobic portion and formed into particles having a nano size.

The addition of the hydrophilic solvent in step 3 results in the interaction between the poorly water-soluble drug and the hydrophobic domains of albumin, thereby placing the poorly water-soluble drug or inorganic nanoparticles for bioimaging, and the hydrophilic and flexible non-peptidyl polymer chain The outermost part of the particle, together with the hydrophilic domain of the protein, forms a core-shell structure, which stabilizes the particle surface and minimizes aggregation by interaction with other particles.

According to one embodiment of the present invention, when the hydrophilic solvent is added, it is confirmed that the poorly water-soluble drug is located in the hydrophobic portion inside the particle.

In addition, the solution containing the nanoparticles of step 3 has the advantage that it has a very stable colloidal properties.

In the present invention, the hydrophilic solvent is water, distilled water, sterile water, phosphate buffered saline (PBS), methanol, purified water, ethanol, 1-propanol, 2-propanol, 1-pentanol, 2-butoxyethanol, ethylene glycol, Acetone, 2-butanone, 4-methyl-2-propanone and combinations thereof. However, it is not limited thereto.

In the present invention, the method of preparing nanoparticles by self-assembly by adding a hydrophilic solvent is preferably a solvent diffusion method or a film formation method, but is not limited thereto.

The size of the aggregate nanoparticles prepared in the present invention is preferably 200nm to 500nm. In the case of 200 nm or less, the amount of poorly soluble drugs or inorganic nanoparticles for bioimaging is collected, and in the case of 500 nm or more, the size of the nanoparticles is large, so that the biotransmission ability is poor.

The association nanoparticles of the non-peptidyl polymer and albumin prepared by the preparation method of the present invention are used as a carrier and thus can safely deliver the drug in vivo.

In another aspect, the present invention provides a non-peptidyl polymer and albumin aggregate nanoparticles, characterized in that it comprises a poorly water-soluble drug or inorganic nanoparticles for biological imaging prepared by the method of the present invention.

In the present invention, since the non-peptide polymer, albumin association and poorly water-soluble drug can be simultaneously dissolved in the organic solvent, the two components are well mixed without additional chemical or physical action. Therefore, the organic solvent is removed and the hydrophilic solvent is removed. To add the nanoparticles can be formed by a solvent diffusion method using a reaction permeable membrane which is a common method for forming nanoparticles or film formation by solvent evaporation.

On the other hand, in the preparation of conventional nanoparticles, the heat-denatured or cross-linked forms in the aqueous phase of the water-in-oil emulsion, so that the albumin derivative and the poorly water-soluble drug are mixed. There was a problem.

The present invention has a high stability of the nanoparticles prepared compared to the conventional method for producing nanoparticles, the manufacturing step is simple bar non-peptidyl including the anticancer agent prepared by the manufacturing method of the present invention has the effect of reducing the cost and cost The combination of the polymer and albumin has an effect that the anticancer agent is well delivered compared to the albumin nanoparticles known in the art, so that the anticancer effect is excellent.

That is, the polyethylene glycol and albumin aggregate nanoparticle composition including paclitaxel prepared by the preparation method according to the present invention is abraxane (IC ₅₀ = 35.3 nM) prepared by chemical crosslinking of albumin and paclitaxel. When compared with the cell activity in the breast cancer cell line (MDA-MB453), it was confirmed that the higher anti-cancer activity because it has a lower IC ₅₀ value (5.7 nM).

In addition, anti-cancer activity was measured in the animal model of SK-BR-3 administration, which is a breast cancer cell line of polyethylene glycol and albumin association nanoparticle composition containing paclitaxel prepared by the method according to the present invention, which contains paclitaxel. Albumin nanoparticles were found to show a similar effect to cancer growth (Abraxane ^® ) to a superior degree.

In the production of conventional nanoparticles, an albumin derivative and a poorly water-soluble drug were mixed by forming a thermodenatured or crosslinked compound in an aqueous phase of a water-in-oil emulsion, resulting in inferior stability of the prepared nanoparticles, long manufacturing time, and increased cost. There was a technical limitation.

In the method for preparing non-peptidyl polymer and albumin association nanoparticles according to the present invention, it is possible to simultaneously dissolve the non-peptidyl polymer, albumin association and poorly water-soluble drug using only one organic solvent. Even if the phosphorus is not applied, the two components are well mixed.

Therefore, after removing the organic solvent, by adding a hydrophilic solvent, it is possible to form nanoparticles containing a poorly water-soluble drug therein by a solvent diffusion method using a reaction permeable membrane which is a general nanoparticle formation method or a film formation method by solvent evaporation. The stability of the particles is increased, the manufacturing method is simple and the cost is reduced.

In addition, the polyethylene glycol and albumin aggregate nanoparticle manufacturing method according to the present invention does not include a surfactant and a solubilizer, and the polyethylene glycol and albumin association having a hydrophobic form while the organic solvent is removed during the preparation of the nanoparticles. Since nanoparticles are formed by self-assembly in which a poorly water-soluble drug is enclosed therein, there is an advantage in that the patient's compliance can be increased without causing hypersensitivity or toxic reactions due to the formulation of poorly water-soluble drugs. In addition, it can be applied to stabilization of inorganic nanoparticles for imaging / diagnosis as well as various poorly water-soluble drugs.

In addition, the association of the non-peptidyl polymer and albumin comprising an anticancer agent prepared by the production method of the present invention has an effect that the anticancer agent is well delivered compared to the albumin nanoparticles known in the prior art has excellent anticancer effect.

Figure 1 shows electrophoresis for confirming the formation of polyethylene glycol and albumin association.

Figure 2 shows that polyethylene glycol and albumin association dissolve on dimethyl sulfoxide (DMSO).

Figure 3 shows a transmission electron microscope (TEM) image (bar = 200 nm) of polyethylene glycol and albumin aggregate nanoparticles containing paclitaxel prepared by the solvent diffusion method.

4 is a transmission electron microscope (TEM) image (bar = 200 nm) and scanning electron microscope (SEM) image (bar) of polyethylene glycol and albumin aggregate nanoparticles including paclitaxel prepared by the film forming method = 100 nm) (inset image). The graph on the left shows the results obtained by measuring the size distribution of albumin aggregate nanoparticles using the light scattering method.

5 shows a transmission electron microscope (TEM) image (TEM) of polyethylene glycol and albumin aggregate nanoparticles containing Kemptothecin prepared by a film forming method (bar = 100 nm).

Figure 6 shows the positional characterization of poorly water-soluble drugs in nanoparticles using Nile red.

Figure 7 shows the release pattern of paclitaxel from polyethylene glycol and albumin association nanoparticles containing paclitaxel prepared by the film forming method (phosphate buffered saline, PBS with 0.1% sodium salicylate).

8 is a polyethylene glycol and albumin association nano-containing hydrophobic fluorescent probes prepared by the film forming method including DiD (red fluorescence, Invitrogen, Carlsbad, CA) and DiI (green fluorescence, Invitrogen, Carlsbad, CA) as model drugs The observation of the particle delivery to the breast cancer cell line MCF-7 was shown by confocal microscopy.

Figure 9 shows the anticancer activity of breast cancer cell lines (SK-BR-3, MDAMB-453, MCF-7) of polyethylene glycol and albumin aggregate nanoparticles containing paclitaxel prepared by the film forming method. Abraxane ^® , a commercially available albumin / paclitaxel formulation, was used as a control.

FIG. 10 is a graph showing the antitumor and anticancer effects of solid tumor growth in animal tumor models (nude mice nu / nu, SK-BR-3 xenograft) of polyethylene glycol and albumin aggregate nanoparticles containing paclitaxel prepared by the film forming method. Results of observation of prolonged survival. Abraxane ^® , a commercially available albumin / paclitaxel formulation, was used as a control.

11 is an animal tumor model of polyethylene glycol and albumin aggregate nanoparticles containing DiD (Invitrogen, Carlsbad, CA), a hydrophobic fluorescent probe prepared by a film forming method, as a model drug (nude mice nu / nu, SK-BR) -3 xenograft) shows the results of the accumulation in solid tumors over time due to the effects of passive tumor targeting.

12 shows a transmission electron microscope (TEM) image (bar = 100 nm) of polyethylene glycol and albumin aggregate nanoparticles containing SPION prepared by the film forming method.

Fig. 13 shows the stability of polyethyleneglycol and albumin aggregate nanoparticles containing SPION prepared by the film forming method (phosphate saline (PBS, pH 7.4), 0.02% sodium azide as a preservative, 37 ° C).

FIG. 14 shows the relaxation (487.5 ± 13.1 mM ^-1 s ^-1 ) of polyethylene glycol and albumin aggregate nanoparticles including SPION prepared by the film forming method. It showed better laxity than Feridex ^® (~ 230 mM ^-1 s ^-1 ), a commercial iron oxide nanoparticle for MR imaging.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example 1 Preparation of Albumin-PEG Association

Activated polyethyleneglycol (NHS-PEG, Mw 5,000, SunBio, Seoul, Korea) activated with N-hydroxysuccimide (NHS) for human albumin 1:10, 1:20, 1:30, 1 The solution was dissolved in PBS at a reaction ratio of: 40, 1:50 (PEG-NHS / albumin) and mixed, followed by stirring for 12 hours. The reaction product was placed in a semipermeable membrane (Spectra / Por, MWCO 10,000), dialyzed in deionized water to remove unreacted PEG, and lyophilized to obtain a uniform white cake.

The obtained powder was dissolved in PBS and diluted in Laemni buffer (Laemni sample buffer, 63 mM Tris-HCl, 10% Glycerol, 2% SDS, 0.0025% Bromophenol blue, pH 6.8) to tri-glysine discontinuous acrylamide. Association formation was confirmed by gel electrophoresis (FIG. 1).

Example 2 Preparation of Paclitaxel Nanoparticles by Solvent Diffusion

0.5 mg of paclitaxel and 10 mg of albumin-PEG conjugate (1:10 association) were dissolved in dimethylsulfoxide (DMSO) to give a clear solution. The mixture was placed in a semipermeable membrane (Spectra / Por, MWCO 10,000) and dialyzed in deionized water to remove the organic solvent through diffusion.

The dispersion was filtered through a 0.45 μm cellulose filter and lyophilized for 48 hours without the addition of the lyophilizer to obtain a uniform white cake, which could be reconstituted into a dispersion by addition of sterile water or PBS. The resulting paclitaxel / albumin-PEG aggregate dispersion was transparent, and transmission electron microscopy (TEM) observation showed that the particles were spherical nanoparticles having a size of 50 to 200 nm (FIGS. 2 and 3).

Example 3: Paclitaxel Nanoparticles Preparation by Film Forming Method

0.5 mg of paclitaxel and 10 mg of albumin-PEG conjugate (1:10 association) were dissolved in 50% THF aqueous solution to obtain a clear solution. The mixture was transferred to a rotary evaporator and the solvent was evaporated at reduced pressure at 100 ° C. to form a thin film on the wall of a round-bottom flask. Distilled water was added to the obtained film and dissolved in a magnetic stirrer or an ultrasonic bath to produce a nanoparticle dispersion, which was then filtered through a 0.45 μm cellulose filter and lyophilized for 48 hours without the addition of a lyophilizer to obtain a uniform white cake. . The obtained cake was reconstituted in a high concentration dispersion by the addition of sterile water or PBS (Fig. 4).

Example 4 Preparation of Kemptothecin Nanoparticles by Film Forming Method

In the same manner as in the above example, 0.5 mg of camptothecin was dissolved in chloroform / methanol (4: 1), and 10 mg of albumin-PEG (80% methanol) conjugate (1:10 association) was dissolved in 80% aqueous methanol solution. After mixing, a clear mixture solution was obtained. The mixture was transferred to a rotary evaporator and the solvent was evaporated at reduced pressure at 100 ° C. to form a thin film on the wall of a round-bottom flask. Distilled water was added to the obtained film and dissolved in a magnetic stirrer or an ultrasonic bath to produce a nanoparticle dispersion, which was then filtered through a 0.45 µm cellulose filter and lyophilized for 48 hours without adding a lyophilizer to obtain a uniform white cake. It could be reconstituted into a dispersion by the addition of sterile water or PBS. The obtained Kemptothecin / albumin-PEG aggregate dispersion was transparent, and transmission electron microscope (TEM) observations showed that the particles were spherical nanoparticles having a size of 50 to 200 nm (FIG. 5).

Example 5 Identification of Albumin-PEG Association Nanoparticles of Water-Soluble Drugs

Nile red, a poorly water-soluble fluorescent substance, causes red-shift of the maximum emission wavelength due to a certain excitation wave depending on the degree of hydrophobicity of the surrounding environment. It can be predicted. Therefore, in order to confirm the location of albumin-PEG aggregate nanoparticles and the properties of their microenvironment, poorly soluble drugs such as paclitaxel and camptothecin are nanoparticles containing nile red, a poorly water-soluble fluorescent substance, using the film forming method described above. Was prepared.

The obtained nanoparticles were observed in scanning mode at the emission wavelength at 530 nm (excitation wavelength), and the maximum emission wavelength was shifted from 655 nm to 635 nm. This suggests that the albumin-PEG aggregate nanoparticles carry the poorly water-soluble drug through self-assembly, which is a hydrophobic domain in the protein (FIG. 6).

Example 6: Quantification of Inclusion Paclitaxel and Trend of Drug Release in Aqueous Solution

To measure the amount of paclitaxel encapsulated in albumin-PEG aggregate nanoparticles, lyophilized nanoparticles were dissolved in DMSO and quantified using HPLC, and when 5% were formulated for target loading, more than 99% of the drugs were nano It was found that the particles were entrapped in the particles.

To observe the trend of drug release from nanoparticles, nanoparticles were dialyzed in phosphate-buffered saline (PBS) containing 0.1% sodium salicylate (MWCO 10,000), sampled over time and the amount of paclitaxel released through HPLC analysis. Was quantified (FIG. 7). At this time, 0.1% sodium salicylate was used to increase the solubility of aqueous solution of paclitaxel, a hydrophobic drug.

As shown in FIG. 7, paclitaxel drug was slowly released from albumin-PEG assembly nanoparticles for a predetermined time.

Example 7: Measurement of intracellular delivery efficiency of albumin-PEG aggregate nanoparticles

Hydrophobic fluorescent probes, DiD (red fluorescence, Invitrogen, Carlsbad, CA) and DiI (green fluorescence, Invitrogen, Carlsbad, CA), were used as poorly water-soluble model drugs for measuring intracellular delivery efficiency of albumin-PEG aggregate nanoparticles. Nanoparticles were prepared by the film forming method as described in Example 3. SK-BR-3 cells, which are breast cancer cells, were cultured in a glass-bottomed dish, treated with nanoparticles carrying fluorescent probes, and immobilized with 10% formaldehyde after 6 hours, and observed using a confocal microscope. As a result, it was confirmed that intracellular delivery of fluorescence nanoparticles occurred efficiently, and yellow (red + green) fluorescence appeared when red and green fluorescence images were superimposed so that red fluorescence and green fluorescence were not present in the same particle. It could be confirmed that it exists. It was found that the nanoparticles exist in a stable form even after intracellular delivery, and thus can release the drug for a predetermined time as in Example 6 (FIG. 8).

Example 8 Determination of Anticancer Activity in Breast Cancer Cell Lines

In order to measure anticancer activity of albumin-PEG aggregate nanoparticles containing paclitaxel, breast cancer cell lines SK-BR-3, MDA-MB453, and MCF-7 were treated, and cell activity was measured by MTT assay. SK-BR-3 and MCF-7 cell lines showed similar cancer cell growth inhibitory effect to Abraxane (Abraxane ^® ), and especially in MDA-MB453 cell line, paclitaxel / albumin-PEG conjugate nanoparticles prepared according to the present invention were control groups. It showed a significantly lower IC ₅₀ value than paclitaxel dissolved in either Abraxane (Abraxane ^® ) or DMSO (FIG. 9). Polyethyleneglycol and albumin aggregate nanoparticle compositions were compared with the cell activity of ABRAXANE (IC ₅₀ = 35.3 nM) and breast cancer cell line (MDA-MB453) prepared by chemical crosslinking of albumin and paclitaxel. As it has a low IC ₅₀ value (5.7 nM), it was confirmed that the higher anticancer activity.

Example 9: Determination of Anticancer Activity in Human Breast Cancer Animal Model

In order to measure the anticancer activity of albumin-PEG conjugate nanoparticles containing paclitaxel in animal models, SK-BR-3, a breast cancer cell line, was cultured and isolated by enzymatic treatment and concentrated by centrifugation (1.5x10 ⁷ cells / ml). ), Animal tumor models were prepared by transdermal injection (100 μl) into immunodeficient mice (nude mouse, nu / nu). For drug administration, albumin nanoparticles containing the same amount of paclitaxel and the control group were diluted in PBS, and then intravenously injected into the mouse tail according to a prescribed dosing schedule, and the solid cancer volume was measured. The formula for calculating the volume of solid rock is as follows.

tumor volume (mm ³ ) = [length × (width) ² ] / 2

Albumin-PEG aggregate nanoparticles containing paclitaxel decreased the volume of solid tumor (tumor) similar to that of Abraxane (Abraxane ^® ), and it was observed that it showed an excellent cancer growth inhibitory effect (FIG. 10).

Example 10 Observation of Solid Cancer Targeting of Albumin-PEG Association Nanoparticles in Human Breast Cancer Animal Models

The accumulation of albumin-PEG aggregate nanoparticles in solid tumors over time by passive tumor targeting effect after intravenous injection was observed using a hydrophobic fluorescent probe as a model drug in real time in vivo imaging technology. After injection of albumin-PEG conjugate nanoparticles containing DiD (Invitrogen, Carlsbad, CA), a hydrophobic fluorescent probe prepared by the film forming method, as a poorly water-soluble model drug, into the animal tumor model shown in Example 9, in In vivo fluorescent imaging equipment was used to visualize the intratumoral distribution of nanoparticles. As a result, it was observed that the nanoparticles accumulated in the tumor even up to 96 hours (FIG. 11). These results show that albumin nanoparticles can circulate stably in the blood for a considerable time.

Example 11 Preparation of Nanoparticles Encapsulated Nanoparticles by Film Forming Method

Based on the above example, 0.5 mg of iron nanoparticles (SPION, 7 nm) were dispersed in 70% THF aqueous solution, and 10 mg of albumin-PEG conjugate (1:10 association) was dissolved in 70% THF aqueous solution and mixed. It was. The mixture was transferred to a rotary evaporator and the solvent was evaporated at reduced pressure at 100 ° C. to form a thin film on the wall of a round-bottom flask. Distilled water was added to the obtained film and dissolved in a magnetic stirrer or an ultrasonic bath to produce a nanoparticle dispersion. The resultant was filtered through a 0.45 μm cellulose filter and lyophilized for 48 hours without adding a lyophilizer to obtain a uniform brown cake. It could be reconstituted into a dispersion by addition of sterile water or PBS. The obtained SPION / albumin-PEG aggregate dispersion was transparent, and the transmission electron microscope (TEM) observation confirmed that 7 nm SPION particles were enclosed in spherical nanoparticles having a size of about 200 nm (FIG. 12).

Claims (15)

1. Mixing the non-peptidyl polymer with albumin to form an association of the non-peptidyl polymer with albumin;

   Preparing a mixture by dissolving a poorly water-soluble drug or inorganic nanoparticles for bioimaging, and an association of the non-peptidyl polymer and albumin on an organic solvent; And

   Removing the organic solvent from the mixture and adding a hydrophilic solvent to prepare nanoparticles by self-assembly,

   Method for producing a conjugated nanoparticles of albumin and non-peptidyl polymer containing a poorly water-soluble drug or inorganic nanoparticles for bioimaging.
2. The method of claim 1, wherein the non-peptidyl polymer is polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylene, polyoxazoline, polyurethane, polyphosphazene, polysaccharide, dextran, poly Vinyl alcohol, polyvinyl pyrrolidone, polyvinyl ethyl ether, polyacrylamide, polyacrylate, polycyanoacrylate, lipid polymer, chitin, hyaluronic acid, heparin and combinations thereof A method of making association nanoparticles of a non-peptidyl polymer with albumin.
3. The method of claim 1, wherein the non-peptidyl polymer is substituted with a reactor capable of reacting with an amine of albumin.
4. The method of claim 3, wherein the reactor capable of reacting with an amine of albumin is N-hydroxysuccinimide, succinimidyl succinate, succinimidyl propionate, succinimidyl butanoate, benzotriazole carbonate, A method for producing a non-peptidyl polymer and albumin aggregate nanoparticles, characterized in that it is selected from the group consisting of aldehydes and catechols.
5. The method of claim 1, wherein the organic solvent is ethanol, methanol, isopropyl alcohol, butanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), acetonitrile, dichloromethane, ethyl acetate, A method for producing association nanoparticles of albumin with a non-peptidyl polymer, characterized in that it is selected from the group consisting of hexane, diethyl ether, benzene, chloroform, acetone and combinations thereof.
6. The method of claim 1, wherein a co-solvent is used by mixing a solvent with the organic solvent, and the co-solvent does not form an emulsion.
7. The method of claim 1, wherein the mole ratio of the non-peptidyl polymer and albumin is 5: 1 to 50: 1.
8. According to claim 1, wherein the molar ratio of the poorly water-soluble drug or bioimaging inorganic nanoparticles and the association of the non-peptidyl polymer and albumin is 5: 1 to 50: 1 of the non-peptidyl polymer and albumin A method of making association nanoparticles.
9. The method of claim 1, wherein the hydrophilic solvent is water, distilled water, sterile water, phosphate buffered saline (PBS), methanol, purified water, ethanol, 1-propanol, 2-propanol, 1-pentanol, 2-butoxyethanol, ethylene writing A method for producing a conjugated nanoparticle of a non-peptidyl polymer and albumin, characterized in that it is selected from the group consisting of lycol, acetone, 2-butanone, 4-methyl-2-propanone and combinations thereof.
10. The method of claim 1, wherein the method of preparing nanoparticles by self-assembly by adding a hydrophilic solvent is a solvent diffusion method or a film forming method.
11. The method of claim 1, wherein the poorly water-soluble drug is an anticancer agent, an antibiotic, an antifungal agent, an anti-inflammatory agent, an immunosuppressive agent, or a nutrient.
12. The method of claim 11, wherein the anticancer agent is paclitaxel or camptothecin.
13. The method of claim 1, wherein the inorganic nanoparticles for biological imaging are diagnostic agents or contrast agents.
14. The method of claim 1, wherein the size of the prepared aggregate nanoparticles is 200 to 500nm, characterized in that the assembly nanoparticles manufacturing method.
15. A non-peptidyl polymer and albumin associated nanoparticles containing a poorly water-soluble drug or inorganic nanoparticles for bioimaging prepared by the method according to any one of claims 1 to 14.

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