CN104622817A - Protein-polymer composite nano-carrier and preparation method thereof - Google Patents

Abstract

The invention relates to a protein-polymer composite nanocarrier and its preparation method, which relates to the field of nanopreparation of antitumor drugs. It is designed and developed based on the structural characteristics of the natural lipoproteins of the human body. The targeting ligand can be flexibly modified according to the needs of the treatment, and a protein-polymer composite nanocarrier with active targeting can be constructed to improve the stability of the drug in vivo and increase the tumor targeting; in order to improve the loading rate of the protein on the polymer core , cationic polymer nanoparticles were prepared by adding cationic additives in the prescription; the nanosuspension was a milky white liquid, and the particles were evenly distributed and spherical in shape through transmission electron microscopy scanning; in order to make it easier to store and improve stability , adding a freeze-drying protective agent to freeze-dry to form a powder; the protein-polymer composite nano-carrier prepared by the invention has uniform particle size and good stability, and can effectively encapsulate fat-soluble antitumor drugs.

Description

A protein-polymer composite nanocarrier and its preparation method

technical field

The invention relates to the field of nano-preparation of anti-tumor drugs, in particular to a protein-polymer composite nano-carrier capable of carrying fat-soluble anti-tumor drugs and a preparation method thereof.

Background technique

Malignant tumor is second only to heart disease, threatening the second largest killer of human health and life in the world. At present, chemotherapy, surgery, and radiotherapy constitute the three major means of tumor treatment.

Chemotherapy is a treatment method that kills tumor cells with chemical drugs. However, most chemotherapeutic drugs are fat-soluble drugs with poor water solubility, which greatly affects the curative effect of the drugs. In addition, due to the strong cytotoxicity of chemical drugs and the lack of targeting properties, they will cause relatively large toxic and side effects, bring pain to patients, and sometimes even cause treatment interruption.

Most of the currently marketed fat-soluble chemotherapeutic drugs use organic solvents and surfactants as solubilization methods, which not only have poor targeting, but also cause irritation or allergies due to the surfactant components used, which increases the suffering of patients. For example, paclitaxel and paclitaxel injection, in order to achieve the purpose of dissolving the drug, respectively need to use surfactant polyoxyethylene castor oil and Tween-80 (polysorbate 80) and ethanol as a cosolvent; another example is etoposide injection , it is necessary to add Tween-80, polyethylene glycol, ethanol, etc. to achieve the purpose of solubilizing the drug. The use of these surfactants and other solubilizing ingredients may cause a series of adverse reactions, such as allergic reactions, neurotoxicity, bone marrow suppression, fluid retention, etc. There are also some anticancer drugs that have shown excellent anticancer activity in cell experiments, but due to the problems of solubility and irritation, no preparations have been used in clinical practice. For example, bufoling is a pharmacological component of the traditional Chinese medicine toad venom, and it is one of the bufadienolides with the strongest anti-tumor effect in toad venom. It has been reported in the literature that bufalin can inhibit tumor cell proliferation, promote tumor cell differentiation, induce apoptosis, inhibit tumor angiogenesis, restore tumor cell sensitivity to chemotherapeutic drugs, and inhibit tumor angiogenesis at very low concentrations. Bufalin has inhibitory effects on a variety of solid tumors, and the inhibitory effect on liver cancer cells is particularly significant. In cell experiments, bufalin has a stronger inhibitory effect on liver tumor cells than the multi-kinase targeting inhibitor Sorafenib (Cao Y, Li HX, Xu LT, et al. Bufalin enhances the anti-proliferative effect of sorafenib on hμman hepatocellular carcinoma cells through downregulation of ERK [J]. Mol Biol Rep, 2012, 39(2):1683). However, bufalin has poor water solubility, short half-life, and is irritating and cardiotoxic to the human body (Ying Li, Hang Zhao, Lin-Rui Duan, et al. Preparation, characterization and evaluation of bufalin liposomes coated with citrus pectin [J] . Colloids and Surfaces A: Physicochem. Eng. Aspects, 2014,444:54–62), various unfavorable factors limit the application of bufalin, and its monomer preparation has not been used clinically. Considering the effectiveness and safety of medication, the development of tumor-targeted nano-preparations of such drugs is the most likely means to solve their solubility and irritation problems, reduce the toxic and side effects of drugs, increase drug safety, and exert their anti-inflammatory properties. tumor effect.

Poly-α‐cyanoacrylate materials have good biocompatibility, non-toxicity, etc., and are biodegradable polymers. They have been widely used in the preparation of nanoparticle carriers. Nanoparticles prepared from poly-α-cyanoacrylate materials can improve the stability of preparations and increase the solubility of drugs. They are currently widely used in the research of compounds and traditional Chinese medicine monomers, as well as protein and polypeptide delivery systems. However, using cyanoacrylate alone as a carrier has similar problems to most polymer carriers. After the polymer nanocarrier enters the blood circulation, it is easy to combine with immunoglobulin G (immunoglobulin G), complement protein C ₃ (complement C ₃ ) and fibronectin (fibronectin) and other plasma opsonins, and are taken up by the mononuclear macrophage system, and the stability in vivo is not good; it does not have tumor targeting.

Albumin is a natural hydrophilic protein, which has the advantages of safety, non-toxicity, non-immunogenicity, biodegradability, good biocompatibility, cheap and easy to obtain, and is quite stable in nature, and can be used in the range of pH4-9 keep it steady. Using albumin as the carrier shell can increase the hydrophilicity of the carrier, improve the stability of the carrier in blood, and make the carrier have a certain tumor tendency. Albumin also contains many surface active groups for modification, which can be used in protein Modified targeting ligands, such as cholic acid, ursodeoxycholic acid, glycyrrhetinic acid, antibodies, etc., a large number of literatures have shown that there are corresponding receptors on the surface of tumor cells, and tumor cells can be enhanced through the mediation of these ligands. The uptake of the carrier makes the carrier active targeting.

This patent uses a hydrophilic protein as the outer shell and a polymer carrier as the inner core, and designs a new type of targeted protein-polymer nanocarrier to carry anti-tumor drugs. This carrier is designed and developed based on the structural characteristics of the human body's natural lipoproteins. Lipoproteins are naturally occurring lipid transport carriers in the human body, with a lipophilic core and a hydrophilic protein shell structure. According to literature reports, lipoproteins can effectively transport fat-soluble anticancer drugs and have tumor targeting (can be targeted to tumor cells that overexpress low-density lipoprotein receptors). However, lipoproteins need to be extracted from fresh plasma, the extraction process is very complicated, and apolipoproteins are unstable, easy to aggregate, and difficult to commercialize; in addition, lipoproteins can only be targeted to tumors with overexpression of low-density lipoprotein receptors. restricted. The protein-polymer carrier designed and constructed by this patent has a hydrophilic protein shell and a polymer carrier core that can accommodate fat-soluble drugs, and can modify different targeting ligands according to needs. It has excellent drug-loading performance and excellent in vivo Compatibility and stability, as well as flexible targeting, is a very functional targeting carrier.

The modified shell with hydrophilic material polyethylene glycol as the surface of the carrier is a commonly used method to increase the stability of nanocarriers such as polymers or liposomes in vivo, and the shell structure with albumin as the carrier, and polyethylene glycol Compared with albumin, it has special advantages, (1) easy to carry out surface modification, each albumin molecule has multiple carboxyl groups and amino groups, and functional groups can be modified according to needs; (2) better biocompatibility and de-opsonization , Albumin is a natural hydrophilic protein that exists in the human body and has no immunogenicity. Albumin can effectively reduce the adsorption of proteins in plasma on the carrier and prolong the circulation time of the carrier in the blood. (3) It has certain tumor tropism, and the vascular permeability of tumor and inflammatory tissue increases. Albumin can pass through the gP60 (60-kDa glycoprotein) receptor of tumor vascular endothelium, or through the combination with cysteine-rich protein in tumor tissue. Secreted protein acid and rich in cysteine (SPARC) promotes the uptake of vectors in tumor tissues.

At present, there are many studies and patents on using albumin alone as a carrier, but using albumin alone as a carrier may have the following problems. (1) Generally, it needs to be cured by heating or chemical crosslinking agent. It may affect the stability of the drug or the hydrophilicity of the protein; (2) The particle size of nanoparticles or microspheres prepared from albumin is relatively large, generally above 500 μm. American Biosciences Corporation has developed Nab technology to prepare pharmaceutical albumin nanoparticles. This technology uses high-pressure homogenization technology to homogenize the mixed system of drug-containing organic phase and protein-containing aqueous phase to prepare nanoparticles. This technology has a great impact on the physical and chemical properties of drugs. The property is highly selective, and the drug needs to have a strong affinity with albumin. In addition, according to the classification of "Pharmaceuticals" (Edited by Cui Fude, "Pharmacy" 2nd Edition, 2011:536.), these carriers are all passive targeting agents, and passive targeting agents can only use different organs in the body , tissue or reticuloendothelial system intercept or phagocytize particles of different sizes, and deliver drugs to the target site, but the targeting effect is not good.

Contents of the invention

In order to solve the above technical problems, the present invention provides a protein-polymer composite nanocarrier, which uses a polymer containing fat-soluble antineoplastic drugs as the inner core, and albumin with targeting effect as the hydrophilic outer shell; the present invention also provides The preparation method of the above protein-polymer composite nanocarrier is provided. According to the needs of treatment, the targeting group can be modified on the albumin in advance to construct the protein-polymer composite nanocarrier with active targeting.

The present invention is achieved through the following technical solutions: a protein-polymer composite nanocarrier, comprising a polymer core and a targeted protein shell; the polymer core is an alkyl cyanoacrylate core, and the alkyl cyanoacrylate core is wrapped The fat-soluble antitumor drug is loaded; the targeting protein shell is wrapped on the surface of the alkyl cyanoacrylate inner core; the targeting protein shell is a hydrophilic protein shell.

The protein-polymer composite nanocarrier includes the following components in terms of mass percentage: 1-10% of antineoplastic drugs, 10-60% of alkyl cyanoacrylate, 0.5-10% of cationic additives, 1-10% % protein substances, 20-60% stabilizers and 5-30% lyoprotectants. 

The antineoplastic drugs are bufalin, etoposide, paclitaxel, paclitaxel, gefitinib, hydroxycamptothecin and doxorubicin, curcumin, fluorouracil, cyclophosphamide, irinotecan, mitoxantrene One of quinones and cisplatin.

The alkyl cyanoacrylates are methyl cyanoacrylate MCA, ethyl cyanoacrylate ECA, n-butyl cyanoacrylate BCA, isobutyl cyanoacrylate IBCA, hexyl cyanoacrylate HCA and isohexyl cyanoacrylate One of the IHCA.

The cationic additive is one of octadecylamine, polyethanolamine, L-lysine and arginine or a combination thereof.

The proteinaceous substance is one or a mixture of natural proteins or synthetic proteins or proteinoids and their derivatives or modified proteins.

The stabilizer is one of Pluronic F68, Pluronic F123, Pluronic F127, Dextran 70, Dextran 40, Dextran 20, polyvinyl alcohol, Tween-80 and Span-80 or a mixture thereof.

The lyoprotectant is one of the following: (1) Sugars/polyols: sucrose, trehalose, mannitol, lactose, glucose, maltose; (2) Polymers: HES, PVP, PEG, dextran Sugar, albumin; (3) amino acids: L-serine, sodium glutamate, alanine, glycine, sarcosine; (4) salts and amines: one of phosphate, acetate, citrate or a mixture thereof.

The proteinaceous substance is albumin.

The modification is one or more of cholic acid, ursodeoxycholic acid, glycyrrhetinic acid, hyaluronic acid, galactose, folic acid and lipoprotein receptor recognition recombinant polypeptide; the modification can be combined with the Amino groups, carboxyl groups or sulfhydryl groups of proteinaceous substances are linked by covalent bonds.

The preparation method of the protein-polymer composite nanocarrier comprises the following steps:

1) preparing an alkyl cyanoacrylate inner core carrying the antitumor drug;

Calculated by mass percentage, weigh 1~10% of antineoplastic drugs, 0.5~8% of cationic additives and 5~40% of stabilizers and dissolve them in the oil phase, and mix the solution with the oil phase. Add 60% alkyl cyanoacrylate monomer dropwise to dilute hydrochloric acid containing 5-40% stabilizer with a concentration of 0-10M to form an aqueous phase system;

Or weigh 1~10% antineoplastic drugs and 5~40% stabilizers and dissolve them in the oil phase, mix the solution with the oil phase, control the speed at 100~1000rpm, slowly add 10~60% alkyl cyanoacrylate monomer drop by drop Contain 5~40% stabilizer and 0.5~5% cationic additives to form an aqueous phase system in dilute hydrochloric acid with a concentration of 0~10M;

Or weigh 1~10% antineoplastic drugs, 0.5~8% cationic additives and dissolve them in the oil phase, mix the solution with the oil phase, control the speed at 100~1000rpm, slowly add 10~60% alkyl cyanoacrylate monomer drop by drop Add 5-50% stabilizer to dilute hydrochloric acid with a concentration of 0-10M to form an aqueous phase system;

Or take 1~10% of antineoplastic drugs and dissolve them in the oil phase, mix the oil phase with the solution, control the speed at 100~1000rpm, slowly add 10~60% alkyl cyanoacrylate monomer dropwise to the 5-50% stabilizer Form an aqueous phase system with 0.5~5% cationic additives in dilute hydrochloric acid with a concentration of 0~10M;

Or weigh 1~10% antineoplastic drugs, 0.5~8% cationic additives and 5~40% stabilizers and dissolve them in the oil phase, mix the solution with the oil phase, control the speed at 100~1000rpm, and slowly mix 10~60% cyano Alkyl acrylate monomers are added dropwise to dilute hydrochloric acid containing 5-40% stabilizers and 0.5-5% cationic additives at a concentration of 0-10M to form an aqueous phase system;

Or weigh 1~10% antineoplastic drugs and 0.5~8% cationic additives and dissolve them in the oil phase, mix the solution with the oil phase, control the speed at 100~1000rpm, slowly add 10~60% alkyl cyanoacrylate monomer drop by drop Adding 5~50% stabilizer and 0.5~5% cationic additives to dilute hydrochloric acid with a concentration of 0~10M to form an aqueous phase system;

Slowly drop the oil phase mixed solution into the water phase system, and after stirring for 2-8 hours, a mixed solution containing the inner core of the alkyl cyanoacrylate is formed;

Calculated by mass percentage, the total amount of cationic additives in the oil phase and the water phase is 0.5-10%; the total amount of stabilizers in the oil phase and water phase is 20-60%.

The oil phase is one of ethyl acetate, dichloromethane, chloroform, acetone, ether or a mixture thereof.

2) The natural protein is extracted and purified or modified on the protein to obtain the targeted protein shell;

Wherein the specific steps of the protein modification method are: adding the modification to tetrahydrofuran or dimethylformamide DMF to dissolve, and then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-Hydroxysuccinimide; Stir at 0°C for 2~6 h, continue to stir at room temperature overnight, recrystallize to obtain the active ester of the modified product, dissolve it in dimethylformamide DMF, and add it dropwise to albumin-dissolved In the solution, stir while adding, and continue to stir at room temperature for 2~6 h; after 48 h of dialysis of the reaction solution, centrifuge at 12,000 rpm for 10 min to remove unreacted modified active ester and its hydrolyzate, and freeze-dry to obtain a white product, which is Modified albumin, the molar ratio of albumin to modifier is 1:5~1:60.

3) Coating the targeted protein shell on the inner core of the alkyl cyanoacrylate to form a suspension containing a protein-polymer composite nanocarrier;

The specific steps are: adjust the pH of the mixed solution containing the inner core of alkyl cyanoacrylate to neutral with sodium hydroxide, add 1-10% protein substances in terms of mass percentage, continue to stir for 0.5-6 h, and rotate to evaporate for 30-120 min , remove the residual organic solvent, centrifuge at 3000-10000 rpm for 2-15 min, pass through a 0.2-0.8 μm microporous membrane for sterile filtration, and obtain the protein-polymer composite nanocarrier suspension.

Proteins are non-covalently bonded to the surface of the alkyl cyanoacrylate core through charge interaction, hydrophobic force, van der Waals force and other intermolecular forces.

4) The freeze-drying agent is added to the suspension to form a powder.

The specific steps are: adding 5-30% freeze-drying protective agent to the protein-polymer composite nano-carrier suspension, and freeze-drying for 24 hours to form a powder.

The temperature of the rotary evaporation is 0-60°C, preferably 20-30°C.

The average particle diameter of the protein-polymer composite nanocarrier is 20-1000 nm, preferably 80-200 nm.

The protein-polymer composite nanocarrier is used for preparing antitumor drugs.

Beneficial effects: Compared with the prior art, the present invention is designed and developed based on the structural characteristics of human natural lipoproteins. It has a polymer core, a hydrophilic protein shell, and can flexibly modify targeting ligands, and can effectively pack lipids Soluble drugs can improve drug stability in vivo and increase tumor targeting; in order to improve the protein loading rate on the polymer core, cationic polymer nanoparticles were prepared by adding cationic additives to the formulation, and the isoelectric point of albumin is 4.5 Left and right, negatively charged in a neutral medium, can be combined on the surface of polymer nanoparticles through intermolecular forces such as charge interaction, hydrophobic force, and van der Waals force; according to the needs of treatment, the targeting group can be modified on the protein in advance, thereby A protein-polymer composite nanocarrier with active targeting is constructed; the nanosuspension is a milky white liquid, and the particle distribution is relatively uniform and spherical in shape when scanned by a transmission electron microscope; in order to make it easier to store and improve stability, Add a lyoprotectant to freeze-dry to form a powder.

Description of drawings

Fig. 1 is a particle size distribution diagram of the bufafolin-ursodeoxycholic acid modified albumin-polymer composite nanoparticles prepared according to Example 1.

FIG. 2 is a transmission electron microscope image of albumin-polymer composite nanoparticles loaded with bufolin-ursodeoxycholic acid modified according to Example 1. FIG.

Fig. 3 is the in vitro release curve of the bufatoxin raw material drug and the drug in the bufatoxin-ursodeoxycholic acid-modified albumin-polymer composite nanoparticles prepared according to Example 1.

Detailed ways

The present invention will be further described below through the embodiments in conjunction with the accompanying drawings, but the protection scope of the present invention is not limited thereto. 

Example 1: Preparation of bufotoxin-loaded ursodeoxycholic acid modified protein-polymer composite nanoparticles.

Weigh 3.5 mg bufalin, 5 mg stearylamine and 30 mg pluronic F127 and dissolve them in dichloromethane. Slowly add 60 mg of n-butyl cyanoacrylate (BCA) dropwise into 5 mL of dilute hydrochloric acid (0.1 M) containing 25 mg each of dextran 70 and Pluronic F68, and slowly add the mixed solution of dichloromethane into the system, Add dropwise while stirring. After stirring for 4 h, adjust the pH to neutral with sodium hydroxide, add 5 mg ursodeoxycholic acid-modified bovine serum albumin, and continue stirring for 1 h. Remove the residual dichloromethane by rotary evaporation, centrifuge at 5000 rpm for 3 min, and pass through a microporous membrane to obtain the bufotoxin-ursodeoxycholic acid modified protein-polymer composite nanocarrier suspension.

Add 30mg of mannitol and 30mg of trehalose to the bufafolin-ursodeoxycholic acid modified protein-polymer composite nanocarrier suspension and freeze-dry for 24 hours to obtain a powder, which can be obtained by adding sterile water or normal saline Re-dispersed to form protein-polymer composite nanocarrier suspension, the particle size is almost unchanged from that before lyophilization.

Example 2: Preparation of bufotoxin-loaded hyaluronic acid modified protein-polymer composite nanoparticles.

Weigh 10 mg bufalin and 10 mg stearylamine respectively and dissolve them in dichloromethane. Slowly add 100 mg of ethyl cyanoacrylate (ECA) dropwise to 10 ml of dilute hydrochloric acid (0.1M) containing 50 mg of dextran 70 and 50 mg of Pluronic F68, and slowly add the mixed solution of dichloromethane into the system, Add dropwise while stirring. After stirring for 4 h, adjust the pH to neutral with sodium hydroxide, add 20 mg of hyaluronic acid-modified bovine serum albumin, and continue stirring for 1 h. Remove the residual dichloromethane by rotary evaporation, centrifuge at 5000 rpm for 3 min, and pass through a microporous membrane to obtain the bufondulin-hyaluronic acid modified protein-polymer composite nanocarrier suspension.

Add 50 mg of sucrose to the bufafolin-hyaluronic acid modified protein-polymer composite nanocarrier suspension and freeze-dry for 24 hours to obtain a powder, which can be redispersed by adding sterile water or saline to form a protein-polymer compound nano-carrier suspension, the particle size is almost unchanged from that before freeze-drying.

Example 3: Preparation of etoposide-glycyrrhetinic acid modified protein-polymer composite nanoparticles.

Weigh 30 mg etoposide, 3 mg stearylamine and 50 mg pluronic F127 and dissolve them in dichloromethane. Slowly add 210 mg of methyl cyanoacrylate (MCA) dropwise into 5 mL of dilute hydrochloric acid (0.1 M) containing 10 mg of dextran 70 and 10 mg of Pluronic F68, and slowly add the dichloromethane mixed solution into the system dropwise. Add and stir. After stirring for 4 h, adjust the pH to neutral with sodium hydroxide, add 5 mg glycyrrhetinic acid-modified bovine serum albumin, and continue stirring for 1 h. Remove the residual dichloromethane by rotary evaporation, centrifuge at 5000 rpm for 3 min, and pass through a microporous membrane to obtain the composite nanocarrier suspension loaded with etoposide-glycyrrhetinic acid-modified protein-polymer.

Add 20 mg PEG 400 and 10 mg glycine to the etoposide-glycyrrhetinic acid-modified protein-polymer composite nanocarrier suspension and freeze-dry for 24 hours to obtain a powder, which can be reconstituted by adding sterile water or normal saline Dispersed to form protein-polymer composite nano-carrier suspension, the particle size is almost unchanged from that before freeze-drying.

Example 4: Preparation of paclitaxel-loaded folic acid modified protein-polymer composite nanoparticles.

Weigh 5 mg paclitaxel, 12 mg stearylamine and 60 mg pluronic F127 and dissolve them in dichloromethane. Slowly add 40 mg of isobutyl cyanoacrylate (IBCA) dropwise to 10 ml of dilute hydrochloric acid (0.1M) containing 10 mg of polyvinyl alcohol PVA 1788 and 1.5 mg of arginine, and slowly add the mixed solution of dichloromethane In the system, add dropwise while stirring. After stirring for 4 h, adjust the pH to neutral with sodium hydroxide, add 15 mg of folic acid-modified bovine serum albumin, and continue stirring for 1 h. Remove residual dichloromethane by rotary evaporation, centrifuge at 5000 rpm for 3 min, and pass through a microporous membrane to obtain paclitaxel-folate-modified protein-polymer composite nanocarrier suspension.

Add 10 mg mannitol and 10 mg trisodium citrate to the paclitaxel-folate-modified protein-polymer composite nanocarrier suspension and lyophilize for 24 hours to obtain a powder, which can be redispersed by adding sterile water or normal saline A protein-polymer composite nanocarrier suspension is formed, and the particle size is almost unchanged from that before lyophilization.

Example 5: Preparation of dophobic paclitaxel-ursodeoxycholic acid modified protein-polymer composite nanoparticles.

Weigh 10 mg of dophytaxel and dissolve in dichloromethane. Slowly add 60 mg n-butyl cyanoacrylate (BCA) dropwise to 10 ml containing 25 mg polyvinyl alcohol PVA 1788, 15 mg Pluronic F127, 20 mg each of dextran 70 and Pluronic F68 and 10 mg polyethanolamine 1200 In dilute hydrochloric acid (0.1 M), the dichloromethane mixed solution was slowly added dropwise to the system, and stirred while dropping. After stirring for 4 h, adjust the pH to neutral with sodium hydroxide, add 10 mg of ursodeoxycholic acid-modified bovine serum albumin, and continue stirring for 1 h. Remove residual dichloromethane by rotary evaporation, centrifuge at 5000 rpm for 3 min, and pass through a microporous membrane to obtain a composite nanocarrier suspension loaded with paclitaxel-ursodeoxycholic acid-modified protein-polymer.

Add 25 mg of mannitol, 20 mg of PVP K30, and 30 mg of sodium glutamate to freeze-dry for 24 hours to obtain a powder in the suspension of loaded dophytaxel-ursodeoxycholic acid modified protein-polymer composite nanocarrier, which can be The protein-polymer composite nanocarrier suspension was formed by redispersing by adding sterile water or normal saline, and the particle size was almost unchanged from that before lyophilization.

Example 6: Preparation of gefitinib-ursodeoxycholic acid modified protein-polymer composite nanoparticles.

Weigh 3 mg gefitinib and 90 mg pluronic F127, respectively, and dissolve them in chloroform. Slowly add 25 mg of hexyl cyanoacrylate (HCA) dropwise to 10 ml of dilute hydrochloric acid (0.1 M) containing 25 mg each of dextran 70 and Pluronic F68, and 10 mg of arginine. Add dropwise to the system and stir while adding dropwise. After stirring for 4 h, adjust the pH to neutral with sodium hydroxide, add 10 mg of ursodeoxycholic acid-modified bovine serum albumin, and continue stirring for 1 h. Remove residual chloroform by rotary evaporation, centrifuge at 5,000 rpm for 3 min, and pass through a microporous membrane to obtain a composite nanocarrier suspension loaded with gefitinib-ursodeoxycholic acid-modified protein-polymer.

Add 25 mg lactose and 20 mg trisodium glycinate to the gefitinib-ursodeoxycholic acid modified protein-polymer composite nanocarrier suspension and lyophilize for 24 hours to obtain a powder, which can be obtained by adding sterile water Or normal saline is redispersed to form a protein-polymer composite nanocarrier suspension, and the particle size is almost unchanged from that before lyophilization.

Example 7: Preparation of hydroxylated camptothecin-hyaluronic acid modified protein-polymer composite nanoparticles.

Weigh 5 mg hydroxycamptothecin and 5 mg octadecylamine, respectively, and dissolve them in dichloromethane. Slowly add 50 mg of isohexyl cyanoacrylate (IHCA) dropwise to 5 ml of dilute hydrochloric acid (0.1M) containing 75 mg of polyvinyl alcohol PVA 1788, 20 mg of Pluronic F127, and 5 mg of lysine. The mixed solution of methane was slowly added dropwise to the system, stirring while adding dropwise. After stirring for 4 h, adjust the pH to neutral with sodium hydroxide, add 5 mg hyaluronic acid-modified bovine serum albumin, and continue stirring for 1 h. Remove residual dichloromethane by rotary evaporation, centrifuge at 5000 rpm for 3 min, and pass through a microporous membrane to obtain a suspension of hydroxy-loaded camptothecin-hyaluronic acid modified protein-polymer composite nanocarrier.

Add 20 mg dextran 70 and 30 mg trehalose to the suspension of hydroxycamptothecin-hyaluronic acid modified protein-polymer composite nanocarrier to obtain a powder by adding sterile Water or saline redispersed to form protein-polymer composite nanocarrier suspension, the particle size was almost unchanged from that before lyophilization.

Example 8: Preparation of doxorubicin-glycyrrhetinic acid modified protein-polymer composite nanoparticles.

Weigh 7.5 mg doxorubicin, 10 mg stearylamine and 20 mg pluronic F127 and dissolve them in dichloromethane. Slowly add 75 mg of isobutyl cyanoacrylate (IBCA) dropwise to 10 ml of dilute hydrochloric acid (0.1M) containing 50 mg of polyvinyl alcohol PVA 1788, slowly add the mixed solution of dichloromethane into the system, dropwise Stir. After stirring for 4 h, adjust the pH to neutral with sodium hydroxide, add 10 mg of glycyrrhetinic acid-modified bovine serum albumin, and continue stirring for 1 h. Remove residual dichloromethane by rotary evaporation, centrifuge at 5000 rpm for 3 min, and pass through a microporous membrane to obtain a suspension of doxorubicin-glycyrrhetinic acid-modified protein-polymer composite nanocarriers.

Add 20 mg glycine, 20 mg PEG400, and 10 mg trisodium citrate to the doxorubicin-glycyrrhetinic acid-modified protein-polymer composite nanocarrier suspension and lyophilize for 24 hours to obtain a powder, which can be obtained by adding sterile Water or saline redispersed to form protein-polymer composite nanocarrier suspension, the particle size was almost unchanged from that before lyophilization.

Example 9: Preparation of paclitaxel-glycyrrhetinic acid modified protein-polymer composite nanoparticles.

Weigh 20 mg paclitaxel, 15 mg stearylamine and 10 mg pluronic F68 and dissolve them in dichloromethane. Slowly add 70 mg of n-butyl cyanoacrylate (BCA) dropwise to 10 ml of dilute hydrochloric acid (0.1M) containing 20 mg of polyvinyl alcohol PVA 1788, 20 mg of dextran 70 and 4 mg of arginine, dichloromethane mixed solution Slowly added dropwise to the system, stirring while adding dropwise. After stirring for 4 h, adjust the pH to neutral with sodium hydroxide, add 20 mg glycyrrhetinic acid-modified bovine serum albumin, and continue stirring for 1 h. Remove residual dichloromethane by rotary evaporation, centrifuge at 5000 rpm for 3 min, and pass through a microporous membrane to obtain paclitaxel-glycyrrhetinic acid modified protein-polymer composite nanocarrier suspension.

Add 10 mg trehalose to the paclitaxel-glycyrrhetinic acid modified protein-polymer composite nanocarrier suspension and freeze-dry for 24 hours to obtain a powder, which can be redispersed by adding sterile water or normal saline to form a protein-polymer The particle size of the composite nanocarrier suspension is almost unchanged from that before freeze-drying.

Experimental example 1: Particle size and distribution.

A Malvern laser particle size analyzer (Zetasizer Nano ZS90 laser particle size analyzer, Malvern, UK) was used to measure the particle size, and the measurement results of Example 1 are shown in Figure 2. The measurement results show that the average particle diameter of the bufalin nano-preparation prepared by the present invention is 149.3 nm, and the polydispersity coefficient is 0.073.

Experimental example 2: Morphological distribution.

Take an appropriate amount of the preparation, dilute it with distilled water, put a drop on a copper grid covered with a carbon support film, place it for a while, add 2% phosphotungstic acid for negative staining, and use a transmission electron microscope (Tecnai 12, Philips company, Holland) Observe particle form, the observation result of embodiment 1 is shown in Fig. 2. It can be seen from Figure 2 that the particle distribution is relatively uniform and spherical.

Experimental Example 3: Protein Loading Rate.

Protein loading rate: protein loading rate (%)=(1-M _free /M _cast )*100

In the formula, M _free refers to the amount of free BSA (modified) (μg), and M _cast refers to the amount of input BSA (modified) (μg). In Example 1, the loading rate of ursodeoxycholic acid-modified albumin on the carrier surface was 94.33%; in Example 2, the loading rate of hyaluronic acid-modified albumin on the carrier surface was 89.72%; in Example 3, glycyrrhetinic acid modified The loading rate of albumin on the carrier surface is 91.19%; the loading rate of folic acid-modified albumin on the carrier surface in Example 4 is 85.42%; the loading rate of albumin modified by ursodeoxycholic acid on the carrier surface in Example 5 It was 92.25%; the loading rate of albumin modified by ursodeoxycholic acid on the carrier surface in Example 6 was 95.18%; the loading rate of albumin modified by hyaluronic acid on the carrier surface in Example 7 was 87.49%; in Example 8 The loading rate of glycyrrhetinic acid-modified albumin on the carrier surface was 92.31%; in Experimental Example 9, the loading rate of glycyrrhetinic acid-modified albumin on the carrier surface was 90.76%.

Experimental Example 4: Release in vitro.

The in vitro release behavior of the preparation was investigated using phosphate buffer at pH 7.4 as the release medium. The release curves of the drug (bufalin) in the bufalin saturated solution and the preparation prepared in Example 1 are shown in FIG. 3 . It can be seen from Figure 3 that the release of the drug in the preparation has a certain sustained release effect compared with the saturated solution, but it can basically achieve complete release within 24 hours.

Experimental Example 5: Cell inhibition rate and targeting evaluation.

Investigate the IC ₅₀ of the bufotoxin-ursodeoxycholic acid modified protein-polymer composite nanoparticles prepared in Example 1 and the ordinary bufotoxin-polymer particles not coated with the modified protein on the liver tumor cell HepG2 were 32.7 nM and 98.6nM, it can be seen that the inhibitory effect of bufalin-ursodeoxycholic acid modified protein-polymer composite nanoparticles on liver tumor cell HepG2 is significantly higher than that of ordinary bufalin-polymer without coated modified protein particles. It shows that after coating the protein modified with ursodeoxycholic acid, it can increase the uptake of tumor cells to the carrier, and has tumor cell targeting. Investigate the IC of the etoposide-glycyrrhetinic acid modified protein-polymer composite nanoparticles prepared in Example 3 and the common etoposide-polymer particles without coated modified protein to the liver tumor cell HepG2 were 97.3 μM and 97.3 _μM respectively. 148.8μM, it can be seen that the inhibitory effect of etoposide-glycyrrhetinic acid modified protein-polymer composite nanoparticles on liver tumor cell HepG2 is significantly higher than that of ordinary etoposide-polymer particles without modified protein coating. It shows that after coating the modified protein, it can increase the uptake of tumor cells to the carrier, and has tumor cell targeting. Investigate the _IC50s of the dotopaxol-ursodeoxycholic acid modified protein-polymer composite nanoparticles prepared in Example 5 and the common dotopetaxel-polymer particles not coated with the modified protein on the liver tumor cell HepG2, respectively: 62.7nM and 128.9nM, it can be seen that the inhibitory effect of the composite nanoparticles loaded with doclitaxel-ursodeoxycholic acid-modified protein-polymer on liver tumor cell HepG2 was significantly higher than that of ordinary dophytaxel-polymerized matter particles. It shows that after coating the modified protein, it can increase the uptake of tumor cells to the carrier, and has tumor cell targeting.

Described embodiment is the preferred embodiment of the present invention, but the present invention is not limited to above-mentioned embodiment, but should admit, those skilled in the art may make various modifications and changes to the present invention, and these modifications and changes belong to all within the scope of the invention as defined by the appended claims.

Claims (10)

1. albumen-composite nano-polymers carrier, is characterized in that, comprises polymer core and targeting proteins matter shell;

Described polymer core is alpha-cyanoacrylate alkane ester kernel, and described alpha-cyanoacrylate alkane ester kernel bag carries fat-soluble antitumor drug;

Described targeting proteins matter shell is wrapped in the surface of described alpha-cyanoacrylate alkane ester kernel;

Described targeting proteins matter shell is hydrophilic protein shell.

2. a kind of albumen-composite nano-polymers carrier according to claim 1; it is characterized in that, by mass percentage calculate comprise following component: the antitumor drug of 1 ~ 10%, the alpha-cyanoacrylate alkane ester of 10 ~ 60%, 0.5 ~ 10% cation additions, the protein matter of 1 ~ 10%, the stabilizing agent of 20 ~ 60% and 5 ~ 30% freeze drying protectant.

3. a kind of albumen-composite nano-polymers carrier according to claim 2, is characterized in that,

Described antitumor drug is the one in Toadpoison Medicine, etoposide, paclitaxel, paclitaxel of dwelling, gefitinib, hydroxy camptothecin and amycin, curcumin, fluorouracil, cyclophosphamide, irinotecan, mitoxantrone, cisplatin more;

Described alpha-cyanoacrylate alkane ester is the one in methyl 2-cyanoacrylate MCA, cyanacrylate ECA, BCA BCA, isobutylcyanoacrylate IBCA, the own ester HCA of alpha-cyanoacrylate and alpha-cyanoacrylate dissident ester IHCA;

Described cation additions is one in 18-amine., PVOH amine, 1B and arginine or its compositions;

Described protein matter is one in the protein modified of native protein, synthetic protein, albuminoid and derivant thereof or modified thing or its mixture;

Described stabilizing agent is one in Pluronic F68, pluronic F123, pluronic F127, macrodex, Dextran 40, Dextran-20, polyvinyl alcohol, tween 80 and Arlacel-80 or its mixture;

Described freeze drying protectant is the one of the following stated:

(1) saccharide/polyhydric alcohol: sucrose, trehalose, mannitol, lactose, glucose, maltose;

(2) polymer: HES, PVP, PEG, glucosan, albumin;

(3) aminoacid: Serine, sodium glutamate, alanine, glycine, sarcosine;

(4) salt and amine: the one in phosphate, acetate, citrate or its mixture.

4. a kind of albumen-composite nano-polymers carrier according to claim 3, it is characterized in that, described protein matter is albumin; Described trim is one or more in cholic acid, ursodesoxycholic acid, enoxolone, hyaluronic acid, galactose, folic acid and lipoprotein receptor identification recombinant polypeptide.

5. the preparation method of a kind of albumen-composite nano-polymers carrier according to claim 1, comprises the following steps:

1) preparation bag carries the alpha-cyanoacrylate alkane ester kernel of described antitumor drug;

2) described native protein extraction purification or modify on protein, obtains targeting proteins matter shell;

3) targeting proteins matter shell described in described alpha-cyanoacrylate alkane ester kernel outer cladding, forms the suspension containing albumen-composite nano-polymers carrier;

4) in suspension, add described freeze drying protectant and carry out lyophilization, form powdery.

6. the preparation method of a kind of albumen-composite nano-polymers carrier according to claim 5, it is characterized in that, described step 1) is specially:

Calculate by mass percentage, take 1 ~ 10% antitumor drug, 0 ~ 8% cation additions and 0 ~ 40% stabilizing agent oil phase to dissolve, oil phase mixed solution, control rotating speed is 100 ~ 1000rpm, and slowly adding 10 ~ 60% alpha-cyanoacrylate alkane ester monomers dropwise containing 0 ~ 5% cation additions and 5 ~ 50% stabilizer concentrations is form aqueous phase system in the dilute hydrochloric acid of 0 ~ 10M;

Oil phase mixed solution is slowly added dropwise in aqueous phase system, after stirring 2 ~ 8h, forms the mixed liquor containing described alpha-cyanoacrylate alkane ester kernel;

Described step 3) is specially: regulated by the mixed liquor sodium hydroxide containing alpha-cyanoacrylate alkane ester kernel pH to neutral; add and calculate 1 ~ 10% protein matter by mass percentage; continue stirring 0.5 ~ 6 h; rotary evaporation 30 ~ 120min; the organic solvent that removing is remaining; centrifugal 2 ~ 15 min of 3000 ~ 10000 rpm, cross 0.2 ~ 0.8 μm of microporous filter membrane and carry out aseptic filtration, obtain albumen-composite nano-polymers vehicle suspension;

Described step 4) is specially: in described albumen-composite nano-polymers vehicle suspension, add the freeze drying protectant of 5 ~ 30%, lyophilizing 24 hours, forms powdery.

7. the preparation method of a kind of albumen-composite nano-polymers carrier according to claim 6, is characterized in that, described oil phase is one in ethyl acetate, dichloromethane, chloroform, acetone, ether or its mixture.

8. the preparation method of a kind of albumen-composite nano-polymers carrier according to claim 6, it is characterized in that, the mean diameter of described albumen-composite nano-polymers carrier is 20 ~ 1000nm.

9. the preparation method of a kind of albumen-composite nano-polymers carrier according to claim 8, it is characterized in that, the mean diameter of described albumen-composite nano-polymers carrier is 80 ~ 200nm.

10. a kind of albumen-composite nano-polymers carrier according to claim 1, is characterized in that, described albumen-composite nano-polymers carrier is for the preparation of antitumor drug.