CN103242517A - Preparation of multifunctional linear-dendritic segmented copolymer and application in pharmaceutics thereof - Google Patents

Abstract

The invention relates to the fields of polymer chemistry and pharmaceutical preparations, in particular to the preparation of a multifunctional linear-dendritic block copolymer and its application in pharmacy. The multifunctional linear-dendritic block copolymer of the present invention has amphiphilicity, can significantly solubilize insoluble drugs, improve drug stability and bioavailability in vivo, especially can carry insoluble or water-soluble drugs, and self-assemble to form nano Advantages of micelles. The modified polyamidoamine structure has an environment-responsive charge reversal property, which makes the surface of the micelles carry negative charges in the in vivo circulation, and after reaching the tumor area, the charge reverses to positive charges in response to the weak acidic environment, which is more conducive to nanomicelles. Cell uptake and intracellular drug delivery can improve the passive targeting of anti-tumor drugs, reduce drug side effects, and provide new ideas for the study of new drug targeted drug delivery.

Description

Preparation of multifunctional linear-dendritic block copolymers and their applications in pharmacy

technical field

The invention relates to the fields of macromolecule chemistry and pharmaceutical preparations, in particular to a preparation method of an amphiphilic linear semipolyamidoamine dendritic and polyester block copolymer and its application in pharmacy. The present invention also relates to the new application of the linear-dendritic block copolymer as a solubilizer for insoluble drugs, especially the application of the multifunctional linear-dendritic block copolymer and its composition in anticancer drug targeting nano drug delivery system .

Background technique

Functional pharmaceutical polymer materials have become a hot field in the research of drug delivery systems. In the past few decades, a large number of functional polymers have been developed and studied, including various surfactants, linear polymers, grafted polymers, etc. However, functional polymers still have many disadvantages such as high critical micelle concentration, weak micelle formation ability and high cytotoxicity; the solubilization range and ability of insoluble drugs are limited, and the stability, targeting and problems such as poor biocompatibility.

Dendrimers are a class of highly symmetrical multi-branched polymer materials with precise and controllable molecular weight, good monodispersity and unique "cavity" structure, which can adsorb or encapsulate drugs and siRNA through non-covalent interactions or active molecules such as diagnostic agents. The surface of dendrimers contains rich functional group residues, which are beneficial for structural modification and endow them with long circulation and targeting effects. Therefore, it is expected to develop multifunctional or intelligent drug polymer carrier materials (Esfand, R., Tomalia, D.A., Drug Discov. Today, 2001, 6(8): 427-436). Due to the defects of the dendritic polymer itself, such as the limited size of the internal cavity of the polymer, it has high selectivity to the structure and molecular weight of the guest; the whole generation of polyamidoamine dendrimers has great hemolysis and cytotoxicity, and cannot be used for injection. Administration (Jansen, J.F.G.A., Meijer, E.W., de Brabander-van Den Berg, E.M.M., J.Am.Chem.Soc., 1995, 117(15): 4417-4418. Jevprasesphant, R., Penny, J., Jalal, R., et al, Int.J.Pharm., 2003, 252(1-2): 263-266.); the particle size of unimolecular micelles formed by the polymer is less than 10nm, which is easily metabolized by the kidneys to cause Renal toxicity, etc. (Kukowska-Latallo, J.F., Candido, K.A., Cao, Z., Cancer Res., 2005, 65(12):5317-5324). In order to solve the above problems, it is necessary to modify and transform the structure of existing dendrimers.

Most of the currently reported linear-dendritic block copolymers are composed of hydrophilic linear materials (such as polyoxypropylene, PPO) and relatively hydrophobic dendritic materials (such as polyamidoamine, PAMAM) (Nguyen, P.M., Hammond, P.T., Langmuir, 2006, 22(18):7825-7832). Due to the hydrophilicity of the dendritic material itself, the hydrophobicity of the whole material is weak, and the concentration required to form micelles is high, which is not suitable for loading hydrophobic drug molecules. And using the dendritic material as the hydrophobic segment, making it be located in the inner layer of micelles, is also not conducive to bringing into play the advantages of the dendritic material in terms of functionalization and cellular uptake (Kitchens, K.M., Foraker, A.B., Kolhatkar, R.B., Pharm.Res , 2007, 24(11), 2138-2145).

Linear-dendritic polymers are a new class of polymers developed in recent years. The polymer includes two parts, a linear polymer segment and a dendrimer. This type of polymer has integrated the respective advantages of linear and dendritic polymers, making it exhibit unique excipient functional properties (Poon, Z., Lee, J.A., Huang, S., Nanomed.Nanotechnol.Biol.Med., 2011, 7(2):201-209). The study found that linear-dendritic polymers are amphiphilic and can self-assemble into micelles with a core-shell structure; dendritic macromolecule "holes" and micellar structures provide a variety of loading methods for drugs to meet different properties The needs of drugs; nano-scale micellar particles can reduce the phagocytosis of the reticuloendothelial system, reduce the renal clearance rate, and improve the bioavailability of drugs; a large number of active amino or hydroxyl residues at the end of the dendritic polymer facilitate the structure Modified to give it a variety of functionalities, combining dendritic materials with polyester materials through covalent bonds to synthesize linear-dendritic block polymers, which have the advantages of biodegradable materials and biocompatibility to achieve specific diagnostics and therapeutic effects, providing new ideas in novel drug delivery systems (Goldberg, D.S., Vijayalakshmi, N., Swaan, P.W., J. Control. Release, 2011, 150(3), 318-325).

Contents of the invention

The purpose of the present invention is to provide a multifunctional amphiphilic linear-dendritic block copolymer, which has the dual advantages of linear and dendritic polymers, wherein the dendritic polymer part is a hydrophilic segment, and the linear The polymer portion is the hydrophobic segment. Using ethanolamine as the core, through the iterative reaction of Michael addition and amination, a semi-dendritic polyamidoamine polymer with a single terminal hydroxyl group was synthesized, and then used as a macromolecular initiator to undergo ring-opening polymerization of hydrophobic polymerized monomers to prepare Amphiphilic linear-dendritic block copolymers. Using the new self-assembly technology to load hydrophobic or hydrophilic drugs and assemble nanomicelles with a core-shell structure, the polyamide amine corona on the outer layer of the polymer has pH-responsive conformational changes and potential reversal properties, which can realize drug Targeted delivery and intracellular drug release in acidic tumor microregions, passively targeting tumor tissue through the EPR effect, improving the cell targeting effect of anti-tumor drugs and reducing drug side effects.

The general formula of the multifunctional linear-dendritic block copolymer provided by the invention is (I):

in:

n is polyamidoamine dendrimer algebra; monomer is linear polymer monomer; m is the degree of polymerization of linear polymer monomer, m is 10-50;

The linear-dendritic block copolymer (I), including (A) containing terminal hydroxyl hydrophilic polyamidoamine sPA; (B) hydrophobic polyester, forming a linear AB type block copolymer, the linear -The weight ratio of dendritic block copolymer semi-dendritic sPA to polyester B is 15:1～1:20, preferably 8:1～1:10;

The (A) polyamidoamine containing terminal hydroxyl groups in the present invention is a half-generation dendritic product, including n=0.5, 1.5, 2.5, 3.5, 4.5, and 5.5 generations.

A kind of linear-dendritic block copolymer provided by the present invention, the preparation steps of its semi-generation dendritic product (sPA) are as follows:

(1) Add ethanolamine and anhydrous methanol into the reactor, slowly add methyl acrylate while stirring, react at 20-30°C for 12-24h under the protection of nitrogen, and evaporate the film under reduced pressure to obtain 0.5 generation polyamidoamine dendrimer molecular;

(2) Dissolve the 0.5 generation polyamidoamine dendrimer obtained in the step (1) in anhydrous methanol, add dropwise ethylenediamine, react at 20-30°C for 12-24h, evaporate the film under reduced pressure, that is Obtain 1.0 generation polyamidoamine dendrimers;

(3) Slowly add anhydrous methanol solution of methyl acrylate to the 1.0 generation polyamidoamine dendrimers obtained in step (2), react at 20-30°C for 12-24h, evaporate the film under reduced pressure, That is, 1.5 generation polyamidoamine dendrimers are obtained;

(4) The 1.5 generation polyamidoamine dendrimer obtained in step (3) is operated according to steps (2) and (3) successively to obtain the 2.5 generation polyamidoamine dendrimer;

(5) The 2.5-generation polyamidoamine dendrimer obtained in step (4) is followed by steps (2) and (3) to obtain a 3.5-generation polyamidoamine dendrimer. The product is dissolved in methanol and purified for 3 to 5 Once, the thin film is evaporated under reduced pressure;

(6) The 3.5 generation polyamidoamine dendrimers obtained in step (5) are operated according to steps (2) and (3) successively to obtain the 4.5 generation polyamidoamine dendrimers, the product is purified 3 times with methanol, distilled water Dissolved in a dialysis bag (molecular weight: 3500) and dialyzed for 3 days, freeze-dried;

(7) The 4.5-generation polyamidoamine dendrimer obtained in step (6) is followed by steps (2) and (3) to obtain the 5.5-generation polyamidoamine dendrimer, which is purified by methanol for 3 times, dissolved in distilled water, and placed Dialyzed (3500 molecular weight) in dialysis bag for 3 days, freeze-dried.

The (B) hydrophobic polyester, its polymerized monomer is at least one or a mixture of lactide, glycolide, ε-caprolactone, and morpholinedione, and the linear-dendritic block copolymerization The substances are sPA-PDLA, sPA-PLLA, sPA-PLGA, sPA-PCL and the like.

The linear-dendritic block copolymer provided by the invention, its preparation method comprises:

(1) Take 1 part by weight and take dry 0.5 to 5.5 generation semi-alternative polyamidoamine dendrimers and 0.01 to 100 parts of B polymerization monomer and mix, add 0.1 to 1.0% stannous octoate, and heat to 20-200°C, react in an oil bath for 0.5-100 hours, stop the reaction, and obtain a crude product;

(2) The crude product is dissolved in 1 to 100 parts by weight of solvent a, and then poured into 1 to 1000 times the volume of solvent b for precipitation. 25h, to obtain the linear-dendritic block copolymer;

Wherein the solvent a is one or a mixture of chloroform and dichloromethane; the solvent b is one or a mixture of methanol, ethanol, petroleum ether, diethyl ether, and ethyl acetate.

The specific synthesis reaction equation is as follows (taking lactide as an example for polymerizing monomer):

In polyamidoamine tree chemical structure modification, polymer micelles encapsulation of insoluble drugs and solubilization, the choice of solvent and dosage are very critical. The linear-dendrimer block copolymer provided by the present invention is characterized in that it is composed of a hydrophilic semipolyamidoamine dendrimer and a hydrophobic linear polymer, and the solvent is selected from water, chloroform, dichloromethane, acetone , dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF); hydrophobic and hydrophilic drugs can be loaded, and the linear-dendritic block polymer of the present invention has amphiphilic and low Surface tension can self-assemble in water to form nano-micelles, which can be used for solubilization and encapsulation of insoluble drugs, prolong the circulation in the body, and reduce the phagocytosis of the reticuloendothelial system. Moreover, the hydrophilic polyamidoamine shell contains a large number of "cavities", which can simultaneously wrap hydrophilic drug molecules to achieve the purpose of synergistic drug loading and release and combined drug delivery, which broadens the scope of application of polymer micelles for drug loading.

The linear-dendritic block copolymer of the present invention is suitable for compounding with drugs or as a drug carrier, has low cytotoxicity, does not produce hemolytic reaction, and can be used as an excellent drug carrier for intravenous injection. The linear-dendritic block copolymer has the solubilizing effect of carrying hydrophobic drugs, and can also be used as an oral drug solubilizer, and can also carry water-soluble drugs, wherein the weight ratio of the drug to the linear-dendritic block polymer is 1:1~1:100, the drug loading can reach 5~20%.

The drug is selected from paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, vincamide, etoposide, gambogic acid, cyclosporine A, etoposide, teniposide, nimodipine, Nifedipine, nitrendipine, doxorubicin, daunorubicin, mitomycin, curcumin, methotrexate, oridonin, harringtonine, homoharringtonine, breviscapine , ginkgolide, silymarin, indirubin, fluorouracil or cisplatin, the weight ratio of the drug to the linear-dendritic block copolymer is 1:2-50.

The pharmaceutical composition of the linear-dendritic block copolymer of the present invention is characterized in that the drug-loaded self-assembles to form polymer nanomicelles, the average particle diameter is 20-120nm, the drug-loading capacity is greater than 8%, and the encapsulation efficiency is More than 87%, wherein the drug is preferably paclitaxel, docetaxel, doxorubicin, fluorouracil, and hydroxycamptothecin, and the weight ratio of the drug to the linear-dendritic block copolymer is 1:5-200.

The pharmaceutical composition of linear-dendritic block copolymer of the present invention, its preparation method is as follows:

(1) Dissolve an appropriate amount of linear-dendritic block copolymer in pure water, dissolve the drug in an organic solvent, stir magnetically for 4 to 8 hours, remove the solvent by thin film evaporation under reduced pressure, sonicate the probe for 5 to 30 minutes, and use a 0.22 μm micrometer to remove the solvent. Filter through a pore membrane to obtain the drug-polymer nanomicelle suspension;

(2) adding a freeze-drying protective agent to the drug polymer nano-micelle suspension, sterile filtering, and freeze-drying to obtain a drug-loaded polymer nano-micelle freeze-dried powder; or adding a filler, using a spray-drying method, The drug-loaded polymer nano-micelle powder is obtained;

The freeze-drying protection agent is selected from one or more of lactose, sucrose, glucose, mannitol, dextran, and trehalose; the amount of the freeze-drying protection agent is based on the weight of the drug-loaded polymer micelle suspension. Add 0.01 to 0.1 part of freeze-drying protective agent to drug-polymer micelle suspension.

In clinical use, it can be redissolved with water for injection, normal saline or glucose. The freeze-dried preparation dissolves quickly and the solution is clear after dissolution.

The filler is selected from lactose, spray-dried lactose, granulated lactose, ground lactose, anhydrous lactose, lactose for dry powder inhalation, microcrystalline cellulose, compressible starch, starch, dextrin, mannitol, powdered sugar, povidone , polyethylene glycol, hypromellose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, calcium sulfate, calcium hydrogen phosphate, crospovidone, croscarmellose sodium, carboxymethyl Sodium starch, low-substituted hydroxypropyl cellulose, one or more of the above two premixed auxiliary materials; 0.02-0.20 parts of filler are added to 1 part of drug-loaded polymer micelle suspension.

The amphiphilic linear-dendritic block copolymers of the present invention have pH-responsive charge-reversal properties. In the long-term circulation in the body, the surface carries a negative charge, and after reaching the outside of the tumor cell or lysosome, the charge reverses to a positive charge in response to the weak acidic environment in which it is located, which is conducive to the stability of the preparation during the in vivo operation, while in the tumor area Uptake by cells and rapid intracellular release.

The polyamidoamine-polylactide block copolymer of the present invention is suitable as an excellent carrier for intravenous injection of antitumor drugs, can be injected clinically, and can also be used as a modifier for oral preparations or mucosal administration preparations to promote Drug absorption, improve efficacy.

Description of drawings:

Figure 1. Effect of pH on polymer particle size (A) and potential (B) (n=3)

Figure 2. In vitro hemolysis experiments of polymer and docetaxel micellar preparations (n=3)

Detailed ways

Example 1

(1) Synthesis of 0.5 generation polyamidoamine dendrimers: Add 10mL ethanolamine and 30mL anhydrous methanol into a 250mL reactor, slowly add 35mL methyl acrylate while stirring, react at 30°C for 12h under nitrogen protection, and depressurize the film Evaporate to obtain 0.5 generation polyamidoamine dendrimer (39.2g, 100%);

(2) Synthesis of 1.5-generation polyamidoamine dendrimers: Dissolve 20.0g of 0.5-generation polyamidoamine dendrimers in 30mL of anhydrous methanol, add 60mL of ethylenediamine dropwise, react at 30°C for 12h, and form a thin film Evaporate under reduced pressure. Dissolve 25.0 g of the product in methanol, slowly add 65 mL of anhydrous methanol solution of methyl acrylate dropwise, react at 30°C for 24 h, and evaporate the film under reduced pressure to obtain a 1.5-generation polyamidoamine dendrimer (54.1 g, 99 %);

(3) Synthesis of 2.5-generation polyamidoamine dendrimers: 45.0 g of 1.5-generation polyamidoamine dendrimers were dissolved in methanol, slowly dripped into 110 mL of ethylenediamine to react for 24 hours, and distilled under reduced pressure, the product Dissolve 26.8 g in methanol, react with 50 mL of methyl acrylate for 24 h, evaporate the film under reduced pressure, and purify with methanol to obtain 2.5-generation polyamidoamine dendrimers (53.4 g, 103%);

(4) Synthesis of 3.5-generation polyamidoamine dendrimers: take 40.0g of 2.5-generation polyamidoamine dendrimers and dissolve them in methanol, add them dropwise to 90mL ethylenediamine to react for 48 hours, absorb concentrated sulfuric acid and reduce pressure Distill to remove excess ethylenediamine, react 25.8 g of the product with 87 mL of methyl acrylate, purify methanol and distill under reduced pressure to obtain 3.5-generation polyamidoamine dendrimers (48.2 g, 102%).

(5) Synthesis of 4.5-generation polyamidoamine dendrimers: take 21.5g of 3.5-generation polyamidoamine dendrimers to react with 45mL of ethylenediamine, react 21.1g of the product with 70mL of methyl acrylate, purify methanol under reduced pressure Distilled 3 times, then dissolved in distilled water and placed in a dialysis bag (molecular weight 3500) for dialysis for 3 days, freeze-dried to obtain 4.5-generation polyamidoamine dendrimers (39.5 g, 105%).

(6) Synthesis of 5.5-generation polyamidoamine dendrimers: take 20.5g of 4.5-generation polyamidoamine dendrimers to react with 50mL of ethylenediamine, react 20.0g of the product with 60mL of methyl acrylate, purify methanol under reduced pressure Distilled 3 times, then dissolved in distilled water and placed in a dialysis bag (molecular weight 3500) for dialysis for 3 days, freeze-dried to obtain 5.5 generation polyamidoamine dendrimers (33.5g, 105%).

The synthesis of embodiment 24.5 generation polyamidoamine-polylactide block copolymer (PALA4.5)

Mix 5.5g of dry 4.5 generation polyamidoamine dendrimer, 4.5g of lactide and 0.05g of stannous octoate, heat to 150°C for 12h, dissolve the product in chloroform, precipitate with petroleum ether, purify twice and then precipitate The starch was redissolved, dialyzed for 50 hours with a 3500 molecular weight dialysis bag, and freeze-dried for 48 hours to obtain it.

PALA4.5: FT-IR: 3449, 2994, 2955, 2847, 1752, 1644, 1556, 1451, 1381, 1271, 1199cm ^-1 ;

¹ HNMR (500MHz, CDCl ₃ , δin ppm): 1.57(m, O-CH- CH ₃ ), 2.33～2.35(m, -CH ₂ CH ₂ CO), 2.42～2.46(m, -CH ₂ COOCH ₃ ), 2.37～2.42(m, -CH ₂ CH ₂ N), 2.74～2.79(m, -CH 2 CH ₂ CO), 3.27～ _3.29 (m, -CH ₂ CH ₂ N), 3.68 (s, -OC H ₃ ), 4.89 (m, O=CC H -OH), 5.22 (m, O=CC H -O), 7.18～8.02 (m, -CON H -);

The PI of the polymer was determined to be 1.12 by gel size exclusion chromatography; the degree of polymerization was calculated to be 76 based on ¹ HNMR peak position integration.

Example 34.5 Synthesis of polyamidoamine-polyglycine block copolymer (PAG4.5)

Take 6.6g of dry 4.5 generation polyamidoamine dendrimer, 3.4g of morpholine-2,5-dione and 0.01g of stannous octoate and mix, heat to 160°C for 10h, the product is dissolved in chloroform, precipitated with ether, Purify twice, redissolve the precipitate, dialyze with a 3500 molecular weight dialysis bag for 50 hours, and freeze-dry for 48 hours to obtain it.

PAG4.5: FT-IR: 3440, 3218, 2994, 2955, 2880, 1766, 1691, 1548, 1112cm ^-1 ;

¹ HNMR (500MHz, CDCl ₃ , δin ppm): 2.32～2.33(m, -CH ₂ CH ₂ CO), 2.41～2.46(m, -CH ₂ COOCH ₃ ), 2.35～2.41(m, -CH ₂ CH ₂ N), 2.72～2.78(m, -CH 2 CH ₂ CO), 3.31～3.33(m, -CH ₂ CH ₂ N), 3.67(s, -OCH ₃ ), _4.42 (m, NH- CH -CH ₃ ), 4.8-4.9 (m, O=CC H ₂ -O), 7.28-8.14 (m, -CON H -).

The PI of the polymer was determined to be 1.13 by gel size exclusion chromatography; the degree of polymerization was calculated to be 52 based on the integration of ¹ HNMR peak positions.

The synthesis of embodiment 44.5 generation polyamidoamine-polylactic acid-glycolic acid block copolymer (PALGA4.5)

Take dry 6.6g 4.5 polyamidoamine dendrimers, 1.7g lactide, 1.7g glycolide and 0.02g stannous octoate, mix, heat to 145°C for 8h, the product is dissolved in dichloromethane, petroleum Precipitate with ether, purify twice, redissolve the precipitate, dialyze with a 3500 molecular weight dialysis bag for 50 hours, and freeze-dry for 48 hours to obtain it.

PALGA4.5:

FT-IR: 3450, 2960, 2870, 1760, 1480, 1271, ^1197cm-1 ;

¹ HNMR (500MHz, CDCl ₃ , δin ppm): 1.57(m, O-CH- CH ₃ ), 2.32～2.37(m, -CH ₂ CH ₂ CO), 2.43～2.45(m, -CH ₂ COOCH ₃ ), 2.33～2.40(m, -CH ₂ CH ₂ N), 2.76～2.79(m, -CH 2 CH ₂ CO), 3.27～ _3.29 (m, -CH ₂ CH ₂ N), 3.68 (s, -OC H ₃ ), 4.68-4.91 (m, O=CC H ₂ -O), 5.13 (m, O=CC H -O), 7.10～8.12 (m, -CON H -);

The PI of the polymer was determined to be 1.20 by gel size exclusion chromatography; the degree of polymerization was calculated to be 56 based on the integration of ¹ HNMR peak positions.

The mensuration of embodiment 5 linear-dendritic block copolymer interfacial tension

Weigh 10.2 mg of linear-dendritic block copolymer (prepared in Examples 2 to 4) and dissolve it in 10 mL of water, stir at room temperature for 1 h to obtain a 1.02 mg/mL polymer stock solution, dilute with distilled water to 100, 10, 1, 0.1 , 0.01 μg/mL polymer solution, placed in a DCAT-21 surface tensiometer to measure the surface tension of the polymer, and when the value drops and stabilizes, it is the minimum interfacial tension value of the polymer. The obtained data are shown in Table 1:

Table 1 The interfacial tension of linear-dendritic block copolymers

Embodiment 6pH responsive experiment

Prepare the PALA4.5 aqueous solution prepared in Example 2 with a concentration of 10 mg/mL, add 1 mL dropwise to 9 mL of phosphate buffer solution with a pH of 7.4, 6.8, 6.0, 5.5, and 5.0, and use a Zetasizer3000HSA particle size analyzer to measure the particle size of the blank micelles and potential. Plot potential and particle size as a function of pH, see Figure 1.

The particle size of PALA4.5 has no obvious change in the range of pH5-6.8, and the particle size increases slightly under physiological pH conditions (pH7.4), which may be caused by the particle aggregation caused by the weakened charge repulsion under neutral conditions. The particle size can be reduced to 50nm by adjusting the pH, which shows that this aggregation has not changed the nature of the colloid. The potential of the blank micelles has a tendency to increase significantly with the decrease of pH. The pH decreases from physiological conditions (pH7.4) to 6.8, and the potential increases from -6.1mV to +14.4mV. When the pH decreases to 5.5 and 5.0, the potential increases respectively. to +26.2mV and +31.3mV. The results showed that a large number of secondary and tertiary amines were present in the polymer, and the decrease in pH caused protonation of the amine groups, resulting in a change in potential. It has been reported in the literature that tumor microregions present a weakly acidic environment (pH6.5), while the pH of endosomes and lysosomes is lower (pH5.0-5.5). The polymer of the present invention has pH responsiveness, which can make the micelles easier to take into cells and accelerate drug release in the cells, thereby improving the therapeutic effect.

Example 7 Preparation of linear-dendritic block copolymer loaded docetaxel micelles (DTX micelles)

Dissolve 150 mg of linear-dendritic block copolymer (prepared in Example 2-4) in 12 mL of distilled water, dissolve 30 mg of docetaxel in 10 mL of ethanol, add the drug solution to the carrier solution, stir for 6 h, and depressurize Remove the solvent, filter aseptically with a 0.22 μm microporous membrane, add 210 mg of a lyoprotectant, and freeze-dry to obtain the docetaxel polymer micellar lyophilized powder. The encapsulation efficiency and drug loading of micelles were determined by HPLC. Zetasizer3000HS particle size analyzer was used to measure particle size and potential. The results are shown in Table 2.

Table 2 The particle size and potential of docetaxel linear-dendrimer block copolymer micelles

Example 8 Preparation of linear-dendritic block copolymer-loaded fluorouracil complex

Put 50mg of block copolymer PALA4.5 (prepared in Example 2) and 50mg of 5-fluorouracil into a beaker, add 10mL of N,N-dimethylformamide-water mixed solution (10:1), and incubate at 40°C for 10min After dissolving, place it in a dialysis bag for dialysis, change distilled water at 2, 4, 6, 8, and 12 hours, and filter with a 0.22 μm filter membrane at 24 hours to obtain the fluorouracil-loaded PALA4.5 complex, add 110 mg of lactose, and freeze-dry. The average particle size of the complex measured by a Malvern particle size analyzer was 58.3nm, and the polydispersity coefficient was 0.25. The drug-loading capacity of the complex was determined to be 10.8% by HPLC, and the encapsulation efficiency was 92.7%.

Example 9 Linear-Dendrimer Block Copolymer Hemolysis Experiment in Vitro

Fresh human whole blood was collected in a vacuum tube containing 7.5% (w/v) EDTA, and centrifuged at 3000 rpm for 10 min. Wash blood cells with isotonic saline three times, and discard the supernatant. The blood cells were diluted to 2% (v/v) with saline, and 2.5 mL of the cell suspension was added to the sample, PALA4.5 (prepared in Example 2), DTX micelles (prepared in Example 7), Tween-80, The concentrations of PAMAM4.0 (commercially available) were 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.5, 5 mg/mL. After incubation at 37°C for 30 minutes, the samples were centrifuged at 3000 rpm for 10 minutes, the supernatant was collected, and the absorbance was measured at a wavelength of 570 nm. Triton X-100 and PBS were used as hemolysis 100% and 0% control groups, respectively. Calculate the hemolysis rate with the following formula: hemolysis rate=(sample absorbance value-0% hemolysis absorbance value)/(100% hemolysis absorbance value-sample absorbance value)×100, the hemolysis result is shown in Figure 2.

中以吐温-80作为增溶剂，临床治疗中存在溶血和过敏毒副作用，而本发明的嵌段共聚物材料溶血发生率低，适用于静脉注射给药。The results showed that PALA4.5 and DTX micelles had no obvious hemolysis phenomenon, and Tween-80 group and PAMAM4.0 (commercially available) group had more obvious hemolysis phenomenon, and the hemolysis rate reached 66% and 100% respectively when 5mg/mL. %. Commercially available docetaxel injection

Tween-80 is used as a solubilizer in the present invention, and there are hemolysis and allergic side effects in clinical treatment, while the block copolymer material of the present invention has a low incidence of hemolysis and is suitable for intravenous administration.

Example 10 Docetaxel linear-dendrimer block copolymer micelles (DTX micelles) in vivo pharmacokinetics experiment

12 SD rats were divided into two groups at random, 6 in every group, injected docetaxel commercially available injection and DTX micelles (prepared in embodiment 7) respectively in the tail vein, and the administration dose was 2.5mg/kg. After 5, 10, 15, 30, 45, 60, 120, 180, 240, 360, 480, 600, and 720 min, 0.5 mL of blood was collected from the retro-orbital venous plexus of rats, placed in a heparinized centrifuge tube, and heated at 10,000 r/min After high-speed centrifugation, 200 μl of plasma was collected, and the plasma drug concentration was determined by HPLC. Kinetic4.4 pharmacokinetic software processes blood drug data, and the pharmacokinetic parameters are as shown in Table 3:

The results show that the drug-loaded micelles (prepared in Example 7) are 1.5 times longer than the commercially available injection, and the area under the time-dependent curve of the blood drug is increased by 61%, which has a better long-term circulation effect. The results show that the linear-dendritic block polymer of the present invention can reduce the uptake of the reticuloendothelial system in vivo, prevent the drug from being degraded in the circulation in the body, and contribute to better drug efficacy.

Table 3 The in vivo pharmacokinetic parameters of docetaxel copolymer micelles (n=3)

Claims (10)

1. the linearity of general formula I-tree-shaped segmented copolymer:

Wherein:

N is the tree-shaped polymkeric substance algebraically of daiamid; Monomer is the linear polymerization monomer; M is the polymerization degree of linear polymerization monomer, and m is 10～50;

Described linearity-tree-shaped segmented copolymer I contains end position hydroxyl hydrophilic daiamid sPA comprising (A); (B) hydrophobicity polyester is formed linear AB block copolymer, and the weight ratio of this linearity-tree-shaped segmented copolymer half branch sPA and polyester B is 15: 1～1: 20, and more excellent weight ratio is 8: 1～1: 10.

2. according to the described linearity of claim 1-tree-shaped segmented copolymer I, it is characterized in that the daiamid that described (A) contains end position hydroxyl is half for the branch product, comprises n=0.5,1.5,2.5,3.5,4.5,5.5 generations.

3. according to the described linearity of claim 1～2-tree-shaped segmented copolymer I, it is characterized in that, described (B) hydrophobicity polyester, its polymerization single polymerization monomer is at least a or mixture in rac-Lactide, glycollide, 6-caprolactone, the morpholine diketone, and described linearity-tree-shaped segmented copolymer is sPA-PDLA, sPA-PLLA, sPA-PLGA, sPA-PCL.

4. according to the described linearity of claim 1～3-tree-shaped segmented copolymer I, its preparation method comprises:

(1) by weight than taking by weighing 1 part of polymerization single polymerization monomer mixing that contains daiamid and 0.01～100 part of B of end position hydroxyl, add 0.1～1.0% stannous octoate, Heating temperature is 20～200 ℃, oil bath reaction 0.5～100h, and stopped reaction gets crude product;

(2) crude product joins in 1～100 weight part a solvent and dissolves, and precipitates in 1～1000 times of volume b of impouring solvent again, and precipitation is redissolved behind solvent a, and with dialysis tubing dialysis 1～100h, lyophilize 10～25h namely gets linear-tree-shaped segmented copolymer;

Wherein said solvent a is chloroform, methylene dichloride is a kind of or mixture; Described solvent b is a kind of or mixture of methyl alcohol, ethanol, sherwood oil, ether, ethyl acetate.

5. according to the described linearity of claim 1～4-tree-shaped segmented copolymer, it is characterized in that can wrap and carry hydrophobicity and hydrophilic medicament, its Chinese traditional medicine is 1: 1～1: 100 with the weight ratio of linearity-tree-shaped block polymer.

6. according to the described linearity of claim 1～5-tree-shaped segmented copolymer and pharmaceutical composition thereof, it is characterized in that, described medicine is wetting ability and hydrophobicity, and its Chinese traditional medicine is selected from taxol, Docetaxel, camptothecine, hydroxycamptothecine, vindesine, etoposide, Fluracil, morellic acid, ciclosporin A, etoposide, Vumon, nimodipine, nifedipine, nitrendipine, Zorubicin, daunorubicin, mitomycin, curcumine, Rheumatrex, rubescensin, harringtonine, homoharringtonine, Breviscarpine, bilobalide, silymarin or Indirubin.

7. according to the described linearity of claim 6-tree-shaped segmented copolymer and pharmaceutical composition thereof, this linearity-tree-shaped segmented copolymer has bag and carries the hydrophobic drug solublization, as the purposes of solubilizing agents for drugs, medicine is 1: 2～50 with linearity-tree-shaped segmented copolymer weight ratio.

8. according to the pharmaceutical composition of the described linearity of claim 6-tree-shaped segmented copolymer, it is characterized in that, the self-assembly of bag medicine carrying thing forms polymer nano micelle, median size is 20～120nm, its Chinese traditional medicine is selected from taxol, Docetaxel, Zorubicin, Fluracil, hydroxycamptothecine, and medicine is 1: 5～200 with the weight ratio of linearity-tree-shaped segmented copolymer.

9. the pharmaceutical composition of described linearity-tree-shaped segmented copolymer according to Claim 8, its preparation method is as follows:

(1) take by weighing linearity-tree-shaped segmented copolymer and be dissolved in right amount in the pure water, with medicine dissolution at organic solvent, magnetic agitation 4～8h, the thin film evaporation removal of solvent under reduced pressure, ultrasonic 5～the 30min of probe with 0.22 μ m filtering with microporous membrane, namely obtains pharmaceutical polymer nano-micelle suspension;

(2) with pharmaceutical polymer nano-micelle suspension, add lyophilized vaccine, sterile filtration, lyophilize namely gets drug-carrying polymer nano-micelle lyophilized powder; Or the adding weighting agent, adopt spray-drying process, namely get drug-carrying polymer nano-micelle powder;

Described lyophilized vaccine is selected from one or more in lactose, sucrose, glucose, N.F,USP MANNITOL, dextran, the trehalose; The lyophilized vaccine consumption is by the drug-carrying polymer micelle weight of suspension, and 1 part of drug-carrying polymer micelle suspension adds 0.01～0.1 part of lyophilized vaccine.

Described weighting agent is selected lactose, spray-dried lactose, particulate lactose, grind lactose, lactose hydrous, dry powder sucks uses lactose, Microcrystalline Cellulose, amylum pregelatinisatum, starch, dextrin, N.F,USP MANNITOL, Icing Sugar, polyvidone, polyoxyethylene glycol, hypromellose, hydroxypropylcellulose, Xylo-Mucine, calcium sulfate, secondary calcium phosphate, polyvinylpolypyrrolidone, croscarmellose sodium, sodium starch glycolate, low-substituted hydroxypropyl cellulose, in above-mentioned two kinds of premixing auxiliary materials one or more; 1 part of drug-carrying polymer micelle suspension adds 0.02～0.20 part of weighting agent.

10. according to the pharmaceutical composition of the described linearity of claim 9-tree-shaped segmented copolymer, it can be made into clinical use formulation, comprises injection, oral solid formulation, powder inhalation or transdermal formulation.

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