CN107266384A - N carboxyl inner-acid anhydride monomers and polyaminoacid based on 2 aminohexadecanoic acids and preparation method thereof - Google Patents

Abstract

The invention discloses an N-carboxyl internal acid anhydride monomer and polyamino acid based on 2-aminohexadecanoic acid and a preparation method thereof. First prepare 2-aminohexadecanoic acid-N-carboxylidene anhydride monomer, and then prepare a polymer from the monomer. The obtained polymer has a narrow molecular weight distribution and excellent biocompatibility, and can be used to control drug release systems. The prepared cRGD-modified polymer micelles and polymer vesicle nano-medicines support stable long-term circulation in the body, are highly enriched in tumor tissues and enter cells efficiently, release drugs in cells, and kill cancer cells efficiently and specifically, effectively Growth of subcutaneous and orthotopic tumors was inhibited without significant toxic side effects.

Description

N-carboxyl internal acid anhydride monomer and polyamino acid based on 2-aminohexadecanoic acid and its preparation preparation method

technical field

The present invention relates to a biodegradable material based on 2-aminohexadecanoic acid, in particular to a kind of 2-aminohexadecanoic acid-N-carboxyl internal anhydride and its synthesis, as well as the polymer prepared by its ring-opening polymerization Polymer micelles and vesicles prepared with this polymer.

Background technique

Polyamino acids are widely used in the fields of drug controlled release, tissue engineering and regenerative medicine due to their low immunogenicity, good degradation performance and mechanical properties, and controllable properties. However, the solubility of hydrophobic polyamino acids (such as polyleucine, polyphenylalanine, polyisoleucine and polymethionine) obtained by polymerization of α-amino acid-N-carboxyl internal anhydride in organic solvents Extremely poor, which in turn leads to uncontrollable polymerization and a smaller molecular weight of the obtained polymer. Therefore, researchers usually modify hydrophobic groups on the side chains of hydrophilic polymers such as polyglutamic acid, polylysine or polyaspartic acid by post-modification to obtain hydrophobic polymer blocks. However, this method generally needs to go through a series of processes such as protection and deprotection, resulting in low final yield and long synthesis time.

Therefore, it is necessary to study the synthesis of hydrophobic polyamino acids with strong hydrophobicity and good solvent selection window; and these polyamino acids have good solvent selectivity and synthesis controllability, and the prepared amphiphilic polymers can be prepared The targeted drug-loaded polymer micelles or vesicles are obtained for targeted therapy of tumors.

Contents of the invention

The object of the present invention is to provide a kind of 2-amino hexadecanoic acid-N-carboxy internal acid anhydride monomer and its preparation method, and use 2-amino hexadecanoic acid-N-carboxy internal acid anhydride to prepare block copolymerization by ring-opening polymerization These polymers can form polymer micelles and polymer vesicles through self-assembly.

In order to achieve the above object, the technical scheme adopted in the present invention is: a kind of 2-aminohexadecanoic acid-N-carboxyl internal acid anhydride with formula I structure:

Formula I.

The invention discloses a preparation method of the above-mentioned 2-aminohexadecanoic acid-N-carboxyl internal anhydride, comprising the following steps, using 2-aminohexadecanoic acid, pinene, and triphosgene as reactants in an organic solvent such as anhydrous react in tetrahydrofuran to prepare the 2-aminohexadecanoic acid-N-carboxylidene anhydride.

In the above-mentioned technical scheme, the molar ratio of 2-aminohexadecanoic acid, pinene and triphosgene is 2: 4～6: 1～2; the time of described reaction is 1～2 hour, and the temperature of reaction is 30～70 ℃. Preferably, the molar ratio of 2-aminohexadecanoic acid, α-pinene and triphosgene is 2:3:1.

In above-mentioned technical scheme, concrete reaction process is as follows:

Add 2-aminohexadecanoic acid, triphosgene and α-pinene into anhydrous tetrahydrofuran, stir well, and react at 50°C for 1 hour, then cool the reaction solution to room temperature naturally, concentrate by rotary evaporation, and then use petroleum The crude product was obtained by ether precipitation; the crude product was recrystallized 2-3 times with anhydrous tetrahydrofuran and petroleum ether, and finally a white powdery solid was obtained, which was 2-aminohexadecanoic acid-N-carboxylidene anhydride (APA -NCA).

The above preparation scheme can be expressed as follows:

The present invention also discloses a polyamino acid, which includes polyamino acid without targeting molecule or polyamino acid containing targeting molecule; It is prepared in the presence of an initiator; the polyamino acid containing the targeting molecule is prepared from the above-mentioned 2-aminohexadecanoic acid-N-carboxyl internal anhydride in the presence of the initiator and the targeting molecule.

In the polyamino acid molecular structure prepared by the present invention, the molecular weight of the initiator segment is 2000-10000, and the molecular weight of the (2-aminohexadecanoic acid) segment is 1000-50000.

The invention also discloses a preparation method of polyamino acid, which is prepared by ring-opening polymerization of the above-mentioned 2-aminohexadecanoic acid-N-carboxyl internal anhydride; or by the above-mentioned 2-aminohexadecanoic acid-N- The carboxyl internal acid anhydride is prepared by coupling targeting molecules after ring-opening polymerization with an initiator.

Preferably, the present invention discloses a block copolymer polyamino acid prepared by polymerization of the above-mentioned 2-aminohexadecanoic acid-N-carboxyl internal anhydride with a polyethylene glycol compound as an initiator, wherein the molecular weight of the polyethylene glycol segment is The molecular weight of the poly(2-aminohexadecanoic acid) segment is 1000-50000.

The polyethylene glycol compound can be methoxy polyethylene glycol amino, methoxy polyethylene glycol silazane, acrylate polyethylene glycol amino, acrylate polyethylene glycol silazane, Allyl Polyethylene Glycol Amino, Allyl Polyethylene Glycol Silazane, Maleimide Polyethylene Glycol Amino, Maleimide Polyethylene Glycol Silazane, Azide Polyethylene Glycol Amino, Azidopolyethylene Glycol Silazane, Alkynyl Polyethylene Glycol Amino, Alkynyl Polyethylene Glycol Silazane, Biotin Polyethylene Glycol Amino, Biotin Polyethylene Glycol Silazane. Preferably, the block copolymer is prepared by ring-opening polymerization of the above-mentioned 2-aminohexadecanoic acid-N-carboxylidene anhydride using polyethylene glycol as an initiator, and its chemical structural formula is shown in formula II:

Formula II.

In the above technical scheme, R is the terminal functional group of the polyethylene glycol compound initiator, and the polyethylene glycol amino initiator is preferred in the present invention. The molecular formula is shown in formula III:

Formula III

Among them, R is , , , or .

The preparation process of the above polyamino acid is carried out in an organic solvent such as N,N-dimethylformamide (DMF), dichloromethane (DCM), chloroform, tetrahydrofuran (THF), and N,N-dimethylformamide is preferred in the present invention. formamide as solvent.

The end of the hydrophilic segment PEG of the polymer obtained in the present invention can be chemically coupled with specific targeting molecules to obtain polyamino acids containing targeting molecules, including short peptides (cRGD, cNGQ, CC-9, CPP33, CPP44, etc.), small molecules Targeting molecules (folate, anisamide, etc.), antibodies and antibody fragments, polysaccharides and monosaccharides, etc.

The polymer prepared by the invention can be applied to prepare polymer nanostructures, including polymer micelles and polymer vesicles, and can be loaded with drugs to obtain nano-medicines, combined with dispersion media such as buffers to obtain nano-medicine systems.

The invention also discloses a polymer nanostructure and a preparation method thereof, prepared from the above-mentioned polyamino acid without targeting molecules and/or polyamino acids containing targeting molecules; or prepared from the above-mentioned polyamino acids without targeting molecules The nanostructure is then coupled with targeting molecules to prepare the polymer nanostructure.

The invention also discloses a nano-medicine, including the polymer nano-structure and medicines, including polypeptide and protein medicines, hydrophobic chemical medicines and hydrophilic chemical medicines.

The present invention also discloses a method for preparing a nano-medicine, wherein the nano-medicine is prepared from the above-mentioned polyamino acid containing no targeting molecule and/or polyamino acid containing a targeting molecule; or the above-mentioned polyamino acid containing no targeting molecule The nanometer drug is prepared by coupling the targeting molecule with the drug-loaded nanostructure prepared by the drug.

The invention also discloses a nano-medicine system and a preparation method thereof, comprising the above-mentioned nano-medicine and a dispersion medium; mixing the above-mentioned nano-medicine and the dispersion medium to obtain the nano-medicine system; the dispersion medium can be a buffer solution or a physiological saline, and the like.

The invention also discloses the application of 2-aminohexadecanoic acid-N-carboxylidene anhydride, polyamino acid, polymer nanostructure or nanomedicine in the preparation of antitumor drugs.

The polymer of the present invention has a suitable hydrophilic-hydrophobic ratio, so a series of size-controllable polymer micelles and polymer vesicles can be prepared by a solvent replacement method. Concrete preparation method can be:

Polymer micelles and vesicles are prepared by solvent displacement method. Dissolve the polymer in N,N-dimethylformamide (DMF) or tetrahydrofuran (THF), then add the polymer solution dropwise to water or PB, and finally dialyze with a dialysis bag with a molecular weight cut-off of 7000 The polymer micelles or vesicles with a particle size of 50-200 nm can be obtained by removing the organic solvent. Specifically targeted polymer micelles or vesicles can be prepared by adjusting the ratio of the above targeting and non-targeting polymers. The introduction of targeting molecules can also be achieved by post-modification methods after the preparation of polymer micelles or vesicles, such as introducing short peptides (cRGD, cNGQ, CC-9, CPP33, CPP44, etc.), small molecule targeting molecules (folate, anisamide, etc.), antibodies and antibody fragments, polysaccharides and monosaccharides, etc.

Due to the application of the above-mentioned technical solution, the present invention has the following advantages compared with the prior art:

The α-amino acid-N-carboxyl internal acid anhydride prepared by the present invention is a long-chain lipid internal acid anhydride monomer, which can obtain polylipopeptides with controllable properties through ring-opening polymerization. Compared with other hydrophobic polyamino acids, this type of polyamino acid Amino acids have a wider solvent selection window and better solubility;

The present invention uses polyethylene glycol as an initiator to obtain polymers with controllable molecular weight and narrow molecular weight distribution through ring-opening polymerization, which enriches the types of amphiphilic biocompatible polymers;

The polymer disclosed in the present invention has excellent biocompatibility, can prepare (tumor targeting) polymer micelles and vesicles, and can be used for efficient loading of polypeptide and protein drugs, hydrophobic and hydrophilic drugs;

The polyamino acid disclosed in the present invention is a polylipopeptide, which can be assembled to form a nanostructure, and can be loaded with various drugs such as hydrophobic chemicals (such as irinotecan) for the treatment of various cancers, and can also be used as an auxiliary material Link to the antigen for the preparation of anti-cancer vaccines, which can be widely used in the field of nanomedicine research;

The preparation method of the invention is simple, and the source of raw materials used is wide, thus having good application prospect.

Description of drawings

Fig. 1 is the proton nuclear magnetic spectrum and the carbon nuclear magnetic spectrum figure of APA-NCA in the embodiment one;

Fig. 2 is the nuclear magnetic hydrogen spectrogram of PEG _5k - b -PAPA _3.5k in embodiment two;

Fig. 3 is the NMR spectrum of AA-PEG _6k - b -PAPA _4.2k in Example 3;

Fig. 4 is the NMR spectrum of cRGD-PEG _6k - b -PAPA _4.2k in Example 3;

Figure 5 is the particle size distribution (A) and transmission electron micrograph (B), serum stability (C) and in vitro release (D) of cRGD-modified polymer micelles in Example 4 and Example 5;

Figure 6 is the particle size distribution (A) and transmission electron micrograph (B), serum stability (C) and in vitro release (D) of cRGD-modified polymersomes in Example 6 and Example 7;

Figure 7 is the result of the cytotoxicity of hollow polymer micelles to L929 mouse fibroblasts and B16F10 mouse melanoma cells (A) and empty polymer vesicles to L929 mouse fibroblasts and A549 human lung cancer cells in Example 8 Figure (B);

Figure 8 shows the cytotoxicity (A) of docetaxel-loaded polymer micelles to B16F10 mouse melanoma cells in Example 9, and the cytotoxicity (B) of doxorubicin-loaded polymer vesicles to A549 human lung cancer cells;

Fig. 9 is a graph showing the blood circulation research results of polymer micelles (A) and vesicles (B) in mice in Example 10;

Figure 10 is the result of the biodistribution study of the drug-loaded polymer micelles in Example 11 in mice bearing B16F10 mouse melanoma (A) and the biodistribution of drug-loaded vesicles in mice bearing A549-Luc human lung cancer Research results map (B);

Figure 11 is a diagram of the tumor inhibition of DTX-loaded targeting polymer micelles DTX-cRGD-PMs in Example 12 in C57/BL6 mice bearing mouse B16F10 melanoma, wherein A is the tumor growth curve, and B is Tumor pictures of mice after treatment, C is body weight change, D is survival curve;

Figure 12 is a diagram of the tumor inhibition of Dox-loaded targeted polymer vesicles Dox-cRGD-LPPs in Example 13 in nude mice bearing human A549-Luc lung cancer orthotopic tumors, where A is the tumor growth curve, and B is the tumor growth curve. Changes in body weight of mice, and C is the survival curve.

detailed description

Below in conjunction with accompanying drawing and embodiment the present invention will be further described:

Example 1 Synthesis of 2-aminohexadecanoic acid-N-carboxylidene anhydride (APA-NCA)

(1) Place the accurately weighed 2-aminohexadecanoic acid (3 g, 11 mmol) into a three-necked round-bottomed flask treated with anhydrous and anaerobic treatment, add anhydrous THF (100 mL), and dissolve the reaction system Stir well in an oil bath at 50°C, then add solid triphosgene (1.64 g, 5.5 mmol) and α-pinene (α-pinene, 2.7 mL, 16.5 mmol) into the reaction solution; The reaction solution was naturally cooled to room temperature and concentrated by rotary evaporation, and then precipitated with petroleum ether to obtain the crude product of APA-NCA; then the crude product was dissolved in THF, and then the solution was spin-dried to obtain a white product, which was then washed with anhydrous THF and petroleum The ether was recrystallized three times, and the solid in bright white powder was finally obtained as APA-NCA (1.65 g, yield 50%).

The NMR characterization of APA-NCA is shown in Figure 1, ¹ H NMR (400 MHz, DMSO- d ₆ ): δ 9.07 (s, 1 H, -CHNHCO-), 4.41 (m, 1 H, -COCHNH-), 1.65 ( d, 2 H, -CH ₂ CH ₂ CH-), 1.29 (d, 2 H, -CH ₂ CH ₂ CH ₃ -), 0.84 (t, 3 H, -CH ₂ CH ₃ ); ¹³ C NMR (100 MHz, DMSO- d ₆ ): δ 171.66,151.96, 57.04, 31.33, 30.95, 29.06, 28.91, 28.76, 28.43, 24.26, 22.12, 13.93; elemental analysis of APA-NCA is C, 68.65; H, 10.51; 4.71 (Theoretical: C, 68.37; H, 10.07; N, 4.65). Mass Spectrum: [M+Na] ⁺ 320.2202; (Theory: 320.2205).

The preparation of embodiment two PEG- b -PAPA polyamino acid block copolymer

The present invention uses amino-terminated polyethylene glycol as an initiator to prepare two polymers PEG- b -PAPA with different PAPA chain lengths through ring-opening polymerization. Take the synthesis of PEG _5k - b _- PAPA _3.5k as an example: under _N2 environment, add the DMF solution of macromolecular initiator polyethylene glycol amino ( Mn =5000 g/mL, 100 mg, 0.02 mmol) into the airtight In the reactor, the DMF solution of APA-NCA (94 mg, 0.32 mmol) was added to the closed reactor under stirring conditions, and reacted in a constant temperature oil bath at 35 °C for 48 h until the APA-NCA monomer was completely polymerized (for the reaction Using infrared monitoring, the carbonyl absorption peak of APA-NCA monomer anhydride at 1841 cm ^-1 disappears, indicating that the polymerization is complete); the polymer solution is precipitated in anhydrous ice ether, filtered, and vacuum-dried at room temperature to obtain the product PEG _5k - b - PAPA _3.5k . Yield: 91%. The NMR characterization of the PEG- b -PAPA block copolymer is shown in Figure 2. By changing the ratio of initiator and monomer, polymers with different molecular weights and compositions can be conveniently prepared (Table 1). Meanwhile, PAPA polyamino acid homopolymer and PEG- b -PAPA copolymer exhibited good solubility in chloroform and THF solvents (Table 2).

Table 1 Characterization of polyamino acid copolymers

a: Calculation results of terminal group analysis by ¹ H NMR; b: Calculation results of GPC.

Table 2 Characterization of Polyamino Acid Solubility

solvent PAPA PEG _5K - b -PAPA _3.5K PEG _5K - b -PAPA _9.8K Chloroform +++ +++ +++ Tetrahydrofuran ++ ++ ++ N,N-Dimethylformamide - + - Dimethyl sulfoxide - - - acetone - - -

+++: very soluble; ++: well soluble; +: soluble under certain conditions; -: insoluble.

Example 3 Synthesis of cRGD-modified PEG- b -PAPA polymer (cRGD-PEG- b -PAPA)

The present invention synthesizes two cRGD-PEG- b -PAPA polymers with different PAPA chain lengths. Take the synthesis of cRGD-PEG _6k - b -PAPA _4.2k as an example: use acrylic acid-modified polyethylene glycol amino group (AA-PEG-NH ₂ , Mw = 6000 g/mol) as a macroinitiator, and then follow the example Two ways to prepare polymers. cRGD-PEG _6k - b -PAPA _4.2k was prepared in two steps. First, the preparation of AA-PEG _6k - b -PAPA _4.2k polymer: under _N2 environment, the macroinitiator acrylic acid polyethylene glycol amino ( M _n = 6000 g/mol, 100 mg, 0.0167 mmol) The DMF solution was added to the closed reactor, and the DMF solution of APA-NCA (79.4 mg, 0.267 mmol) was added to the closed reactor under stirring conditions, and the APA-NCA monomer was reacted in a constant temperature oil bath at 35 °C for 48 h. Complete polymerization (infrared monitoring is used for the reaction, the carbonyl absorption peak of APA-NCA monomer anhydride at 1841 cm ^-1 disappears, indicating that the polymerization is complete); the polymer solution is precipitated in anhydrous ice ether, and the product is obtained after filtration and vacuum drying at room temperature AA-PEG _6k - b -PAPA _4.2k . The NMR characterization of the AA-PEG _6k - b -PAPA _4.2k block copolymer is shown in Figure 3. Then, cRGD-PEG _6k - b -PAPA _4.2k was prepared by photoclick chemistry. Dissolve the above polymer in DMF under N ₂ environment, then add cRGD-SH and photoinitiator I2959 into the polymer solution; then transfer the reaction to a UV curing box under ice bath conditions and react in 365 nm UV light 20 minutes. After the reaction, the polymer solution was dialyzed in DMF for 24 h with a dialysis bag with a molecular weight cut-off of 3500, then permeated with distilled water for 48 h, and finally freeze-dried to obtain a white solid. Yield: 83%. The NMR characterization of cRGD-PEG _6k - b -PAPA _4.2k block copolymer is shown in Figure 4.

The reactions of Example 2 and Example 3 can be expressed as follows, A is Example 2, and B is Example 3.

The preparation of embodiment four PEG- b -PAPA polymer micelles

In the state of stirring, drop the PB buffer solution or ultrapure water into the N,N-dimethylformamide (DMF) solution of PEG _5k - b -PAPA _3.5k , and then use a dialysis bag with a molecular weight cut-off of 7000 The above solution was dialyzed to remove the organic solvent, and the dialyzing process was carried out in PB buffer solution or pure water with pH=7.4. Finally, the size of polymer micelles measured by dynamic light scattering particle size analyzer (DLS) was 78 nm, and the particle size distribution was narrow.

The cRGD targeting micelles were prepared by dissolving PEG _5k - b -PAPA _3.5k and cRGD-PEG _6k - b -PAPA _4.2k in 1 mL DMF at a certain molar ratio, and then using the above-mentioned dialysis method. The PEG molecular weight of the targeting polymer is longer than that of the non-targeting polymer to ensure better exposure of the targeting molecule on the surface of the micelles. The cRGD density on the surface of polymer micelles can be controlled by adjusting the ratio of cRGD-containing cRGD-PEG _6k - b -PAPA _4.2k polymer. The targeting molecule-containing micelles prepared with 20% molar concentration of cRGD-PEG _6k - b -PAPA _4.2k had a smaller size (80 nm) and a narrower particle size distribution (Fig. 5A). It can be seen from Figure 5B that the polymer micelles were solid spherical structures as measured by TEM. Moreover, the polymer micelles had good stability in the environment of 10% fetal bovine serum (Fig. 5C).

Example 5 Loading of docetaxel in polymer micelles and its release in vitro

The preparation of drug-loaded micelles is similar to the preparation of polymer micelles by solvent displacement. Specifically, mPEG _5k - b -PAPA _3.5k and cRGD-PEG _6k - b -PAPA _4.2k were dissolved in DMF (5 mg/mL) at a molar ratio of 4:1. The polymer solution and docetaxel (DTX, 10 mg/mL, DMF) were mixed and placed in a small bottle, and then PB (pH=7.4, 10 mM) buffer solution or ultrapure water was dropped into it, and then The above solution was dialyzed to remove organic solvents and free drugs with a dialysis bag with a molecular weight cut off of 7000, and the dialysis process was carried out in PB buffer solution or pure water with pH=7.4. Finally, the particle size of the drug-loaded micelles was measured by DLS to be about 60 nm, and the particle size distribution was 0.18-0.19. The loading efficiency of DTX determined by HPLC was 74% (Table 3). The obtained drug-loaded micelles were named DTX-cRGD-PMs, indicating that the drug loaded was DTX, and the targeting molecule was cRGD.

The in vitro release experiment of DTX was carried out in a constant temperature shaker at 37°C (200 rpm), and three parallel samples were made for each group. DTX-loaded targeting micelles were in PB (10 mM, pH 7.4), the concentration of micelles was 0.2 mg/mL, 0.5 mL was put into release dialysis bags (MWCO: 12,000), and each test tube was added The corresponding dialysis medium is 25 mL, and 5.0 mL of the external medium of the dialysis bag is taken out at predetermined time intervals for testing, and 5.0 mL of the response medium is added to the test tube at the same time. The drug concentration in solution was determined using HPLC. Figure 5D shows the relationship between the cumulative release of DTX and time. It can be seen from the figure that the drug-loaded micelles can release the drug through diffusion.

Table 3 Characterization of docetaxel-loaded micelles (theoretical drug loading is 15%)

a: Measured by dynamic light scattering; b: Measured by HPLC; c: Measured by electrophoresis.

Example 6 Preparation of PEG- b -PAPA polymersomes

In the stirring state, drop the THF solution of PEG _5k - b -PAPA _9.8k into PB buffer solution or ultrapure water, and then use a dialysis bag with a molecular weight cut-off of 7000 to dialyze the above solution to remove the organic solvent. The dialysis process is at pH =7.4 PB buffer or pure water. Finally, the size of the polymersomes measured by a dynamic light scattering particle size analyzer (DLS) was 80 nm, and the particle size distribution was narrow.

For cRGD-targeted polymersomes, PEG _5k - b -PAPA _9.8k and cRGD-PEG _6k - b -PAPA _10k were dissolved in THF at a molar ratio of 4:1 to prepare a 2 mg/mL solution. The polymer solution was dropped dropwise into PB or pure water under stirring, and then dialyzed to remove the organic solvent. The size of the polymersomes measured by a dynamic light scattering particle size analyzer (DLS) was 80 nm, and the particle size distribution was narrow (Figure 6A). It can be seen from Fig. 6B that the polymersomes measured by TEM are hollow spherical structures. Moreover, polymersomes remained stable in the environment of 10% fetal bovine serum (Fig. 6C).

Example 7 Polymersomes Carrying Hydrophilic Drug DOX·HCl and Its Release in Vitro

PEG _5k - b -PAPA _9.8k and cRGD-PEG _6k - b -PAPA _10k were dissolved in THF at a molar ratio of 4:1 to prepare a 2 mg/mL solution. Add 200 μL of the polymer solution dropwise to 800 μL of citric acid buffer (10 mM, pH 4.0), then adjust the pH to 7.8~8.0 with supersaturated Na ₂ HPO ₃ , and then add DOX·HCl into the solution, and then placed in a 37°C shaker (200rpm) overnight; finally dialyzed in dialysis medium (PB, 10 mM, pH 7.4) for 8 h, changing the dialysis solution five times. The particle size of polymersomes loaded with different proportions of drugs (10-20 wt.%) was 80-90 nm, and the particle size distribution was 0.17-0.20. The encapsulation efficiency of DOX·HCl measured by fluorescence spectrometer was 68-84%, and Dox-cRGD-LPPs were obtained (Table 4). The in vitro release experiment of DOX·HCl is the same as in Example 5. The relationship between cumulative release of DOX·HCl and time shows that DOX·HCl can be released across the membrane through self-diffusion (Figure 6D). The PEG molecular weight of the targeting polymer is longer than that of the non-targeting polymer to ensure that the targeting molecule can be better exposed on the surface of the polymersome. Mixing the two in different ratios can prepare polymer micelles with different densities of targeting molecules. In the present invention, the targeting ratio of both the micellar system and the vesicle system is preferably 20 wt.%.

Table 4. Characterization of doxorubicin-targeted vesicles

a: Measured by fluorescence spectrometer; b: Measured by dynamic light scattering; c: Measured by electrophoresis.

Example 8 MTT method to test the cytotoxicity of empty polymer micelles and vesicles

MTT method uses murine melanoma cells (B16F10), human lung cancer cells (A549) and murine fibroblasts (L929). The cells were seeded in a 96-well plate at 5×10 ³ cells/mL, 100 μL per well, and cultured to about 70% of the cells after 24 hours. Then, micelles or vesicle samples containing different concentrations (0.1-1.0 mg/mL) were added to each well of the experimental group (taking the empty polymer micelles of Example 4 and the empty polymer vesicles of Example 6 as examples ), and a cell blank control well and a medium blank well (complex 4 wells). After culturing for 24 hours, add 10 μL of MTT (5.0 mg/mL) to each well, continue to cultivate for 4 hours, add 150 μL DMSO to each well to dissolve the generated crystals, measure the absorbance value at 570 nm with a microplate reader, and use the culture medium Blank wells were adjusted to zero, and the cell viability was calculated. Accompanying drawing 7A is the cytotoxicity result of polymer micelle to L929 and B16F10, it can be seen that when the concentration of polymer micelle increases from 0.1 to 1.0 mg/mL, the survival rate of B16F10 is still higher than 90%, indicating that the Polymeric micelles have good biocompatibility. The determination of the cytotoxicity of the polymersomes is similar to this, as shown in Figure 7B, the toxicity is very small and has good biocompatibility.

Example 9 Toxicity of drug-loaded polymer micelles to B16F10 melanoma cells and drug-loaded polymer vesicles to A549 lung cancer cells measured by MTT method

The test objects were the DTX-cRGD-PMs of Example 5 and the Dox-cRGD-LPPs of Example 7, the non-targeted DTX-PMs and free DTX, the non-targeted Dox-LPPs and Lipo-DOX groups were respectively used as the control group . The culture of the cells is the same as in Example 8. After the nanoparticles or drugs are co-cultured with the cells for 4 hours, the culture medium is renewed for 44 hours and then MTT is added. The treatment and absorbance measurement are the same as in Example 8. See Figure 8 for the experimental results. The results in Figure 8A show that the half-lethal concentration (IC ₅₀ ) of DTX-cRGD-PMs on B16F10 cells is 0.15 μg/mL, which is 2.6 times smaller than that of DTX-PMs; Figure 8B shows the effect of drug-loaded vesicles on A549 cells From the results of toxicity experiments, it can be seen from the figure that the half-lethal concentration of Dox-cRGD-LPPs on A549 cells is 15.17 μg/mL, which is much lower than that of Lipo-Dox and twice as small as the half-lethal concentration of Dox-LPPs. It shows that the micelles and vesicles of the present invention can well deliver drugs into cells, and release them effectively, and finally kill cancer cells, while the effect of targeting micelles and targeting vesicles is better.

Example 10 Blood circulation of targeted and non-targeted micelles and vesicles

All animal experiments were performed in accordance with the regulations of the Animal Experiment Center of Soochow University. In the experiment, Balb/C mice aged 4-6 weeks were selected with a body weight of about 18-20 grams. Cy5-labeled micelles PMs-Cy5 were prepared by mixing PEG _5k - b _{-PAPA 3.5k} and PEG _5k - b -PAPA _{3.5k -Cy5} at a ratio of 1:1, and the particle size of Cy5-labeled micelles formed in this ratio was 60 nm. The particle size distribution was 0.19. cRGD-PMs-Cy5 micelles were prepared by mixing cRGD-PEG _6k - b -PAPA _4.2k and PEG _5k - b -PAPA _{3.5k -Cy5} at a ratio of 1:1, and the particle size of the formed polymer micelles was about 60 nm. The particle size distribution was 0.18. PMs-Cy5 and cRGD-PMs-Cy5 were injected into the mice through the tail vein (the concentration of Cy5 was 4 μM), and blood was collected at fixed points for about 10 hours at 0, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours. μL, the blood weight was accurately calculated by the differential method, and then 100 μL of 1% Triton and 600 μL of DMF were extracted for 24 h; after centrifugation (20000 rpm, 20 min), the supernatant was taken and measured by a fluorescence spectrometer. The amount of Cy5 at each time point was obtained. In FIG. 9A , the abscissa is time, and the ordinate is Cy5 per gram of blood accounted for the total injection amount of Cy5 (ID %/g). It can be seen from Figure 9A that the elimination half-lives of targeting polymer micelles and non-targeting polymer micelles in mice are 2.73 and 2.33 hours respectively, so the polymer micelles of the present invention are stable in mice and have a longer Cycle Time.

As above, Dox-LPPs and Dox-cRGD-LPPs were injected into mice through the tail vein (the dosage of DOX was 10 mg/kg), at 0, 0.25, 0.5, 1, 2, 4, 8, About 10 μL of blood was drawn at fixed points at 12 and 24 hours, and the blood weight was accurately calculated by the differential method, and then 100 μL of 1% Triton and 600 μL of DMF were extracted for 24 hours; then centrifuged (20000rpm, 20 min), The supernatant was taken, and the amount of DOX at each time point was measured by a fluorescence spectrometer. In Fig. 9B, the abscissa is time, and the ordinate is DOX per gram of blood as a percentage of the total injected DOX (ID %/g). It can be seen from Figure 9B that the elimination half-lives of targeted polymersomes and non-targeted polymersomes in mice are 3.74 and 3.33 hours, respectively, so the polymersomes of the present invention are stable in mice and have a longer Cycle Time.

Example 11 DTX-PMs and DTX-cRGD-PMs polymer micelles in mice bearing B16F10 melanoma and Dox-LPPs and Dox-cRGD-LPPs polymer vesicles in mice bearing human A549-Luc orthotopic lung cancer Biodistribution in vivo

Animals were the same as in Example 10. C57BL/6 black mice were subcutaneously injected with 8×10 ⁷ mouse melanoma cells, and the experiment started when the tumor size was about 200 mm ³ about 7 to 8 days later. DTX-PMs, DTX-cRGD-PMs and free DTX were injected into the mice through the tail vein (DTX: 10 mg/kg), and the mice were sacrificed 6 hours later, and the tumors and heart, liver, spleen, lung and kidney tissues were Take it out, wash and weigh it, add 500 μL of anhydrous methanol, grind it through a homogenizer, and then add 500 μL of anhydrous methanol for extraction for 24 hours. Finally, centrifuge (20000 rpm, 20 min), take the supernatant, and use high performance liquid chromatography to measure the amount of DTX in all samples. In Fig. 10A, the abscissa is the tissue and organ, and the ordinate is the DTX per gram of tumor or tissue as a percentage of the total DTX injection (ID%/g). DTX-PMs, DTX-cRGD-PMs and free DTX injected 6 hours of DTX accumulation in the tumor were 2.9, 7.9 and 1.3 ID%/g, DTX-cRGD-PMs were 2.5 times and 5.5 times that of DTX-PMs and DTX times, indicating that DTX-cRGD-PMs are more enriched in tumor sites through active targeting.

8×10 ⁷ A549-Luc human lung cancer cells were orthotopically injected into the left lung lobe of nude mice, and the experiment began when the bioluminescent intensity of the mouse lung was detected to be 10 ⁶ about 10 days later. Dox-LPPs, Dox-cRGD-LPPs and commercial Lipo-Dox were injected into mice through the tail vein (Dox·HCl: 10 mg/kg), and the mice were sacrificed 6 hours later. And kidney tissue was taken out, washed and weighed, added 500 μL of 1% Triton solution, ground through a homogenizer, and then added 500 μL N,N-dimethylformamide for extraction for 24 h. Finally, centrifuge (20000 rpm, 20 min), take the supernatant, and measure the amount of Dox in all samples with a fluorescence spectrometer. In Figure 10B, the abscissa is the tissue and organ, and the ordinate is the Dox per gram of tumor or tissue as a percentage of the total Dox injection (ID%/g). Dox-LPPs, Dox-cRGD-LPPs and Lipo-Dox accumulated Dox amounts in tumors 6 hours after injection were 3.4, 6.1 and 4.3 ID%/g, indicating that Dox-cRGD-LPPs were enriched in tumor sites through active targeting more.

Example 12 Antitumor effect, body weight change and survival rate of DTX-cRGD-PMs and DTX-PMs administered with multiple low doses and single high dose in mice bearing B16F10 subcutaneous melanoma

Tumor inoculation and tail vein administration were the same as in Example 11, and the experiment started when the tumor size was 30-50 mm ³ four days after inoculation. DTX-cRGD-PMs (single-dose), DTX-cRGD-PMs (multi-dose 40&80 mg/kg), DTX-PMs, DTX and PBS were administered on days 0, 2, 4 and 6 (single administration The group was only given on day 0) into the mice via tail vein injection (DTX dose was 10 mg/kg). On days 0-10, the body weight and tumor volume of the mice were measured every two days. The calculation method of the tumor volume was: V=(L×W ² )/2, (where L and W are the length and width of the tumor, respectively). The survival of the mice was continuously observed up to 40 days. It can be seen from Figure 11 that on the tenth day, the tumor in the DTX-cRGD-PMs treatment group was significantly inhibited, while the tumor in the DTX-PMs group had a certain growth, and the inhibitory effect of free DTX on the tumor was not obvious; In the high-dose administration group, the 40 mg/kg group had poor tumor inhibitory effect, while the 80 mg/kg group showed excellent tumor inhibitory effect, and the tumor size photos of each group further verified this conclusion. Moreover, the body weights of the mice in each treatment group did not change significantly, indicating that the mice tolerated the dosage well and had low toxic and side effects. In terms of survival rate of mice, DTX-cRGD-PMs (multi-dose), DTX-cRGD-PMs (single-dose 40), DTX-cRGD-PMs (single-dose 80), DTX-PMs, DTX and PBS The median survival values of the groups were 35 d, 13 d, 23 d, 27 d, 9 d, and 9 d, respectively.

Example 13. Tumor inhibitory effect, body weight change and survival rate of DOX-cRGD-LPPs and DOX-LPPs in mice bearing A549-Luc orthotopic lung cancer

Animals are the same as in Example 11. Inject 5×10 ⁶ A549-Luc human lung cancer cells on the left lung lobe of 5-week-old nude mice, and start when the tumor fluorescence value is about 105-106 after about 2 to 3 weeks. experiment. Dox-cRGD-LPPs, Dox-LPPs, Lipo-Dox and PBS were injected into mice through the tail vein on the 0th, 4th, 8th and 12th day respectively (DOX·HCl injection amount: Dox-cRGD-LPPs, Dox- LPPs group was 10.0 mg/kg; Lipo-Dox group was 7.5 mg/kg). On days 0-20, the mice were weighed every 4 days, and the mice were subjected to bioluminescent imaging, and the tumor size was monitored by the fluorescence value. It can be seen from Figure 12 that on the 20th day, the tumors in the Dox-cRGD-LPPs treatment group were significantly inhibited, while the tumors in the Dox-LPPs and Lipo-Dox groups increased to a certain extent. After observation, the median survival values of Dox-cRGD-LPPs and Dox-LPPs mice were about 36 days and 24 days, respectively.

Polymersomes based on PEG ₃ . _5k - b -PAPA _7k , PEG _5k - b -PAPA _13k , PEG _7.5k - b -PAPA _21k , PEG _10k - b -PAPA _50k , targeted polymersomes , the survival rate of the test cells is greater than 90%, the toxicity is very small, and it has good biocompatibility; the particle size of the doxorubicin-loaded polymer vesicles is about 80 nanometers, and the encapsulation efficiency when the theoretical drug loading is 20% reached more than 83%; the half-lethal concentration for A549 cells was about 15 μg/mL; the elimination half-life in mice was greater than 3.2 hours; Dox-LPPs and Dox-cRGD-LPPs polymer vesicle nano-medicines were injected 6 hours later The amount of Dox accumulated in the tumor was greater than 3.2 ID%/g and 6.0 ID%/g, respectively, and the median survival of the mice was about 36 days and 24 days, respectively.

Polymer micelles and targeted polymer micelles prepared on the basis of PEG _10k - b -PAPA _8k , PEG _7.5k - b -PAPA _6k , PEG _5k - b -PAPA _5k , PEG _3.5k - b -PAPA _4k , The survival rate of the test cells is greater than 90%, the toxicity is very small, and it has good biocompatibility; the particle size of the polymer micelles loaded with docetaxel is about 60 nanometers, and the theoretical drug loading is 15%. The half-lethal concentration (IC ₅₀ ) of B16F10 cells is about 0.15 μg/mL; the elimination half-life in mice is greater than 2.2 hours; DTX-PMs and DTX-cRGD-PMs polymer micellar nano Six hours after drug injection, the amount of DTX accumulated in the tumor was greater than 2.8 ID%/g and 7.8 ID%/g, respectively, and the median survival of the mice was about 35 days and 26 days, respectively.

Claims (10)

1. a kind of 2- aminohexadecanoic acid-N- carboxyl inner-acid anhydrides with structure shown in formula I：

Formula I.

2. the preparation method of 2- aminohexadecanoic acids-N- carboxyl inner-acid anhydrides described in claim 1, it is characterised in that including as follows Step, using 2- aminohexadecanoic acids, firpene, triphosgene as reactant, reacts in organic solvent, prepares the 2- amino Hexadecanoic acid-N- carboxyl inner-acid anhydrides.

3. a kind of polyaminoacid, it is characterised in that the polyaminoacid is included without targeted molecular polyaminoacid or containing targeting Molecule polyaminoacid；The targeted molecular polyaminoacid that is free of is as in the 2- aminohexadecanoic acid-N- carboxyls described in claim 1 Acid anhydrides is prepared in the presence of initiator；The polyaminoacid containing targeted molecular is as the 2- amino 16 described in claim 1 Alkanoic acid-N- carboxyls inner-acid anhydride is prepared in the presence of initiator and targeted molecular.

4. a kind of preparation method of polyaminoacid, it is characterised in that as the 2- aminohexadecanoic acid-N- carboxylics described in claim 1 Base inner-acid anhydride is prepared with initiator ring-opening polymerisation；Or as in the 2- aminohexadecanoic acid-N- carboxyls described in claim 1 Acid anhydrides is prepared with coupling targeted molecular after initiator ring-opening polymerisation.

5. a kind of polymer nanostructures, it is characterised in that the polymer nanostructures described in claim 4 as being free of target Prepared to molecule polyaminoacid and/or polyaminoacid containing targeted molecular；Or dividing described in claim 4 without targeting Sub- polyaminoacid is prepared to be coupled targeted molecular and prepares the polymer nanostructures again after nanostructured.

6. a kind of preparation method of polymer nanostructures, it is characterised in that as gathering described in claim 4 without targeted molecular Amino acid and/or polyaminoacid containing targeted molecular prepare the polymer nanostructures；Or described in claim 4 Prepared without targeted molecular polyaminoacid and be coupled targeted molecular after nanostructured again and prepare the polymer nanocomposite knot Structure.

7. a kind of Nano medication, it is characterised in that the Nano medication include polymer nanostructures described in claim 5 and Medicine.

8. a kind of preparation method of Nano medication, it is characterised in that as being free of targeted molecular polyaminoacid described in claim 4 And/or polyaminoacid containing targeted molecular, medicine preparation obtain the Nano medication；Or be free of target described in claim 4 Obtain being coupled targeted molecular after medicament-carried nano structure again to molecule polyaminoacid and medicine preparation and prepare the Nano medication.

9. a kind of Nano medication system, it is characterised in that the Nano medication system include Nano medication described in claim 7 with And decentralized medium.

10. 2- aminohexadecanoic acids-N- carboxyls inner-acid anhydride described in claim 1, polyaminoacid, right described in claim 3 will Ask application of the Nano medication in antineoplastic is prepared described in polymer nanostructures described in 5 or claim 7.