CN108392471B

CN108392471B - Nano-drug based on terminal sulfur-containing octanoyl star polymer

Info

Publication number: CN108392471B
Application number: CN201810172582.XA
Authority: CN
Inventors: 程茹; 王秀秀; 钟志远
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2018-03-01
Filing date: 2018-03-01
Publication date: 2020-06-16
Anticipated expiration: 2038-03-01
Also published as: CN108392471A

Abstract

The invention discloses a nano-drug based on a terminal sulfur-containing octanoyl star polymer. The prepared cancer-targeted reduction-sensitive reversible crosslinked polymer nanoparticle nano-drug support is highly enriched in cancer tissues, efficiently enters cells, is rapidly de-crosslinked in the cells to release the drugs, efficiently and specifically kills cancer cells, and effectively inhibits the growth of cancers without causing toxic and side effects.

Description

Nano-drug based on terminal sulfur-containing octanoyl star polymer

Technical Field

The invention relates to a biocompatible polymer material and application thereof, in particular to a nano-drug based on a star-shaped biocompatible polymer containing sulfur octanoyl at the tail end, belonging to the field of medical materials.

Background

The polymer nano-drug prepared by the prior art has the problems of unstable in vivo circulation, low tumor cell uptake, low intracellular drug concentration and slow intracellular drug release speed, so that the drug effect of the nano-drug is not high, and toxic and side effects caused by drug leakage also exist; for example, BIND-014 did not achieve the expected results in the second clinical stage, due to insufficient stability of the nano-drug in vivo.

Cancer is a major killer threatening human health, and its morbidity and mortality rate are on an increasing trend year by year. The appearance of nano-drugs brings new hope for treating cancers, but in the prior art, high-efficiency nano-drugs which are stable in vivo circulation, specifically target cancers, quickly respond to and release drugs in cells and have small toxic and side effects are not available, and particularly, polymer nano-carriers which can keep stable in the in vivo circulation process and quickly release drugs in cells are not available. Based on this, the development of a nano-carrier which can prolong the circulation time of the drug in vivo and can quickly release the drug in tumor cells is urgent.

Disclosure of Invention

The invention aims to provide a nano-drug based on a terminal sulfur-containing octanoyl radical star-shaped biocompatible polymer.

In order to achieve the purpose, the specific technical scheme of the invention is as follows: the nano-drug based on the terminal sulfur-containing octanoyl star polymer is obtained by reversibly crosslinking a polymer nanoparticle loaded drug with reduction responsiveness;

the reversible crosslinked polymer nano particle with reduction responsiveness is prepared by assembling a star polymer with a sulfur octanoyl-containing terminal and a second polymer together; the second polymer is an amphiphilic polymer and/or a targeting amphiphilic polymer;

the chemical structural formula of the star polymer with the terminal sulfur octanoyl group is as follows:

wherein x and y are (1-3) to 1.

The star polymer with the terminal containing the sulfur octanoyl group can be called sP-LA, and the molecular weight is 10000-75000.

The preparation method of the star-shaped polymer with the terminal sulfur octanoyl group comprises the following steps:

(1) under the nitrogen environment and the ice-water bath condition, dropwise adding the N, N-dicyclohexylcarbodiimide solution into the lipoic acid solution; after the dropwise addition is finished, sealing and reacting at room temperature for 10-15 hours; obtaining lipoic acid anhydride solution;

(2) under the vacuum environment, under the catalytic action of stannous octoate, taking polyhydroxy glucose as an initiator, and reacting lactide and glycolide for 8 hours at 160 ℃; obtaining a star polymer;

(3) adding a lipoic anhydride solution into an organic solvent containing 4-dimethylaminopyridine and a star polymer under a nitrogen environment, and reacting for 1-3 days at 30 ℃ under a sealed condition; the star polymer containing the sulfur octanoyl group at the terminal is obtained.

Specifically, the preparation method can be as follows:

(1) under the nitrogen environment, dissolving thioctic acid in an organic solvent to prepare a thioctic acid solution; putting N, N-dicyclohexylcarbodiimide in an organic solvent to prepare a N, N-dicyclohexylcarbodiimide solution; then, dropwise adding the N, N-dicyclohexylcarbodiimide solution into the thioctic acid solution in an ice-water bath; after the dropwise addition is finished, sealing and reacting at room temperature for 10-15 hours; after the reaction is finished, filtering the reaction solution to obtain a lipoic acid anhydride solution;

(2) in N₂Under the environment, adding polyhydroxy glucose, lactide and glycolide into a closed reaction bottle, then adding a catalyst stannous octoate into the reaction bottle, and uniformly mixing all the materials; the reaction flask was then evacuated to replace N₂Thirdly, finally vacuumizing the reaction bottle for 30 minutes, and sealing the reaction bottle; the polymerization reaction is carried out for 8 hours in a vacuum box at the temperature of 160 ℃; after the reaction is finished, dissolving the product in dichloromethane, then precipitating in ice methanol, filtering and drying in vacuum to obtain a star polymer;

(3) adding a lipoic anhydride solution into an organic solvent containing 4-dimethylaminopyridine and a star polymer under a nitrogen environment, and reacting for 1-3 days at 30 ℃ under a sealed condition; then the reactant is precipitated in glacial ethyl ether, and the star polymer (sP-LA) containing the sulfur octanoyl group at the terminal is obtained after suction filtration and vacuum drying of filter cakes.

The preparation method of the nano-drug comprises the following steps:

(3) adding a lipoic anhydride solution into an organic solvent containing 4-dimethylaminopyridine and a star polymer under a nitrogen environment, and reacting for 1-3 days at 30 ℃ under a sealed condition; obtaining a star polymer with the tail end containing sulfur octanoyl;

(4) assembling the star polymer with the terminal containing the sulfur octanoyl group, a second polymer and a small molecular drug in a solvent to obtain the nano drug.

In the invention, the solvent in the N, N-dicyclohexylcarbodiimide solution is dichloromethane; the solvent in the thioctic acid solution is dichloromethane; the organic solvent in the organic solvent containing the 4-dimethylaminopyridine and the star-shaped polymer is dichloromethane.

In the invention, the molar ratio of polyhydroxy glucose to lactide to glycolide is 1 to (50-55) to (60-65); the structural formula of the prepared star polymer is as follows:

in the invention, the molar ratio of the N, N-dicyclohexylcarbodiimide to the lipoic acid is 2: 1; the mol ratio of the sulfur caprylic anhydride, the 4-dimethylaminopyridine and the star polymer is 7.5: 10: 1.

The star polymer with the terminal containing the sulfur octanoyl group can be assembled with polypeptides with different targeting functions or amphiphilic polymers modified by polysaccharide molecules, such as cRGD-PEG-PDLLA, GE11-PEG-PDLLA, TAT-PEG-PDLLA and HA-b-PDLLA, so that the nanoparticles modified by the polypeptides and the polysaccharides with different targeting molecules, such as cRGD, GE11, TAT or HA, are prepared, and different cancer cell specific targeting polymer nanoparticles are obtained, and have targeting property and biocompatibility. The nanoparticles formed by the polymer have good stability and higher loading efficiency, and can be specifically targeted to cancer cells.

The invention also discloses a polymer nano particle with reversible crosslinking and reduction responsiveness, which is prepared by the co-assembly of the star polymer with the side chain containing the sulfur octanoyl group and a second polymer; the second polymer is an amphiphilic polymer and/or a targeting amphiphilic polymer; preferably, the second polymer is used in an amount of 10 to 60 percent of the mass of the star-shaped polymer containing the sulfur octanoyl group at the terminal; when the second polymer is an amphiphilic polymer and a targeting amphiphilic polymer, the mass percentage of the targeting amphiphilic polymer is less than 70%; the amphiphilic polymer is free of targeting molecules, and the targeting amphiphilic polymer is provided with polypeptides and polysaccharides such as cRGD, GE11, TAT or HA and the like; preferably the second polymer is an amphiphilic polymer and a targeted amphiphilic polymer. The polymer nano-particle is prepared by assembling the star polymer with the side chain containing the sulfur octanoyl group and the amphiphilic polymer together; or the star polymer with the side chain containing the sulfur octanoyl group and the amphiphilic polymer of the grafting targeting molecule are assembled together to prepare the polymer; or the side chain-containing caprylyl-sulfonyl star polymer, the amphiphilic polymer of the grafting targeting molecule and the amphiphilic polymer are assembled together to prepare the drug-loaded polymer nanoparticle, for example, the side chain-containing caprylyl-sulfonyl star polymer, the amphiphilic polymer of the grafting targeting molecule and the amphiphilic polymer are mixed according to different proportions to prepare polymer nanoparticles with different targeting densities, so that the polymer nanoparticles with the active targeting function of cancer cells can be obtained, and the uptake of the drug-loaded polymer nanoparticles in the cancer cells can be increased.

The star polymer with the side chain containing the sulfur octanoyl group disclosed by the invention has excellent biocompatibility and biodegradability, and can be assembled together with a second polymer to form a polymer nanoparticle, so that the cross-linked polymer nanoparticle or the cross-linked polymer nanoparticle with an active targeting function on cancer cells can be prepared in the presence of a catalytic amount of a reducing agent such as Dithiothreitol (DTT) or Glutathione (GSH), the particle size of the cross-linked polymer nanoparticle is 70-180 nanometers, and the cross-linked polymer nanoparticle can be used as a carrier of a medicament for treating cancer; hydrophobic small-molecule anti-lung cancer drugs such as adriamycin (DOX), Paclitaxel (PTX), Docetaxel (DTX) and the like can be loaded in the polymer nanoparticles, so that the bioavailability of the hydrophobic drugs in vivo is improved, the circulation time of the drugs is prolonged, and the enrichment amount of the drugs in tumor parts is improved. Therefore, the nano-drug disclosed by the invention is obtained by loading the cross-linked polymer nano-particles with the small-molecule drug. The cross-linked polymer nano particles prepared by the star polymer with the side chain containing the sulfur octanoyl group disclosed by the invention form stable chemical cross-linking in the inner core, so that the star polymer can be stably and circularly used in vivo; but after endocytosis enters cancer cells, the cross-linking can be rapidly released in the reductive environment in the cells, the drug can be rapidly released, and the cancer cells can be efficiently killed. Therefore, the invention claims the application of the star polymer, the polymer nano particle, the cross-linked polymer nano particle and the nano drug with the side chain containing the sulfur octanoyl in the preparation of drugs for treating tumors, such as melanoma, lung cancer and triple negative breast cancer.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:

1. the invention utilizes the reaction of the star polymer and lipoic acid anhydride to obtain the star polymer with the side chain with controllable degree of substitution containing the sulfur octanoyl group, endows the star polymer with new functions and enriches the variety of the star polymer.

2. The star polymer with the side chain containing the sulfur octanoyl group disclosed by the invention has excellent biocompatibility and biodegradability, can be used for preparing polymer nanoparticles and polymer nanoparticles with cancer cell active targeting functions, is loaded with different drugs, can form disulfide bond crosslinking, and obtains a stable crosslinked polymer nano-drug, thereby overcoming the defects of unstable in vivo circulation, easy early release of the drugs and toxic and side effects of the nano-drug in the prior art.

3. The cross-linked polymer nano-drug disclosed by the invention has reversible cross-linking, namely, supports the internal long circulation of the body, and can be highly enriched in cancer cells; but can be rapidly crosslinked after entering cancer cells to release the drug, thereby realizing efficient and specific killing of the cancer cells without toxic and side effects; overcomes the defects that the chemically cross-linked nano-drug is too stable in the prior art, and the drug is slowly released in cells to cause drug resistance.

4. The biocompatible polymer nanoparticles and the polymer nanoparticles with the cancer cell active targeting function disclosed by the invention can form reduction-sensitive disulfide bond cross-linking in the preparation process, and the preparation method is simple and convenient, so that the defects that complex operation and purification process are required in the preparation of cross-linked nano-drugs in the prior art are overcome.

5. The cross-linked polymer nano particle prepared by assembling the star polymer with the side chain containing the sulfur octanoyl group and the amphiphilic polymer together can be used for a controlled release system of a hydrophobic anticancer drug, so that the defect that the hydrophobic anticancer drug which can be efficiently loaded and can stabilize the internal circulation is not available in the prior art is overcome; furthermore, the polypeptide can be bonded with targeting molecules, and has wider application value in the aspect of efficient targeting treatment of cancer.

Drawings

FIG. 1 is a hydrogen nuclear magnetic spectrum diagram of a side chain thioctanoyl-containing star polymer sP-LA of an example;

FIG. 2 shows the nuclear magnetic spectra of the amphiphilic polymers PEG-PDLLA (A), cRGD-PEG-PDLLA (B), GE11-PEG-PDLLA (C) and TAT-PEG-PDLLA (D) in examples II, III, IV and V;

FIG. 3 shows the amphiphilic polymer HA-b-nuclear magnetic spectrum of PDLLAPEG-PDLLA;

FIG. 4 is a graph of the particle size distribution and transmission electron microscopy of the crosslinked nanoparticles sLy XNPs of example seven (A), the crosslinked nanoparticle stability (B), the reduction responsiveness test (C) and the in vitro release profile (D);

FIG. 5 shows the particle size distribution (A) and TEM image (B), the reduction responsiveness test image (C) and the in vitro release image (D) of the cross-linked nanoparticles cRGD-XNPs using cRGD as the targeting molecule in example eight;

FIG. 6 shows the particle size distribution (A) and the TEM image (B), the reduction responsiveness test image (C) and the in vitro release image (D) of the cross-linked nanoparticle HA-sPLY XNPs using HA as the targeting molecule in the eleventh example;

FIG. 7 is a graph showing the uptake of different nanoparticles, sLy XNPs, in example seventeen in B16F10 cells (A), cRGD-XNPs in B16F10 cells (B), GE11/TAT-XNPs in MDA-MB-231 cells (C) and HA-sLy XNPs in A549 cells (D);

FIG. 8 is a graph showing the results of toxicity of blank nanoparticles of example eighteen, sLy XNPs on B16F10 cells (A), cRGD-XNPs on B16F10 cells (B), GE11/TAT-XNPs on MDA-MB-231 cells (C), and HA-sLy XNPs on A549 cells (D);

FIG. 9 is a graph showing the results of toxicity of the loaded nanoparticles DOX-sLy XNPs on B16F10 cells (A), DOX-cRGDXNPs on B16F10 cells (B), DTX-GE11/TAT XNPs on MDA-MB-231 cells, and DTX-HA-sLy XNPs on A549 cells in nineteen example;

FIG. 10 is a graph showing the results of the study of blood circulation in mice of the twenty, twenty-one, and twenty-three drug-loaded nanoparticles DOX-sPLY XNPs (A), DOX-cRGD XNPs (B), and DTX-HA-sPLY XNPs (C) of examples;

FIG. 11 is a biological profile of the loaded nanoparticles DOX-cRGD XNPs in the B16F10 melanoma (A) and DTX-HA-sLy XNPs in A549 lung carcinoma subcutaneous tumor (B) mice of examples twenty-four and twenty-six;

FIG. 12 is a graph of tumor suppression in B16F10 melanoma-bearing mice by DOX-loaded targeted cross-linked nanoparticles DOX-cRGDXNPs using cRGD as the targeting molecule in twenty-seventh example, wherein A is a tumor growth curve, B is the change in body weight of the mice, and C is a survival curve;

FIG. 13 is a graph of tumor suppression of DTX-loaded targeted cross-linked nanoparticles DTX-GE11/TAT XNPs using GE11 and TAT as targeting molecules in nude mice with triple negative breast cancer MDA-MB-231 subcutaneous tumor in example twenty-eight, wherein A is a tumor growth curve, B is a tumor picture of mice after treatment, C is weight change, and D is a survival curve;

FIG. 14 is a graph of tumor inhibition in nude mice with subcutaneous tumor of A549 lung cancer by DTX-loaded targeting cross-linked nanoparticles DTX-HA-sLy XNPs using HA as targeting molecule in twenty-nine examples, wherein A is tumor growth curve, B is weight change, C is tumor inhibition rate, and D is tumor picture after mouse treatment.

Detailed Description

The invention is further described below with reference to examples and figures:

example one Synthesis of Star Polymer having a side chain containing a Sulfur octanoyl group

Synthesis of Star-shaped and Linear polymers

The star polymer can be synthesized by initiating the ring-opening polymerization reaction of lactide and glycolide under the catalytic action of stannous octoate by using polyhydroxy glucose as an initiator. In N₂Under ambient conditions, 0.18 g (1 mmol) of a polyglucose polyol, 7.5g (52 mmol) of lactide and 7.5g (65 mmol) of glycolide were added to a closed reaction flask, followed by 4.73 mg of the catalyst stannous octoate to the reaction flask and mixing all materials well. The reaction flask was then evacuated to replace N₂And thirdly, finally vacuumizing the reaction bottle for 30 minutes, and sealing the reaction bottle. The polymerization reaction was carried out in a vacuum oven at 160 ℃ for 8 hours. The crude product was dissolved in dichloromethane and subsequently precipitated in ice-methanol, filtered off with suction and dried in vacuo to give the star polymer.¹H NMR(600 MHz, CDCl₃): δ 5.22 (-OOCCH ₂O-), 4.83 (-OOCCH(CH₃)O-), 2.45 (, -OCCH ₂-),1.6 (-CH(CH ₃)O-)。

The linear polymer is synthesized by taking 1, 4-butanediol as an initiator and initiating the ring-opening polymerization reaction of lactide and glycolide under the catalytic action of stannous octoate. In N₂0.9 g (1 mmol) of 1, 4-butanediol, 7.5g (52 mmol) of lactide and 7.5g (65 mmol) of glycolide were added to the closure under ambient conditionsIn a reaction flask, 4.73 mg of catalyst stannous octoate was then added to the reaction flask and all materials were mixed well. The reaction flask was then evacuated to replace N₂And thirdly, finally vacuumizing the reaction bottle for 30 minutes, and sealing the reaction bottle. The polymerization reaction was carried out in a vacuum oven at 200 ℃ for 5 hours. The crude product was dissolved in dichloromethane and subsequently precipitated in ice-methanol, filtered off with suction and dried in vacuo to give a linear polymer.

Synthesis of liponic anhydride LAA: under nitrogen, 30mg (0.15 mmol) of Lipoic Acid (LA) was dissolved in 1mL of dichloromethane, added to a two-necked flask and stirred until dissolved, and 60 mg (0.3 mmol) of N, N-Dicyclohexylcarbodiimide (DCC) was added to 0.5mL of dichloromethane dropwise to the LA solution under ice-water bath. Continuously introducing nitrogen for 5 minutes, sealing the two neck bottles, and reacting for 12 hours at room temperature; after the reaction, the precipitate produced by the reaction was filtered off to obtain lipoic anhydride solution, and the lipoic anhydride (LAA) solution was concentrated to 0.5 ml.

Synthesizing a star polymer (sP-LA) with a side chain containing sulfur octanoyl: adding 30mg (0.075 mmol) of thiooctanoic anhydride (LAA) solution into a solution of 150 mg (0.01 mmol) of star polymer and 12 mg (0.1 mmol) of DMAP in 2 mL of dichloromethane under a nitrogen atmosphere, continuously introducing nitrogen for 5 minutes, sealing the flask, and placing the flask in an oil bath at 30 ℃ for reaction for 48 hours; then the product is precipitated in glacial ethyl ether, and is filtered and dried in vacuum to obtain star polymer sP-LA with the side chain containing sulfur octanoyl, and the yield is 86.7%.¹HNMR (600 MHz, CDCl₃): δ 5.22 (-OOCCH ₂O-), 4.83 (-OOCCH(CH₃)O-), 3.57 (-CH₂CHCH₂CH₂S₂), 3.17 (-CH₂CHCH₂CH ₂S₂), 2.45 (-OCCH ₂-), 1.6 (-CH(CH ₃) O-). The nuclear magnetism showed that the LA was grafted at 90%, as shown in FIG. 1.

EXAMPLE two Synthesis of amphiphilic Polymer PEG-PDLLA

The amphiphilic polymer PEG-PDLLA can be prepared by initiating ring-opening polymerization of D, L-lactide by a macroinitiator PEG. In N₂To 2.5 mL of PEG(s) under ambient conditionsM _n0.5mL (0.2 mol/L) of a toluene stock of stannous octoate was added rapidly to an anhydrous toluene solution of 5.0 kg/mol,0.5 g, 0.1 mmol) and D, L-lactide (0.4g, 2.8 mmol). After reacting for 48 h in a constant temperature oil bath at 110 ℃, glacial acetic acid is added to stop the reaction. The product was then precipitated in glacial ethyl ether, filtered off with suction and dried in vacuo to give PEG-PDLLA in the following yield: 88.9 percent.¹H NMR (600 MHz, CDCl₃): δ 5.16 (-CH(CH₃)O- ), 3.65 (-CH ₂CH ₂O-), 3.38 (CH ₃O-), 1.56 (-CH(CH ₃) O-), see FIG. 2 (A).M _n(¹HNMR) = 8.9 kg/mol，M _n(GPC) = 15.9 kg/mol，M _w/M _n(GPC) = 1.3。

EXAMPLE three Synthesis of amphiphilic targeting Polymer cRGD-PEG-PDLLA

The targeting polymer cRGD-PEG-PDLLA is obtained through two-step reaction. Firstly, synthesizing maleimide functionalized amphiphilic polymer MAL-PEG-PDLLA, and then further synthesizing the amphiphilic polymer cRGD-PEG-PDLLA modified by cRGD polypeptide through Michael addition of MAL and sulfhydrylated polypeptide cRGD-SH. The maleimide functionalized MAL-PEG-PDLLA is prepared by initiating ring-opening polymerization of D, L-lactide by MAL-PEG. In N₂Under the circumstance, 2.5 mL of MAL-PEG (M _n0.5mL (0.2 mol/L) of a toluene stock of stannous octoate was added rapidly to an anhydrous toluene solution of 5.0 kg/mol,0.5 g, 0.1 mmol) and D, L-lactide (0.4g, 2.8 mmol). After reacting for 48 h in a constant temperature oil bath at 110 ℃, glacial acetic acid is added to stop the reaction. The product was then precipitated in glacial ethyl ether, filtered off with suction and dried in vacuo to give MAL-PEG-PDLLA. Then dissolving MAL-PEG-PDLLA and cRGD-SH in DMF, and reacting at room temperature for 24 h. The product was dialyzed against DMF for 48 h, then against deionized water for 24 h and finally freeze-dried. Yield: 85.4 percent.¹H NMR (600 MHz, DMSO-d ₆): δ 7.0-7.4 cRGD, 5.16 (-CH(CH₃)O- ), 3.65 (-CH ₂CH ₂O-), 1.56 (-CH(CH ₃) O-), see FIG. 2 (B).M _n(¹H NMR) = 8.8 kg/mol。M _n(GPC) = 13.9 kg/mol。M _w/M _n(GPC) = 1.3. The grafting yield of cRGD was 94% by BCA test.

EXAMPLE four Synthesis of amphiphilic targeting Polymer GE11-PEG-PDLLA

The synthesis of the targeting polymer GE11-PEG-PDLLA modified by GE11 polypeptide is similar to the synthesis process of cRGD modified cRGD-PEG-PDLLA in example III. Dissolving MAL-PEG-PDLLA and GE11-SH in DMF, and reacting at room temperature for 24 h. The product was dialyzed against DMF for 48 h, then against deionized water for 24 h and finally freeze-dried. Yield: 89.6 percent.¹H NMR (600 MHz, DMSO-d ₆): δ 6.5-7.1 GE11, 5.16 (-CH(CH₃)O- ),3.65 (-CH ₂CH ₂O-), 1.56 (-CH(CH ₃) O-), see FIG. 2 (C).M _n(¹H NMR) = 8.8 kg/mol。M _n(GPC) = 13.9 kg/mol。M _w/M _n(GPC) = 1.3. The grafting yield of cRGD was 96% by BCA test.

EXAMPLE five Synthesis of amphiphilic targeting Polymer TAT-PEG-PDLLA

The synthesis of the targeting polymer TAT-PEG-PDLLA modified by TAT polypeptide is similar to the synthesis process method of the cRGD modified cRGD-PEG-PDLLA in the third embodiment. Dissolving MAL-PEG-PDLLA and TAT-SH in DMF, and reacting at room temperature for 24 h. The product was dialyzed against DMF for 48 h, then against deionized water for 24 h and finally freeze-dried. Yield: 84.3 percent.¹H NMR (600 MHz, DMSO-d ₆): δ 7.0-7.4 TAT, 5.16 (-CH(CH₃)O- ),3.65 (-CH ₂CH ₂O-), 1.56 (-CH(CH ₃) O-), see FIG. 2 (D).M _n(¹H NMR) = 8.8 kg/mol。M _n(GPC)= 13.9 kg/mol。M _w/M _n(GPC) = 1.3. The grafting ratio of TAT was 98% by BCA test.

EXAMPLE six Synthesis of targeting Polymer HA-b-PDLLA

HA-b-PDLLA encapsulation of HA with Azide by alkynylationEnd PDLLA (N)₃-PDLLA) by a click chemistry reaction. Firstly, aldehyde group on hyaluronic acid and amino group on propynylamine are subjected to aldehyde-amine condensation reaction to generate Schiff base, and then the Schiff base is reduced into imine by cyano sodium borohydride to prepare a product Alkyn-HA; the method comprises the following specific steps: under the protection of nitrogen, oligomeric hyaluronic acid (HA, 1360 mg, 0.17 mmol), propynylamine (37.4 mg, 0.68 mmol) and sodium cyanoborohydride (42.8 mg, 0.68 mmol) were added to deionized water (15 mL), and the reaction was stirred at 60 ℃ for two days under sealed conditions, followed by adjusting the temperature to 40 ℃ and continuing the reaction for two days. Dialyzing the reaction solution, and freeze-drying to obtain a white solid which is the intermediate product Alkyn-HA. Yield: 89 percent.¹H NMR (600 MHz,D₂O)：1.94 (s, -OCH₃), 2.91 (t, -CH₂CH₂N-), 4.38-4.48 (m, HA)。

Under the protection of nitrogen in a glove box, D, L-lactide was placed in a closed reactor, and 1.2 mL of methylene chloride was added to dissolve it sufficiently. Followed by the sequential addition of azidoethanol (4.35 mg, 0.05 mmol) and 1,5, 7-triazabicyclo [4.4.0]Dec-5-ene (TBD, 6.95 mg, 0.05 mmol) was used as initiator and catalyst. The system was stirred thoroughly and transferred out of the glove box after sealing. The reaction was carried out at 30 ℃ for 4 h and then quenched by the addition of two drops of glacial acetic acid. Precipitating the product in frozen anhydrous ether with the volume 15-20 times of the volume of the reaction solution in excess. Filtering, vacuum drying to obtain viscous light yellow solid as intermediate product N₃-PDLLA). Yield: 91 percent.¹H NMR (600 MHz, CDCl₃): 5.16 (-CH(CH₃)O- ), 1.56 (-CH(CH ₃) O-). Nuclear magnetic calculation gives the molecular weight: 5.9 kDa. Measuring the molecular weight by GPC: 11.0 kDa, molecular weight distribution: 1.2.

amphiphilic polymers are prepared by Click chemistry (Click chemistry) of alkynyl and azide. Taking the alkynyl-modified hyaluronic acid derivative Alkyn-HA (500 mg, 0.06 mmol) synthesized in the experiment and N₃PDLLA (416 mg, 76. mu. mol) was dissolved in DMSO, respectively, and transferred to a two-necked flask after it was dissolved sufficiently. The total volume of DMSO was kept at 15 mL. Subsequently, nitrogen was introduced to remove oxygen from the solution. Mixing copper sulfate pentahydrate (1.5 mg, 0.006 mmol) and sodium ascorbate (2.4 mg, 0.012 mm)ol) are respectively dissolved in 10 mu L of deionized water, and are sequentially added into a reaction system after being dissolved. Setting the reaction temperature at 50 ℃, and the reaction time for 24 hours under the protection of nitrogen in the reaction process. After the reaction was completed, a yellow transparent liquid was obtained. Dialyzing with a dialysis bag having a molecular weight cutoff of 15000 in DMSO to remove unreacted N₃PDLLA, followed by dialysis in EDTA salt solution and deionized water sequentially for 2 days. After the dialysis is finished, the mixture is concentrated, frozen and dried, and finally the white solid which is the final product HA-b-PDLLA. After drying, a sample was taken for nuclear magnetism. Yield: 86 percent.¹H NMR (600 MHz, D₂O/DMSO-d ₆= 1/9) delta (ppm) 1.79,3.03-3.67, and 4.41-4.49 (HA) (= 1/9) delta (ppm) (= PDLLA) 1.56 and 5.16 (PDLLA), see FIG. 3.

EXAMPLE seventhly preparation of crosslinked polymeric and non-crosslinked nanoparticles

10 mg of sP-LA and 10 mg of PEG-PDLLA are respectively dissolved in 1mL of DMSO to prepare a solution of 10 mg/mL, 100 mL of sP-LA solution and 30 mL of PEG-PDLLA are uniformly mixed, the mixture is dripped into 870mL of phosphate buffer solution (PB, 10mM, pH 7.4), the mixture is stirred for 0.5 h at room temperature, 10mM of DTT solution is added under the condition of nitrogen, a small bottle is sealed, and the mixture is incubated for 10 h in a constant temperature shaking table at 37 ℃ and 100 rpm. The solution was then placed in dialysis bags (MWCO 3500) and dialyzed in a large volume of dialysis medium (PB, 10mM, pH 7.4) for 24 hours, with five water changes, to give the crosslinked polymeric nanoparticles, sPLY XNPs. The size of the obtained cross-linked polymer nanoparticles is 73nm measured by a dynamic light scattering particle size analyzer (DLS), the particle size distribution is very narrow, as shown in FIG. 4A, as can be seen from FIG. 4A, the nanoparticles are in a solid spherical structure measured by TEM, the cross-linked nanoparticles still keep unchanged particle size and particle size distribution under high dilution and the presence of fetal calf serum (FIG. 4B), but rapidly swell and un-cross-link under the environment simulating the reduction of tumor cells (FIG. 4C). Therefore, the obtained crosslinked nanoparticles have the property of reducing sensitivity and decrosslinking, and are suitable for drug carriers.

Non-crosslinked nanoparticles were prepared similarly to crosslinked nanoparticles, but without the need for DTT crosslinking. Two sets of controls were set for non-crosslinked nanoparticles, each prepared by co-assembling the star polymer and linear polymer of example one with PEG-PDLLA. The method specifically comprises the following steps: 100 mL of star polymer or linear polymer solution (10 mg/mL in DMSO) and 30 mL of PEG-PDLLA solution (10 mg/mL in DMSO) are uniformly mixed, dropped into 870mL of phosphate buffer solution (PB, 10mM, pH 7.4), stirred at room temperature for 0.5 h, and then the solution is placed into a dialysis bag (MWCO 3500), dialyzed in a large amount of dialysis medium (PB, 10mM, pH 7.4) for 24 h, and replaced with five times of water to obtain non-crosslinked polymer nanoparticles sPLY NPs and lplyNPs. The size of the obtained polymer nanoparticles was 85 nm as measured by dynamic light scattering particle size analyzer (DLS), and the particle size distribution was narrow.

EXAMPLE eight preparation of Targeted crosslinked and non-crosslinked nanoparticles with cRGD as targeting molecule

Preparing the targeted cross-linked nano particles with cRGD as the targeted molecules: dissolving PEG-PDLLA and cRGD-PEG-PDLLA in 30 mL DMSO according to a certain mass ratio, and preparing the targeted cross-linked nanoparticles with the PEG-PDLLA and the cRGD-PEG-PDLLA as second polymers by referring to the method of the seventh example, wherein the targeted cross-linked nanoparticles are of a solid spherical structure. PEG-PDLLA and cRGD-PEG-PDLLA are mixed according to different proportions to prepare the cross-linked nano particles with targeting molecules with different mass ratios on the surface, and when the content of the cRGD-PEG-PDLLA in the second polymer is 50 wt.%, the size of the targeted cross-linked vesicle measured by DLS is about 85 nm, and the particle size distribution is narrow. The target cross-linked nano particles taking the cRGD as the target molecules are abbreviated as cRGD XNPs.

Preparing targeted non-crosslinked nanoparticles with cRGD as targeted molecules: dissolving PEG-PDLLA and cRGD-PEG-PDLLA in 30 mL DMSO according to a certain mass ratio, and respectively assembling the PEG-PDLLA and the cRGD-PEG-PDLLA which are used as second polymers with the star polymer and the linear polymer in the first embodiment, and preparing the targeted non-crosslinked nanoparticles by the method in the seventh embodiment. PEG-PDLLA and cRGD-PEG-PDLLA are mixed according to different proportions to prepare the cross-linked nano particles with targeting molecules with different mass ratios on the surface, and when the content of the cRGD-PEG-PDLLA in the second polymer is 50 wt.%, the size of the targeted cross-linked vesicle measured by DLS is about 90 nm, and the particle size distribution is narrow. The targeted non-crosslinked nanoparticles taking cRGD as the targeted molecules are referred to as cRGD NPs for short.

EXAMPLE nine preparation of Targeted Cross-Linked nanoparticles with GE11 Polypeptides as targeting molecules

Assembling the second polymer of PEG-PDLLA and GE11-PEG-PDLLA with sP-LA, and preparing the targeted cross-linked nanoparticles by the method of example seven. When the content of GE11-PEG-PDLLA in the second polymer is 20 wt.%, the size of the targeted cross-linked vesicle is about 87 nm by DLS measurement, and the particle size distribution is narrow.

EXAMPLE Ten preparation of GE11/TAT Dual-Targeted Cross-Linked Polymer nanoparticles

The second polymer, PEG-PDLLA, GE11-PEG-PDLLA and TAT-PEG-PDLLA, was assembled with sP-LA to prepare crosslinked nanoparticles as in example seven. In the second polymer, the content of GE11-PEG-PDLLA is 20 wt.%, the content of TAT-PEG-PDLLA is 20 wt.%, the size of the targeted cross-linked vesicle is about 104 nm by DLS measurement, and the particle size distribution is narrow.

EXAMPLE eleventh preparation of targeted crosslinked and non-crosslinked nanoparticles with HA as targeting molecule

Referring to the method of example seven, PEG-PDLLA was replaced with HA-bPDLLA and sP-LA are co-assembled to obtain cross-linked polymer nanoparticles HA-sPLY XNPs, the formed nanoparticles are 90 nm according to DLS, and the particle size distribution is narrow, as shown in FIG. 6A; as shown in FIG. 6B, when TEM determines that the nanoparticles are solid spherical structures, the cross-linked nanoparticles still maintain unchanged particle size and particle size distribution in the presence of high-power dilution and fetal calf serum (FIG. 6C), but are rapidly released and de-cross-linked in a simulated tumor cell reduction environment (FIG. 6D). Therefore, the obtained crosslinked nanoparticles have the property of reducing sensitivity and decrosslinking, and are suitable for drug carriers.

Referring to the method of example seven, PEG-PDLLA was replaced with HA-bAnd (3) self-assembling PDLLA with the star polymer and the linear polymer in the first embodiment to obtain non-crosslinked polymer nanoparticles HA-sPLY NPs and HA-lPLy NPs, wherein the formed nanoparticles are 105 nm and 110 nm respectively, and the particle size distribution is narrow.

Examples dodeca-crosslinked and non-crosslinked nano-drug loaded doxorubicin and in vitro release

The preparation method of the targeting cross-linked nano-particle, the preparation method of the drug-loaded cross-linked nano-particle and the preparation method of the empty nano-particle are similar. Specifically, 100 mL of sP-LA solution and 30 mL of PEG-PDLLA were added to 15 mL of DOX in DMSO (10 mg/mL) and mixed well, and the mixture was added dropwise to 850mL of phosphate buffer solution (PB, 10mM, pH 7.4) and stirred at room temperature for 0.5 h. Under nitrogen, 10mM DTT solution was added, the vial was sealed, and incubated at 37 ℃ for 10 h on a constant temperature shaker at 100 rpm. The solution was then placed in dialysis bags (MWCO 3500) and dialyzed in a large volume of dialysis medium (PB, 10mM, ph 7.4) for 24 hours, and water was changed five times to obtain drug-loaded (DOX) cross-linked nanoparticles with a size of 85 nm as measured by dynamic light scattering particle size analyzer (DLS) and a narrow particle size distribution. In addition, non-crosslinked nanoparticles were prepared as a control, in the same manner as the crosslinked nanoparticles, but without the need for DTT crosslinking. The obtained nano-drugs are named DOX-sPLY XNPs, DOX-sPLY NPs and DOX-lPLy NPs, the carried drugs are DOX, the XNPs are cross-linked nanoparticles, the NPs are non-cross-linked nanoparticles, and the other names are analogized.

The particle size of the cross-linked polymer nano particles loaded with drugs with different proportions (10-30 wt.%) obtained by adopting DOX DMSO solutions with different concentrations is 150 nm, and the particle size distribution is 0.15-0.19; the encapsulation efficiency of the polymer nanoparticles to DOX is 85-98% as determined by a fluorescence spectrometer.

In vitro release experiments of DOX with drug-loaded cross-linked nanoparticles (DOX-sPLY XNPs) were performed by shaking (200 rpm) in a 37 ℃ constant temperature shaker, with three replicates per group. First, crosslinked DOX-loaded nanoparticles were added to 10mM GSH simulated intracellular reducing environment PB (10mM, pH 7.4); second, DOX-loaded cross-linked nanoparticles in PB (10mM, pH 7.4); the concentration of the drug-loaded cross-linked nanoparticles was 100 mg/L, 0.5mL was placed in dialysis bags (MWCO: 12,000), 25 mL of the corresponding dialysis solvent was added to each tube, 5.0 mL of the medium outside the dialysis bags was removed at predetermined time intervals for testing, and 5.0 mL of the corresponding medium was added to the tubes. The concentration of the drug in the solution was determined using a fluorimeter. FIG. 4D is a graph showing the relationship between the cumulative release amount of DOX and time, and it can be seen that after GSH in the simulated tumor cells is added, the release is significantly faster than that of the sample without GSH, thus indicating that the drug-loaded cross-linked nanoparticles can effectively release the drug in the presence of 10mM GSH.

The in vitro release experiment of the drug-loaded non-crosslinked nanoparticles on DOX is carried out under the same condition as that of the crosslinked nanoparticles. FIG. 4D is a graph showing the relationship between the cumulative release amount of DOX and time, and it can be seen that, after GSH in the tumor-mimicking cells is added, DOX-sPLY NPs and DOX-lPLy NPs have no significant enhancement effect on the release amount of DOX, and exhibit slow sustained release behavior similar to the release amount under normal physiological conditions.

Example in vitro Release of DOX by thirteen cRGD Targeted Cross-Linked/non-Cross-Linked nanoparticles

Referring to the method of example eight and example twelve, cRGD targeted cross-linked drug loaded (DOX) nanoparticles (DOX-cRGD XNPs) and cRGD targeted non-cross-linked drug loaded nanoparticles (DOX-cRGD NPs) were prepared, and an in vitro release experiment of DOX was performed according to the method of example twelve.

FIG. 5D is a graph showing the relationship between the cumulative release amount of DOX and time, and it can be seen from the graph that, after GSH in the simulated tumor cells is added to DOX-cRGD XNPs, the release is obviously faster than that of the sample without GSH, which indicates that the drug-loaded cross-linked nanoparticles can effectively release the drug in the presence of 10mM GSH. The introduction of cRGD does not affect the release behavior of the nanoparticles on DOX.

FIG. 5D is a graph showing the relationship between the cumulative release amount of DOX and time, and it can be seen that, after the simulated tumor cell GSH was added, DOX-cRGD NPs did not significantly enhance the release amount of DOX, and exhibited slow sustained release behavior similar to the release amount under normal physiological conditions, and the release behavior was not changed by the introduction of cRGD.

Example in vitro release of Docetaxel (DTX) by fourteen crosslinked nanoparticles

Cross-linked drug-loaded nanoparticles (DTX-sly XNPs) were prepared according to the method of example twelve, and an in vitro release experiment of DTX was performed according to the method of example twelve, and the concentration of DTX drug in solution was determined using high performance liquid chromatography. It can be seen from the relationship between the cumulative release amount of DTX and time that after GSH in the simulated tumor cells is added, the release is obviously faster than that of a sample without GSH, which indicates that the drug-loaded cross-linked nanoparticles can effectively release the drug in the presence of 10mM GSH.

Example fifteen polypeptide Targeted Cross-Linked nanoparticle Release of Docetaxel (DTX)

Referring to the methods of example nine, example ten and example twelve, GE11 targeted cross-linked drug-loaded nanoparticles (DTX-GE11 XNPs), GE11/TAT targeted cross-linked drug-loaded nanoparticles (DTX-GE11/TAT XNPs) were prepared, and an in vitro release experiment of DTX was performed according to the method of example twelve, and DTX drug concentration in the solution was determined using a high performance liquid chromatograph. It can be seen from the relationship between the cumulative release amount of DTX and time that after GSH in the simulated tumor cells is added, the release is obviously faster than that of a sample without GSH, which indicates that the drug-loaded cross-linked nanoparticles can effectively release the drug in the presence of 10mM GSH. The introduction of GE11 and TAT did not affect the release behavior of the nanoparticles on DOX.

Example release of Docetaxel (DTX) by sixteen HA targeted cross-linked/non-cross-linked nanoparticles

Referring to the methods of example nine, example ten and example twelve, HA-targeted cross-linked drug-loaded nanoparticles (DTX-HA-sLy XNPs), HA-targeted non-cross-linked drug-loaded nanoparticles (DTX-HA-sLy NPs, DTX-HA-lPLyNPs) were prepared, and in vitro release experiments for DTX were performed according to the method of example twelve, and DTX drug concentration in solution was determined using high performance liquid chromatography.

FIG. 6D is a graph showing the relationship between DTX cumulative release amount and time, and it can be seen that, after GSH in the simulated tumor cells is added, the release is significantly faster than that of the sample without GSH, which indicates that the drug-loaded cross-linked nanoparticles can effectively release the drug in the presence of 10mM GSH.

FIG. 6D is a graph showing the cumulative amount of DTX released over time, and it can be seen that DTX-HA-sPLY NPs and DTX-HA-lPLy NPs release significantly more DTX than cross-linked nanoparticles under the same conditions under normal physiological conditions, indicating that the non-cross-linked nanoparticles have poor colloidal stability.

Example effect of polypeptide Density on the surface of seventeen-Nano drugs on endocytosis of nanoparticles

At 5X 10⁶And (4) planting the cells in a 6-well plate with each well being 1mL, and culturing the cells until the cells are attached to the wall by about 70% after 24 hours. Then, adding nanoparticle samples containing different polypeptide target densities into each hole of the experimental group, incubating for 4 h, digesting and centrifuging each group of cells, re-dispersing with 500 μ L PBS, placing into a special flow measurement tube, and measuring by a flow cytometer; wherein the experimental group is set as Lipo-DOX (existing), DOX-cRGD XNPs, DOX-sPLY XNPs, DOX-cRGD NPs and DOX-sPLYNPs, and a sample blank control hole is set.

Determining the endocytosis behavior of the targeted nanoparticles with cRGD as the targeting molecule in B16F10 cells, and fig. 7 (A) shows that the crosslinked nanoparticles show stronger fluorescence signals due to quick response release; the cRGD-modified cross-linked nanoparticles had the highest fluorescence intensity in cells, see fig. 7 (B);

measuring the endocytosis of targeted and Cy 5-labeled nanoparticles with GE11 and TAT as targeting molecules in MDA-MB-231 breast cancer cells, wherein experimental groups are Cy 5-labeled double-targeted cross-linked nanoparticles (GE 11/(10, 15, 20, 25, 30)% TAT XNPs) with different TAT ratios, Cy 5-labeled cross-linked nanoparticles (GE 11 XNPs) with GE11 targeting molecules and Cy 5-labeled cross-linked nanoparticles (XNPs), and FIG. 7 (C) shows that the endocytosis of the targeted cross-linked nanoparticles with the GE11 targeting molecules is obviously higher than that of the cross-linked nanoparticles, and the intracellular fluorescence intensity is increased along with the increase of the TAT amount; the following cellular and animal experiments selected groups were GE11/20% TAT XNPs (hereinafter abbreviated as GE11/TAT XNPs), GE11XNPs, XNPs.

Specific binding of Cy 5-labeled cross-linked nanoparticles Cy 5-HA-spllyxnps to a549 cells was determined in a549 cells with HA as the targeting molecule fig. 7 (D).

Example seventeen-seven MTT method A blank Polymer was tested for cytotoxicity

The MTT method uses mouse melanoma cells (B16F 10), human non-small cell lung cancer cells (A549), triple negative breast cancer cells (MDA-MB-231) to test the cytotoxicity of the blank nanoparticles. At 4X 10³One/well, cells were seeded in 96-well plates, 100 per wellMu L, and culturing after 24 hours until the cell adheres to the wall by about 70%. Then, the nanoparticle samples with different concentrations (0.1-1 mg/mL) were added to each well of the experimental group, and a cell blank control well and a culture medium blank well (duplicate 4 wells) were separately set. After 24 hours of incubation, 10. mu.L of MTT (5.0 mg/mL) was added to each well, and after 4 hours of incubation, 150. mu.L of DMSO was added to each well to dissolve the crystals formed, and the absorbance value (A) was measured at 490 nm using a microplate reader, and the cell viability was calculated by zeroing out the blank wells of the medium.

FIG. 8 (A) shows the cytotoxicity results of sPLY XNPs, sPLY NPs and lPLy NPs on B16F10, and it can be seen that when the concentration of the cross-linked polymer nanoparticles is increased from 0.1 to 0.5 mg/mL, the survival rate of B16F10 is still higher than 90%, which indicates that the side chain thioctanoyl group-containing star-shaped polymer has good biocompatibility; fig. 8 (B) shows that both cRGD XNPs, cRGD NPs incubated B16F10 cell viability was above 95%; FIG. 8 (C) shows that all nanoparticles with TAT on the surface were removed, and that MDA-MB-231 cell viability was higher than 90% when other groups of nanoparticles (GE 11/TAT XNPs, GE11XNPs, XNPs) were incubated; FIG. 8 (D) shows that the cell viability of HA-sPLY XNPs and HA-sPLY NPs, HA-lPLy NPs was above 90% after incubation of A549 cells.

The blank nanoparticles have little toxicity to cells, which indicates that the carriers have good biocompatibility.

Example eighteen MTT method for measuring toxicity of drug-loaded polymer nanoparticles to cells

The cytotoxicity of DOX-sPLY XNPs, DOX-sPLY NPs, DOX-lPLy NPs (DOX concentrations of 0.001 to 20. mu.g/mL) prepared in example twelve against B16F10 was tested. The cells were cultured as in example fourteen, and after 4 hours of co-culture, the aspirated samples were replaced with fresh medium and incubated for a further 44 hours, after which time MTT was added, processed and absorbance measured as in example seventeen.

The results in FIG. 9(A) show that the drug-loaded cross-linked nanoparticles DOX-sLy XNPs have the highest cell proliferation inhibition effect, and the half-lethal concentration (IC) of the drug-loaded cross-linked nanoparticles on the cells₅₀) 1.8 mg/mL, IC of the respective products compared with those of the non-crosslinked control DOX-sPLY NPs and DOX-lPLy NPs₅₀The values are 2.4 and 4.2 times lower, indicating that the crosslinked nanoparticles areThe medicine is well delivered into cells and effectively released, and finally cancer cells are killed.

The same methods and conditions were used to test the cytotoxicity of DOX-cRGD XNPs and DOX-cRGD NPs against B16F 10. The results show that the targeted cross-linked nanoparticles have high cytotoxicity on B16F10 cells and IC (integrated circuit) of the targeted cross-linked nanoparticles₅₀0.92 mg/mL, 1.95 times, 3.48 times and 4.70 times lower than DOX-XNPs, DOX-cRGD NPs and DOX NPs, respectively, as shown in FIG. 9 (B); the targeted cross-linked nano particles have good targeting effect, can effectively transmit the medicament into cells, and can effectively release the medicament to finally kill cancer cells.

The same method and conditions test the killing ability of the DTX-loaded cross-linked nanoparticles with GE11 and TAT bipolypeptides as targeting molecules to MDA-MB-231 cells. The experimental results showed that DTX-GE11/TAT XNPs have the strongest ability to inhibit cell proliferation, IC, of MDA-MB-231₅₀0.05 mg/mL, 4.6 times, 9.8 times and 16.2 times lower for DTX-GE11 XNPs, DTX-XNPs and free DTX, respectively, see FIG. 9 (C); the double-targeting cross-linked nanoparticles have good targeting effect on MDA-MB-231 cells, can efficiently transmit the drugs into the cells, and can be rapidly released to finally kill cancer cells.

The same methods and conditions were used to test the in vitro anti-tumor activity of DTX-HA-sPLY XNPs, DTX-HA-sPLY NPs, DTX-HA-lPLy NPs on A549 cells; MTT results showed that DTX-HA-sLy XNPs have the strongest anti-cell proliferation effect on A549, and IC thereof₅₀0.18 mg/mL, which is 2.1 times, 6.6 times and 4.6 times lower than DTX-HA-sLy NPs, DTX-HA-lPLy NPs and free DTX, respectively, see FIG. 9 (D); the targeted cross-linked nanoparticles have good targeting effect on A549 cells, can efficiently transmit the medicament into the cells, and can be rapidly released to finally kill cancer cells.

Example blood circulation of nineteen drug-loaded particles

All animal experimental procedures were in compliance with the provisions of the animal experimental centre of the university of suzhou. Balb/C nude mice with the weight of about 18-20 g and the age of 4-6 weeks are selected for the experiment. DOX-sPLY XNPs, DOX-sPLY NPs, DOX-lPLy NPs were all prepared according to example twelve. Injecting each group of drug-loaded cross-linked nanoparticles into mice through tail vein (DOX dosage is 10 mg/kg), performing fixed-point blood sampling at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours, centrifuging blood samples (3000 rpm, 6 min), taking 20 mu L of serum, and adding 100 mu L, 1% triton and 500 mu L of anhydrous DMSO for extraction (containing 20 mM DTT); the amount of DOX at each time point was measured by fluorescence spectroscopy. In FIG. 10 (A), the abscissa represents time, and the ordinate represents DOX concentration. As can be seen, DOX-sPLY XNPs have longer circulation time, about 4.94 h, higher than that of non-crosslinked nanoparticles (DOX-sPLY NPs: 4.43h, DOX-lPLy NPs: 3.46 h), so that the drug-loaded crosslinked polymer vesicles are more stable in mice and have longer circulation time than the non-crosslinked nanoparticles.

Examples blood circulation of the twenty-DOX-cRGD XNPs, DOX-cRGD NPs

Animals are as in example seventeen. DOX-cRGD XNPs and DOX-cRGD NPs are prepared as in example eight and tested as in example nineteen. FIG. 10 (B) shows time on the abscissa and DOX concentration on the ordinate. As can be seen from the figure, DOX-cRGD XNPs have longer circulation time, which is about 5.16 h and is higher than that of non-crosslinked nanoparticles DOX-cRGD NPs, so that the drug-loaded crosslinked polymer vesicles are more stable in mice compared with the non-crosslinked nanoparticles and have longer circulation time, and the introduction of cRGD polypeptide does not have great influence on the in-vivo circulation time of the nanoparticles.

Example twenty-one DTX particle-loaded blood circulation

Animals are as in example seventeen. DTX-GE11/TAT XNPs, DTX-GE11 XNPs and DTX-sLy XNPs were prepared as in example nine and tested in the same manner as in example nineteen by high performance liquid chromatography to determine the amount of DTX at each time point. In FIG. 10(C), the abscissa represents time and the ordinate represents DTX concentration. As can be seen, DTX-GE11/TAT XNPs, DTX-GE11 XNPs, DTX-XNPs have similar cycle times, all being significantly longer than free DTX (0.34 h) for about 5 h. Therefore, the drug-loaded cross-linked polymer nanoparticles have longer circulation time in a mouse, and the introduction of the polypeptide, particularly the introduction of TAT, has no great influence on the circulation of the nanoparticles.

Example blood circulation of twenty-two HA Targeted drug loaded particles

Animals are as in example seventeen. DTX-HA-sLy XNPs, DTX-HA-sLy NPs and DTX-HA-lPLGA NPs were prepared as in example eight and tested in the same manner as in example nineteen, and the amount of DTX at each time point was determined by high performance liquid chromatography. In fig. 10(D), the abscissa represents time, and the ordinate represents the concentration of DTX. As can be seen, DTX-HA-sLy XNPs have a longer cycle time, about 4.18 h, higher than that of the non-crosslinked nanoparticles DTX-HA-sLy NPs (3.5), DTX-HA-lPLGA NPs (2.97) and free DTX (0.23); therefore, the drug-loaded cross-linked polymer nanoparticles are more stable in a mouse body and have longer circulation time compared with non-cross-linked nanoparticles.

Example in vivo biodistribution of Twenty-three DOX particles in B16F10 melanoma-bearing mice

Animals were injected subcutaneously with 1X 10 as in seventeen examples⁵After about 7 days, the tumor size of the B16F10 human lung cancer cell is 100-200 mm³The experiment was started. DOX-cRGD XNPs, DOX-cRGD NPs, tail vein injection mice (DOX: 10 mg/kg) were prepared according to example eight, the mice were sacrificed after 8 hours, the tumors and heart, liver, spleen, lung and kidney tissues were removed, washed, weighed, 500. mu.L of 1% triton was added and ground by a homogenizer, and 900. mu.L of anhydrous DMSO (containing 20 mM DTT) was added and extracted. After centrifugation (20000 rpm, 20 min), the supernatant was taken and the amount of DOX in each tissue was determined by fluorescence spectroscopy. In FIG. 11(A), the abscissa is the tissue organ and the ordinate is the total DOX injection per gram of tumor or tissue (ID%/g). The DOX amount accumulated in the tumor after the DOX-cRGD XNPs are injected for 8 hours is 10.96ID%/g, which is 2.1 times of that of the DOX-cRGD XNPs, so that the medicament-loaded DOX-cRGD XNPs are accumulated more in the tumor part through active targeting, and have better tumor specific targeting effect.

Example in vivo biodistribution of Twenty-four DTX particles in MDA-MB-231 Breast cancer-bearing mice

Tumor inoculation and tail vein administration in biodistribution experiments were as in eighteen examples. First, DTX-GE11/TAT XNPs, DTX-GE11 XNPs, DTX-sLy XNPs were prepared according to example nine. The amount of DTX in each tissue was measured by high performance liquid chromatography using the method of example twenty three in tail vein injection mice (DTX: 10 mg/kg). In fig. 11(B) the abscissa is tissue organ and the ordinate is DTX to total DOX injection per gram of tumor or tissue (ID%/g). DTX-GE11/TAT XNPs are injected for 8 hours, the DTX amount accumulated in the tumor is 9.72 ID%/g, and is respectively 1.88, 3.12 and 8.75 times of DTX-GE11 XNPs, DTX-sPLYXNPs and free DTX, so that the DTX-GE11/TAT XNPs are accumulated more in the tumor site through active targeting, and the tumor specific targeting effect is better.

Example in vivo biodistribution of twenty-five HA-targeted DTX-loaded particles in A549 lung cancer-bearing mice

Tumor inoculation and tail vein administration in biodistribution experiments were as in eighteen examples. DTX-HA-sPLY XNPs, DTX-HA-sPLY NPs and DTX-HA-lPLGA NPs were prepared according to example nine, using the method of example twenty-four. In fig. 11(C) the abscissa is tissue organ and the ordinate is DTX to total DOX injection per gram of tumor or tissue (ID%/g). DTX amount of DTX-HA-sPLY XNPs accumulated in the tumor after 8 hours of injection is 9.48ID%/g which is 1.5,2.0 and 3.9 times of DTX-HA-sPLY NPs, DTX-HA-lPLGA NPs and free DTX respectively, which shows that the DTX-HA-sPLY XNPs are accumulated more in the tumor part through active targeting and have better tumor specific targeting effect.

Example therapeutic Effect of hexacosanol particles in B16F10 melanoma-bearing mice

Tumor inoculation and tail vein administration as in eighteen examples, the tumor size was 30-50 mm after about two weeks³The experiment was started. DOX-cRGD XNPs, DOX-sPLY XNPs, DOX-cRGD NPs, Lipo-DOX and PBS were injected into mice via tail vein on

days

0, 2, 4, 6 and 8, respectively (DOX dose of 10 mg/kg), and a high dose administration group (DOX dose of 20 mg/kg) was set for DOX-cRGD XNPs. Weighing the weight of the mouse every three days for 0-12 days, measuring the tumor volume by using a vernier caliper, wherein the tumor volume calculation method comprises the following steps: v = (L × W)/2, (where L, W, H is the length, width, and thickness of the tumor, respectively). Mice were observed continuously for 45 days of survival. As can be seen from FIG. 12, the DOX-cRGD XNPs (DOX: 20 mg/kg) treatment group showed significant tumor suppression at day 12, which was equivalent to the Lipo-DOX group, and miceThere was no significant change in weight, and it was noted that the mice in the Lipo-DOX group had a weight loss of about 15%. The DOX-cRGD XNPs, DOX-sPLY XNPs and DOX-cRGD NPs have certain tumor growth, but the growth speed and the growth amount are obviously lower than those of the PBS group, and the weight change of the mouse is not caused, so that the drug-loaded polymer nanoparticles have no toxic or side effect on the mouse. Median survival for DOX-cRGD XNPs (DOX: 20 mg/kg) mice was 43 days, median survival for DOX-cRGD XNPs, DOX-sPLY XNPs, DOX-cRGD NPs and Lipo-DOX and PBS group mice was 30, 26, 25, 21 and 14 days, respectively. In addition, all treatment groups except the Lipo-DOX group did not cause obvious damage to the main organs of the mice, and the liver, spleen and kidney of the mice in the Lipo-DOX group were obviously damaged.

Example therapeutic Effect of twenty-seven DTX particles in MDA-MB-231 Breast cancer-bearing mice

Tumor inoculation and tail vein administration were as in nineteen examples. DTX-GE11/TAT XNPs, DTX-GE11 XNPs, DTX-XNPs and free DTX and PBS were injected into mice via tail vein on

days

0,3, 6 and 9, respectively (DTX dose was 5 mg/kg). Mice were weighed every three days on days 0-18, tumor volumes were measured as in example twenty-six and mice were observed for 60 days. As can be seen from FIG. 13, the tumors in DTX-GE11/TAT XNPs, DTX-GE11 XNPs, DTX-XNPs and free DTX treated groups were inhibited to different degrees at 18 days, while the tumors in PBS group were significantly increased. It is noted that DTX-GE11/TAT XNPs, DTX-GE11 XNPs and DTX-XNPs can obviously and effectively inhibit the growth of tumors, and the body weight of the mice is not obviously changed. In the free DTX group, although tumor growth was somewhat inhibited, the weight loss of the mice was significant, indicating that free DTX caused severe systemic toxicity to the mice.

Example therapeutic Effect of twenty-eight HA-targeted DTX-loaded particles on A549 lung cancer-bearing mice

Inoculation of a549 subcutaneous tumors and tail vein administration were as in nineteen examples. DTX-HA-sLy XNPs, DTX-HA-sLy NPs, DTX-HA-lPLGA NPs and free DTX and PBS were injected into mice via tail vein at 0, 4, 8 and 12 days, respectively (DTX dose was 5 mg/kg). Mice were weighed as in example twenty in 0-16 days, tumor volume quantified and observed for 60 days. As shown in FIG. 14, DTX-HA-sPLY XNPs, DTX-HA-sPLY NPs, DTX-HA-lPLGA NPs and free DTX treatment resulted in some inhibition of the tumor, while the tumor growth was evident in the PBS group. It is worth noting that the body weight of the mice has no obvious change except for the free DTX group, the body weight of the mice is seriously reduced by about 15% in 16 days due to the strong toxic and side effect of DTX, and the results show that the drug-loaded nanoparticles have good biocompatibility and have no toxic and side effect on the mice. In addition, the DTX-HA-sPLY XNPs treatment group HAs significant difference relative to the DTX-HA-sPLY NPs and DTX-HA-lPLGA NPs treatment group, and shows the most excellent effect of inhibiting tumor growth.

Claims

1. The nano-drug based on the terminal sulfur-containing octanoyl star polymer is characterized in that the nano-drug based on the terminal sulfur-containing octanoyl star polymer is obtained by reversibly crosslinking a polymer nanoparticle with reduction responsiveness and loading a drug;

the reversible crosslinked polymer nano particle with reduction responsiveness is prepared by assembling a star polymer with a sulfur octanoyl-containing terminal and a second polymer together; the second polymer is an amphiphilic polymer and/or a targeting amphiphilic polymer; the amphiphilic polymer is PEG-PDLLA; the target amphiphilic polymer is cRGD-PEG-PDLLA, GE11-PEG-PDLLA, TAT-PEG-PDLLA and HA-b-one or more of PDLLA;

wherein x and y are (1-3) to 1;

the preparation method of the nano-drug based on the terminal sulfur-containing octanoyl star polymer comprises the following steps:

(2) under the vacuum environment, under the catalytic action of stannous octoate, taking polyhydroxy glucose as an initiator, and reacting lactide and glycolide for 8 hours at 160 ℃; obtaining a star polymer; the molar ratio of the polyhydroxy glucose to the lactide to the glycolide is 1 to (50-55) to (60-65);

2. The nano-drug based on the terminal sulfur-containing octanoyl star polymer of claim 1, wherein after the reaction of the step (1) is completed, the reaction solution is filtered to obtain lipoic acid anhydride solution; the solvent in the N, N-dicyclohexylcarbodiimide solution is dichloromethane; the solvent in the thioctic acid solution is dichloromethane; the mol ratio of the N, N-dicyclohexylcarbodiimide to the lipoic acid is 2: 1.

3. The nano-drug based on the terminal sulfur-containing octanoyl star polymer of claim 1, wherein the star polymer is obtained by dissolving the product obtained by the reaction in the step (2) in dichloromethane, and then precipitating in ice methanol, filtering, and drying in vacuum.

4. The nano-drug based on the terminal sulfur-containing octanoyl star polymer of claim 1, wherein the star polymer containing the terminal sulfur-containing octanoyl is obtained by precipitating the reactant in glacial ethyl ether after the reaction in the step (3), and then performing suction filtration and vacuum drying on a filter cake; the organic solvent in the organic solvent containing the 4-dimethylaminopyridine and the star-shaped polymer is dichloromethane; the mol ratio of the sulfur caprylic anhydride, the 4-dimethylaminopyridine and the star polymer is 7.5: 10: 1.

5. The nano-drug based on the terminal sulfur-containing octanoyl star polymer of claim 1, wherein: the dosage of the second polymer is 10-60% of the mass of the star polymer with the terminal containing the sulfur octanoyl group.

6. The nano-drug based on the terminal sulfur-containing octanoyl star polymer of claim 1, wherein: the medicine comprises adriamycin, paclitaxel and docetaxel.

7. The nano-drug based on the terminal sulfur-containing octanoyl star polymer of claim 1, wherein: the molecular weight of the star polymer with the terminal containing the sulfur octanoyl group is 10000-75000.

8. The nano-drug based on the terminal sulfur-containing octanoyl star polymer of claim 1, wherein: when the second polymer is an amphiphilic polymer and a targeting amphiphilic polymer, the mass percentage of the targeting amphiphilic polymer is less than 70%.