CN108392471B - Nanomedicines based on terminal lipoyl star polymers - Google Patents

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

Nanomedicines based on terminal lipoyl star polymers

technical field

The invention relates to a biocompatible polymer material and an application thereof, in particular to a nanomedicine based on a terminal lipoyl-containing star-shaped biocompatible polymer, which belongs to the field of medical materials.

Background technique

Polymer materials with good biocompatibility and biodegradability have been widely used in biomedical fields including tissue engineering and drug controlled release. The polymer nano-drugs prepared by the technology have the problems of unstable circulation in vivo, low tumor cell uptake, low intracellular drug concentration, and slow intracellular drug release rate, resulting in low efficacy of nano-drugs and toxic side effects caused by drug leakage. ; For example, the results of BIND-014 in the second clinical phase did not meet expectations, due to the insufficient stability of the nanodrug in vivo.

Cancer is the main killer that threatens human health, and its incidence and mortality are increasing year by year. The emergence of nano-drugs has brought new hope for the treatment of cancer, but in the existing technology, there is still a lack of high-efficiency nano-drugs with stable circulation in vivo, cancer-specific targeting, rapid intracellular response to release drugs, and less toxic and side effects. There is a lack of polymeric nanocarriers that can maintain stability during in vivo circulation and rapidly release drugs within cells. Based on this, it is imminent to develop a nanocarrier that can prolong the circulation time of drugs in vivo and rapidly release drugs in tumor cells.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a nanomedicine based on a star-shaped biocompatible polymer containing lipoyl at the end.

In order to achieve the above object, the specific technical scheme of the present invention is: a nanomedicine based on a star-shaped polymer containing lipoyl at the end, wherein the nanomedicine based on the star-shaped polymer containing lipoyl at the end is reversible cross-linking and has reduction properties. Responsive polymer nanoparticles loaded with drugs are obtained;

The reversibly cross-linked polymer nanoparticles with reduction responsiveness are prepared by co-assembly of a star-shaped polymer containing a lipoyl group at the end and a second polymer; the second polymer is an amphiphilic polymer and/or targeting amphiphilic polymers;

The chemical structural formula of the star-shaped polymer containing lipoyl at the end is as follows:

Wherein, x:y is (1-3):1.

The star-shaped polymer containing lipoyl group at the end disclosed in the present invention can be called sP-LA, and the molecular weight is 10000-75000.

The preparation of the above-mentioned star-shaped polymer containing lipoyl group at the end comprises the following steps:

(1) Under nitrogen environment and ice-water bath, add N,N-dicyclohexylcarbodiimide solution dropwise to lipoic acid solution; after the dropwise addition is completed, seal and react at room temperature for 10-15 hours; obtain lipoic anhydride solution ;

(2) In a vacuum environment, polyhydroxyglucose is used as an initiator, and under the catalysis of stannous octoate, lactide and glycolide are reacted at 160 ° C for 8 hours; a star polymer is obtained;

(3) Under nitrogen environment, add lipoic anhydride solution to the organic solvent containing 4-dimethylaminopyridine and star polymer, and react at 30°C for 1 to 3 days under sealed conditions; Star polymers of acyl groups.

Specifically, the preparation method can be as follows:

(1) In a nitrogen environment, lipoic acid is dissolved in an organic solvent to prepare a lipoic acid solution; N,N-dicyclohexylcarbodiimide is prepared in an organic solvent to prepare N,N-dicyclohexylcarbodiimide Then, under the ice-water bath, the N,N-dicyclohexylcarbodiimide solution was added dropwise to the lipoic acid solution; after the dropwise addition was completed, the reaction was sealed at room temperature for 10 to 15 hours; after the reaction was completed, the reaction solution was filtered to obtain sulfur Caprylic anhydride solution;

(2) In the _N2 environment, polyhydroxyglucose, lactide and glycolide were added to the closed reaction flask, then the catalyst stannous octoate was added to the reaction flask and all the materials were mixed uniformly; then the reaction flask was pumped out. Vacuum-substitute N for _three times, and finally the reaction flask was evacuated for 30 minutes, and the reaction flask was sealed; the polymerization reaction was carried out in a vacuum box at 160 °C for 8 hours; after the reaction, the product was dissolved in dichloromethane, and then in ice methanol. Precipitation, suction filtration and vacuum drying to obtain star polymer;

(3) Under nitrogen environment, add lipoic anhydride solution to the organic solvent containing 4-dimethylaminopyridine and star polymer, and react at 30°C for 1 to 3 days under sealed conditions; Precipitation in glacial ether, suction filtration and vacuum drying of the filter cake to obtain a star polymer (sP-LA) containing a lipoyl group at the end.

The preparation method of above-mentioned nanometer medicine, comprises the following steps:

(1) Under nitrogen environment and ice-water bath, add N,N-dicyclohexylcarbodiimide solution dropwise to lipoic acid solution; after the dropwise addition is completed, seal and react at room temperature for 10-15 hours; obtain lipoic anhydride solution ;

(2) In a vacuum environment, polyhydroxyglucose is used as an initiator, and under the catalysis of stannous octoate, lactide and glycolide are reacted at 160 ° C for 8 hours; a star polymer is obtained;

(3) Under nitrogen environment, add lipoic anhydride solution to the organic solvent containing 4-dimethylaminopyridine and star polymer, and react at 30°C for 1 to 3 days under sealed conditions; Star polymers of acyl groups;

(4) The nano-drug is obtained by assembling the star-shaped polymer containing a lipoyl group at the end with the second polymer and the small molecule drug in a solvent.

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

In the present invention, the molar ratio of polyhydroxyglucose, lactide and glycolide is 1:(50-55):(60-65); the prepared star-shaped polymer has the following structural formula:

In the present invention, the molar ratio of N,N-dicyclohexylcarbodiimide and lipoic acid is 2:1; the molar ratio of lipoic anhydride, 4-dimethylaminopyridine and star polymer is 7.5:10: 1.

The star-shaped polymer containing lipoyl group at the end of the present invention can be modified with amphiphilic polymers such as cRGD-PEG-PDLLA, GE11-PEG-PDLLA, TAT-PEG-PDLLA with polypeptides or polysaccharide molecules with different targeting functions , HA-b-PDLLA are assembled together to prepare nanoparticles with different targeting molecules such as cRGD, GE11, TAT or HA and other polypeptides and polysaccharide-modified nanoparticles to obtain different cancer cell-specific targeting polymer nanoparticles, with target tropism and biocompatibility. Nanoparticles formed by such polymers have good stability, high loading efficiency, and can be specifically targeted to cancer cells.

The invention also discloses a reversibly cross-linked polymer nanoparticle with reduction responsiveness, which is prepared by co-assembly of the above-mentioned star-shaped polymer containing lipoyl groups in the side chain and a second polymer; the second polymer is Amphiphilic polymer and/or targeted amphiphilic polymer; preferably, the amount of the second polymer is 10% to 60% of the mass of the star polymer containing lipoyl groups at the end; when the second polymer When the substance is an amphiphilic polymer and a targeting amphiphilic polymer, the mass percentage of the targeting amphiphilic polymer is less than 70%; in the present invention, the amphiphilic polymer does not have a targeting molecule, and the targeting amphiphilic polymer The polymer carries targeting molecules such as cRGD, GE11, TAT or HA and other polypeptides and polysaccharides; preferably the second polymer is an amphiphilic polymer and a targeting amphiphilic polymer. That is, the polymer nanoparticles of the present invention are prepared by co-assembling the above-mentioned star-shaped polymer containing lipoyl group in the side chain and an amphiphilic polymer; or by the above-mentioned star-shaped polymer containing lipoyl group in the side chain and a graft target It is prepared by co-assembly of the amphiphilic polymer of the molecule; or prepared by co-assembly of the above-mentioned star-shaped polymer containing lipoyl group in the side chain and the amphiphilic polymer and the amphiphilic polymer of the grafted target molecule, For example, the above-mentioned star-shaped polymer containing lipoyl group in the side chain and the amphiphilic polymer of the grafted targeting molecule and the amphiphilic polymer can be mixed in different proportions to prepare polymer nanoparticles with different targeting densities, that is, Obtaining polymer nanoparticles with active targeting function of cancer cells can increase the uptake of drug-loaded polymer nanoparticles in cancer cells.

The star-shaped polymer containing lipoyl group in the side chain disclosed in the present invention has excellent biocompatibility and biodegradability. By co-assembling with the second polymer to form polymer nanoparticles, the polymer nanoparticle can be prepared in a catalytic amount of a reducing agent such as In the presence of dithiothreitol (DTT) or glutathione (GSH), cross-linked polymer nanoparticles or cross-linked polymer nanoparticles with active targeting function to cancer cells were prepared, with a particle size of 70 ~180 nanometers, can be used as a carrier of drugs for the treatment of cancer; hydrophobic small molecule anti-lung cancer drugs doxorubicin (DOX), paclitaxel (PTX), docetaxel (DTX), etc. can be loaded in polymer nanoparticles to improve the hydrophobicity The bioavailability of the drug in the body, prolonging the circulation time of the drug, and improving the enrichment of the drug in the tumor site. Therefore, the nanomedicine disclosed in the present invention is obtained by loading small molecule medicines with cross-linked polymer nanoparticles. The cross-linked polymer nanoparticles prepared from the star-shaped polymer containing lipoyl groups in the side chain disclosed in the present invention form stable chemical cross-links in the inner core, so that they can be stably circulated in the body for a long time; In the intracellular reducing environment, the cross-linking is quickly de-linked, the drug is quickly released, and the cancer cells are efficiently killed. Therefore, the present invention claims the use of the above-mentioned star-shaped polymer containing lipoyl group in the side chain, polymer nanoparticles, cross-linked polymer nanoparticles, and nanomedicines in the preparation of drugs for the treatment of tumors such as melanoma, lung cancer and triple-negative breast cancer. application.

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

1. The present invention utilizes a star-shaped polymer to react with lipoic anhydride to obtain a star-shaped polymer containing a lipoyl group in a side chain with a controllable degree of substitution, which gives the star-shaped polymer new functions and enriches the types of the star-shaped polymer. .

2. The star-shaped polymer containing lipoyl group in the side chain disclosed in the present invention has excellent biocompatibility and biodegradability, and can prepare polymer nanoparticles and polymer nanoparticles with cancer cell active targeting function, Different drugs can be loaded and cross-linked by disulfide bonds to obtain stable cross-linked polymer nano-drugs, thereby overcoming the defects of the prior art that nano-drugs are unstable in in vivo circulation, easy to release drugs early, and cause toxic and side effects.

3. The cross-linked polymer nanomedicine disclosed in the present invention has reversible cross-linking, that is, it supports long-term circulation in the body and can be highly enriched in cancer cells; It kills cancer cells with high efficiency and specificity without toxic and side effects; it overcomes the defects of chemically cross-linked nano-drugs in the prior art that are too stable, slow drug release in cells, and cause drug resistance.

4. The biocompatible polymer nanoparticles and the polymer nanoparticles with the active targeting function of cancer cells disclosed in the present invention can form reduction-sensitive disulfide bond crosslinks during the preparation process, and the preparation method is simple and convenient, thereby overcoming the existing problems. In the prior art, the preparation of cross-linked nanomedicines requires complicated operations and purification processes.

5. The cross-linked polymer nanoparticles prepared by the co-assembly of the star-shaped polymer containing lipoyl in the side chain and the amphiphilic polymer disclosed in the present invention can be used for the controlled release system of hydrophobic anticancer drugs, thereby overcoming the prior art. There is no defect of hydrophobic anticancer drugs that can efficiently load and stabilize the circulation in vivo; further, it can be bonded to target molecules, which has wider application value in the efficient targeted therapy of cancer.

Description of drawings

Fig. 1 is the hydrogen nuclear magnetic spectrogram of the star polymer sP-LA containing lipoyl group in the side chain of the embodiment;

Fig. 2 is the amphiphilic polymer PEG-PDLLA (A), cRGD-PEG-PDLLA (B), GE11-PEG-PDLLA (C) and TAT-PEG-PDLLA (D) in embodiment two, three, four and five. ) of the NMR spectrum;

Fig. 3 is the nuclear magnetic spectrum of the amphiphilic polymer HA- b -PDLLAPEG-PDLLA in Example 6;

Figure 4 shows the particle size distribution and transmission electron microscope image of the cross-linked nanoparticle sPLy XNPs in Example 7 (A), the cross-linked nanoparticle stability (B), the reduction responsiveness test (C) and the in vitro release image (D) ;

Figure 5 shows the particle size distribution (A) and transmission electron microscopy (B), reduction responsiveness test (C) and in vitro release ( D);

Figure 6 shows the particle size distribution (A) and transmission electron microscope (B), reduction responsiveness test (C) and in vitro release of the cross-linked nanoparticle HA-sPLy XNPs with HA as the targeting molecule in Example 11 Figure (D);

Figure 7 shows the effect of different nanoparticles on B16F10 cells (A), cRGD-XNPs on B16F10 cells (B), GE11/TAT-XNPs on MDA-MB-231 cells (C) and HA-sPLy XNPs in Example 17 Graph of uptake in A549 cells (D);

Figure 8 shows blank nanoparticles sPLy XNPs on B16F10 cells (A), cRGD-XNPs on B16F10 cells (B), GE11/TAT-XNPs on MDA-MB-231 cells (C) and HA-sPLy XNPs in Example 18 Toxicity to A549 cells (D);

Figure 9 shows drug-loaded nanoparticles DOX-sPLy XNPs on B16F10 cells (A), DOX-cRGDXNPs on B16F10 cells (B), DTX-GE11/TAT XNPs on MDA-MB-231 cells and DTX-HA in Example 19 - Graph of the toxicity results of sPLy XNPs on A549 cells;

Figure 10 shows the drug-loaded nanoparticles DOX-sPLy XNPs (A), DOX-cRGD XNPs (B), DTX-HA-sPLy XNPs (C) in mice in Example 20, 21, and 23. Diagram of the results of blood circulation research;

Figure 11 shows the biodistribution of drug-loaded nanoparticles DOX-cRGD XNPs in Example 24 and 26 in B16F10 melanoma (A) and DTX-HA-sPLy XNPs in A549 lung cancer subcutaneous tumor (B) mice picture;

Figure 12 is a graph showing the tumor inhibition situation of DOX-loaded cross-linked nanoparticle DOX-cRGDXNPs with cRGD as the targeting molecule in B16F10 melanoma-bearing mice in Example 27, wherein A is the tumor growth curve, and B is the small Changes in mouse body weight, C is the survival curve;

Figure 13 shows the expression of DTX-loaded cross-linked nanoparticles DTX-GE11/TAT XNPs with GE11 and TAT as targeting molecules in Example 28 in nude mice bearing triple-negative breast cancer MDA-MB-231 subcutaneous tumor. Tumor inhibition graph, in which A is the tumor growth curve, B is the tumor picture of mice after treatment, C is the body weight change, and D is the survival curve;

Figure 14 is a graph showing the tumor inhibition of the DTX-targeted cross-linked nanoparticle DTX-HA-sPLy XNPs with HA as the targeting molecule in Example 29 in nude mice bearing A549 lung cancer subcutaneous tumor, wherein A is the tumor growth Curve, B is body weight change, C is tumor inhibition rate, D is tumor picture of mice after treatment.

Detailed ways

Below in conjunction with embodiment and accompanying drawing, the present invention is further described:

Example 1 Synthesis of star polymer containing lipoyl in side chain

Synthetic star polymers and linear polymers

Star polymers can be synthesized by the ring-opening polymerization of lactide and glycolide under the catalysis of stannous octoate by using polyhydroxyglucose as an initiator. Under N atmosphere, 0.18 _g (1 mmol) polyhydroxyglucose, 7.5 g (52 mmol) lactide and 7.5 g (65 mmol) glycolide were added to a closed reaction flask, followed by 4.73 mg of catalyst octoate Tin was added to the reaction vial and all contents were mixed well. The reaction vial was then evacuated - displaced N ₃ three times, and finally the reaction vial was evacuated for 30 min and the reaction vial was sealed. The polymerization was carried out in a vacuum oven at 160°C for 8 hours. The crude product was dissolved in dichloromethane, then precipitated in glacial methanol, filtered off with suction and dried in vacuo to give the star polymer. ¹ H NMR (600 MHz, CDCl ₃ ): δ 5.22 (-OOCC H ₂ O-), 4.83 (-OOCC H (CH ₃ )O-), 2.45 (, -OCC H ₂ -), 1.6 (-CH ( CH3 ₎ O-).

The linear polymer was synthesized by the ring-opening polymerization of lactide and glycolide under the catalysis of stannous octoate with 1,4-butanediol as the initiator. Under N atmosphere, 0.9 _g (1 mmol) 1,4-butanediol, 7.5 g (52 mmol) lactide and 7.5 g (65 mmol) glycolide were added to a closed reaction flask, followed by 4.73 g 1 mg of catalyst stannous octoate was added to the reaction flask and all contents were mixed well. The reaction vial was then evacuated - displaced N ₃ three times, and finally the reaction vial was evacuated for 30 min and the reaction vial was sealed. The polymerization was carried out in a vacuum oven at 200°C for 5 hours. The crude product was dissolved in dichloromethane, then precipitated in glacial methanol, filtered off with suction and dried in vacuo to give a linear polymer.

Synthesis of lipoic anhydride LAA: under nitrogen atmosphere, 30 mg (0.15 mmol) of lipoic acid (LA) was dissolved in 1 mL of dichloromethane, added to a two-neck flask and stirred until dissolved, 60 mg (0.3 mmol) ) N,N-dicyclohexylcarbodiimide (DCC) in 0.5 mL of dichloromethane, under an ice-water bath, was added dropwise to the LA solution. Continue to pass nitrogen for 5 minutes, seal the two-necked flask, and react at room temperature for 12 hours; after the reaction, filter out the precipitate produced by the reaction to obtain a lipoic anhydride solution, and concentrate the lipoic anhydride (LAA) solution to 0.5 ml.

Synthesis of lipoyl-containing star polymer (sP-LA) in the side chain: under nitrogen atmosphere, 30 mg (0.075 mmol) solution of lipoic anhydride (LAA) was added to 150 mg (0.01 mmol) star polymer and 12 mg (0.1 mmol) DMAP in 2 mL of dichloromethane solution, continue to pass nitrogen for 5 minutes, seal the flask, and place it in an oil bath at 30 °C to react for 48 h; then the product is precipitated in ice ether, suction filtered and vacuum dried to obtain side The star polymer sP-LA containing lipoyl groups in the chain was obtained in 86.7% yield. ¹ HNMR (600 MHz, CDCl ₃ ): δ 5.22 (-OOCC H ₂ O-), 4.83 (-OOCC H (CH ₃ )O-), 3.57 (-CH ₂ CH CH ₂ CH ₂ S ₂ ), 3.17 ₍ -CH2CHCH2CH2S2 ₎ , 2.45 ( _-OCCH2- ) , 1.6 (-CH _{( CH3} ) _O- ). The grafting rate of LA calculated by NMR is 90%, as shown in Figure 1.

Example 2 Synthesis of amphiphilic polymer PEG-PDLLA

The amphiphilic polymer PEG-PDLLA can be prepared by the ring-opening polymerization of D,L-lactide initiated by the macroinitiator PEG. To 2.5 mL of a solution of PEG ( Mn =5.0 kg/mol, 0.5 g, 0.1 mmol) and D,L _- lactide (0.4 g, 2.8 mmol) in anhydrous toluene under _N2 , 0.5 mL (0.2 mol/L) stannous octoate stock solution in toluene. After reacting in a constant temperature oil bath at 110 °C for 48 h, glacial acetic acid was added to terminate the reaction. The product was subsequently precipitated in glacial ether, filtered with suction and dried in vacuo to obtain PEG-PDLLA, yield: 88.9%. ¹ H NMR (600 MHz, CDCl ₃ ): δ 5.16 (-CH ( CH ₃ )O- ), 3.65 (- CH ₂ CH ₂ O-), 3.38 ( CH ₃ O-), 1.56 (- CH( CH3 ₎ O-), see Figure 2(A). Mn ( ¹ HNMR) = 8.9 kg/mol, _Mn (GPC) = 15.9 kg/mol, M w _{/ Mn} ( _GPC ) = 1.3.

Example 3 Synthesis of amphiphilic targeting polymer cRGD-PEG-PDLLA

The targeting polymer cRGD-PEG-PDLLA was obtained by a two-step reaction. First, the maleimide-functionalized amphiphilic polymer MAL-PEG-PDLLA was synthesized, and then the cRGD polypeptide-modified amphiphilic polymer cRGD-PEG- was further synthesized by Michael addition of MAL to the thiolated polypeptide cRGD-SH. PDLLA. Maleimide-functionalized MAL-PEG-PDLLA was prepared by ring-opening polymerization of D,L-lactide initiated by MAL-PEG. To _2.5 mL of MAL-PEG ( M _n =5.0 kg/mol, 0.5 g, 0.1 mmol) and D,L-lactide (0.4 g, 2.8 mmol) in anhydrous toluene under N atmosphere, rapidly Add 0.5 mL (0.2 mol/L) of a toluene stock solution of stannous octoate. After reacting in a constant temperature oil bath at 110 °C for 48 h, glacial acetic acid was added to terminate the reaction. The product was then precipitated in glacial ether, filtered with suction and dried in vacuo to give MAL-PEG-PDLLA. Then MAL-PEG-PDLLA and cRGD-SH were dissolved in DMF and reacted 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%. ¹ H NMR (600 MHz, DMSO- d ₆ ): δ 7.0-7.4 cRGD , 5.16 (-CH ( CH ₃ )O- ), 3.65 (-CH ₂ CH ₂ O-), 1.56 (-CH ( CH ₃ )O-) , see Figure 2(B). Mn ^{( 1} _H NMR) = 8.8 kg/mol. M _n (GPC) = 13.9 kg/mol. M _w / M _n (GPC) = 1.3. The grafting rate of cRGD was 94% by BCA test.

Example 4 Synthesis of Amphiphilic Targeting Polymer GE11-PEG-PDLLA

The synthesis of GE11 polypeptide-modified targeting polymer GE11-PEG-PDLLA is similar to the synthesis process of cRGD-modified cRGD-PEG-PDLLA in Example 3. MAL-PEG-PDLLA and GE11-SH were dissolved in DMF and reacted 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%. ¹ H NMR (600 MHz, DMSO- d ₆ ): δ _6.5-7.1 GE11, 5.16 (-CH( CH3 _{) O-} ), 3.65 ( -CH2CH2O- ), 1.56 (-CH( CH ₃ )O-), see Figure 2(C). Mn ^{( 1} _H NMR) = 8.8 kg/mol. M _n (GPC) = 13.9 kg/mol. M _w / M _n (GPC) = 1.3. The grafting rate of cRGD was 96% by BCA test.

Example 5 Synthesis of amphiphilic targeting polymer TAT-PEG-PDLLA

The synthesis of TAT polypeptide-modified targeting polymer TAT-PEG-PDLLA is similar to the synthesis process of cRGD-modified cRGD-PEG-PDLLA in Example 3. MAL-PEG-PDLLA and TAT-SH were dissolved in DMF and reacted 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%. ¹ H NMR (600 MHz, DMSO- d ₆ ): δ 7.0-7.4 TAT, 5.16 (-CH (CH ₃ )O- ), 3.65 (-CH ₂ CH ₂ O-), 1.56 (-CH ( CH ₃ )O-) , see Figure 2(D). Mn ^{( 1} _H NMR) = 8.8 kg/mol. M _n (GPC) = 13.9 kg/mol. M _w / M _n (GPC) = 1.3. The grafting rate of TAT was 98% by BCA test.

Example 6 Synthesis of targeted polymer HA- b -PDLLA

HA- b -PDLLA was obtained by click chemistry of alkynylated HA with azide-terminated PDLLA (N3 _- PDLLA). First, the aldehyde group on hyaluronic acid and the amino group on propargylamine undergo aldehydramine condensation reaction to form Schiff base, which is then reduced to imine by sodium cyanoborohydride to obtain the product Alkyn-HA; the specific steps are: nitrogen gas Protected, oligomeric hyaluronic acid (HA, 1360 mg, 0.17 mmol), propargylamine (37.4 mg, 0.68 mmol), sodium cyanoborohydride (42.8 mg, 0.68 mmol) were added to deionized water (15 mL) , the reaction was stirred at 60 °C for two days under airtight conditions, and then the temperature was adjusted to 40 °C to continue the reaction for two days. The reaction solution was dialyzed and freeze-dried to obtain a white solid, which was the intermediate product Alkyn-HA. Yield: 89%. ¹ H NMR (600 MHz, D ₂ O): 1.94 (s, -OCH ₃ ), 2.91 (t, -CH ₂ CH ₂ N-), 4.38-4.48 (m, HA).

Under the protection of nitrogen in the glove box, D,L-lactide was placed in a closed reactor, and 1.2 mL of dichloromethane was added to make it fully dissolved. 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) were subsequently added as initiators and catalysts . Stir the system well, seal it and transfer it out of the glove box. The reaction was carried out at 30 °C for 4 h, and then two drops of glacial acetic acid were added to stop the reaction. The product was precipitated in ice-cold anhydrous ether with an excess of 15-20 times the volume of the reaction solution. Filtration and vacuum drying to finally obtain a viscous pale yellow solid, which is the intermediate product N ₃ -PDLLA). Yield: 91%. ¹ H NMR (600 MHz, CDCl ₃ ): 5.16 (-CH( CH ₃ )O- ), 1.56 (-CH( CH ₃ )O-). NMR calculated molecular weight: 5.9 kDa. Molecular weight measured by GPC: 11.0 kDa, molecular weight distribution: 1.2.

Amphiphilic polymers were prepared by click chemistry of alkynyl and azide groups. The alkynyl-modified hyaluronic acid derivatives Alkyn-HA (500 mg, 0.06 mmol) and N ₃ -PDLLA (416 mg, 76 μmol) synthesized in the above experiments were dissolved in DMSO, respectively. necked flask. Keep the total volume of DMSO at 15 mL. Nitrogen was then purged to remove oxygen from the solution. Copper sulfate pentahydrate (1.5 mg, 0.006 mmol) and sodium ascorbate (2.4 mg, 0.012 mmol) were dissolved in 10 μL of deionized water, respectively, and then added to the reaction system in sequence. The reaction temperature was set to 50 °C, nitrogen protection was performed during the reaction, and the reaction time was 24 h. After the reaction, a yellow transparent liquid was obtained. The unreacted N ₃ -PDLLA was removed by dialysis against DMSO using a dialysis bag with a molecular weight cut-off of 15,000, followed by dialysis against EDTA salt solution and deionized water for 2 days. After the dialysis, it was concentrated, freeze-dried, and finally a white solid was obtained, which was the final product HA- b -PDLLA. After drying, samples were taken for NMR. Yield: 86%. ¹ H NMR (600 MHz, D ₂ O/DMSO- d ₆ = 1/9) δ (ppm): 1.79, 3.03-3.67, and 4.41-4.49 (HA); 1.56 and 5.16 (PDLLA ), see Figure 3.

Example 7 Preparation of cross-linked polymer nanoparticles and non-cross-linked nanoparticles

Dissolve 10 mg of sP-LA and 10 mg of PEG-PDLLA in 1 mL of DMSO to prepare a 10 mg/mL solution, take 100 mL of sP-LA solution and 30 mL of PEG-PDLLA, mix well, and add dropwise to 870 mL of phosphate buffer. The solution (PB, 10 mM, pH 7.4) was stirred at room temperature for 0.5 h. Under nitrogen, 10 mM DTT solution was added, the vial was sealed, and incubated at 37 °C, 100 rpm in a constant temperature shaker for 10 h. The solution was then placed in a dialysis bag (MWCO3500) and dialyzed against a large amount of dialysis medium (PB, 10 mM, pH 7.4) for 24 hours with five water changes to obtain cross-linked polymer nanoparticles sPLy XNPs. The size of the obtained cross-linked polymer nanoparticles was measured by dynamic light scattering particle size analyzer (DLS), and the formed nanoparticles were 73 nm, and the particle size distribution was very narrow. Solid spherical structure, the cross-linked nanoparticles still maintain the same particle size and size distribution in the presence of high dilution and fetal bovine serum (Fig. 4C). It can be seen that the obtained cross-linked nanoparticles have reduction-sensitive properties of de-cross-linking, and are suitable for drug carriers.

The preparation of non-crosslinked nanoparticles is similar to that of crosslinked nanoparticles, but does not require the addition of DTT crosslinking. Two sets of controls were set for the non-crosslinked nanoparticles, which were prepared by co-assembly of the star polymer and linear polymer of Example 1 with PEG-PDLLA. Specifically: mix 100 mL star polymer or linear polymer solution (10 mg/mL in DMSO) and 30 mL PEG-PDLLA solution (10 mg/mL in DMSO), and add dropwise to 870 mL phosphate buffer solution ( PB, 10 mM, pH 7.4), stirred at room temperature for 0.5 h, then put the solution in a dialysis bag (MWCO3500), and dialyzed it in a large amount of dialysis medium (PB, 10 mM, pH 7.4) for 24 hours, then changed Water five times to obtain non-crosslinked polymer nanoparticles sPLy NPs, lPLy NPs. The size of the obtained polymer nanoparticles was measured by dynamic light scattering particle size analyzer (DLS). The nanoparticles formed were 85 nm with a narrow particle size distribution.

Example 8 Preparation of targeted cross-linked nanoparticles and non-cross-linked nanoparticles with cRGD as targeting molecule

Preparation of targeted cross-linked nanoparticles with cRGD as targeting molecule: PEG-PDLLA and cRGD-PEG-PDLLA were dissolved in 30 mL DMSO according to a certain mass ratio, and PEG-PDLLA and cRGD-PEG-PDLLA were used as the second polymerization The target cross-linked nanoparticle was prepared by referring to the method of Example 7, which was a solid spherical structure. Mixing PEG-PDLLA and cRGD-PEG-PDLLA in different proportions can prepare cross-linked nanoparticles with different mass ratios of targeting molecules on the surface. When the content of cRGD-PEG-PDLLA in the second polymer is 50 wt.%, The size of the targeted cross-linked vesicles determined by DLS was about 85 nm, and the particle size distribution was narrow. Targeted cross-linked nanoparticles with cRGD as the targeting molecule are abbreviated as cRGD XNPs.

Preparation of targeted non-crosslinked nanoparticles with cRGD as targeting molecule: PEG-PDLLA and cRGD-PEG-PDLLA were dissolved in 30 mL DMSO in a certain mass ratio, and PEG-PDLLA and cRGD-PEG-PDLLA were used as the second The polymer was assembled with the star polymer and the linear polymer of Example 1, respectively, and the targeted non-crosslinked nanoparticles were prepared by referring to the method of Example 7. Mixing PEG-PDLLA and cRGD-PEG-PDLLA in different proportions can prepare cross-linked nanoparticles with different mass ratios of targeting molecules on the surface. When the content of cRGD-PEG-PDLLA in the second polymer is 50 wt.%, The size of the targeted cross-linked vesicles determined by DLS was about 90 nm, and the particle size distribution was narrow. Targeted non-crosslinked nanoparticles with cRGD as the targeting molecule are abbreviated as cRGD NPs.

Example 9 Preparation of targeted cross-linked nanoparticles with GE11 polypeptide as targeting molecule

Using PEG-PDLLA and GE11-PEG-PDLLA as the second polymer to assemble with sP-LA, the targeted cross-linked nanoparticles were prepared as in Example 7. When the content of GE11-PEG-PDLLA in the second polymer was 20 wt.%, the size of the targeted cross-linked vesicles determined by DLS was about 87 nm, and the particle size distribution was narrow.

Example 10 Preparation of GE11/TAT dual-targeted cross-linked polymer nanoparticles

PEG-PDLLA, GE11-PEG-PDLLA and TAT-PEG-PDLLA were used as the second polymer to assemble with sP-LA, and cross-linked nanoparticles were prepared by the method of Example 7. In the second polymer, the content of GE11-PEG-PDLLA was 20 wt.%, and the content of TAT-PEG-PDLLA was 20 wt.%. The size of the targeted cross-linked vesicles determined by DLS was about 104 nm, and the particle size distribution was relatively high. narrow.

Example 11 Preparation of targeted cross-linked nanoparticles and non-cross-linked nanoparticles with HA as targeting molecule

With reference to the method of Example 7, PEG-PDLLA was replaced by HA- b -PDLLA and co-assembled with sP-LA to obtain cross-linked polymer nanoparticles HA-sPLy XNPs. The distribution is very narrow, as shown in Figure 6A; from Figure 6B, it can be seen that the nanoparticles are solid spherical structures measured by TEM, and the cross-linked nanoparticles still maintain the same particle size and particle size distribution in the presence of high dilution and fetal bovine serum (Fig. 6C), but rapidly released in a simulated tumor cell reducing environment, de-crosslinking (Fig. 6D). It can be seen that the obtained cross-linked nanoparticles have reduction-sensitive properties of de-cross-linking, and are suitable for drug carriers.

Referring to the method of Example 7, replace PEG-PDLLA with HA- b -PDLLA and self-assemble with the star polymer and linear polymer of Example 1 to obtain non-crosslinked polymer nanoparticles HA-sPLy NPs, HA- lPLy NPs, the formed nanoparticles are 105 nm and 110 nm, respectively, with a narrow particle size distribution.

Example 12 Cross-linked nanomedicine and non-crosslinked nanomedicine loaded with doxorubicin and released in vitro

The preparation of targeted cross-linked nanoparticles by solvent displacement method, preparation of drug-loaded cross-linked nanoparticles, and preparation of empty nanoparticles are similar. Specifically, take 100 mL of sP-LA solution and 30 mL of PEG-PDLLA, add 15 mL of DOX in DMSO solution (10 mg/mL), mix well, and add the mixture dropwise to 850 mL of phosphate buffer solution (PB, 10 mM, pH 7.4), stirred at room temperature for 0.5 h. Under nitrogen, add 10 mM DTT solution, seal the vial, and incubate for 10 h at 37 °C, 100 rpm incubator. The solution was then placed in a dialysis bag (MWCO3500), and it was dialyzed in a large amount of dialysis medium (PB, 10 mM, pH 7.4) for 24 hours, and the water was changed five times to obtain drug-loaded (DOX) cross-linked nanoparticles, The size was measured by dynamic light scattering particle size analyzer (DLS) to be 85 nm and the particle size distribution was narrow. In addition, non-cross-linked nanoparticles were also prepared as a control group, and the preparation method was the same as that of cross-linked nanoparticles, but without the addition of DTT for cross-linking. The obtained nanomedicines were named as DOX-sPLy XNPs, DOX-sPLy NPs, DOX-lPLy NPs, which indicated that the loaded drug was DOX, XNPs indicated cross-linked nanoparticles, NPs indicated non-cross-linked nanoparticles, and so on.

The cross-linked polymer nanoparticles with different proportions of drugs (10%-30wt.%) obtained by using DOX DMSO solutions of different concentrations have a particle size of 130-150 nm and a particle size distribution of 0.15-0.19; the fluorescence spectrometer determines the invention. The encapsulation efficiency of DOX by polymer nanoparticles is 85%-98%.

The in vitro release experiments of DOX by drug-loaded cross-linked nanoparticles (DOX-sPLy XNPs) were performed at 37 °C in a constant temperature shaker with shaking (200 rpm), and each group had three parallel samples. The first group, DOX-loaded cross-linked nanoparticles in PB (10 mM, pH 7.4) simulated intracellular reducing environment by adding 10 mM GSH; the second group, DOX-loaded cross-linked nanoparticles in PB (10 mM, pH 7.4) ; The concentration of drug-loaded cross-linked nanoparticles is 100 mg/L, take 0.5 mL and put it into a dialysis bag (MWCO: 12,000), add 25 mL of the corresponding dialysis solvent to each test tube, and take out 5.0 mL at predetermined time intervals The medium outside the dialysis bag was used for the test, and 5.0 mL of the corresponding medium was added to the test tube. Use a fluorometer to measure the drug concentration in the solution. Figure 4D shows the relationship between the cumulative release of DOX and time. It can be seen from the figure that after adding GSH in the simulated tumor cells, its release is significantly faster than that of the sample without GSH, indicating that the drug-loaded cross-linked nanoparticles are at 10 mM. In the presence of GSH, the drug can be effectively released.

The in vitro release experiments of DOX from drug-loaded non-cross-linked nanoparticles were performed under the same conditions as cross-linked nanoparticles. Figure 4D shows the relationship between the cumulative release of DOX and time. It can be seen from the figure that after adding GSH in simulated tumor cells, DOX-sPLy NPs and DOX-lPLy NPs did not significantly enhance the release of DOX, which was different from that in The amount of release under normal physiological conditions is similar, showing a slow sustained release behavior.

Example 13 In vitro release of DOX by cRGD-targeted cross-linked/non-cross-linked nanoparticles

Refer to the methods of Example 8 and Example 12 to prepare cRGD-targeted cross-linked drug-loaded (DOX) nanoparticles (DOX-cRGD XNPs) and cRGD-targeted non-cross-linked drug-loaded nanoparticles (DOX-cRGD NPs). The method of Example 12 was used to conduct the in vitro release experiment of DOX.

Figure 5D shows the relationship between the cumulative release of DOX and time. It can be seen from the figure that for DOX-cRGD XNPs, after adding GSH in simulated tumor cells, the release is significantly faster than that of samples without GSH, indicating that the drug-loaded The linked nanoparticles can effectively release the drug in the presence of 10 mM GSH. The introduction of cRGD did not affect the release behavior of DOX from nanoparticles.

Figure 5D shows the relationship between the cumulative release of DOX and time. It can be seen from the figure that after adding GSH in simulated tumor cells, DOX-cRGD NPs did not significantly enhance the release of DOX, which was different from that under normal physiological conditions. The release amount was similar, showing a slow sustained release behavior, and its release behavior was not changed by the introduction of cRGD.

Example 14 In vitro release of docetaxel (DTX) by cross-linked nanoparticles

The cross-linked drug-loaded nanoparticles (DTX-sPLy XNPs) were prepared with reference to the method of Example 12, the in vitro release experiment of DTX was carried out according to the method of Example 12, and the drug concentration of DTX in the solution was measured by high performance liquid chromatography. From the relationship between the cumulative release of DTX and time, it can be seen that the release of GSH in the simulated tumor cells is significantly faster than that of the sample without GSH, indicating that the drug-loaded cross-linked nanoparticles in the presence of 10 mM GSH, Can effectively release drugs.

Example 15 Release of Docetaxel (DTX) by Polypeptide Targeted Cross-linked Nanoparticles

Preparation of GE11 targeted cross-linked drug-loaded nanoparticles (DTX-GE11 XNPs) and GE11/TAT targeted cross-linked drug-loaded nanoparticles (DTX-GE11/TAT XNPs) with reference to the methods of Example 9, Example 10 and Example 12 ), the in vitro release experiment of DTX was carried out according to the method of Example 12, and the drug concentration of DTX in the solution was measured by high performance liquid chromatography. From the relationship between the cumulative release of DTX and time, it can be seen that the release of GSH in the simulated tumor cells is significantly faster than that of the sample without GSH, indicating that the drug-loaded cross-linked nanoparticles in the presence of 10 mM GSH, Can effectively release drugs. The introduction of GE11 and TAT did not affect the release behavior of DOX from nanoparticles.

Example 16 Release of docetaxel (DTX) by HA-targeted cross-linked/non-cross-linked nanoparticles

HA targeted cross-linked drug-loaded nanoparticles (DTX-HA-sPLy XNPs) and HA-targeted non-cross-linked drug-loaded nanoparticles (DTX-HA-sPLy NPs, DTX-HA-1PLyNPs), the in vitro release experiment of DTX was carried out according to the method of Example 12, and the drug concentration of DTX in the solution was measured by high performance liquid chromatography.

Figure 6D shows the relationship between the cumulative release of DTX and time. It can be seen from the figure that after adding GSH in the simulated tumor cells, the release is significantly faster than that of the sample without GSH, indicating that the drug-loaded cross-linked nanoparticles are at 10 mM. In the presence of GSH, the drug can be effectively released.

Figure 6D shows the relationship between the cumulative release of DTX and time. It can be seen from the figure that under normal physiological conditions, the release of DTX from DTX-HA-sPLy NPs and DTX-HA-lPLy NPs is significantly more than that of cross-linked nanoparticle. The release amount of the particles under the same conditions indicates that the colloidal stability of the non-crosslinked nanoparticles is poor.

Example 17 The effect of nanomedicine surface polypeptide density on endocytic nanoparticles

Cells were seeded in a 6-well plate at 5×10 ⁶ cells/well, 1 mL per well, and cultured until about 70% of the cells adhered after 24 hours. Then, nanoparticle samples containing different peptide targeting densities were added to each well of the experimental group. After incubation for 4 h, the cells in each group were digested and centrifuged, redispersed with 500 μL PBS, and placed in a dedicated flow assay tube. Cytometry assay; the experimental group was set as Lipo-DOX (existing), DOX-cRGD XNPs, DOX-sPLy XNPs, DOX-cRGD NPs, DOX-sPLyNPs, and sample blank control wells were set.

The endocytosis behavior of targeting nanoparticles with cRGD as targeting molecule was measured in B16F10 cells. Figure 7(A) shows that the cross-linked nanoparticles showed a strong fluorescence signal due to the rapid response release; cRGD-modified cross-linked nanoparticles The linked nanoparticles have the highest fluorescence intensity in cells, as shown in Figure 7(B);

The endocytosis of Cy5-labeled nanoparticles with GE11 and TAT as targeting molecules was determined in MDA-MB-231 breast cancer cells. The experimental group was Cy5-labeled and double-targeted cross-linking with different TAT ratios. Nanoparticles (GE11/(10, 15, 20, 25, 30)% TAT XNPs), Cy5-labeled cross-linked nanoparticles with GE11 targeting molecules (GE11 XNPs), Cy5-labeled cross-linked nanoparticles (XNPs), Figure 7(C) shows that the endocytosis of the targeted cross-linked nanoparticles with the GE11 targeting molecule is significantly higher than that of the cross-linked nanoparticles, in addition, with the increase of the amount of TAT, the intracellular fluorescence intensity is also enhanced; the following The experimental groups selected for cell experiments and animal experiments were GE11/20%TAT XNPs (hereinafter referred to as GE11/TAT XNPs), GE11XNPs, and XNPs.

The specific binding of HA-targeted, Cy5-labeled cross-linked nanoparticles Cy5-HA-sPLyXNPs to A549 cells was determined in A549 cells (Figure 7(D).

Example 17 Cytotoxicity of blank polymer tested by MTT method

The cytotoxicity of blank nanoparticles was tested by MTT assay using mouse melanoma cells (B16F10), human non-small cell lung cancer cells (A549), and triple negative breast cancer cells (MDA-MB-231). Cells were seeded in a 96-well plate at 4×10 ³ cells/well, 100 μL per well, and cultured until about 70% of the cells adhered after 24 hours. Then, nanoparticle samples containing different concentrations (0.1-1 mg/mL) were added to each well of the experimental group, and blank control wells and medium blank wells (4 wells) were set separately. After culturing for 24 hours, 10 μL of MTT (5.0 mg/mL) was added to each well, and after culturing for 4 hours, 150 μL of DMSO was added to each well to dissolve the resulting crystals, and the absorbance value (A) was measured at 490 nm with a microplate reader. The medium blank wells were zeroed and cell viability was calculated.

Figure 8(A) shows the cytotoxicity results of sPLy XNPs, sPLy NPs, and lPLy NPs on B16F10. It can be seen that when the concentration of cross-linked polymer nanoparticles increased from 0.1 to 0.5 mg/mL, the survival rate of B16F10 remained unchanged. higher than 90%, indicating that the star-shaped polymer containing lipoyl in the side chain has good biocompatibility; Figure 8(B) shows that the survival rate of B16F10 cells incubated with cRGD XNPs and cRGD NPs is higher than 95%; Figure 8(B) 8(C) shows that the survival rate of MDA-MB-231 cells incubated with other groups of nanoparticles (GE11/TAT XNPs, GE11 XNPs, XNPs) was higher than 90% after removing the nanoparticles whose surfaces were all TAT; Figure 8(D) ) showed that the cell viability of HA-sPLy XNPs and HA-sPLy NPs and HA-lPLy NPs was higher than 90% after incubation in A549 cells.

The above blank nanoparticles have little toxicity to cells, indicating that these carriers have good biocompatibility.

Example 18 Determination of the toxicity of drug-loaded polymer nanoparticles to cells by MTT method

The DOX-sPLy XNPs, DOX-sPLy NPs, and DOX-lPLy NPs (with DOX concentrations ranging from 0.001 to 20 μg/mL) prepared in Example 12 were tested for their cytotoxicity to B16F10. The cells were cultured in the same way as in Example 14. After co-cultivation for 4 hours, the samples were aspirated and replaced with fresh medium for 44 hours of incubation. Then, MTT was added, processed, and measured for absorbance as in Example 17.

Figure 9(A) shows that the drug-loaded cross-linked nanoparticle DOX-sPLy XNPs has the highest inhibitory effect on cell proliferation, and its half-lethal concentration (IC ₅₀ ) on cells is 1.8 mg/mL, which is higher than that of the non-cross-linked control, respectively. The _IC50 values of the groups DOX-sPLy NPs and DOX-lPLy NPs were 2.4 and 4.2 times lower, indicating that the cross-linked nanoparticles could well deliver the drug into the cells and release them effectively, eventually killing cancer cells.

The same methods and conditions were used to test the cytotoxicity of DOX-cRGD XNPs and DOX-cRGD NPs against B16F10. The results showed that the targeted cross-linked nanoparticles had high cytotoxicity against B16F10 cells with an IC ₅₀ of 0.92 mg/mL, which was 1.95-fold, 3.48-fold and 4.70-fold lower than that of DOX-XNPs, DOX-cRGD NPs and DOX NPs, respectively. times, as shown in Figure 9(B); it shows that the targeted cross-linked nanoparticles have a good targeting effect, and can effectively deliver the drug into the cell and release it effectively, and finally kill the cancer cells.

The same method and conditions were used to test the killing ability of DTX-loaded cross-linked nanoparticles with GE11 and TAT double polypeptides as targeting molecules on MDA-MB-231 cells. The experimental results showed that DTX-GE11/TAT XNPs had the strongest ability to inhibit cell proliferation on MDA-MB-231, and its IC ₅₀ was 0.05 mg/mL, which was 4.6 times lower than that of DTX-GE11 XNPs, DTX-XNPs and free DTX, respectively. , 9.8 times, and 16.2 times, as shown in Figure 9(C); it shows that the dual-targeted cross-linked nanoparticles have a good targeting effect on MDA-MB-231 cells, can efficiently deliver drugs into cells, and quickly release, eventually killing the cancer cells.

The same method and conditions were used to test the in vitro antitumor activity of DTX-HA-sPLy XNPs, DTX-HA-sPLy NPs, and DTX-HA-lPLy NPs on A549 cells; MTT results showed that DTX-HA-sPLy XNPs had the strongest resistance to A549. The cell proliferation effect, its IC ₅₀ was 0.18 mg/mL, which was 2.1 times, 6.6 times and 4.6 times lower than that of DTX-HA-sPLy NPs, DTX-HA-lPLy NPs and free DTX, respectively, see Figure 9(D); The targeted cross-linked nanoparticles have a good targeting effect on A549 cells, and can efficiently deliver the drug into the cells and release them quickly, finally killing the cancer cells.

Example 19 Blood circulation of drug-loaded particles

All animal experiments comply with the regulations of the Animal Experiment Center of Soochow University. Balb/C nude mice weighing 18-20 grams and 4-6 weeks old were used in the experiment. DOX-sPLy XNPs, DOX-sPLy NPs, and DOX-lPLy NPs were all prepared according to Example 12. The drug-loaded cross-linked nanoparticles in each group were injected into mice through the tail vein (DOX dose of 10 mg/kg), and blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours. The blood samples were centrifuged (3000 rpm, 6 min), and 20 μL of serum was taken, followed by extraction with 100 μL, 1% Triton and 500 μL anhydrous DMSO (containing 20 mM DTT); each time point was measured by fluorescence spectrometer The amount of DOX. In Fig. 10(A), the abscissa is time, and the ordinate is the concentration of DOX. It can be seen from the figure that DOX-sPLy XNPs have a longer cycle time of about 4.94 h, which is higher than that of non-crosslinked nanoparticles (DOX-sPLy NPs: 4.43 h, DOX-lPLy NPs: 3.46 h), so the drug-loaded cross-linked Polymersomes are more stable in mice than non-crosslinked nanoparticles and have longer circulation times.

Example 20 Blood circulation of DOX-cRGD XNPs, DOX-cRGD NPs

Animals are the same as in Example 17. The preparation method of DOX-cRGD XNPs and DOX-cRGD NPs is as in Example 8, and the test method is the same as that in Example 19. Figure 10(B) The abscissa is time, and the ordinate is the concentration of DOX. It can be seen from the figure that the DOX-cRGD XNPs have a longer circulation time, about 5.16 h, which is higher than that of the non-crosslinked nanoparticles DOX-cRGD NPs, so the drug-loaded cross-linked polymer vesicles in mice are relatively longer than the non-cross-linked nanoparticles. Nanoparticles are more stable and have longer circulation times, and the introduction of cRGD polypeptides does not have a particularly large effect on the in vivo circulation time of nanoparticles.

Example 21 DTX particle-loaded blood circulation

Animals are the same as in Example 17. The preparation method of DTX-GE11/TAT XNPs, DTX-GE11 XNPs, DTX-sPLy XNPs is as in Example 9, the test method is the same as that in Example 19, and the amount of DTX at each time point is measured by high performance liquid chromatography. In Fig. 10(C), the abscissa is time, and the ordinate is the concentration of DTX. It can be seen from the figure that DTX-GE11/TAT XNPs, DTX-GE11 XNPs, and DTX-XNPs have similar cycle times, about 5 h, which are all significantly longer than free DTX (0.34 h). Therefore, the drug-loaded cross-linked polymer nanoparticles have a long circulation time in mice, and the introduction of polypeptides, especially the introduction of TAT, does not have a great impact on the circulation of nanoparticles.

Example 22 Blood circulation of HA-targeted drug-loaded particles

Animals are the same as in Example 17. The preparation method of DTX-HA-sPLy XNPs, DTX-HA-sPLy NPs, and DTX-HA-lPLGA NPs is as in Example 8, and the test method is the same as that in Example 19. The amount of DTX at each time point is measured by high performance liquid chromatography. In Fig. 10(D), the abscissa is time, and the ordinate is the concentration of DTX. It can be seen from the figure that the DTX-HA-sPLy XNPs have a longer cycle time of about 4.18 h, which is higher than that of the non-crosslinked nanoparticles DTX-HA-sPLy NPs (3.5), DTX-HA-lPLGA NPs (2.97) and free DTX (0.23); therefore, drug-loaded cross-linked polymer nanoparticles are more stable in mice than non-cross-linked nanoparticles, and have longer circulation times.

Example 23 Biodistribution of DOX particles in B16F10 melanoma-bearing mice

Animals were the same as in Example 17, and were injected subcutaneously with 1×10 ⁵ B16F10 human lung cancer cells. About 7 days later, the experiment started when the tumor size was 100-200 mm ³ . DOX-cRGD XNPs and DOX-cRGD NPs were prepared according to Example 8, and injected into mice through tail vein (DOX: 10 mg/kg). After 8 hours, the mice were sacrificed, and the tumors and heart, liver, spleen, lung and kidney tissues were removed. After washing and weighing, add 500 μL of 1% triton and grind it through a homogenizer, and then add 900 μL of anhydrous DMSO for extraction (which contains 20 mM DTT). After centrifugation (20,000 rpm, 20 minutes), the supernatant was taken, and the amount of DOX in each tissue was measured by fluorescence spectroscopy. In Figure 11(A), the abscissa is the tissues and organs, and the ordinate is the DOX per gram of tumor or tissue to the total DOX injection (ID%/g). DOX-cRGD XNPs injected DOX-cRGD XNPs for 8 hours, and the amount of DOX accumulated in the tumor was 10.96ID%/g, which was 2.1 times that of DOX-cRGDNPs, indicating that drug-loaded DOX-cRGD XNPs accumulated more in tumor sites through active targeting and had better tumor performance. specific targeting.

Example 24 Biodistribution of DTX particles in MDA-MB-231 breast cancer-bearing mice

The tumor inoculation and tail vein administration in the biodistribution experiment are the same as in Example 18. Firstly, DTX-GE11/TAT XNPs, DTX-GE11 XNPs and DTX-sPLy XNPs were prepared according to Example IX. The mice were injected into the tail vein (DTX: 10 mg/kg), and the method of Example 23 was used to measure the amount of DTX in each tissue by high performance liquid chromatography. In Figure 11(B), the abscissa is the tissues and organs, and the ordinate is the total DOX injection volume (ID%/g) per gram of tumor or tissue. DTX-GE11/TAT XNPs injected 8 hours into the tumor with an accumulated DTX amount of 9.72 ID%/g, which was 1.88, 3.12, and 8.75 times that of DTX-GE11 XNPs, DTX-sPLyXNPs and free DTX, respectively, indicating that DTX-GE11/TAT XNPs Through active targeting, it accumulates more at the tumor site and has better tumor-specific targeting effect.

Example 25 Biodistribution of HA-targeted DTX-loaded particles in A549 lung cancer-bearing mice

The tumor inoculation and tail vein administration in the biodistribution experiment are the same as in Example 18. Firstly, DTX-HA-sPLy XNPs, DTX-HA-sPLy NPs and DTX-HA-lPLGA NPs were prepared according to Example 9, and the method of Example 24 was adopted. In Figure 11(C), the abscissa is the tissues and organs, and the ordinate is the total DOX injection amount (ID%/g) per gram of tumor or tissue. DTX-HA-sPLy XNPs injected 8 hours into the tumor to accumulate 9.48ID%/g of DTX, which was 1.5, 2.0, and 3.9 times that of DTX-HA-sPLy NPs, DTX-HA-lPLGA NPs and free DTX, respectively, indicating that DTX- HA-sPLy XNPs accumulate more in tumor sites through active targeting, and have better tumor-specific targeting effect.

Example 26 Therapeutic effect of DOX particles in B16F10 melanoma-bearing mice

Tumor inoculation and tail vein administration were the same as in Example 18, and the experiment was started when the tumor size was 30-50 mm ³ after about two weeks. DOX-cRGD XNPs, DOX-sPLy XNPs, DOX-cRGD NPs, Lipo-DOX and PBS were injected into mice via tail vein at 0, 2, 4, 6 and 8 days, respectively (DOX dose was 10 mg/kg) , a high-dose group (DOX dose of 20 mg/kg) was set up for DOX-cRGD XNPs. From 0 to 12 days, the mice were weighed every three days, and the tumor volume was measured with a vernier caliper. The tumor volume was calculated as: V=(L×W×W)/2, (where L, W, and H were length, width and thickness). Mice were continuously observed for survival up to 45 days. As can be seen from Figure 12, the DOX-cRGD XNPs (DOX: 20 mg/kg) treatment group significantly inhibited the tumor at 12 days, and its effect was comparable to the Lipo-DOX group, and there was no significant change in the body weight of the mice. It is worth noting It was the Lipo-DOX group that lost about 15% of the body weight. The DOX-cRGD XNPs, DOX-sPLy XNPs, and DOX-cRGD NPs groups all had a certain growth in tumors, but the growth rate and amount were significantly lower than those in the PBS group, and none of them caused any changes in the body weight of the mice, indicating that the expression The drug polymer nanoparticles have no toxic side effects on mice. The median survival of DOX-cRGD XNPs (DOX: 20 mg/kg) mice was 43 days, the median survival of DOX-cRGD XNPs, DOX-sPLy XNPs, DOX-cRGD NPs and Lipo-DOX and PBS groups The periods are 30, 26, 25, 21 and 14 days, respectively. Moreover, 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 Lipo-DOX group were significantly damaged.

Example 27 Therapeutic effect of DTX particles in MDA-MB-231 breast cancer-bearing mice

Tumor inoculation and tail vein administration were the same as in Example 19. 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). On days 0-18, the body weight of the mice was weighed every three days, the tumor volume was measured as in Example 26 and the mice were observed for 60 days. It can be seen from Figure 13 that the tumors in the DTX-GE11/TAT XNPs, DTX-GE11 XNPs, DTX-XNPs and free DTX treatment groups were inhibited to varying degrees at 18 days, while the tumors in the PBS group increased significantly. It is worth noting that DTX-GE11/TAT XNPs, DTX-GE11 XNPs, and DTX-XNPs can effectively inhibit tumor growth, and the body weight of mice did not change significantly. For the free DTX group, although the tumor growth was inhibited to a certain extent, the weight of the mice decreased significantly, indicating that the free DTX brought serious systemic toxicity to the mice.

Example 28 Therapeutic effect of HA-targeted DTX-loaded particles in mice bearing A549 lung cancer

A549 subcutaneous tumor inoculation and tail vein administration were the same as in Example 19. DTX-HA-sPLy XNPs, DTX-HA-sPLy NPs, DTX-HA-lPLGA NPs and free DTX and PBS were injected into mice via tail vein on 0, 4, 8 and 12 days, respectively (DTX dose was 5 mg /kg). As in Example 20, the body weight of the mice, the tumor volume were measured and the mice were observed for 60 days within 0-16 days. It can be seen from Figure 14 that when DTX-HA-sPLy XNPs, DTX-HA-sPLy NPs, DTX-HA-lPLGA NPs and free DTX were treated for 16 days, the tumors were inhibited to a certain extent, while the tumors in the PBS group increased significantly. It is worth noting that except for the free DTX group, the body weight of the mice did not change significantly. Due to the strong toxic and side effects of DTX, the body weight of the mice decreased severely, and decreased by about 15% at 16 days. The above results show that the drug-loaded nanoparticles have Good biocompatibility, no toxic side effects to mice. In addition, the DTX-HA-sPLy XNPs treatment group had significant differences compared with the DTX-HA-sPLy NPs and DTX-HA-lPLGA NPs treatment groups, showing the most excellent tumor growth inhibition effect.

Claims (8)

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;

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 preparation method of the nano-drug based on the terminal sulfur-containing octanoyl star polymer 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; the molar ratio of the polyhydroxy glucose to the lactide to the glycolide is 1 to (50-55) to (60-65);

(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.

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%.

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