Bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride and preparation method and application thereof

Abstract

The present invention relates to a bicyclic thioketal cysteine-N-ring carboxylic anhydride and its preparation method and application, bicyclic thioketal cysteine-N-ring carboxylic anhydride, its structural formula is as follows: the bicyclic thioketal cysteine-N-ring carboxylic anhydride containing thioketal group designed and synthesized by the present invention contains two N-carboxyl ring anhydride units and a thioketal connecting unit, the N-carboxyl ring anhydride unit can self-polymerize, and can also be copolymerized with other amino acid ring anhydride monomers, using amino-terminated polyethylene glycol monomethyl ether as an initiator for the polymerization reaction, an amphiphilic block copolymer can be obtained, which can be self-assembled into a nanostructure and efficiently coated with hydrophobic drugs. Because the thioketal structure is sensitive to ROS, once the drug carrier reaches the lesion site, the thioketal structure is immediately oxidized and broken, causing the disintegration of the carrier and the release of drug molecules, thereby exerting its functional effect.

Description

Technical Field

[0001] The invention belongs to the technical field of organic polymer compounds, and particularly relates to bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride and a preparation method and application thereof. Background Art

[0002] To cope with the threat posed by cancer, a variety of treatment options are used clinically, including surgery, chemotherapy, and radiotherapy. Although some emerging treatment options have emerged in recent years, such as targeted therapy, immunotherapy, and gene therapy, chemotherapy remains one of the common methods for treating malignant tumors. Cisplatin (CDDP), doxorubicin (DOX), and paclitaxel (PTX) are the most widely used chemotherapy drugs in clinical practice. However, as a systemic treatment, chemotherapy drugs will diffuse to most organs and tissues throughout the body through the blood circulation, inevitably causing systemic toxicity. The emergence of nano-drug carriers has improved the distribution of chemotherapy drugs in the human body and the metabolic kinetics of drugs, which helps to improve the therapeutic effect of chemotherapy drugs.

[0003] Tumor tissues are usually characterized by hypoxia, acidic pH, increased oxidative stress, high concentrations of glutathione (GSH), and overexpression of enzymes. The excessive production of reactive oxygen species (ROS) in tumor tissues is the result of the combined action of multiple cancer-promoting events such as the loss of tumor suppressor function and hypoxia-induced aerobic respiration. In addition, the more malignant the tumor is, the higher its degree of hypoxia. A variety of ROS-responsive smart nanodrug delivery carriers designed and developed using the difference in ROS concentrations between normal physiological and pathological states have been used for anti-tumor drug delivery. For example, CN117551118A provides a polyamino acid containing phenylboronic acid, which achieves efficient loading of hydrophilic / hydrophobic small molecule drugs, peptide drugs, protein drugs, and nucleic acid drugs through the reactive oxygen response of the phenylboronic acid group, as well as their controlled release at the lesion site. CN114958008A provides a polyamino acid-bonded glycosaminoglycan hydrogel that responds to active oxygen, whose polymer side chains contain thioether groups, which can respond to active oxygen and reduce the content of active oxygen. The glycosaminoglycans in the hydrogel have the function of actively eliminating excessive inflammatory responses, thereby solving the problems of persistent inflammatory state of chronic wounds, drug tolerance and difficulty in healing. CN114409607A provides a polyamino acid containing thioether groups. This material can self-assemble into nanoparticles with oxidation responsiveness in water. Nanoparticles loaded with anticancer drugs can release the loaded drugs in the high active oxygen environment of tumors, thereby killing tumor cells. In these schemes, the polyamino acid side chains all use thioether bonds or phenylboronic acid ester bonds as active oxygen responsive chemical bonds, and then release drugs at high active oxygen lesions to achieve selective treatment of the disease. However, these drugs are not stable enough under physiological conditions, and there are disadvantages such as drug leakage and difficulty in effectively protecting drugs. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies in the prior art and provide a bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride and a preparation method and application thereof. The bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride is polymerized to obtain a ROS-responsive polyamino acid. The ROS-responsive polyamino acid can form stable micelles to encapsulate components such as hydrophobic drugs, imaging agents or dye molecules, thereby protecting the drugs before reaching the tumor site and utilizing the high ROS level at the tumor site for responsive release, thereby effectively solving the problem of specific release of chemotherapy drugs at the tumor site and, at the same time, reducing to a certain extent the destructive effect of drug leakage on normal tissues.

[0005] The present invention provides a bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride, the structural formula of which is as follows:

[0006]

[0007] The present invention also provides a method for preparing the bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride, which comprises the following steps:

[0008] 1) L-cysteine ​​hydrochloride and acetone are added to a dilute hydrochloric acid solution for condensation reaction, and post-treatment is performed to obtain cysteine ​​containing thioketal (3,3'-(propane-2,2-diylbis(sulfinyl))bis(2-aminopropionic acid)), the structure of which is

[0009] The formula is as follows:

[0010]

[0011] 2) adding the cysteine ​​containing thioketal obtained in step 1) and triphosgene into a solvent for condensation reaction, and post-treating to obtain bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride.

[0012] According to the above scheme, the mass ratio of L-cysteine ​​hydrochloride to acetone in step 1) is 1:0.1-1.

[0013] According to the above scheme, the concentration of the dilute hydrochloric acid solution in step 1) is 0.5-12 mol / L, preferably 5-7 mol / L, and the mass volume ratio of the L-cysteine ​​hydrochloride to the dilute hydrochloric acid solution is 1 g: 2-15 mL.

[0014] According to the above scheme, the condensation reaction conditions in step 1) are: under an inert atmosphere (nitrogen, helium or argon), the reaction is carried out at 0-80°C for 3-36 hours, preferably at 25-40°C for 10-15 hours.

[0015] According to the above scheme, the mass ratio of the cysteine ​​containing thioketal to triphosgene in step 2) is 1:0.4-6.0.

[0016] According to the above scheme, the solvent in step 2) is one of tetrahydrofuran (THF), dichloromethane, ethyl acetate, and N,N-dimethylformamide (DMF), preferably tetrahydrofuran or dichloromethane, and the mass volume ratio of the cysteine ​​containing thioketal to the solvent is 0.05 to 0.5 g / mL.

[0017] According to the above scheme, the condensation reaction conditions in step 2) are: under an inert atmosphere, the reaction is carried out at 5-70° C. for 1-10 hours, preferably at 45-55° C. for 4-5 hours.

[0018] The present invention also includes the use of the bicyclic thioketal cysteine-N-intra-ring carboxylic acid anhydride in the field of drug delivery carriers.

[0019] The present invention also includes a ROS-responsive polyamino acid prepared according to the above-mentioned bicyclic thioketal cysteine-N-intra-ring carboxylic anhydride, the structural formula of which is shown below:

[0020]

[0021] Wherein n is 18 to 460, m is 3 to 20, and y is 0 or 3 to 50;

[0022] R is selected from the following groups:

[0023] The present invention also includes a method for preparing the above-mentioned ROS-responsive polyamino acid, the specific steps of which are as follows:

[0024] The bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride and amino-terminated polyethylene glycol monomethyl ether are dissolved in a solvent, and copolymerized amino acid monomers are added as needed, and ROS-responsive polyamino acids are obtained through polymerization reaction and purification.

[0025] According to the above scheme, the number average molecular weight (M n ) is 800~20000.

[0026] According to the above scheme, the copolymerized amino acid monomer is one of glutamic acid 5-benzyl ester N-carboxyl ring anhydride, L-phenylalanine-N-ring carboxylic anhydride, N6-benzyloxycarbonyl-L-lysine ring anhydride or methionine N-ring carboxylic anhydride, and its structure is shown below:

[0027]

[0028] According to the above scheme, the mass ratio of the bicyclic thioketal cysteine-N-intracyclic carboxylic acid anhydride to the amino-terminated polyethylene glycol monomethyl ether and the copolymerized amino acid monomer is 0.2-3.4:1:0.4-5.8.

[0029] According to the above scheme, the solvent is one of N,N-dimethylformamide, dimethyl sulfoxide (DMSO), chloroform, and tetrahydrofuran, preferably N,N-dimethylformamide or dimethyl sulfoxide. The mass volume ratio of the amino-terminated polyethylene glycol monomethyl ether to the solvent is 1g:5-30mL.

[0030] According to the above scheme, the polymerization reaction conditions are: under an inert atmosphere, the reaction is carried out at 0-60°C for 24-120 hours, preferably at 25-30°C for 60-96 hours.

[0031] The present invention also provides the use of the above-mentioned ROS-responsive polyamino acid as a drug delivery carrier, wherein the polymer is assembled into nanoparticles, which are coated with hydrophobic drugs: such as anti-tumor drugs (including but not limited to doxorubicin, paclitaxel, etc.), anti-inflammatory drugs such as dexamethasone, as well as hydrophobic fluorescent probes, imaging agents, etc.

[0032] The specific application method is: dispersing the ROS-responsive polyamino acid and the hydrophobic drug in an organic solvent, adding water to obtain an emulsion, removing the organic solvent, freeze-drying to obtain nanoparticles, and storing them for use.

[0033] The beneficial effects of the present invention are:

[0034] 1. The bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride containing a thioketal group designed and synthesized by the present invention contains two N-carboxyl intracyclic anhydride units and a thioketal linker unit. The N-carboxyl intracyclic anhydride units can self-polymerize or copolymerize with other amino acid intracyclic anhydride monomers. Using amino-terminated polyethylene glycol monomethyl ether as an initiator for the polymerization reaction, an amphiphilic block copolymer can be obtained, which can self-assemble into a nanostructure and efficiently encapsulate hydrophobic drugs. The thioketal linker unit does not participate in the polymerization reaction, but can promote the cross-linking of polyamino acid molecular chains, making the self-assembled nanostructure very dense and stable, thereby protecting the encapsulated drug from leakage during transportation and causing no damage to non-target organs. Because the thioketal structure is sensitive to ROS, once the drug carrier reaches the lesion site, the thioketal structure is immediately oxidized and broken, causing the carrier to disintegrate and release the drug molecules, thereby exerting its functional effect.

[0035] 2. The raw materials for preparing bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride of the present invention are L-cysteine ​​hydrochloride, acetone and triphosgene, which are abundant in source, and the reaction steps are simple and easy to control. The by-products are basically gases, which are easy to separate and purify, and are convenient for mass production and popularization and application.

[0036] 3. The present invention uses amino acid cyclic N-carboxyl intracyclic anhydride monomers as raw materials to polymerize ROS-responsive polyamino acids. The polymerization efficiency of such monomers is very high, and the only by-product is CO2, which is easy to separate. In addition, the hydrophilic and hydrophobic chain lengths of the polymerized products can be adjusted by changing the dosage ratio of bicyclic thioketal cysteine-N-intracyclic carboxylic acid anhydride and amino-terminated polyethylene glycol monomethyl ether, thereby achieving polymer self-assembly and efficient drug encapsulation.

[0037] 4. The ROS-responsive polyamino acids prepared by the present invention have no restrictions on the types of drugs that can be encapsulated. They only need to have a certain degree of hydrophobicity. Therefore, they can be small molecule anticancer drugs such as paclitaxel or doxorubicin, small molecule anti-inflammatory drugs such as dexamethasone, or fluorescent, phosphorescent, nuclear magnetic or radioactive reagents for imaging and diagnostic purposes, realizing multiple uses of one carrier.

[0038] 5. The method of using the ROS-responsive polyamino acid of the present invention to coat drugs is self-assembly in water, which can be achieved by simple dissolution at room temperature, stirring evaporation at room temperature, and freeze-drying generally accepted in the pharmaceutical industry, which is easy to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the H NMR spectrum of the cysteine ​​containing thioketal prepared in Example 1 of the present invention;

[0040] Figure 2 This is the H NMR spectrum of the bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride prepared in Example 1;

[0041] Figure 3 This is the C NMR spectrum of the bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride prepared in Example 1;

[0042] Figure 4 This is the H NMR spectrum of PEG-PCYT prepared in Example 2;

[0043] Figure 5 This is the H NMR spectrum of PEG-bP(CYT-co-BLG) prepared in Example 2;

[0044] Figure 6 Comparison of the H NMR spectra of the PEG-PCYT micelle dispersion in Example 5 and after reaction with H2O2 aqueous solution;

[0045] Figure 7 Comparison of the H NMR spectra of the PEG-bP (CYT-co-BLG) micelle dispersion and the micelle dispersion after reaction with H2O2 aqueous solution in Example 5;

[0046] Figure 8Comparison of DLS and TEM tests of the PEG-PCYT micelle dispersion, the PEG-bP(CYT-co-BLG) micelle dispersion, and the micelle dispersion after reaction with H2O2 aqueous solution in Example 5;

[0047] Figure 9 Comparison of DLS and TEM tests of the PEG-PCYT@DOX aqueous dispersion, the PEG-bP(CYT-co-BLG)@DOX aqueous dispersion, and the reaction with H2O2 aqueous solution in Example 6;

[0048] Figure 10 2 is a comparative graph showing the drug release curves of the aqueous dispersion of PEG-PCYT micelles encapsulating Nile Red in Example 8 in PBS buffer, PBS buffer containing 50 mM H2O2, and PBS buffer containing 100 mM H2O2;

[0049] Figure 11 These are photos of PEG-PCYT@DOX micelles being taken up by cells at 2 h, 4 h, and 6 h, as observed using an inverted fluorescence microscope in Example 9;

[0050] Figure 12 This is a comparison chart of the cell viability of 4T1 and L929 cells in Example 10 in PEG-PCYT micelle dispersions and PEG-bP (CYT-co-BLG) micelle dispersions at different concentrations;

[0051] Figure 13 This is a test graph of the killing effect of PEG-PCYT@DOX, PEG-bP(CYT-co-BLG)@DOX and pure doxorubicin on 4T1 cells in Example 11. DETAILED DESCRIPTION

[0052] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below using embodiments and in conjunction with the accompanying drawings.

[0053] Example 1

[0054] Synthesis of bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride (CysTK NCA):

[0055] The synthetic route and typical reaction conditions are as follows:

[0056]

[0057] The synthesis process is as follows:

[0058] 1) L-cysteine ​​hydrochloride (2 g, 12.7 mmol) was dissolved in 10 mL of dilute hydrochloric acid solution (6 mol / L). Acetone (1.48 g, 25.4 mmol) was added and stirred at 30°C under a nitrogen atmosphere for 12 h. The solvent was then removed by rotary evaporation, washed with dilute hydrochloric acid solution, and recrystallized three times from pure water at 95°C. The product was dried to obtain white crystalline product, cysteine ​​containing thioketal (CysTK, 1.39 g, yield: 71.53%). Step 1) was repeated several times to collect more product.

[0059] 2) Cysteine ​​containing thioketal (2.8 g, 10 mmol) was suspended in 30 mL of anhydrous tetrahydrofuran. Under a nitrogen atmosphere, triphosgene (1.24 g, 4.17 mmol) was added portionwise to the reaction system and reacted at 55°C for 4 h. After the reaction, unreacted starting material was removed by filtration. The filtrate was precipitated in n-hexane and filtered. The product was dissolved in 500 mL of ethyl acetate and washed sequentially with ice-saturated sodium carbonate solution and pure water (10 mL × 3), dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was recrystallized three times from a mixed solvent of ethyl acetate and n-hexane in a volume ratio of 4:1 to obtain 0.43 g of a light yellow product (CysTK NCA, yield 13%).

[0060] Figure 1 This is the H NMR spectrum of the cysteine ​​containing thioketal synthesized in step 1) of this example.

[0061] Figure 2 and Figure 3 The following are the hydrogen and carbon nuclear magnetic resonance spectra of the bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride prepared in this example, respectively. The characteristic peaks of the methyl group on the thioketal appear in the spectra, and the chemical shifts and integrated intensities of each proton are analyzed, confirming the synthesis of the bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride.

[0062] Example 2

[0063] Synthesis of ROS-responsive polyamino acid polymer PEG-PCYT and copolymer PEG-bP (CYT-co-BLG):

[0064] The synthetic route and typical reaction conditions are as follows:

[0065]

[0066] The synthesis process is as follows:

[0067] Weigh 0.5 g of amino-terminated polyethylene glycol monomethyl ether (PEG-NH2, 0.1 mmol) in a polymerization bottle, remove water with toluene azeotropy for 2 h, cool to 30-40 ° C, then add the bicyclic thioketal cysteine-N-ring carboxylic anhydride (CysTK NCA, 0.167 g, 0.5 mmol) prepared in Example 1, add 10 mL of DMF as solvent under nitrogen protection, stir the reaction at room temperature for 72 h, and precipitate the reactant in anhydrous ether at 0 ° C. Filter to obtain a white solid, and vacuum dry to obtain 0.61 g of a white product PEG 113 -PCYT5 (denoted as PEG-PCYT, yield 90.7%).

[0068] 0.5 g of amino-terminated polyethylene glycol monomethyl ether (PEG-NH2, 0.1 mmol) was weighed into a polymerization bottle, and water was removed by azeotropic distillation with toluene for 2 h. The mixture was cooled to 30-40 ° C., and then 0.526 g of 5-benzyl glutamic acid N-carboxyl ring anhydride (BLG NCA, 2 mmol) monomer and 0.167 g of CysTK NCA (0.5 mmol) monomer prepared in Example 1 were added. Under nitrogen protection, 12 mL of N, N-dimethylformamide was added as a solvent, and the reaction was stirred at room temperature (25 ° C) for 72 h. The reactant was precipitated in ice anhydrous ether, filtered to obtain a white solid, and vacuum dried to obtain 1.09 g of a white product PEG 113 -bP(CYT 5- co-BLG 20 ) (denoted as PEG-bP(CYT-co-BLG), yield 91.6%).

[0069] The nuclear magnetic resonance hydrogen spectrum of PEG-PCYT synthesized in this example is as follows Figure 4 shown.

[0070] The nuclear magnetic resonance hydrogen spectrum of PEG-bP (CYT-co-BLG) synthesized in this example is as follows Figure 5 shown.

[0071] By adjusting the amount of raw materials used in this example and keeping other conditions unchanged, products with different degrees of polymerization were obtained. The amount of raw materials used and the products with different degrees of polymerization are shown in Table 1 below.

[0072] Table 1

[0073]

[0074] Example 3

[0075] Synthesis of copolymer PEG-bP(CYT-co-Met):

[0076] The synthetic route and typical reaction conditions are as follows:

[0077]

[0078] The synthesis process is as follows:

[0079] 0.5 g PEG-NH2 (0.1 mmol) was weighed into a polymerization bottle, and water was removed by azeotropic co-extraction with toluene for 2 h. The mixture was cooled to 30-40°C, and then 0.35 g L-methionine-N-intracyclic carboxylic anhydride (L-Met NCA, 2 mmol) monomer and 0.167 g CysTK NCA (0.5 mmol) monomer prepared in Example 1 were added. Under nitrogen protection, 15 mL dimethyl sulfoxide was added as a solvent, and the reaction was stirred at 30°C for 80 h. The reactants were precipitated in ice-cold anhydrous ether and filtered to obtain a white solid. The white solid was dried in vacuo to obtain 0.94 g of a white product PEG-bP (CYT-co-Met) (yield 92.4%).

[0080] Example 4

[0081] Synthesis of copolymer PEG-bP(CYT-co-Lys):

[0082] The synthetic route and typical reaction conditions are as follows:

[0083]

[0084] The synthesis process is as follows:

[0085] 0.5 g of PEG-NH2 (0.1 mmol) was weighed into a polymerization bottle and azeotroped with toluene for 2 h to remove water. The mixture was cooled to 30-40°C, and then 0.344 g of N6-benzyloxycarbonyl-L-lysine cyclic anhydride (Lys NCA, 2 mmol) monomer and 0.167 g of CysTK NCA (0.5 mmol) monomer prepared in Example 1 were added. Under nitrogen protection, 50 mL of chloroform was added as a solvent, and the reaction was stirred at 27°C for 96 h. The reactants were precipitated in ice-cold anhydrous ether and filtered to obtain a white solid. The white solid was dried in vacuo to obtain 0.89 g of a white product, PEG-bP(CYT-co-Lys) (yield 88.1%).

[0086] Example 5

[0087] Preparation of ROS-responsive polyamino acid micelles:

[0088] The polymer was self-assembled into micelles using an oil-in-water (O / W) emulsion method: 10 mg of the PEG-PCYT prepared in Example 2 was dispersed in 100 μL of chloroform, and then added dropwise to 4 mL of ultrapure water and ultrasonically dispersed for 15 min to form an emulsion. The chloroform was completely evaporated at room temperature, and the mixture was centrifuged, filtered through a microporous filter membrane (pore size: 0.225 μm), and then diluted to obtain a polymer PEG-PCYT micelle dispersion (1 mg / mL).

[0089] The polymer PEG-bP (CYT-co-BLG) prepared in Example 2 was used as a raw material, and the above method was used to prepare a polymer PEG-bP (CYT-co-BLG) micelle dispersion (1 mg / mL).

[0090] ROS response performance test:

[0091] 100 μL of 10 mM H2O2 aqueous solution was added to 1 mL of the two micelle dispersions prepared above, and the mixture was stirred for 24 h. The H NMR spectra of the two micelle dispersions and the H2O2 aqueous solution after reaction were compared. Figure 6 and Figure 7 As shown, it can be seen that the nuclear magnetic resonance hydrogen spectrum after the reaction did not detect the characteristic peak of the proton at the methyl group on the thioketal, proving that the chemical bond was broken at the thioketal.

[0092] Dynamic light scattering (DLS) was used to test the changes in the size of the two micelle dispersions and the micelles after reaction with H2O2 aqueous solution, and transmission electron microscopy (TEM) was used to test the morphology changes of the micelles before and after the reaction. Figure 8 As shown, the average particle size of PEG-PCYT micelles is 57.51±5.28nm, and the average particle size of PEG-bP(CYT-co-BLG) micelles is 90.54±5.87nm. After reacting with H2O2, the average particle sizes of the two micelles are 751±88.71nm and 652±51.58nm, respectively. This shows that the micelle particle size of the two micelles increases significantly after reacting with H2O2 at room temperature, proving that both polymers have good ROS response characteristics. Figure 8 As can be seen from the inset, both micelles have spherical morphology, but the spherical morphology of the micelles collapses after reacting with H2O2.

[0093] Example 6

[0094] Preparation of ROS-responsive polyamino acid micelles encapsulating doxorubicin (DOX):

[0095] 10 mg of PEG-PCYT prepared in Example 2 was dissolved in 100 μL of chloroform to obtain a PEG-PCYT dispersion. 12 mg of doxorubicin hydrochloride was dissolved in 500 μL of dimethyl sulfoxide, and triethylamine was added to remove the hydrochloric acid. 50 μL of the dehydrochlorinated doxorubicin solution was mixed with 100 μL of the PEG-PCYT dispersion and then added to 4 mL of ultrapure water for ultrasonic dispersion for 15 min to form an emulsion. The organic solvent was completely evaporated at room temperature, and the solvent was removed by dialysis (MWCO 3.5 kDa). After centrifugation (3500 rpm), filtration, and dilution, a fluorescently labeled micellar PEG-PCYT@DOX aqueous dispersion (1 mg / mL) was obtained.

[0096] Polymer PEG with different polymerization degrees prepared in Example 2 113 -PCYT 10 PEG 113 -bP(CYT5-co-BLG 20 ), PEG 113 -bP(CYT 10 -co-BLG 20 ) as raw materials, and the above method was used to prepare different drug-loaded micelle aqueous dispersions (1 mg / mL).

[0097] Determination of drug loading capacity (DLC) and drug loading efficiency (DLE): The DOX content in the ROS-responsive polyamino acid micelles encapsulating doxorubicin (DOX) was determined by measuring the doxorubicin absorbance value at 585 nm using a UV-visible spectrophotometer, and the drug loading capacity (DLC) and drug loading efficiency (DLE) were calculated using the formula.

[0098]

[0099] The performance test results of the ROS-responsive polyamino acid micelles encapsulating doxorubicin (DOX) are shown in Table 2.

[0100] Table 2

[0101]

[0102] The test results show that the drug loading capacity and efficiency of the material are closely related to the degree of polymerization of the bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride. The drug loading efficiency of the drug-loaded micelles is above 70%, indicating that the micelles have good drug loading capacity.

[0103] The particle size changes of the two dispersions prepared in this example and the blank micelle dispersion without drug coating (the two micelle dispersions prepared in Example 5) were tested by dynamic light scattering, and the morphology changes of the micelles before and after the reaction were tested by transmission electron microscopy. Figure 9 As shown in the figure, it can be seen that due to hydrophobic interaction, the micelle particle size after encapsulating the drug is significantly increased compared with the blank micelle, and the PDI value is reduced. This is mainly because doxorubicin exists inside the micelle, and the micelle particle size is increased based on hydrophobic interaction.

[0104] Example 7

[0105] Preparation of ROS-responsive polyamino acid micelles encapsulating dexamethasone (DEX):

[0106] 15 mg of PEG-PCYT prepared in Example 2 was dissolved in 150 μL of chloroform to obtain a PEG-PCYT dispersion. 1 mg of DEX was dissolved in 100 μL of dimethyl sulfoxide to obtain a DEX dispersion. 50 μL of the DEX dispersion was mixed with 150 μL of the PEG-PCYT dispersion and added to 4 mL of ultrapure water for 15 min to form an emulsion. The organic solvent was completely evaporated at room temperature, and the solvent was removed by dialysis (MWCO 3.5 kDa). The solution was centrifuged (3500 rpm) and then diluted to obtain a fluorescently labeled micellar PEG-PCYT@DEX aqueous dispersion (1 mg / mL).

[0107] The polymer PEG-bP(CYT-co-BLG) prepared in Example 2 was used as a raw material to prepare a PEG-bP(CYT-co-BLG)@DEX aqueous dispersion (1 mg / mL) using the above method.

[0108] Example 8

[0109] Drug release performance test:

[0110] Nile red was encapsulated in micelles as a model drug and incubated with H2O2 solutions of varying concentrations to simulate drug release behavior from responsive micelles. A PEG-PCYT micelle aqueous dispersion encapsulating Nile red (Nile red concentration 0.1 mg / mL) was prepared using the same method as in Example 6. 100 μL of the Nile red-encapsulated micelle aqueous dispersion was incubated in 2 mL of PBS buffer (pH 7.0), 2 mL of PBS buffer containing 50 mM H2O2, and 2 mL of PBS buffer containing 100 mM H2O2 at 37°C. Three parallel control samples were set up for each experimental group. Nile red present in the micelles exhibited fluorescence, with a maximum emission peak at 570 nm. Released Nile red present in aqueous solution did not exhibit fluorescence. Samples were taken at 0h, 3h, 6h, 12h, 24h, 36h, 48h, 72h, 96h, and 120h, and the emission peak of Nile Red at 570nm was measured using a fluorescence spectrometer. The excitation and emission slit widths were set to 5nm. The cumulative release of Nile Red was calculated as follows:

[0111]

[0112] FI0 is the fluorescence intensity at the beginning of the experimental group, FI t The fluorescence intensity at the time of measurement was used for the experimental group.

[0113] The drug release curves of the micellar aqueous dispersion containing Nile Red in this example were incubated in PBS buffer, PBS buffer containing 50 mM H2O2, and PBS buffer containing 100 mM H2O2 as a function of time. Figure 10As shown in the figure, Control represents incubation in PBS buffer. It can be seen that the drug release amount is positively correlated with the H2O2 concentration. After adding H2O2, the drug-loaded micelles continuously release the drug within 120 hours, achieving the purpose of sustained drug release and continuous treatment. In the Control group, only about 5% of the drug is released, indicating that the micelles have good sealing and no leakage.

[0114] Example 9

[0115] In vitro cell uptake capacity test:

[0116] 4T1 cells (mouse breast cancer cells) were counted and expressed as approximately 2×10 5 The cells were seeded at a density of 100 cells / well in a 6-well plate and cultured at 37°C and 5% CO2 for 24 hours. The cells were then incubated with the aqueous dispersion of PEG-PCYT@DOX micelles encapsulating doxorubicin described in Example 6 (doxorubicin concentration was 10 μg / mL) for 2 hours, 4 hours, and 6 hours. The culture medium was then removed, and the cells were washed three times with PBS to remove uninternalized micelles. The cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, and the uptake of PEG-PCYT@DOX micelles by 4T1 cells was observed under an inverted fluorescence microscope. Figure 11 Shown are photos of fluorescently labeled micelles PEG-PCYT@DOX being taken up by cells at 2h, 4h, and 6h, as observed by an inverted fluorescence microscope. Bright Field is the bright field image, Fluorescence is the fluorescence image, and Merged is the overlay image of bright field and fluorescence. The red fluorescence intensity in the cells gradually increased at the 2h, 4h, and 6h time points, indicating that the micelles were continuously internalized by the cells as time went on, proving that PEG-PCYT micelles can be continuously taken up by cells as drug delivery carriers.

[0117] Example 10

[0118] In vitro cell safety testing:

[0119] The cytotoxicity of the micelles to 4T1 and L929 cells was detected by the methylthiazolium tetrazolium (MTT) assay.

[0120] 4T1 or L929 cells were grown at a rate of approximately 8 × 10 3Cells were seeded at a density of 1000 μg / mL per well in a 96-well plate and cultured at 37°C in a 5% CO2 atmosphere. When the cell confluency reached approximately 80%, a concentration gradient of 1000 μg / mL, 200 μg / mL, 40 μg / mL, 8 μg / mL, 1.6 μg / mL, and 0.32 μg / mL of PEG-PCYT micelle dispersion and PEG-bP(CYT-co-BLG) micelle dispersion (prepared using the method of Example 5 and diluted with purified water to the desired concentration) was added. After incubation for 24 hours, the culture medium was removed, and 110 μL of culture medium containing 10 μL of 5 mg / mL MTT assay solution was added to each well. After incubation at 37°C for 4 hours, 100 μL of DMSO was added to each well, and the cells were incubated in an incubator for 2 hours to dissolve the resulting formazan crystals. The absorbance at 570 nm was measured using a microplate reader, and the cell viability was calculated.

[0121]

[0122] where N s 、N c 、N b The absorbances of the sample well, control well (without micelle solution), and blank well are shown in Figure 2. Figure 12 As shown, Figure 12 a is a comparison of cell survival rates of L929 cells and 4T1 cells in PEG-PCYT micelle dispersions at different concentrations. Figure 12 b is a comparison chart of the cell survival rates of L929 cells and 4T1 cells in PEG-bP (CYT-co-BLG) micelle dispersions at different concentrations. At micelle solution concentrations of 0.32 to 1000 μg / mL, the cell survival rates of both 4T1 and L929 cell lines in the two micelle solutions were above 90%, indicating that the micelles have low cytotoxicity and exhibit good cell safety as in vivo drug delivery carriers.

[0123] Example 11

[0124] In vitro cell killing test:

[0125] 4T1 cells were grown at a rate of approximately 8 × 10 3Cells were seeded at a density of 100 cells / well in a 96-well plate and cultured at 37°C in a 5% CO2 atmosphere. When the cell confluence reached approximately 80%, the cells were treated with aqueous dispersions of PEG-PCYT@DOX and PEG-bP(CYT-co-BLG)@DOX (prepared using the method of Example 6 and diluted with water to the desired concentration) at drug DOX concentrations of 160 μg / mL, 80 μg / mL, 40 μg / mL, 20 μg / mL, 10 μg / mL, and 5 μg / mL, respectively. At the same time, cells were treated with an aqueous solution of free DOX (Free DOX) at the same equivalent concentration as a control. The cells were incubated in a cell culture incubator for 24 h. The culture medium was removed, and 110 μL of culture medium containing 10 μL of 5 mg / mL MTT detection solution was added to each well. 4 h later, 100 μL of DMSO was added to each well, and the cells were incubated in an incubator for 2 h to dissolve the produced formazan crystals. The absorbance at 570 nm was measured using an enzyme-labeled instrument to determine cell viability. Figure 13 As shown in the figure, it can be seen that both polymer drug-loaded micelles have good killing effects on 4T1 cells, and are better than free DOX at the same equivalent concentration. In addition, the killing effect on cells shows a certain correlation with the concentration within the test concentration range.

Claims

1. A bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride, characterized in that Its structural formula is as follows: 。 2. The method for preparing the bicyclic thioketal cysteine-N-intra-ring carboxylic anhydride according to claim 1, wherein: The specific steps are as follows: 1) L-cysteine ​​hydrochloride and acetone are added to a dilute hydrochloric acid solution for condensation reaction, and post-treatment is performed to obtain cysteine ​​containing thioketal, the structural formula of which is shown below: ； 2) adding the cysteine ​​containing thioketal obtained in step 1) and triphosgene into a solvent for condensation reaction, and post-treating to obtain bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride.

3. The method for preparing the bicyclic thioketal cysteine-N-intra-ring carboxylic anhydride according to claim 2, wherein: In step 1), the mass ratio of L-cysteine ​​hydrochloride to acetone is 1:0.1-1; the concentration of the dilute hydrochloric acid solution in step 1) is 0.5-12 mol / L, and the mass volume ratio of L-cysteine ​​hydrochloride to the dilute hydrochloric acid solution is 1 g:2-15 mL; and the condensation reaction conditions in step 1) are: under an inert atmosphere, at 0-80° C. for 3-36 hours.

4. The method for preparing the bicyclic thioketal cysteine-N-intra-ring carboxylic anhydride according to claim 2, wherein: In step 2), the mass ratio of the cysteine ​​containing the thioketal to triphosgene is 1:0.4-6.0; in step 2), the solvent is one of tetrahydrofuran, dichloromethane, ethyl acetate, and N,N-dimethylformamide, and the mass volume ratio of the cysteine ​​containing the thioketal to the solvent is 0.05-0.5 g / mL; and in step 2), the condensation reaction conditions are: under an inert atmosphere, at 5-70° C. for 1-10 hours.

5. Use of the bicyclic thioketal cysteine-N-intra-ring carboxylic anhydride according to claim 1 in the preparation of a drug delivery carrier.

6. A ROS-responsive polyamino acid prepared based on the bicyclic thioketal cysteine-N-intra-ring carboxylic anhydride according to claim 1, characterized in that: Its structural formula is shown below: ； Where n is 18~460, m is 3~20, and y is 0 or 3~50; R is selected from the following groups: , , , .

7. The method for preparing the ROS-responsive polyamino acid according to claim 6, wherein: The specific steps are as follows: dissolving the above-mentioned bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride and amino-terminated polyethylene glycol monomethyl ether in a solvent, adding copolymerized amino acid monomers as needed, and performing polymerization reaction and purification to obtain ROS-responsive polyamino acid.

8. The method for preparing a ROS-responsive polyamino acid according to claim 7, wherein The number average molecular weight of the amino-terminated polyethylene glycol monomethyl ether is 800-20,000; the copolymerized amino acid monomer is one of glutamic acid 5-benzyl ester N-carboxyl intracyclic anhydride, L-phenylalanine-N-intracyclic carboxylic anhydride, N6-benzyloxycarbonyl-L-lysine intracyclic anhydride, or methionine N-intracyclic carboxylic anhydride; the mass ratio of the bicyclic thioketal cysteine-N-intracyclic carboxylic anhydride to the amino-terminated polyethylene glycol monomethyl ether to the copolymerized amino acid monomer is 0.2-3.4:1:0.4-5.8; and the polymerization reaction conditions are: reaction at 0-60°C under an inert atmosphere for 24-120 hours.

9. Use of the ROS-responsive polyamino acid according to claim 6 as a drug delivery carrier, characterized in that: The ROS-responsive polyamino acids are assembled into nanoparticles, in which hydrophobic drugs are encapsulated.

10. Use of the ROS-responsive polyamino acid as a drug delivery carrier according to claim 9, characterized in that: The specific application method is: dispersing the ROS-responsive polyamino acid and the hydrophobic drug in an organic solvent, adding water to obtain an emulsion, removing the organic solvent, freeze-drying to obtain nanoparticles, and storing them for use.

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