A drug delivery system for photosensitizer and nitric oxide prodrug and preparation method thereof

Abstract

The invention provides a drug delivery system for a photosensitizer and a nitric oxide prodrug. The drug delivery system comprises a functionalized amphiphilic polymer loaded with chemical bonds for carrying photosensitizer prodrugs pheophytin A and nitric oxide prodrugs, wherein two ends of the PheoA-CSSE-JSK are respectively a hydrophilic segment polyethylene glycol and a hydrophobic segment polycaprolactone, wherein the PheoA is connected to one end of the hydrophilic segment polyethylene glycol, the JSK is connected to the other end of the hydrophobic segment polycaprolactone, and a disulfide bond coupling agent alkynyl cystamine is used to connect the PheoA-polyethylene glycol block and the JSK-polyester through a click reaction, and the amphiphilic prodrug functionalized polymer is used to prepare nanoparticles through a solvent evaporation method; the polyester of the hydrophobic segment of the drug delivery system of the invention has good biocompatibility, the hydrophilic polyethylene glycol segment can increase the solubility of the photosensitizer PheoA, and the amphiphilic block copolymer composed of polyethylene glycol and polyester has high thermal stability and kinetic stability, and maintains drug activity during drug delivery.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a drug delivery system for a photosensitizer and a nitric oxide prodrug and a preparation method thereof. Background Art

[0002] Malignant tumors are common and frequently occurring diseases that seriously threaten human health. Currently, the main clinical treatments for cancer include surgical resection, radiotherapy, chemotherapy, and immunotherapy. Although these treatments can control the disease and prolong the patient's survival to varying degrees, there are still huge challenges in conquering cancer due to the complex mechanisms of tumor occurrence and development.

[0003] Photodynamic therapy (PDT) has gained great attention in cancer treatment due to its unique spatiotemporal specificity and minimally invasiveness, which can remove local tumor tissue and avoid damage to normal organs. The production process of PDT involves three key factors: photosensitizer (PS), light and oxygen, among which photosensitizer can convert oxygen into phototoxic ROS under the irradiation of light. ROS can cause strong damage to tumor cells by inducing intracellular oxidation processes, including lysosomes, mitochondria, plasma membranes and nuclear membranes, thereby exerting the anti-tumor effect of PDT. However, the hydrophobicity and non-targeted nature of photosensitizers can reduce the efficiency of PDT and have potential side effects on healthy organs, which hinders the further application of PDT in clinical practice. To solve this problem, scientists have developed a variety of PS delivery systems, incorporating PS into its hydrophobic core, including liposomes, micelles, inorganic nanomaterials, etc., but all of them have the problem of decreased phototoxicity of photosensitizers caused by aggregation-induced quenching. Therefore, there is an urgent need to develop an effective PS delivery nanosystem that can keep PS in a self-quenching state during circulation in the body to reduce damage to normal tissues, but can be locally and specifically released and activated in the unique tumor microenvironment (TME) to allow the photosensitizer to produce a large amount of phototoxic ROS for PDT treatment.

[0004] In addition, hypoxia is another prominent feature of most solid tumors, which greatly hinders the PDT efficiency of oxygen consumption. Collagen, as the main component of dense tumor extracellular matrix (ECM), is one of the biggest culprits of hypoxia, so bioactive molecules with the ability to degrade collagen are urgently needed to relieve hypoxia and sensitize PDT to improve the anti-tumor efficiency of PDT. Nitric oxide (NO) is the most important gas signaling molecule, which can mediate various physiological functions in tumors, especially regulating the tumor microenvironment. Recent studies have shown that nitric oxide can promote the degradation of collagen in tumors through reactive nitrogen radicals (RNS). Therefore, NO may bring a new perspective to relieve hypoxia to improve the efficiency of PDT. However, both NO and photosensitizers are small molecules with no targeting to tumors, and their off-target behavior to tumors often leads to damage to normal tissues and organs, thus limiting their application in tumor treatment. Therefore, the nanoprodrug drug delivery system based on tumor microenvironment (TME) response provides a new strategy to achieve the controlled release of NO and photosensitizers, utilizing the tumor-specific cell microenvironment to control the in situ release of photosensitizers and NO, thereby improving the bioavailability and biosafety of photosensitizers and NO.

[0005] The present invention mainly aims at the problem of reduced photodynamic efficiency of photosensitizer caused by photosensitizer aggregation quenching and tumor tissue hypoxia, and prepares a polymer nano drug delivery system loaded with photosensitizer and NO prodrug. The phototoxicity of photosensitizer is activated by the tumor microenvironment, and the photodynamic efficiency is enhanced by overcoming tumor hypoxia, thereby improving the effect of photodynamic therapy on tumors. Summary of the invention

[0006] In order to make up for the above shortcomings, the present invention provides a drug delivery system of a photosensitizer and a nitric oxide prodrug and a preparation method thereof, which activates the phototoxicity of the photosensitizer through the tumor microenvironment and overcomes tumor hypoxia to sensitize the photodynamic efficiency, thereby improving the effect of photodynamic therapy for tumors.

[0007] The present invention is achieved in that:

[0008] A drug delivery system for a photosensitizer and a nitric oxide prodrug, the drug delivery system comprising a functionalized amphiphilic polymer loaded with a chemical bond for a photosensitizer prodrug pheophytin A and a nitric oxide prodrug, the chemical bond loaded photosensitizer prodrug pheophytin A being PheoA, the nitric oxide prodrug being JSK, the amphiphilic polymer being PheoA-CSSE-JSK, the two ends of the PheoA-CSSE-JSK being a hydrophilic segment polyethylene glycol and a hydrophobic segment polycaprolactone, wherein the PheoA A is connected to one end of the hydrophilic segment polyethylene glycol, the JSK is connected to the other end of the hydrophobic segment polycaprolactone, and the PheoA-polyethylene glycol block and the JSK-polyester are connected by a click reaction using a disulfide bond coupling agent alkynyl cystamine, and the amphiphilic prodrug functionalized polymer is used to prepare self-assembled nanoparticles by a solvent evaporation method, the PheoA and the JSK are distributed on the hydrophilic surface and the hydrophobic inner cavity of the nanoparticles, respectively, the particle size of the nanoparticles is 50-300nm, and the drug loading rate is 8%-40%.

[0009] Preferably, when the amphiphilic polymer is used as a carrier material, the mass ratio of the carrier material to the photosensitizer prodrug and the NO prodrug is 1:0.1:0.5 respectively.

[0010] Preferably, the drug photosensitizer in the chemical bond-loaded photosensitizer prodrug pheophorbide A is a combination of one or more of chlorins, pheophorbide a, chlorophyll-a, chlorin e6 and pyropheophorbide-a.

[0011] Preferably, the donor of the nitric oxide prodrug is one or more combinations of nitramine alcohols, nitrosothiols, metal-nitroso and trifluoronitrobenzene.

[0012] Preferably, the drug delivery system characterized by the photosensitizer and the nitric oxide prodrug is prepared from the following compounds as raw materials:

[0013] Photosensitizer: pheophorbide A;

[0014] Nitric oxide prodrug: alkynylated O2-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)piperazin-1-yl]azo-1-ium-1,2-diol;

[0015] Compounds containing disulfide bonds: cystamine hydrochloride;

[0016] Alkyne-containing compounds: pentynoic acid;

[0017] Hydrophilic polymer: amino-polyethylene glycol-azide with a molecular weight of 2000-10000;

[0018] Hydrophobic polymer: Azide-polycaprolactone-azide with a molecular weight of 2000-10000.

[0019] A method for preparing a drug delivery system of a photosensitizer and a nitric oxide prodrug, comprising the preparation of the following carrier materials:

[0020] Preparation of coupling agent dialkynyl-Cys, preparation of hydrophobic segment JSK-PCL, preparation of hydrophilic segment PheoA-PEG, preparation of JSK-CSSE-PheoA;

[0021] The preparation method of the coupling agent dialkynyl-Cys is as follows:

[0022] Cystamine dihydrochloride and 4-pentenoic acid were dissolved in a round-bottom flask, and catalysts dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added. Under the protection of inert gas, the mixture was stirred at room temperature for 24-48 hours. The reactants were filtered to remove the white precipitate, and the product dialkynyl-Cys was separated by column chromatography:

[0023] The molar ratio of cystamine dihydrochloride to 4-pentynoic acid is 1:2-3;

[0024] The molar ratio of the catalyst dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 1:0.8-1.2;

[0025] The molar ratio of cystamine dihydrochloride to dicyclohexylcarbodiimide is 1:2.5-4;

[0026] The solvent is anhydrous dichloromethane, and 2-3 mmol of cystamine dihydrochloride is added to every 50 ml of anhydrous dichloromethane.

[0027] Preferably, the preparation of the hydrophobic segment JSK-PCL is:

[0028] The NO prodrug alkynyl-JSK was connected to one side of azide-polycaprolactone-azide (N3-PCL-N3) by click reaction, using copper sulfate pentahydrate and sodium ascorbate as catalysts, and reacted at room temperature for 24-48 hours under inert gas protection. After the reaction was completed, it was placed in a dialysis bag with a molecular weight of 1000, dialyzed with deionized water for 3-4 days, and freeze-dried to obtain JSK-PCL:

[0029] The molar ratio of the azide-polycaprolactone-azide (N3-PCL-N3) and the NO prodrug JSK is 1:0.8-1.2, dissolved in tetrahydrofuran, 0.1-0.2mmol azide-polycaprolactone-azide (N3-PCL-N3) is added to 8mL tetrahydrofuran;

[0030] The catalyst copper sulfate pentahydrate and sodium ascorbate have a molar ratio of 1:1.8-2.4, which are dissolved in deionized water, and 0.02-0.04mmol copper sulfate pentahydrate is added to 2mL of deionized water;

[0031] The volume ratio of tetrahydrofuran to water is 5-3:1.

[0032] Preferably, the preparation of the hydrophilic segment PheoA-PEG is:

[0033] Pheophorbide A is connected to one side of amino-polyethylene glycol-azide (NH2-PEG-N3) by amidation reaction, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as catalysts, dissolved in a solvent and stirred at room temperature in the dark for 24-48 hours, and dialyzed and freeze-dried after the reaction to obtain PheoA-PEG;

[0034] The molar ratio of amino-polyethylene glycol-azide (NH2-PEG-N3) to pheophorbide A is 1:1-1.5;

[0035] The molar ratio of the catalyst 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is 3:2;

[0036] The molar ratio of the pheophorbide A to the EDC is 1:2.8-3.2;

[0037] The solvent is dimethyl sulfoxide (DMSO), and 200 mg of amino-polyethylene glycol-azide is added to every 10 mL of DMSO.

[0038] Preferably, the preparation of JSK-CSSE-PheoA:

[0039] Through click reaction, copper sulfate pentahydrate and sodium ascorbate were used as catalysts, and dialkynyl-Cys was used to connect the hydrophobic segment JSK-PCL and the hydrophilic segment PheoA-PEG to form an amphiphilic block polymer. The reaction was carried out in two steps.

[0040] First, PheoA-PEG reacts with dialkynyl-Cys to form PheoA-PEG-Cys, and then reacts to form JSK-CSSE-PheoA. Both steps are dissolved in a mixed solvent and reacted at room temperature for 24-48 hours under the protection of an inert gas. After the reaction is completed, the product is dialyzed and freeze-dried.

[0041] The molar ratio of PheoA-PEG to dialkynyl-Cys is 1:1-1.5;

[0042] The molar ratio of PheoA-PEG to the catalyst copper sulfate pentahydrate and sodium ascorbate is 1:0.2-0.4:0.4-0.8;

[0043] The molar ratio of PheoA-PEG-Cys to JSK-PCL is 1:1-1.4;

[0044] The molar ratio of the JSK-PCL to the catalyst copper sulfate pentahydrate and sodium ascorbate is 1:0.2-0.4:0.4-0.8;

[0045] The mixed solvent is tetrahydrofuran and water, the volume ratio of tetrahydrofuran to water is 5:1, 0.05-0.1 mmol PheoA-PEG is added to every 10 mL tetrahydrofuran, and 0.05-0.1 mmol PheoA-PEG-Cys is added to every 10 mL tetrahydrofuran.

[0046] Preferably, the volatilization method of the mixed solvent comprises the following steps:

[0047] Dissolve JSK-CSSE-PheoA in an organic solvent to form an oil phase, slowly drop the oil phase into an aqueous medium under ultrasound or stirring, evaporate the organic solvent under reduced pressure or naturally, and then remove impurities with a 0.45 μM filter;

[0048] The organic solvent is tetrahydrofuran, dichloromethane, and acetone, the aqueous medium is a phosphate buffer solution with a pH of 7.4 and pure water, and the ratio of oil phase to water phase is: oil phase: aqueous medium = 1:10-60.

[0049] The beneficial effects of the present invention are:

[0050] The polyester of the hydrophobic segment of the drug delivery system of the present invention has good biocompatibility, can be self-degraded, metabolized, absorbed and excreted in the body, and the hydrophilic polyethylene glycol segment can increase the solubility of the photosensitizer PheoA, and is excreted by the kidneys after entering the body. In addition, the amphiphilic block copolymer composed of polyethylene glycol and polyester has high thermal stability and kinetic stability, can be stably present for more than two months, and can fully maintain the drug activity during the drug delivery process;

[0051] The hydrophilic polyethylene glycol segments and hydrophobic polyester can self-assemble into nanoparticles. The nitric oxide prodrug JSK is encapsulated in the hydrophobic cavity. The polyethylene glycol improves the solubility of the photosensitizer, making the photosensitizer dispersed on the surface of the nanoparticles in a self-quenching state. Under normal circumstances, the light effect is weak. In the tumor environment, the disulfide bonds between the hydrophilic and hydrophobic segments react with glutathione, the disulfide bonds break, and the photosensitizer is activated. A large amount of reactive oxygen species (ROS) can be generated under near-infrared light irradiation.

[0052] The nitric oxide prodrug JSK releases nitric oxide under the high glutathione and glutathione transferase in the tumor environment. Nitric oxide can consume solitary GSH, inhibit the mitochondrial respiratory chain, and increase the content of intracellular reactive oxygen species.

[0053] The generated nitric oxide (NO) and reactive oxygen species (ROS) can further generate reactive nitrogen species (RNS), which can promote the expression of matrix metalloproteinase (MMP-2). Matrix metalloproteinase can degrade collagen in tumor tissue, overcome tumor hypoxia, increase the oxygen content in the tumor, and enhance the efficacy of photodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0055] Figure 1 Schematic diagram of the synthesis structure of Example 1 of the present invention;

[0056] Figure 2 1H NMR spectra of dialkynyl-Cys, JSK-PCL, PheoA-PEG and JSK-CSSE-PheoA in Example 1 of the present invention;

[0057] Figure 3 TEM image of the nanoparticles prepared in Example 1 of the present invention;

[0058] Figure 4 is a hydrated particle size diagram of the nanoparticles prepared in Example 1 of the present invention;

[0059] Figure 5 is a graph showing the change in average particle size of nanoparticles over time in Example 3;

[0060] Figure 6 It is a graph showing the changes of the absorbance value of DBPF and JSK-CSSE-PheoA or JSK-CSSE-PheoA+glutathione under different near-infrared light irradiation in Example 4;

[0061] Figure 7 This is the nitric oxide release graph of JSK-CSSE-PheoA nanoparticles in Example 5. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0063] See also Figure 1-7 The present invention provides a technical solution: a drug delivery system of photosensitizer and nitric oxide prodrug, wherein the nanoparticles have a high anti-tumor efficiency: on the one hand, under the condition of high glutathione content in the tumor, the nanoparticles can stimulate the release of PheoA induced by aggregation quenching on the surface of the nanoparticles in response to activation of the phototoxicity of PheoA. On the other hand, the JSK prodrug in the hydrophobic core of the nanoparticle releases nitric oxide under the stimulation of glutathione and glutathione transfer highly expressed in the tumor; nitric oxide alleviates the hypoxia problem of solid tumors by mediating collagen degradation, and sensitizes the photodynamic anti-tumor effect of PheoA. The invention is simple and easy to repeat, with mild preparation conditions and cheap raw materials, and has broad application prospects.

[0064] The drug delivery system comprises a functionalized amphiphilic polymer having a chemical bond loaded photosensitizer prodrug pheophytin A and a nitric oxide prodrug, wherein the chemical bond loaded photosensitizer prodrug pheophytin A is PheoA, the nitric oxide prodrug is JSK, the amphiphilic polymer is PheoA-CSSE-JSK, and the two ends of the PheoA-CSSE-JSK are respectively a hydrophilic segment polyethylene glycol and a hydrophobic segment polycaprolactone, wherein the PheoA is connected to the hydrophilic segment The invention discloses a novel self-assembled nanoparticle comprising a poly(ethylene glycol) block and a hydrophobic segment polycaprolactone, wherein the poly(ethylene glycol) block is connected to one end of the hydrophobic segment polycaprolactone, and the JSK is connected to the other end of the hydrophobic segment polycaprolactone, and the PheoA-poly(ethylene glycol) block and the JSK-polyester are connected by a click reaction using a disulfide bond coupling agent alkynyl cystamine, and the amphiphilic prodrug functionalized polymer is prepared by a solvent evaporation method. The PheoA and the JSK are respectively distributed on the hydrophilic surface and the hydrophobic inner cavity of the nanoparticles, and the particle size of the nanoparticles is 50-300 nm, and the drug loading rate is 8%-40%.

[0065] The small molecule photosensitizer pheophorbide A (PheoA) and the NO prodrug O2-(2,4-dinitrophenyl)-1-〔(4-ethoxycarbonyl)piperazine-1-yl〕azo-1-ium-1,2-diol (JSK) were modified onto a biodegradable amphiphilic block copolymer, and the block copolymer was connected with a disulfide bond-containing conjugate to achieve glutathione (GSH) redox cleavage.

[0066] In this embodiment, preferably, when the amphiphilic polymer is used as a carrier material, the mass ratio of the carrier material to the photosensitizer prodrug and the NO prodrug is 1:0.1:0.5 respectively.

[0067] In this embodiment, preferably, the drug photosensitizer in the chemical bond-loaded photosensitizer prodrug pheophorbide A is a combination of one or more of chlorins, pheophorbide a, chlorophyll-a, chlorin e6 and pyropheophorbide-a.

[0068] In this embodiment, preferably, the donor of the nitric oxide prodrug is one or more combinations of nitramine alcohols, nitrosothiols, metal-nitroso and trifluoronitrobenzene.

[0069] In this embodiment, preferably, the drug delivery system of the characteristic photosensitizer and nitric oxide prodrug is prepared from the following compounds as raw materials:

[0070] Photosensitizer: pheophorbide A;

[0071] Nitric oxide prodrug: alkynylated O2-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)piperazin-1-yl]azo-1-ium-1,2-diol;

[0072] Compounds containing disulfide bonds: Cystamine hydrochloride;

[0073] Alkyne-containing compounds: pentynoic acid;

[0074] Hydrophilic polymer: amino-polyethylene glycol-azide with a molecular weight of 2000-10000;

[0075] Hydrophobic polymer: Azide-polycaprolactone-azide with a molecular weight of 2000-10000.

[0076] A method for preparing a drug delivery system of a photosensitizer and a nitric oxide prodrug, comprising the preparation of the following carrier materials:

[0077] Preparation of coupling agent dialkynyl-Cys, preparation of hydrophobic segment JSK-PCL, preparation of hydrophilic segment PheoA-PEG, preparation of JSK-CSSE-PheoA;

[0078] The preparation method of the coupling agent dialkynyl-Cys is as follows:

[0079] Cystamine dihydrochloride and 4-pentenoic acid were dissolved in a round-bottom flask, and catalysts dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added. Under the protection of inert gas, the mixture was stirred at room temperature for 24-48 hours. The reactants were filtered to remove the white precipitate, and the product dialkynyl-Cys was separated by column chromatography:

[0080] The molar ratio of cystamine dihydrochloride to 4-pentynoic acid is 1:2-3;

[0081] The molar ratio of the catalyst dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 1:0.8-1.2;

[0082] The molar ratio of cystamine dihydrochloride to dicyclohexylcarbodiimide is 1:2.5-4;

[0083] The solvent is anhydrous dichloromethane, and 2-3 mmol of cystamine dihydrochloride is added to every 50 ml of anhydrous dichloromethane.

[0084] Preferably, the preparation of the hydrophobic segment JSK-PCL is:

[0085] The NO prodrug alkynyl-JSK was connected to one side of azide-polycaprolactone-azide (N3-PCL-N3) by click reaction, using copper sulfate pentahydrate and sodium ascorbate as catalysts, and reacted at room temperature for 24-48 hours under inert gas protection. After the reaction was completed, it was placed in a dialysis bag with a molecular weight of 1000, dialyzed with deionized water for 3-4 days, and freeze-dried to obtain JSK-PCL:

[0086] The molar ratio of the azide-polycaprolactone-azide (N3-PCL-N3) and the NO prodrug JSK is 1:0.8-1.2, dissolved in tetrahydrofuran, 0.1-0.2mmol azide-polycaprolactone-azide (N3-PCL-N3) is added to 8mL tetrahydrofuran;

[0087] The catalyst copper sulfate pentahydrate and sodium ascorbate have a molar ratio of 1:1.8-2.4, which are dissolved in deionized water, and 0.02-0.04mmol copper sulfate pentahydrate is added to 2mL of deionized water;

[0088] The volume ratio of tetrahydrofuran to water is 5-3:1.

[0089] In this embodiment, preferably, the preparation of the hydrophilic segment PheoA-PEG is:

[0090] Pheophorbide A is connected to one side of amino-polyethylene glycol-azide (NH2-PEG-N3) by amidation reaction, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as catalysts, dissolved in a solvent and stirred at room temperature in the dark for 24-48 hours, and dialyzed and freeze-dried after the reaction to obtain PheoA-PEG;

[0091] The molar ratio of amino-polyethylene glycol-azide (NH2-PEG-N3) to pheophorbide A is 1:1-1.5;

[0092] The molar ratio of the catalyst 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is 3:2;

[0093] The molar ratio of the pheophorbide A to the EDC is 1:2.8-3.2;

[0094] The solvent is dimethyl sulfoxide (DMSO), and 200 mg of amino-polyethylene glycol-azide is added to every 10 mL of DMSO.

[0095] In this embodiment, preferably, the preparation of the JSK-CSSE-PheoA is:

[0096] Through click reaction, copper sulfate pentahydrate and sodium ascorbate were used as catalysts, and dialkynyl-Cys was used to connect the hydrophobic segment JSK-PCL and the hydrophilic segment PheoA-PEG to form an amphiphilic block polymer. The reaction was carried out in two steps.

[0097] First, PheoA-PEG reacts with dialkynyl-Cys to form PheoA-PEG-Cys, and then reacts to form JSK-CSSE-PheoA. Both steps are dissolved in a mixed solvent and reacted at room temperature for 24-48 hours under the protection of an inert gas. After the reaction is completed, the product is dialyzed and freeze-dried.

[0098] The molar ratio of PheoA-PEG to dialkynyl-Cys is 1:1-1.5;

[0099] The molar ratio of PheoA-PEG to the catalyst copper sulfate pentahydrate and sodium ascorbate is 1:0.2-0.4:0.4-0.8;

[0100] The molar ratio of PheoA-PEG-Cys to JSK-PCL is 1:1-1.4;

[0101] The molar ratio of the JSK-PCL to the catalyst copper sulfate pentahydrate and sodium ascorbate is 1:0.2-0.4:0.4-0.8;

[0102] The mixed solvent is tetrahydrofuran and water, the volume ratio of tetrahydrofuran to water is 5:1, 0.05-0.1 mmol PheoA-PEG is added to every 10 mL tetrahydrofuran, and 0.05-0.1 mmol PheoA-PEG-Cys is added to every 10 mL tetrahydrofuran.

[0103] In this embodiment, preferably, the volatilization method of the mixed solvent comprises the following steps:

[0104] Dissolve JSK-CSSE-PheoA in an organic solvent to form an oil phase, slowly drop the oil phase into an aqueous medium under ultrasound or stirring, evaporate the organic solvent under reduced pressure or naturally, and then remove impurities with a 0.45 μM filter;

[0105] The organic solvent is tetrahydrofuran, dichloromethane, and acetone, the aqueous medium is a phosphate buffer solution with a pH of 7.4 and pure water, and the ratio of oil phase to water phase is: oil phase: aqueous medium = 1:10-60.

[0106] Example 1

[0107] Preparation of dialkynyl-Cys:

[0108] 0.416 g (2.70 mmol) of cystamine dihydrochloride and 0.582 g (5.94 mmol) of 4-pentenoic acid were placed in a 100 ml round-bottom flask, 50 ml of anhydrous dichloromethane was added to dissolve, and then 1.33 g (6.46 mmol) of dicyclohexylcarbodiimide (DCC) and 0.792 g (6.48 mmol) of 4-dimethylaminopyridine (DMAP) were added, and stirred at room temperature for 48 hours under nitrogen protection. The reactants were filtered to remove white precipitates, and the product dialkynyl-Cys was isolated by column chromatography.

[0109] Synthesis of hydrophobic segment JSK-PCL

[0110] 200 mg (0.1 mmol) of azide-polycaprolactone-azide (N3-PCL-N3) and 40 mg (0.1 mmol) of alkynyl-JSK were dissolved in 8 ml of tetrahydrofuran, 5 mg (0.02 mmol) of copper sulfate pentahydrate and 8 mg (0.4 mmol) of sodium ascorbate were dissolved in 2 ml of deionized water, and the two solutions were mixed. The mixture was reacted at room temperature for 24 hours under nitrogen protection, and then placed in a dialysis bag with a molecular weight of 1000, dialyzed with deionized water for 3 days, and freeze-dried.

[0111] Synthesis of PheoA-PEG

[0112] 60 mg (0.1 mmol) of pheophorbide A (PheoA) and 200 mg (0.1 mmol) of amino-polyethylene glycol-azide (NH2-PEG-N3) were dissolved in 10 ml of dimethyl sulfoxide, and 47 mg (0.3 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 23 mg (0.2 mmol) of N-hydroxysuccinimide (NHS) were added. Stir at room temperature in the dark for 24 hours. The product was dialyzed against dimethyl sulfoxide for 2 days, then dialyzed against deionized water for 3 days, and lyophilized.

[0113] Synthesis of JSK-CSSE-PheoA

[0114] PheoA-PEG (129 mg, 0.05 mmol) and 35 mg (0.11 mmol) of dialkynyl-Cys were dissolved in 10 ml of tetrahydrofuran. 2.5 mg (0.01 mmol) of copper sulfate pentahydrate and 4 mg (0.2 mmol) of sodium ascorbate were dissolved in 2 ml of water and reacted at room temperature for 24 hours under nitrogen protection. After the reaction, the product PheoA-PEG-Cys was dialyzed with deionized water for 3 days and freeze-dried. Similarly, 145 mg (0.05 mmol) of PheoA-PEG-Cys and 120 mg (0.05 mmol) of JSK-PCL were dissolved in 10 ml of tetrahydrofuran. 2.5 mg (0.01 mmol) and 4 mg (0.2 mmol) of sodium ascorbate were dissolved in 2 ml of water and reacted at room temperature for 24 hours under nitrogen protection. After the reaction, the product JSK-CSSE-PheoA was obtained by dialyzing with deionized water for 3 days and freeze-drying. Figure 2 .

[0115] Preparation of drug-loaded nanoparticle drug delivery system

[0116] 5 mg of JSK-CSSE-PheoA was dissolved in 0.5 ml of tetrahydrofuran; then, it was dropped into 5 ml of deionized water under magnetic stirring and stirred in a fume hood until the tetrahydrofuran was completely evaporated. The solution was then filtered with a 0.45 μM filter. The morphology of the nanoparticles was observed by TEM. The results are shown in the attached figure. Figure 3 ; The particle size was analyzed by nanometer particle size analyzer. The results are shown in the attached Figure 4 , the average particle size is 167nm.

[0117] Example 2 Calculation of drug loading rate of drug-loaded nanoparticle drug delivery system

[0118] Different concentrations of JSK and PheoA were measured by UV-visible spectrophotometer to draw a standard curve. 1 mg of JSK-CSSE-PheoA was dissolved in 4-40 ml of tetrahydrofuran, and the absorbance at 300 nm and 670 nm was tested by UV-visible spectrophotometer. The concentrations of JSK and PheoA in JSK-CSSE-PheoA were calculated by the standard curves of JSK and PheoA. The drug loading rate calculation formula of the nanoparticle drug delivery system is as follows:

[0119] JSK loading rate = encapsulated JSK weight / nanoparticle weight × 100%;

[0120] PheoA loading rate = encapsulated PheoA weight / nanoparticle weight × 100%.

[0121] The JSK loading rate is 9.34% and the PheoA loading rate is 9.60%.

[0122] Example 3 Stability experiment of drug-loaded nanoparticles

[0123] The JSK-CSSE-PheoA nanoparticles prepared in Example 1.5 were tested by a nanoparticle size analyzer and found to have an average particle size of 167 nm. The particle size of the samples was tested every two days, and no significant change in the particle size was found within 9 days of testing, indicating that the nanoparticle drug delivery system can be stably present. The results are shown in the attached Figure 5 .

[0124] Example 4 Photosensitizer activation experiment

[0125] The singlet oxygen probe 1,3-diphenylisobenzofuran (DPBF) was used to detect the activation of the photosensitizer. 5 mg of DPBF was dissolved in 1 ml of dimethyl sulfoxide. Take the nanoparticles prepared in Example 1.5, add 3 mg of glutathione to every 1 ml of nanoparticles with a concentration of 1 mg / mL, and incubate at 37°C for 6 to 12 hours to lyse the nanoparticles. Subsequently, 200 μL of the above-mentioned lysed nanoparticles and 200 μL of the nanoparticles prepared in Example 1.5 were added to a 96-well plate, and 1 μL of DPBF solution was added. Three parallel samples were added to each group and irradiated with 670 nm near-infrared light with a power of 0.2 W for different periods of time. The absorbance at 404 nm was tested using an enzyme reader. The results show that the cleavage of the nanoparticles contributes to the activation of the photosensitizer. The results are shown in the attached figure. Figure 6 .

[0126] Example 5 In vitro NO release experiment

[0127] We used a nitric oxide kit to test the NO release behavior of JSK-CSSE-PheoA nanoparticles. The nanoparticles prepared in Example 1 were added with glutathione and glutathione transferase, with a final concentration of 10 mmol / L of glutathione and a final concentration of 5 μg / L of glutathione transferase. Nanoparticles without glutathione and glutathione transferase were used as a control group. Incubate in a shaker at 37°C, take 50 μL of working solution and add it to a 96-well plate at 0, 0.16, 0.5, 1, 2, 4, 8, 10, 12, 24, 32, 48, and 72 hours, respectively, and add 50 μL of Griess reagent I and 50 μL of Griess reagent II. Use an enzyme reader to test the absorbance at 540 nm, and test the nitric oxide content released by the JSK-CSSE-PheoA nanoparticles based on the nitric oxide standard curve. The results are attached. Figure 7 .

[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A drug delivery system for a photosensitizer and a nitric oxide prodrug, characterized in that: The drug delivery system comprises a functionalized amphiphilic polymer loaded with a chemical bond photosensitizer pheophorbide A and a nitric oxide prodrug, wherein the chemical bond loaded photosensitizer pheophorbide A is PheoA, the nitric oxide prodrug is JSK, the functionalized amphiphilic polymer is JSK-CSSE-PheoA, the two ends of the JSK-CSSE-PheoA are respectively a hydrophilic segment polyethylene glycol and a hydrophobic segment polycaprolactone, wherein the PheoA is connected to the hydrophilic segment The PheoA-modified polyethylene glycol block and the JSK-modified polyester are connected at one end of the hydrophobic segment polycaprolactone, and the JSK is connected to the other end of the hydrophobic segment polycaprolactone, and the PheoA-modified polyethylene glycol block and the JSK-modified polyester are connected by a click reaction using a disulfide bond coupling agent alkynyl cystamine, and the amphiphilic prodrug functionalized polymer is prepared by a solvent evaporation method to prepare self-assembled nanoparticles, wherein the PheoA and the JSK are distributed on the hydrophilic surface and the hydrophobic inner cavity of the nanoparticles, respectively, and the particle size of the nanoparticles is 50-300 nm, and the drug loading rate is 8%-40%; The drug delivery system of photosensitizer and nitric oxide prodrug is prepared from the following compounds as raw materials: Photosensitizer: pheophorbide A; Nitric oxide prodrug: alkynylated O2-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)piperazin-1-yl]azo-1-ium-1,2-diol; Compounds containing disulfide bonds: Cystamine hydrochloride; Alkyne-containing compounds: pentynoic acid; Hydrophilic polymer: amino-polyethylene glycol-azide with a molecular weight of 2000-10000; Hydrophobic polymer: Azide-polycaprolactone-azide with a molecular weight of 2000-10000.

2. A method for preparing the drug delivery system of a photosensitizer and a nitric oxide prodrug according to claim 1, characterized in that: The preparation includes the following carrier materials: preparation of coupling agent dialkynyl-Cys, preparation of hydrophobic segment JSK-PCL, preparation of hydrophilic segment PheoA-PEG, preparation of JSK-CSSE-PheoA The preparation method of the coupling agent dialkynyl-Cys is as follows: Cystamine dihydrochloride and 4-pentynoic acid were dissolved in a round-bottom flask, and catalysts dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added. Under the protection of inert gas, the mixture was stirred at room temperature for 24-48 hours. The reactants were filtered to remove the white precipitate, and the product dialkynyl-Cys was separated by column chromatography: The molar ratio of cystamine dihydrochloride to 4-pentynoic acid is 1:2-3; The molar ratio of the catalyst dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 1:0.8-1.2; The molar ratio of cystamine dihydrochloride to dicyclohexylcarbodiimide is 1:2.5-4; The solvent is anhydrous dichloromethane, and 2-3 mmol of cystamine dihydrochloride is added to every 50 ml of anhydrous dichloromethane.

3. The method for preparing a drug delivery system of a photosensitizer and a nitric oxide prodrug according to claim 2, characterized in that: Preparation of the hydrophobic segment JSK-PCL: The NO prodrug alkynyl-JSK was connected to one side of azide-polycaprolactone-azide (N3-PCL-N3) by click reaction, using copper sulfate pentahydrate and sodium ascorbate as catalysts. The reaction was carried out at room temperature for 24-48 hours under the protection of inert gas. After the reaction was completed, it was placed in a dialysis bag with a molecular weight of 1000, dialyzed with deionized water for 3-4 days, and freeze-dried to obtain JSK-PCL: The molar ratio of the azide-polycaprolactone-azide (N3-PCL-N3) and the NO prodrug JSK is 1:0.8-1.2, dissolved in tetrahydrofuran, 0.1-0.2mmol azide-polycaprolactone-azide (N3-PCL-N3) is added to 8mL tetrahydrofuran; The catalyst copper sulfate pentahydrate and sodium ascorbate have a molar ratio of 1:1.8-2.4 and are dissolved in deionized water, 0.02-0.04 mmol copper sulfate pentahydrate is added to 2 mL of deionized water; The volume ratio of tetrahydrofuran to water is 5-3:

1.

4. The method for preparing a drug delivery system of a photosensitizer and a nitric oxide prodrug according to claim 2, characterized in that: Preparation of the hydrophilic segment PheoA-PEG: Pheophorbide A was connected to one side of amino-polyethylene glycol-azide (NH2-PEG-N3) by amidation reaction, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as catalysts, dissolved in a solvent and stirred at room temperature in the dark for 24-48 hours, and dialyzed and freeze-dried after the reaction to obtain PheoA-PEG; The molar ratio of amino-polyethylene glycol-azide (NH2-PEG-N3) to pheophorbide A is 1:1-1.5; The molar ratio of the catalyst 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is 3:2; The molar ratio of the pheophorbide A to the EDC is 1:2.8-3.2; The solvent is dimethyl sulfoxide (DMSO), and 200 mg of amino-polyethylene glycol-azide is added to every 10 mL of dimethyl sulfoxide.

5. The method for preparing a drug delivery system of a photosensitizer and a nitric oxide prodrug according to claim 2, characterized in that: The preparation of JSK-CSSE-PheoA includes: Through click reaction, copper sulfate pentahydrate and sodium ascorbate were used as catalysts, and dialkynyl-Cys was used to connect the hydrophobic segment JSK-PCL and the hydrophilic segment PheoA-PEG to form an amphiphilic block polymer. The reaction was carried out in two steps. First, PheoA-PEG reacts with dialkynyl-Cys to form PheoA-PEG-Cys, and then reacts to form JSK-CSSE-PheoA. Both steps are dissolved in a mixed solvent and reacted at room temperature for 24-48 hours under the protection of an inert gas. After the reaction is completed, the product is dialyzed and freeze-dried. The molar ratio of PheoA-PEG to dialkynyl-Cys is 1:1-1.5; The molar ratio of PheoA-PEG to the catalyst copper sulfate pentahydrate and sodium ascorbate is 1:0.2-0.4:0.4-0.8; The molar ratio of PheoA-PEG-Cys to JSK-PCL is 1:1-1.4; The molar ratio of the JSK-PCL to the catalyst copper sulfate pentahydrate and sodium ascorbate is 1:0.2-0.4:0.4-0.8; The mixed solvent is tetrahydrofuran and water, the volume ratio of tetrahydrofuran to water is 5:1, 0.05-0.1 mmol PheoA-PEG is added to every 10 mL tetrahydrofuran, and 0.05-0.1 mmol PheoA-PEG-Cys is added to every 10 mL tetrahydrofuran.

6. The method for preparing a drug delivery system of a photosensitizer and a nitric oxide prodrug according to claim 2, characterized in that: The mixed solvent volatilization method comprises the following steps: JSK-CSSE-PheoA is dissolved in an organic solvent to form an oil phase, the oil phase is slowly added dropwise to an aqueous medium under ultrasound or stirring, the organic solvent is evaporated under reduced pressure or naturally, and impurities are removed with a 0.45 μM filter to obtain a drug delivery system of a photosensitizer and a nitric oxide prodrug; The organic solvent is tetrahydrofuran, dichloromethane and acetone, the aqueous medium is a phosphate buffer solution with a pH value of 7.4 and pure water, and the ratio of oil phase to water phase is: oil phase: aqueous medium = 1:10-60.

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