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CN-121971635-A - DOX/H of three sulfur bond response2S-release-cooperated nano-carrier and preparation method and application thereof

CN121971635ACN 121971635 ACN121971635 ACN 121971635ACN-121971635-A

Abstract

The invention discloses a DOX/H 2 S synergistic release nano-carrier with a trisulfide bond response, and a preparation method and application thereof, and belongs to the technical field of nano-drug delivery. The nano-carrier comprises an amphiphilic random copolymer and Doxorubicin (DOX) which is covalently loaded on the copolymer through an active ester bond, wherein the amphiphilic random copolymer is formed by copolymerizing a hydrophilic unit and a hydrophobic unit, the hydrophilic unit is OEGMA or PCB, the hydrophobic unit is a hydrophobic monomer containing a trisulfide bond, the amphiphilic random copolymer and DOX loaded on the copolymer are self-assembled in a phosphate buffer solution to form a micelle, and the nano-carrier is obtained, and the drug loading rate of the DOX is more than 25%. When the nano-carrier is used for high-concentration GSH (10 mM) in a tumor microenvironment, the trisulfide bond is broken and the DOX and H 2 S are released synchronously, so that the chemotherapy-gas cooperative treatment is realized. In vivo and in vitro experiments prove that the nano-carrier has remarkable killing effect on HeLa cells and H22 liver cancer, and provides a new strategy for tumor cooperative treatment.

Inventors

  • GE ZHISHEN
  • ZHAO MENG

Assignees

  • 嵊州市长三角新能源产教融合研究院
  • 西安交通大学

Dates

Publication Date
20260505
Application Date
20251225

Claims (10)

  1. 1. A trisulfide-responsive DOX/H 2 S co-release nanocarrier characterized in that the nanocarrier comprises an amphiphilic random copolymer and Doxorubicin (DOX) covalently supported on the copolymer via an active ester linkage; the amphiphilic random copolymer is formed by copolymerizing a hydrophilic unit and a hydrophobic unit, wherein: The hydrophilic unit is polyethylene glycol methyl ether methacrylate (OEGMA) or carboxyl betaine methacrylate (PCB); the hydrophobic unit is a functional monomer containing a trisulfide bond, and the hydrophobic monomer containing the trisulfide bond is 2- ((2- (((4-nitrophenoxy) carbonyl) oxy) ethyl) trisulfide group) ethyl methacrylate, which is called 3S-2 for short; And the amphiphilic random copolymer and Doxorubicin (DOX) loaded on the copolymer are self-assembled in Phosphate Buffer (PBS) to form a micelle, so that the nano-carrier is obtained.
  2. 2. The nanocarrier of claim 1, wherein the molar ratio of the hydrophobic units in the amphiphilic random copolymer is between 15% and 30%.
  3. 3. The nanocarrier of claim 1, wherein the PCB is deprotected from a PCB precursor monomer CB-tBu synthesized by quaternization of 2- (dimethylamino) ethyl methacrylate with t-butyl bromoacetate.
  4. 4. The nano-carrier according to claim 1, wherein the trisulfide functional monomer 3S-2 is prepared by nucleophilic substitution reaction of 2-bromoethanol and sodium sulfide as raw materials to generate 2,2' -trisulfide diethanol, and then sequentially reacting with methacryloyl chloride and 4-nitrobenzoate.
  5. 5. A method of preparing the nanocarrier of any of claims 1-4, comprising the steps of: (1) Synthesis of trisulfide functional monomer 3S-2: dissolving sodium sulfide and elemental sulfur in deionized water, stirring for 2 hours at 50 ℃, then dropwise adding 2-bromoethanol, reacting for 12 hours under the protection of nitrogen, extracting and drying a reaction liquid to obtain 2,2' -trisulfodiethanol; Dissolving 2,2' -trithiodiethanol and triethylamine in dichloromethane, dropwise adding methacryloyl chloride under ice bath, reacting at room temperature for 12 hours, and purifying to obtain an intermediate 2- ((2-hydroxyethyl) trithioalkyl) ethyl methacrylate, namely 3S-1; Dissolving 3S-1 and 4-nitrobenzoic acid ester in dichloromethane, adding triethylamine, reacting for 24 hours at 25 ℃, and purifying the reaction liquid to obtain 3S-2; (2) Preparation of amphiphilic random copolymer: the amphiphilic random copolymer is formed by copolymerizing a hydrophilic unit and a hydrophobic unit; ① If the hydrophilic unit is OEGMA, 3S-2 and OEGMA are reacted in 1, 4-dioxane under the initiation of AIBN at 80 ℃ for 24 hours, POEGMA-3S is obtained by vacuum drying, ② if the hydrophilic unit is PCB, 3S-2, CB-tBu, AIBN and chain transfer agent 2-cyano-2-propylbenzene disulfide are dissolved in 1, 4-dioxane, the reaction is carried out for 24 hours at 80 ℃ after freeze thawing and deoxidization, the precipitation is dried to obtain PCB-tBu-3S, the PCB-tBu-3S is dissolved in dichloromethane, trifluoroacetic acid (TFA) is added for deprotection at room temperature for 1 hour, and the PCB-3S is obtained by vacuum drying; (3) DOX drug covalent coupling DOX.HCl reacts with the copolymer (POE GMA-3S or PCB-3S) obtained in the step (2) under the activation of triethylamine, and 4-nitrophenoxycarbonyl groups of side chains of the copolymer are reacted, DOX covalent loading is realized through an active ester bond, and DOX coupling copolymer (POE GMA-3S-DOX or PCB-3S-DOX) is obtained; (4) Self-assembly of nanocarriers Dissolving the DOX coupling copolymer obtained in the step (3) in dimethyl sulfoxide (DMSO), injecting the DOX coupling copolymer into PBS, and self-assembling to form a micelle with the particle size of 25-85nm, thus obtaining the nano-carrier.
  6. 6. The method of preparing nanocarriers of claim 5, wherein the molar ratio of the raw materials in step (1) is: sodium sulfide, elemental sulfur and 2-bromoethanol are in a ratio of 1:2:2; 2,2' -trithiodiethanol, triethylamine and methacryloyl chloride are 2:3:2; 3S-1:4-nitrobenzene chloroformate and triethylamine are 5:6:6.
  7. 7. The method of claim 5, wherein in the step (2), if the hydrophilic unit is OEGMA 3S-2, OEGMA, AIBN, 2-cyano-2-propylbenzodisulfide The molar ratio of (2) is 175:420:1:7.
  8. 8. The method according to claim 5, wherein in the step (2), if the hydrophilic unit is PCB, the molar ratio of 3S-2, CB-tBu, AIBN and chain transfer agent 2-cyano-2-propylbenzenedisulphide is 370:2800:7:30.
  9. 9. The method of preparing nanocarriers of claim 5, wherein in step (3), DOX The molar ratio of HCl to copolymer is 30:1, and the mol ratio of triethylamine to DOX is 30:1 The molar ratio of HCl was 2:1.
  10. 10. Use of the nanocarrier of any of claims 1-4 in the preparation of a medicament for treating a tumor, wherein the tumor comprises a tumor derived from HeLa cells and a tumor derived from liver cancer cells.

Description

DOX/H 2 S synergistic release nano-carrier with trisulfide bond response and preparation method and application thereof Technical Field The invention belongs to the technical field of nano drug delivery systems, and particularly relates to a DOX/H 2 S synergistic release nano carrier with a trisulfide bond response function, a preparation method and application thereof. Background Cancer is one of the major causes of death worldwide, and its clinical treatment has long been limited by the inherent deficiency [1] of chemotherapeutic drugs. Classical chemotherapeutic drugs represented by Doxorubicin (DOX) have three major bottlenecks of poor water solubility, insufficient tumor targeting accumulation and obvious systemic toxic and side effects, severely restrict the curative effect and influence the survival quality [2] of patients. In recent years, intelligent response type nano-carriers based on Tumor Microenvironment (TME) characteristics become a core strategy for breaking through the dilemma, and accurate controlled release [3] of drugs is realized by identifying signals such as reduced Glutathione (GSH), acidic pH or specific enzymes and the like which are over-expressed in TME. Among them, the nanocarrier using disulfide bond as a response unit attracts attention [4] by utilizing the remarkable difference between high concentration GSH (2-10 mM) in tumor cells and extracellular environment (2-20 μm). The carrier can generate disulfide bond rupture at the tumor part to release the encapsulated medicine, and reduce systemic toxicity to a certain extent. However, the traditional disulfide bond system has the fundamental limitations that firstly, the response mechanism is single, only the delivery of chemotherapeutic drugs can be realized, and other treatment modes cannot be integrated, secondly, the drug loading rate is generally lower than 20%, the physical encapsulation is easy to cause the early leakage of the drugs, thirdly, the response sensitivity of the disulfide bond to GSH is insufficient, the drug release rate is lower than 15% under the physiological concentration GSH (10 mu M), and the requirement of high-efficiency treatment (the problem is faced by a polyurethane vesicle system reported by Wu and the like) is difficult to meet [5]. Of interest, gas molecular therapy provides a new perspective for tumor treatment. Endogenous gas signaling molecule hydrogen sulfide (H 2 S) can selectively induce apoptosis [6] in cancer cells by inhibiting mitochondrial respiratory chain complex IV to block ATP synthesis, deplete intracellular GSH libraries, and induce oxidative stress storm triplet mechanisms. However, the half-life of H 2 S is extremely short (second order), and systemic administration is prone to toxic side effects such as blood pressure dip, the development of a tumor-targeted H 2 S precise delivery system [7] is highly needed. Recent studies have attempted to introduce trisulfide bonds into nanocarriers that not only have a higher GSH response sensitivity than disulfide bonds, but also release therapeutic doses of H 2 S simultaneously at break, providing the potential [8] for chemo-gas co-therapy. However, the existing three-sulfur bond carrier still takes over the traditional design, namely, the traditional three-sulfur bond carrier is loaded with a medicine through physical encapsulation (the medicine loading rate is less than 15 percent), or a small molecular prodrug dimer is constructed, the former is faced with the problem of medicine leakage, and the latter is poor in self-assembly stability and quick in-vivo clearance due to molecular rigidity, so that long-acting circulation and tumor enrichment [9] are difficult to realize. At the same time, the hydrophilic chain structure of the nanocarriers has a decisive influence on the in vivo behavior. Oligomeric Ethylene Glycol Methacrylate (OEGMA) can prolong blood circulation time by "stealth effects", but is prone to induce immunogenicity and produce Accelerated Blood Clearance (ABC) phenomena, limiting repeat dosing effects [10]. Zwitterionic polymers (such as carboxybetaine methacrylate, PCB) can form a more compact hydration layer than OEGMA by virtue of super hydration capability and an electrically neutral surface, so that ultra-low protein adsorption is realized, and the polymer has better long-cycle potential [11] theoretically. However, no research system for comparing the performance difference of PCB and PEG in the trisulfide carrier exists so far, and further research on carrier stability and therapeutic efficacy is lacking in hydrophobic proportion regulation. In summary, the existing tumor treatment nano-carrier has three challenges, namely, firstly, the traditional disulfide bond system has low response efficiency and single function, and can not cooperatively release various therapeutic agents, secondly, the trisulfide bond carrier can synchronously release drugs and H 2 S, but the physical encapsulation or dimer strategy