CN113813402B - Preparation method and application of nanogel with anti-tumor function of hunger combined gas therapy - Google Patents

Preparation method and application of nanogel with anti-tumor function of hunger combined gas therapy Download PDF

Info

Publication number
CN113813402B
CN113813402B CN202111167823.XA CN202111167823A CN113813402B CN 113813402 B CN113813402 B CN 113813402B CN 202111167823 A CN202111167823 A CN 202111167823A CN 113813402 B CN113813402 B CN 113813402B
Authority
CN
China
Prior art keywords
nanogel
alkynyl
starvation
combined gas
gas therapy
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111167823.XA
Other languages
Chinese (zh)
Other versions
CN113813402A (en
Inventor
陈维
牛亚凡
戴琳
黄德春
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
China Pharmaceutical University
Original Assignee
China Pharmaceutical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by China Pharmaceutical University filed Critical China Pharmaceutical University
Priority to CN202111167823.XA priority Critical patent/CN113813402B/en
Publication of CN113813402A publication Critical patent/CN113813402A/en
Application granted granted Critical
Publication of CN113813402B publication Critical patent/CN113813402B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/14—Esterification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03004—Glucose oxidase (1.1.3.4)

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Polymers & Plastics (AREA)
  • Medicinal Preparation (AREA)

Abstract

The invention discloses a preparation method and application of nanogel with a starvation combined gas therapy anti-tumor function, wherein the nanogel is prepared by forming a nanoenzyme compound through electrostatic interaction of azido polyethylenimine grafted with L-arginine and glucose oxidase, then covering the surface of the nanogel with an alkynyl-containing polycarbonate block copolymer, and performing click chemical crosslinking on the azido and alkynyl. Compared with the prior art, the invention consumes endogenous glucose and releases nitric oxide gas by utilizing specific enzymatic reaction, kills cancer cells by starvation and gas therapy, and simultaneously, the nitric oxide gas can redistribute oxygen in the cancer cells, thereby relieving the hypoxia of tumors, and having wide application prospect in the aspects of resisting tumors and inhibiting the metastasis of tumors.

Description

Preparation method and application of nanogel with anti-tumor function of hunger combined gas therapy
Technical Field
The invention belongs to a preparation method and application of materials and biological medicines, and particularly relates to a preparation method and application of nanogel with an anti-tumor function of hunger combined gas therapy.
Background
In recent years, cancer metabolism, particularly glucose metabolism, has received increasing attention in the design of cancer treatments. Based on the Yu Wabo lattice effect, proliferating cancer cells consume more glucose to produce energy than normal tissue. Several cancer treatment strategies have been proposed to prevent glucose consumption. Combining this strategy with other treatments may be an attractive option because the blocking of the glucose supply can only slow down tumor growth, but not kill cancer cells completely.
Glucose oxidase (Gox) catalyzes the oxidation of glucose and produces gluconic acid and hydrogen peroxide (H) in the presence of oxygen 2 O 2 ). The process can effectively consume glucose and oxygen in tumor, thereby increasing hypoxia, acidity and H of tumor microenvironment 2 O 2 An environment. Hypoxia is a significant feature of many solid tumors, however, often associated with tumor growth, lung metastasis, and resistance to most standard therapeutic approaches. For example, upregulation of hypoxia-inducible factor-1α (HIF-1α) is significantly positively correlated with tumor lung metastasis. Gox faces the problem of exacerbating tumor hypoxia during its function, thereby promoting HIF-1α stabilization. Meanwhile, gox has the disadvantages of poor stability, short half-life in vivo, immunogenicity and systemic toxicity.
NO plays the most prominent role in regulating neuronal communication, vascular regulation, wound healing and other physiological and pathological activities, and meanwhile, NO is used as a respiratory inhibitor, can redistribute oxygen to non-respiratory targets and promotes degradation of HIF-1 alpha. However, highly active NO gas cannot be used directly in clinical trials because of its short lifetime and concentration dependence. Accordingly, a large number of NO donors have been developed for storing and releasing NO under certain conditions. L-arginine (L-Arg) has good biocompatibility as nitric oxide donor, and can be used as inducible nitric oxide synthase or H 2 O 2 Can precisely release nitric oxide in the presence of the nitric oxide. The reaction of L-Arg with reactive oxygen species to form nitric oxide derives new mechanisms for tumor therapy. In tumor cells enriched with active oxygen, L-Arg is expected to produce nitric oxide for use in gas therapy. Cancer cells contain a greater amount of H than normal cells 2 O 2 But endogenous H 2 O 2 The level still does not allow an effective reaction. Increasing H in tumor cells 2 O 2 Concentration oxidation of L-Arg to NO remains challenging for use in gas therapy.
At present, medicines such as Gox, vitamin C and the like are commonly used for improving H in tumor cells 2 O 2 Is a concentration of (3). Given the important role of glucose in providing energy for tumor metabolism, we pass through glucoseGlucose metabolism consumes glucose in tumors and uses Gox's catalysis to oxidize glucose to gluconic acid and H 2 O 2 Thereby strategically starving the tumor. In addition, H 2 O 2 The increase in concentration helps to accelerate the oxidation of L-Arg to NO. NO production can inhibit HIF-1α accumulation caused by Gox, thereby inhibiting tumor proliferation and lung metastasis.
However, few reports are currently relevant concerning techniques and methods for starvation therapy and NO gas therapy simultaneously for inhibiting tumor proliferation and lung metastasis.
Disclosure of Invention
The invention aims to: the invention provides a preparation method of a nanogel with an anti-tumor function.
Another object of the present invention is to provide the use of the nanogel with anti-tumor function.
The technical scheme is as follows: the preparation method of the nanogel with the anti-tumor function of hunger combined gas therapy mainly comprises the steps of forming a nano enzyme compound with uniform particle size through self-assembly of functional polyethyleneimine and glucose oxidase (Gox) through electrostatic interaction, and covering the surface of the nano enzyme compound with a cross-linked poly-carbonate block copolymer through click chemistry of alkynyl and azide to obtain the nanogel.
Preferably, the method for preparing the positively charged azide polymer comprises the following steps: sequentially carrying out amidation reaction on the polyethyleneimine and carboxylic acid monomers containing azido and L-arginine to obtain the functional polyethyleneimine.
Preferably, the functionalized polyethyleneimine containing azide groups is synthesized by the following method: firstly, dissolving carboxylic acid monomers containing azido into high-purity water, adding a catalyst of 1-ethyl- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide to activate carboxyl, then adding polyethylenimine PEI, regulating pH to be neutral, and carrying out amidation reaction to obtain azido polyethylenimine; and then dissolving L-arginine (Arg) in high-purity water, regulating the pH to be neutral, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide to activate carboxyl, and adding azido polyethyleneimine into a reaction system to perform amidation reaction after activation to obtain the functionalized polyethyleneimine containing azido groups.
Preferably, the carboxylic acid monomer containing an azide group is selected from compounds having the following structure:wherein R is 1 Selected from C2-C5 alkyl.
Preferably, the molecular weight of the polyethyleneimine is 1.8-25kDa.
Preferably, the preparation method of the polycarbonate block copolymer containing alkynyl comprises the following steps: firstly, using polyethylene glycol (PEG) as an initiator to obtain a triblock copolymer containing acrylate functional groups through ring-opening polymerization with a cyclic carbonate monomer containing acrylate functional groups; and modifying the double bond of the sulfhydryl-containing carboxylic monomer into carboxyl by a Michael addition reaction, and modifying the carboxyl into alkynyl by an esterification reaction of the hydroxyl-containing alkyne monomer to obtain the alkynyl polycarbonate block polymer.
Preferably, the alkynyl-containing polycarbonate block copolymer is synthesized by the following method: using polyethylene glycol PEG as an initiator, methylene dichloride as a solvent, and bis (bistrimethylsilyl) amine zinc as a catalyst, and carrying out ring-opening copolymerization on the bis (bistrimethylsilyl) amine zinc and a cyclic carbonate monomer (AC) containing an acrylic ester functional group to prepare a triblock copolymer containing the acrylic ester functional group; n, N-Dimethylformamide (DMF) is taken as a solvent, triethylamine is taken as a catalyst, and a double bond is modified into carboxyl by a carboxyl monomer containing sulfhydryl through Michael addition reaction; and (3) using DMF as a solvent and dicyclohexylcarbodiimide/4-dimethylaminopyridine as a catalyst, and modifying carboxyl into alkynyl by using an alkyne monomer containing hydroxyl through esterification reaction to obtain the alkynyl polycarbonate block polymer.
Preferably, the cyclic carbonate monomer (AC) containing an acrylate functional group is selected from the group consisting of compounds having the structure shown below:
the sulfhydryl-containing carboxylic monomer is characterized by being selected from compounds with the following structures:wherein R is 2 Selected from C2-C4 alkyl, or C4-C8 aryl.
A hydroxyl-containing alkyne monomer, characterized by a compound selected from the group consisting of the structures shown below:wherein R is 3 Selected from C1-C5 alkyl,/and>
preferably, the nano-enzyme complex is self-assembled in water by the action of charges by the functional polyethyleneimine and glucose oxidase to form the nano-enzyme complex with uniform particle size.
Preferably, encapsulation of glucose oxidase in different proportions can be accurately achieved by adjusting the pH of the solution.
Preferably, the preparation method of the nanogel comprises the following steps:
(1) Firstly, respectively dissolving the functional polyethyleneimine and the Gox in high-purity water, quickly dripping a certain amount of Gox into the functional polyethyleneimine solution, standing after vortex to obtain a nano enzyme compound, and finely adjusting the pH of the aqueous solution through sodium hydroxide to accurately realize the encapsulation of glucose oxidase with different proportions;
(2) Dissolving the polycarbonate block copolymer containing alkynyl in dimethyl sulfoxide (DMSO), slowly dripping the polycarbonate block copolymer into the nano-composite obtained in the step (1) under a stirring state, stirring and reacting for a period of time to crosslink, and finally dialyzing to remove the DMSO.
The cross-linked drug-loaded nano gel obtained by the method can improve the stability of nano particles.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:
1. the invention discloses a loading mode of protein drugs for the first time, and the encapsulation of drugs with different proportions can be accurately realized through the charge effect between materials and the protein drugs, so that the encapsulation efficiency of nano carriers on macromolecular drugs is greatly improved.
2. The invention provides a method for stabilizing a nano-composite formed by charge interaction for the first time, wherein an alkynyl-containing polycarbonate block copolymer covers the surface of a nano-enzyme composite for crosslinking, so that the stability of the nano-enzyme composite is enhanced, the nano-enzyme composite is not easy to dissociate in the outside of cells and blood, and the defects of easy leakage and low carrying efficiency of medicines in the body in the prior art are overcome.
3. The loading mode of the glucose oxidase can keep the enzyme activity to the greatest extent, and the grafted L-arginine still has the property of generating nitric oxide by oxidation in the presence of active oxygen, so that the product of starvation therapy is utilized to achieve secondary treatment. Meanwhile, the nitric oxide can relieve the tumor hypoxia environment aggravated by glucose oxidase, and has very effective inhibition effect on tumor metastasis.
4. The invention consumes endogenous glucose and releases nitric oxide gas by utilizing specific enzymatic reaction, kills cancer cells by starvation and gas therapy, and simultaneously, the nitric oxide gas can redistribute oxygen in the cancer cells, thereby relieving the hypoxia of tumors, having wide application prospect in the aspects of resisting tumors and inhibiting metastasis of tumors and being used for synergetic cancer starvation and gas therapy.
Drawings
FIG. 1 is an infrared spectrum of functionalized polyethyleneimine containing azide groups (PEI-AATA-Arg) of example 1;
FIG. 2 is a graph showing the particle size and stability of the crosslinked nanogel obtained in example 3;
FIG. 3 is a graph showing the cytotoxicity effect on mouse breast cancer 4T1 cells of the nanogel obtained in example 5.
Detailed Description
EXAMPLE 1 Synthesis of PEI-AATA-Arg
(1) Synthesis of azidated polyethylenimine PEI-AATA
The synthetic route pattern for PEI-AATA is as follows:
azidoacetic acid (48. Mu.L, 0.64 mmol) was dissolved in high purity water, and the catalyst 1-ethyl- (3-dimethylaminopropyl) carbodiimide N-hydroxysuccinimide was added to activate the carboxyl group; simultaneously, polyethylenimine (300 mg,0.16 mmol) is dissolved in high-purity water, concentrated hydrochloric acid is added to adjust the pH, an activated azidoacetic acid solution is added to react for 24 hours, and after dialysis and freeze drying, light yellow powder azidated polyethylenimine with the structure marked PEI-AATA is obtained.
(2) Synthesis of functionalized polyethyleneimine containing azido groups (PEI-AATA-Arg)
The synthetic route pattern for PEI-AATA-Arg is as follows:
l-arginine (278 mg,1.6 mmol) was dissolved in high purity water, concentrated hydrochloric acid was added to adjust the pH, and then the catalyst 1-ethyl- (3-dimethylaminopropyl) carbodiimide N-hydroxysuccinimide was added to activate the carboxyl group; finally adding polyethylenimine azide (200 mg,0.1 mmol) for reaction for 24 hours, dialyzing, freeze drying to obtain light yellow powder polyethylenimine azide, the structure of which is marked as PEI-AATA-Arg, and the infrared spectrum is shown in figure 1.
EXAMPLE 2 Synthesis of BCN-MPA-PAC-PEG-PAC-MPA-BCN
(1) Synthesis of triblock copolymer PAC-PEG-PAC containing acrylate functional group
In a glove box, 500mg of PEG and 120mg of AC monomer are dissolved in methylene dichloride, the mixture is added into a sealed reactor, then catalytic amount of bis (bistrimethylsilyl) amine zinc is added, the reactor is sealed, the reactor is transferred out of the glove box and is placed into an oil bath at 40 ℃ for reaction for 24 hours, glacial acetic acid is used for stopping the reaction, precipitation is carried out in glacial diethyl ether, finally, the supernatant is removed by centrifugation, the vacuum drying is carried out to obtain a product, and a nuclear magnetic result shows that the proportion of the AC unit in the triblock copolymer is 16.6 percent, and the structure of the triblock copolymer is PAC-PEG-PAC.
(2) The double bonds of the AC units of the triblock copolymers being completely substituted by carboxyl groups
0.1g of triblock copolymer PAC-PEG-PAC is dissolved in Dimethylformamide (DMF), mercaptopropionic acid MPA (40 mu L,0.46 mmol) and catalytic amount of triethylamine are added for reaction for 6h, the reaction solution is collected in a dialysis bag and dialyzed in dichloromethane, then is precipitated in glacial diethyl ether, finally, the solution is centrifuged, and the supernatant is discarded, so that a product with the structure of MPA-PAC-PEG-PAC-MPA is obtained by vacuum drying.
(3) Synthesis of alkynyl-containing polycarbonate triblock copolymer BCN-MPA-PAC-PEG-PAC-MPA-BCN
0.1g of triblock copolymer MPA-PAC-PEG-PAC-MPA is dissolved in anhydrous DMF, after vacuum pumping, bicyclo [6.1.0] non-4-alkyne-9-yl methanol BCN (38 mg,0.4 mu mol) is added under the protection of nitrogen, a catalytic amount of dicyclohexylcarbodiimide/4-dimethylaminopyridine is added for reaction for 24 hours, the reaction solution is collected in a dialysis bag and dialyzed overnight in dichloromethane, precipitation is carried out in glacial diethyl ether, finally, centrifugation is carried out, supernatant liquid is removed, and vacuum drying is carried out to obtain the product, and the structure of the product is BCN-MPA-PAC-PEG-PAC-MPA-BCN.
Example 3 preparation of nanogels
(1) Preparation of nano-enzyme complex
Glucose oxidase Gox and PEI-AATA-Arg are respectively dissolved in high-purity water with the concentration of 1mg/mL, and the two solutions are mixed in equal proportion by vortex and then are stood, so that the nano enzyme compound is obtained. After the pH of the PEI-AATA-Arg solution is finely adjusted by sodium hydroxide, two solutions with different proportions can be mixed to obtain the nano enzyme compound with uniform particle size, the average particle size is 190nm, and the particle size distribution is 0.18. This nanoenzyme complex was stable on standing but dissociated in PBS.
(2) Preparation of nanogels
Dissolving BCN-MPA-PAC-PEG-PAC-MPA-BCN in dimethyl sulfoxide, slowly dripping the mixture into the nanocomposite obtained in the step (1) (the molar quantity of azide groups and alkynyl groups is equal), stirring and reacting for a period of time to crosslink, and finally dialyzing to remove DMSO to obtain the nanogel with the average particle size of 190nm and the particle size distribution of 0.18. The stability of the nano particles after crosslinking is greatly improved, the particle size is not obviously changed in standing and PBS, and the particle size characterization diagram is shown in figure 2.
Example 4 nanogels release hydrogen peroxide and nitric oxide in glucose solutions
The prepared nanogel solution is divided into two parts and placed in two different media respectively: (i) PBS, pH 7.4, 37deg.C; (ii) PBS solution of glucose (1 mg/mL), pH 7.4, 37 ℃. The solutions were rapidly transferred to dialysis bags, respectively, and placed in a thermostatic shaker at 37 ℃. The former was immersed in the corresponding PBS solution, the latter in the corresponding glucose PBS solution, 0.5mL of dialysis medium was taken from the delivery system at the indicated time, and then the same volume of fresh medium was replenished. The released hydrogen peroxide and nitric oxide are measured with an enzyme-labeled instrument.
Example 5 glucose oxidase polymer nanogels were tested for 4T1 cytotoxicity (MTT)
Toxicity of the glucose oxidase polymer nanogel in 4T1 cells was determined by the MTT method. Firstly, 100 mu L of DMEM suspension (the sugar-free DMEM culture medium contains 10% of fetal calf serum, 100IU/mL of penicillin and 100 mu g/mL of streptomycin) is spread in a 96-well culture plate, and the culture is carried out at 37 ℃ under the condition of 5% carbon dioxide for 24 hours to ensure that the coverage rate of single-layer cells reaches 70-80%. Then 10. Mu.L of NPs-Gox and NPs-Gox-Arg were added to each well to give final concentrations of 2.5, 5, 7.5 and 10. Mu.g/mL in the cell well, and glucose solution was added after uptake by the cells to give final concentrations of 1mg/mL in the 96-well plate. After further incubation for 24h, 10. Mu.L of PBS solution (5 mg/mL) of 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide (MTT) was added to each well, and the mixture was placed into an incubator for further incubation for 4h to allow MTT to act on living cells. The MTT-containing culture broth was then removed, 150 μl DMSO was added to each well to solubilize the living cells with the purple formazan crystals produced by MTT, and the absorbance at 570nm was measured for each well using a microplate reader (SpectraMax i3 x). Cell relative viability was obtained by absorbance at 570nm compared to control wells with only blank cells. The experimental data were all performed in triplicate.
Cell viability (%) = (OD 570 sample/OD 570 control) ×100%
As shown in FIG. 3, the empty NPs show more than 90% of cell survival rate, and the cytotoxicity of NPs-Gox is also enhanced along with the increase of the concentration of the entrapped Gox, and the significant difference of the cytotoxicity of NPs-Gox-Arg compared with NPs-Gox is more obvious along with the increase of the concentration of the entrapped Gox, so that the survival rate of 4T1 cells can be obviously reduced, the double-load treatment effect is higher than that of single starvation therapy, the NPs-Gox-Arg can realize good cooperation, the starvation therapy and the gas therapy are integrated, and a new idea is brought to the treatment of cancers.

Claims (9)

1. A preparation method of nanogel with anti-tumor function of hunger combined gas therapy is characterized in that a positive charge azide polymer and glucose oxidase are firstly subjected to electrostatic interaction to prepare a nanoenzyme compound, then an alkynyl-containing polycarbonate block copolymer is covered on the surface of the nanoelectrode compound, and the nanogel is obtained by crosslinking through click chemistry reaction of azide and alkynyl; the positively charged azide polymer is functionalized polyethyleneimine PEI-AATA-Arg containing azide groups, and the alkynyl-containing polycarbonate block copolymer is an alkynyl-containing polycarbonate triblock copolymer BCN-MPA-PAC-PEG-PAC-MPA-BCN; wherein AATA is azidoacetic acid, BCN is bicyclo [6.1.0] non-4-alkyne-9-yl methanol, and MPA is mercaptopropionic acid.
2. The method for preparing nanogel with anti-tumor function of starvation combined gas therapy according to claim 1, wherein the method for preparing the positively charged azide polymer comprises the following steps: sequentially carrying out amidation reaction on the polyethyleneimine and carboxylic acid monomers containing azido and L-arginine to obtain the functional polyethyleneimine.
3. The method for preparing nanogel with anti-tumor function of starvation combined gas therapy according to claim 2, wherein the molecular weight of polyethyleneimine is 1.8-25kDa.
4. The method for preparing nanogel with anti-tumor function by starvation combined gas therapy according to claim 2, wherein the carboxylic acid monomer containing azido group is azidoacetic acid.
5. The method for preparing the nanogel with the anti-tumor function of the starvation combined gas therapy according to claim 1, wherein the preparation method of the polycarbonate block copolymer containing alkynyl comprises the following steps: firstly, using polyethylene glycol as an initiator and a cyclic carbonate monomer containing acrylate functional groups to obtain a triblock copolymer containing the acrylate functional groups through ring-opening polymerization; and modifying the double bond of the sulfhydryl-containing carboxylic monomer into carboxyl by a Michael addition reaction, and modifying the carboxyl into alkynyl by an esterification reaction of the hydroxyl-containing alkyne monomer to obtain the alkynyl polycarbonate block polymer.
6. The method for preparing nanogel with anti-tumor function by starvation combined gas therapy according to claim 5, wherein,
the cyclic carbonate monomer containing acrylate functional groups is selected from compounds with the following structures:
；
the sulfhydryl-containing carboxylic monomer is mercaptopropionic acid;
the hydroxyl-containing alkyne monomer is selected from compounds with the following structures:。
7. the method for preparing nanogel with anti-tumor function of starvation combined gas therapy according to claim 1 or 2, wherein the nano-enzyme complex is self-assembled in water by the action of charges from functional polyethyleneimine and glucose oxidase to form the nano-enzyme complex with uniform particle size.
8. The method for preparing nanogel with anti-tumor function of starvation combined gas therapy according to claim 7, wherein encapsulation of glucose oxidase with different proportions can be accurately achieved by adjusting the pH of the solution.
9. Use of the nanogel prepared by the preparation method of any one of claims 1 to 8 in preparation of anti-tumor drugs.
CN202111167823.XA 2021-09-30 2021-09-30 Preparation method and application of nanogel with anti-tumor function of hunger combined gas therapy Active CN113813402B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111167823.XA CN113813402B (en) 2021-09-30 2021-09-30 Preparation method and application of nanogel with anti-tumor function of hunger combined gas therapy

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111167823.XA CN113813402B (en) 2021-09-30 2021-09-30 Preparation method and application of nanogel with anti-tumor function of hunger combined gas therapy

Publications (2)

Publication Number Publication Date
CN113813402A CN113813402A (en) 2021-12-21
CN113813402B true CN113813402B (en) 2024-02-27

Family

ID=78920139

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111167823.XA Active CN113813402B (en) 2021-09-30 2021-09-30 Preparation method and application of nanogel with anti-tumor function of hunger combined gas therapy

Country Status (1)

Country Link
CN (1) CN113813402B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114767656A (en) * 2022-04-20 2022-07-22 中国药科大学 Preparation method and application of nitric oxide donor nano-medicament for targeting atherosclerotic plaques
CN115252644B (en) * 2022-07-18 2023-12-29 华南理工大学 Preparation method and application of anti-tumor nano-drug enhanced by synergistic starvation therapy/chemodynamic therapy

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008127423A2 (en) * 2006-11-14 2008-10-23 Cornell Research Foundation, Inc. Microencapsulated catalyst systems
CN108210931A (en) * 2016-12-15 2018-06-29 深圳大学 Nanometer diagnosis and treatment agent, preparation method and application
CN111840564A (en) * 2020-07-31 2020-10-30 曲阜师范大学 Manganese dioxide-based nano-drug carrier and preparation method and application thereof
CN113166391A (en) * 2018-09-24 2021-07-23 沙特阿美技术公司 Polycarbonate block copolymers and methods thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008127423A2 (en) * 2006-11-14 2008-10-23 Cornell Research Foundation, Inc. Microencapsulated catalyst systems
CN108210931A (en) * 2016-12-15 2018-06-29 深圳大学 Nanometer diagnosis and treatment agent, preparation method and application
CN113166391A (en) * 2018-09-24 2021-07-23 沙特阿美技术公司 Polycarbonate block copolymers and methods thereof
CN111840564A (en) * 2020-07-31 2020-10-30 曲阜师范大学 Manganese dioxide-based nano-drug carrier and preparation method and application thereof

Also Published As

Publication number Publication date
CN113813402A (en) 2021-12-21

Similar Documents

Publication Publication Date Title
CN113813402B (en) Preparation method and application of nanogel with anti-tumor function of hunger combined gas therapy
Song et al. A cooperative polymeric platform for tumor-targeted drug delivery
CN107596380B (en) Polyethylene glycol-polycarbonate-based reduction-sensitive camptothecin prodrug and preparation method and application thereof
CN109810092A (en) Cyclic carbonate monomer and its preparation and application containing nitric oxide donors
CN111870579B (en) Tumor-targeted nano micelle, preparation method and application of nano micelle as drug carrier
CN111249253B (en) Preparation method of core-shell structure stimulus response type drug carrier and drug release method
CN111388450A (en) CO gas-chemodynamic therapy-assisted anti-tumor nano delivery carrier, preparation method and application in anti-tumor drugs
CN112121174B (en) Heparin nano drug carrying system for loading amino antitumor drug and preparation method thereof
CN111514114A (en) Modified nickel cobalt Prussian blue analogue nano material and application thereof, and targeted drug
CN114533671A (en) Preparation method and application of biodegradable hyperbranched polycarbonate shell-core polymer micelle
Yadav et al. Chondroitin sulphate decorated nanoparticulate carriers of 5-fluorouracil: development and in vitro characterization
CN110917349B (en) Bowl-shaped ISP (internet service provider) composite functional nano particle as well as preparation method and application thereof
CN112121177B (en) Carboxylic acid antitumor drug-loaded PEG (polyethylene glycol) heparin nano micelle and preparation method thereof
CN113143867B (en) CMCS-DSP-IPI549 anti-tumor nano-delivery system and preparation method thereof
CN111423571B (en) Biodegradable zwitterionic polycarbonate and application thereof
CN112274495B (en) H2O2Preparation method and application of self-supply type calcium peroxide loaded curcumin nanoparticles
CN115252790A (en) Double-targeting multifunctional nano delivery system responding to tumor microenvironment as well as preparation method and application thereof
CN114652699A (en) Size-transition type nano drug delivery carrier and preparation method and application thereof
CN108721636B (en) Drug delivery material with dual responsiveness connected by diselenide bond and preparation method and application thereof
CN108815536B (en) Drug delivery material with pH and dual redox responsiveness and preparation method and application thereof
CN113456587A (en) Preparation and application of glutathione-responsive nano-drug carrier targeting breast cancer stem cells
CN113827593A (en) Squalene Sidapamide prodrug self-assembly nanoparticles and preparation method and application thereof
CN111956617A (en) Targeting microsphere with long-acting anti-tumor characteristic and preparation method thereof
CN112661958A (en) Inositol and arginine-based polyesteramide and preparation method and application thereof
CN113559275B (en) Method for preparing macromolecule/combretastatin A4/BLZ945 nano bonding medicine by one-pot method

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant