CN113813402A - Preparation method and application of nanogel with hunger combined gas therapy anti-tumor function - Google Patents

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

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CN113813402A
CN113813402A CN202111167823.XA CN202111167823A CN113813402A CN 113813402 A CN113813402 A CN 113813402A CN 202111167823 A CN202111167823 A CN 202111167823A CN 113813402 A CN113813402 A CN 113813402A
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nanogel
gas therapy
tumor function
preparing
starvation
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CN113813402B (en
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陈维
牛亚凡
戴琳
黄德春
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China Pharmaceutical University
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    • C12Y101/03004—Glucose oxidase (1.1.3.4)

Abstract

The invention discloses a preparation method and application of nanogel with hunger combined gas therapy anti-tumor function. Compared with the prior art, the invention consumes endogenous glucose and releases nitric oxide gas by using specific enzymatic reaction, kills cancer cells by starvation and gas therapy, and meanwhile, the nitric oxide gas can redistribute oxygen in the cancer cells, relieves hypoxia of tumors, and has wide application prospect in the aspects of resisting tumors and inhibiting tumor metastasis.

Description

Preparation method and application of nanogel with hunger combined gas therapy anti-tumor function
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 a hunger combined gas therapy anti-tumor function.
Background
In recent years, cancer metabolism, particularly glucose metabolism, has received increasing attention in the design of cancer treatments. Based on the Palboge effect, proliferating cancer cells consume more glucose to produce energy than normal tissues. Several cancer treatment strategies have been proposed to prevent glucose consumption. Since the blockade of glucose supply only slows down tumor growth, but does not completely kill cancer cells, combining this strategy with other therapeutic approaches may be an attractive option.
Glucose oxidase (Gox) catalyzes the oxidation of glucose and produces gluconic acid and hydrogen peroxide (H) in the presence of oxygen2O2). This process effectively depletes glucose and oxygen from the tumor, thereby increasing hypoxia, acidity and H of the tumor microenvironment2O2And (4) environment. Hypoxia is a significant feature of many solid tumors, however, and is often associated with tumor growth, lung metastasis, and resistance to most standard therapeutic approaches. For example, upregulation of hypoxia inducible factor-1 alpha (HIF-1 alpha) is significantly positively correlated with tumor lung metastasis. Gox faces the problem of increasing tumor hypoxia in its functioning, thereby promoting HIF-1 α stabilization. Meanwhile, Gox has the defects of poor stability, short in-vivo half-life, immunogenicity and systemic toxicity.
NO plays the most prominent role in regulating neuronal communication, vascular regulation, wound healing and other physiological and pathological activities, while NO, as a respiratory inhibitor, redistributes oxygen to non-respiratory targets, promoting the degradation of HIF-1 α. However, the highly reactive NO gas cannot be directly used in clinical trials becauseIt has a short lifetime and is concentration dependent. Therefore, 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 in inducible nitric oxide synthase or H2O2Can accurately release nitric oxide in the presence of nitric oxide. The reaction of L-Arg with active oxygen to generate nitric oxide derives a new mechanism for tumor treatment. In tumor cells rich in active oxygen, L-Arg is expected to produce nitric oxide for gas therapy. Cancer cells contain a large amount of H compared to normal cells2O2But endogenous H2O2The level still does not allow an efficient reaction. Increasing H in tumor cells2O2The concentration oxidation of L-Arg to NO for gas therapy remains challenging.
At present, Gox, vitamin C and other medicines are commonly used for increasing H in tumor cells2O2The concentration of (c). Considering the important role of glucose in providing energy for tumor metabolism, the glucose in the tumor is consumed through glucose metabolism reaction, and the glucose is oxidized into gluconic acid and H by utilizing the catalytic action of Gox2O2Thereby strategically starving the tumor. Furthermore, H2O2The increase in concentration helps to accelerate the oxidation of L-Arg to NO. The production of NO can inhibit the accumulation of HIF-1 alpha caused by Gox, thereby inhibiting the proliferation of tumor and lung metastasis.
However, there are currently few reports of techniques and methods involving starvation therapy and NO gas therapy for inhibiting both tumor proliferation and lung metastasis.
Disclosure of Invention
The purpose of the invention is as follows: the invention provides a preparation method of nanogel with an anti-tumor function.
The invention also aims to provide the application of the nanogel with the anti-tumor function.
The technical scheme is as follows: the preparation method of the nanogel with the hunger combined gas therapy anti-tumor function is mainly characterized in that functional polyethyleneimine and glucose oxidase (Gox) are subjected to self-assembly through electrostatic interaction to form a nanoenzyme compound with uniform particle size, and then cross-linked polycarbonate block copolymers are covered on the surface of the nanogel through click chemistry of alkynyl and azide to obtain the nanogel.
Preferably, the preparation method of the positively charged azide polymer comprises the following steps: and the functionalized polyethyleneimine is obtained by amidation reaction of polyethyleneimine with carboxylic acid monomer containing azido and L-arginine in sequence.
Preferably, the azide group-containing functionalized polyethyleneimine is synthesized by the following method: firstly, carboxylic acid monomers containing azido are dissolved in high-purity water, a catalyst 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine/N-hydroxysuccinimide is added to activate carboxyl, then polyethyleneimine PEI is added, the pH is regulated to be neutral, and amidation reaction is carried out to obtain azido polyethyleneimine; dissolving L-arginine (Arg) in high-purity water, adjusting the pH value to be neutral, adding 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine/N-hydroxysuccinimide to activate carboxyl, and adding azido polyethyleneimine into a reaction system after activation to carry out amidation reaction, thereby obtaining the functional polyethyleneimine containing azido groups.
Preferably, the carboxylic acid monomer containing the azide group is selected from compounds with the structures shown as follows:
Figure BDA0003289774720000021
wherein R is1Selected from C2-C5 alkyl.
Preferably, the polyethyleneimine has a molecular weight of 1.8-25 kDa.
Preferably, the method for preparing the alkynyl group-containing polycarbonate block copolymer comprises the following steps: firstly, polyethylene glycol (PEG) is used as an initiator to be subjected to ring-opening polymerization with a cyclic carbonate monomer containing an acrylate functional group to obtain a triblock copolymer containing the acrylate functional group; and modifying double bonds of carboxylic acid monomers containing sulfydryl into carboxyl through Michael addition reaction, and finally modifying carboxyl into alkynyl through esterification reaction of acetylene monomers containing hydroxyl, thereby obtaining the alkynyl polycarbonate block polymer.
Preferably, the alkynyl group-containing polycarbonate block copolymer is synthesized by the following method: the method comprises the following steps of (1) performing ring-opening copolymerization on a cyclic carbonate monomer (AC) containing an acrylate functional group by using polyethylene glycol (PEG) as an initiator, dichloromethane as a solvent and bis (trimethyl silicon) amine zinc as a catalyst to prepare a triblock copolymer containing the acrylate functional group; modifying double bonds into carboxyl by carboxylic acid monomers containing sulfydryl through Michael addition reaction by using N, N-Dimethylformamide (DMF) as a solvent and triethylamine as a catalyst; DMF is taken as a solvent, dicyclohexylcarbodiimide/4-dimethylaminopyridine is taken as a catalyst, and hydroxyl-containing alkyne monomers are subjected to esterification reaction to modify carboxyl into alkynyl, so that the alkynylated polycarbonate block polymer is obtained.
Preferably, the acrylate functional group-containing cyclic carbonate monomer (AC) is selected from compounds having a structure shown in the following:
Figure BDA0003289774720000031
the carboxylic acid monomer containing sulfydryl is characterized by being selected from compounds with the structures shown as follows:
Figure BDA0003289774720000032
wherein R is2Selected from C2-C4 alkyl, or C4-C8 aryl.
A hydroxyl-containing acetylenic monomer characterized by being selected from the group consisting of compounds of the structures shown below:
Figure BDA0003289774720000033
wherein R is3Selected from C1-C5 alkyl,
Figure BDA0003289774720000034
preferably, the nanoenzyme complex is formed by self-assembly of functionalized polyethyleneimine and glucose oxidase in water through charge action, and the nanoenzyme complex has 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 functional polyethyleneimine and Gox in high-purity water, quickly dropwise adding a certain amount of Gox into a functional polyethyleneimine solution, vortexing and standing to obtain a nano enzyme complex, finely adjusting the pH value of an aqueous solution by using sodium hydroxide, and accurately encapsulating glucose oxidase with different proportions;
(2) and (2) dissolving the alkynyl-containing polycarbonate block copolymer in dimethyl sulfoxide (DMSO), slowly dropwise adding the solution into the nano-composite obtained in the step (1) under the stirring state, carrying out stirring reaction for a period of time to generate crosslinking, and finally dialyzing to remove DMSO.
The cross-linked drug-loaded nanogel obtained by the method can improve the stability of the nanoparticles.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages:
1. the invention discloses a protein drug loading mode for the first time, which can accurately realize the encapsulation of drugs with different proportions through the charge action between materials and protein drugs, and greatly improve the encapsulation efficiency of nano-carriers to macromolecular drugs.
2. The invention provides a method for stabilizing a nano-composite formed by charge interaction for the first time, which is characterized in that the surface of the nano-enzyme composite is covered with a polycarbonate block copolymer containing alkynyl for crosslinking, so that the stability of the nano-enzyme composite is enhanced, the nano-enzyme composite is not easy to dissociate outside cells and in blood, and the defects of easy leakage and low carrying efficiency of a medicament in vivo in the prior art are overcome.
3. The loading mode of the glucose oxidase can retain the enzyme activity to the maximum 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 the starvation therapy is utilized to achieve secondary treatment. Meanwhile, nitric oxide can relieve tumor hypoxia environment aggravated by glucose oxidase, and has very effective inhibition effect on tumor metastasis.
4. According to the invention, endogenous glucose is consumed by using a specific enzymatic reaction and nitric oxide gas is released, cancer cells are killed by starvation and gas therapy, and meanwhile, the nitric oxide gas can redistribute oxygen in the cancer cells to relieve hypoxia of tumors, so that the preparation method has wide application prospects in the aspects of resisting tumors and inhibiting tumor metastasis, and is used for cooperating cancer starvation and gas therapy.
Drawings
FIG. 1 is an IR spectrum of azide group-containing functionalized polyethyleneimine (PEI-AATA-Arg) in example 1;
FIG. 2 shows the particle size and stability of the crosslinked nanogel obtained in example 3;
FIG. 3 is a graph showing the cytotoxic effect of the nanogel obtained in example 5 on mouse breast cancer 4T1 cells.
Detailed Description
EXAMPLE 1 Synthesis of PEI-AATA-Arg
(1) Synthesis of Azide polyethyleneimine PEI-AATA
The synthetic scheme for PEI-AATA is as follows:
Figure BDA0003289774720000051
dissolving azidoacetic acid (48 mu L, 0.64mmol) in high-purity water, and adding a catalyst 1-ethyl- (3-dimethylaminopropyl) carbodiimide N-hydroxysuccinimide to activate carboxyl; and meanwhile, dissolving polyethyleneimine (300mg, 0.16mmol) in high-purity water, adding concentrated hydrochloric acid to adjust the pH, adding an activated azido acetic acid solution, reacting for 24 hours, dialyzing, and freeze-drying to obtain light yellow powder azido polyethyleneimine, wherein the structural label of the azido polyethyleneimine is PEI-AATA.
(2) Synthesis of azido group-containing functionalized polyethyleneimine (PEI-AATA-Arg)
The synthetic scheme for PEI-AATA-Arg is as follows:
Figure BDA0003289774720000052
dissolving L-arginine (278mg, 1.6mmol) in high-purity water, adding concentrated hydrochloric acid to adjust pH, and adding a catalyst 1-ethyl- (3-dimethylaminopropyl) carbodiimide N-hydroxysuccinimide to activate carboxyl; finally, adding azidation polyethyleneimine (200mg, 0.1mmol) to react for 24h, dialyzing, and freeze-drying to obtain light yellow powder azidation polyethyleneimine, wherein the structure of the azidation polyethyleneimine is 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 dichloromethane and added into a sealed reactor, then a catalytic amount of bis (bis-trimethylsilyl) amine zinc is added, then the reactor is sealed and transferred out of the glove box, the reactor is put into an oil bath at 40 ℃ for reaction for 24 hours, the reaction is stopped by glacial acetic acid, the precipitation is carried out in glacial ethyl ether, finally the centrifugation is carried out, the supernatant liquid is removed, and the vacuum drying is carried out to obtain the product, and the nuclear magnetic result shows that the proportion of AC units in the triblock copolymer is 16.6 percent, and the structure of the triblock copolymer is marked as PAC-PEG-PAC.
(2) The double bonds of the AC units of the triblock copolymer being completely substituted by carboxyl groups
Dissolving 0.1g triblock copolymer PAC-PEG-PAC in Dimethylformamide (DMF), adding mercaptopropionic acid MPA (40 mu L, 0.46mmol) and a catalytic amount of triethylamine to react for 6h, collecting the reaction solution in a dialysis bag, dialyzing in dichloromethane, precipitating in glacial ethyl ether, finally centrifuging, removing the supernatant, and drying in vacuum to obtain the product with the structure labeled as MPA-PAC-PEG-PAC-MPA.
(3) Synthesis of alkynyl-containing polycarbonate triblock copolymer BCN-MPA-PAC-PEG-PAC-MPA-BCN
Dissolving 0.1g of triblock copolymer MPA-PAC-PEG-PAC-MPA in anhydrous DMF, vacuumizing, adding bicyclo [6.1.0] non-4-alkyne-9-methanol BCN (38mg, 0.4 mu mol) under the protection of nitrogen, adding a catalytic amount of dicyclohexylcarbodiimide/4-dimethylaminopyridine, reacting for 24 hours, collecting the reaction solution in a dialysis bag, dialyzing overnight in dichloromethane, precipitating in glacial ethyl ether, centrifuging, removing the supernatant, and drying in vacuum to obtain the product with the structure of BCN-MPA-PAC-PEG-PAC-MPA-BCN.
EXAMPLE 3 preparation of nanogels
(1) Preparation of nanoenzyme complexes
Respectively dissolving glucose oxidase Gox and PEI-AATA-Arg in high-purity water with the concentration of 1mg/mL, mixing the two solutions in an equal proportion by vortex, and standing to obtain the nano enzyme compound. After the pH value of the PEI-AATA-Arg solution is finely adjusted by sodium hydroxide, the 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 is stable on standing, but disassociates in PBS.
(2) Preparation of nanogels
And (2) dissolving BCN-MPA-PAC-PEG-PAC-MPA-BCN in dimethyl sulfoxide, slowly dropwise adding the mixture into the nano compound (azide groups and alkynyl are equal in molar quantity) obtained in the step (1) under the stirring state, carrying out stirring reaction for a period of time to generate crosslinking, and finally dialyzing to remove DMSO, so that the nanogel is obtained, wherein the average particle size is 190nm, and the particle size distribution is 0.18. After crosslinking, the stability of the nanoparticles is greatly improved, the particle size is not obviously changed in PBS after standing, and a particle size characterization chart is shown in FIG. 2.
Example 4 Nanogel Release of Hydrogen peroxide and nitric oxide in glucose solution
Dividing the prepared nanogel solution into two parts, and respectively placing the two parts in two different media: (i) PBS, pH 7.4, 37 ℃; (ii) glucose in PBS (1mg/mL), pH 7.4, 37 ℃. The solutions were quickly transferred to dialysis bags separately and placed in a 37 ℃ constant temperature shaker. The former was immersed in the corresponding PBS solution and the latter in the corresponding glucose PBS solution, 0.5mL of dialysis medium was removed from the release system at the indicated time, and then the same volume of fresh medium was replenished. The released hydrogen peroxide and nitric oxide were measured with a microplate reader.
Example 5 glucose oxidase Polymer nanogels cytotoxicity test against 4T1 (MTT)
Toxicity of glucose oxidase polymer nanogels in 4T1 cells was determined by the MTT method. Firstly, 100 mu L of DMEM suspension of cells (10% of fetal calf serum, 100IU/mL penicillin and 100 mu g/mL streptomycin in a sugar-free DMEM culture medium) is paved in a 96-hole culture plate and is cultured for 24 hours at 37 ℃ under the condition of 5% carbon dioxide, so that the coverage rate of the monolayer cells reaches 70-80%. Then, 10. mu.L of NPs-Gox and NPs-Gox-Arg at different concentrations were added to each well so that the final concentrations of the drugs in the wells of the cells were 2.5, 5, 7.5 and 10. mu.g/mL, and a glucose solution was added after uptake of the cells so that the final concentration of glucose in the 96-well culture plate was 1 mg/mL. After further incubation for 24h, 10. mu.L of 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide (MTT) in PBS (5mg/mL) was added to each well, and the mixture was placed in an incubator for further incubation for 4h to allow the MTT to react with living cells. The MTT-containing culture solution was then removed, 150 μ L DMSO was added to each well to dissolve viable cells and purple formazan crystals produced by MTT, and the absorbance at 570nm was measured for each well using a plate reader (SpectraMax i3 x). Cell relative viability was obtained by comparing the absorbance at 570nm of control wells with only blank cells. The experimental data were performed in three parallel groups.
Cell viability (%) - (OD570 samples/OD 570 control) × 100%
As shown in fig. 3, unloaded NPs show a cell survival rate of more than 90%, the cytotoxicity of NPs-Gox is enhanced with the increase of the concentration of the encapsulated Gox, and the significant difference of the cytotoxicity of NPs-Gox-Arg compared with NPs-Gox is more obvious with the increase of the concentration of the encapsulated Gox, so that the survival rate of 4T1 cells can be obviously reduced, the double-load treatment effect is higher than that of single-load starvation treatment, and the NPs-Gox-Arg can realize good cooperation, so that the starvation treatment and the gas therapy are integrated, and a new idea is brought to the treatment of cancer.

Claims (9)

1. A preparation method of nanogel with hunger combined gas therapy anti-tumor function is characterized in that firstly, a nano enzyme compound is prepared by using azide polymer with positive charges and glucose oxidase through electrostatic interaction, then, polycarbonate block copolymer containing alkynyl is covered on the surface of the nano enzyme compound, and the nanogel is obtained by utilizing click chemistry reaction of azide and alkynyl to crosslink.
2. The method for preparing nanogel with anti-tumor function by starvation combination gas therapy according to claim 1, wherein the method for preparing the positively charged azide polymer comprises the following steps: and the functionalized polyethyleneimine is obtained by amidation reaction of polyethyleneimine with carboxylic acid monomer containing azido and L-arginine in sequence.
3. The method for preparing nanogel with anti-tumor function by starvation combination gas therapy according to claim 2, wherein the molecular weight of polyethyleneimine is 1.8-25 kDa.
4. The method for preparing nanogel with anti-tumor function by combined starvation gas therapy according to claim 2, wherein the azido-containing carboxylic acid monomers are selected from compounds having the structures shown as follows:
Figure FDA0003289774710000011
wherein R is1Selected from C2-C5 alkyl.
5. The method for preparing nanogel with anti-tumor function by starvation combination gas therapy according to claim 1, wherein the method for preparing the alkynyl group-containing polycarbonate block copolymer comprises the following steps: firstly, polyethylene glycol (PEG) is used as an initiator to be subjected to ring-opening polymerization with a cyclic carbonate monomer containing an acrylate functional group to obtain a triblock copolymer containing the acrylate functional group; and modifying double bonds of carboxylic acid monomers containing sulfydryl into carboxyl through Michael addition reaction, and finally modifying carboxyl into alkynyl through esterification reaction of acetylene monomers containing hydroxyl, thereby obtaining the alkynyl polycarbonate block polymer.
6. The method for preparing nanogel with anti-tumor function by starvation combination gas therapy according to claim 5,
the cyclic carbonate monomer containing the acrylate functional group is selected from the group consisting of the compounds with the structures shown in the specificationCompound (a):
Figure FDA0003289774710000012
the carboxylic acid monomer containing sulfydryl is selected from compounds with the structures shown as follows:
Figure FDA0003289774710000021
wherein R is2Selected from C2-C4 alkyl or C4-C8 aryl;
the hydroxyl-containing acetylene monomer is selected from compounds with the structures shown as follows:
Figure FDA0003289774710000022
wherein R is3Selected from C1-C5 alkyl,
Figure FDA0003289774710000023
7. the method for preparing nanogel with hunger-associated gas therapy anti-tumor function according to claim 1 or 2, wherein the nanoenzyme complex is formed by self-assembly of functionalized polyethyleneimine and glucose oxidase in water through charge action, and the nanoenzyme complex has uniform particle size.
8. The method for preparing nanogel with hunger combined gas therapy anti-tumor function according to claim 7, wherein encapsulation of glucose oxidase with different proportions can be accurately realized by adjusting pH of solution.
9. Use of nanogel prepared by the preparation method of any one of claims 1 to 8 in preparation of antitumor drugs.
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