CN111249473B - Delivery system and preparation method of polymerized chloroquine fluorene methyl carbonyl nanogel - Google Patents

Delivery system and preparation method of polymerized chloroquine fluorene methyl carbonyl nanogel Download PDF

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CN111249473B
CN111249473B CN202010086613.7A CN202010086613A CN111249473B CN 111249473 B CN111249473 B CN 111249473B CN 202010086613 A CN202010086613 A CN 202010086613A CN 111249473 B CN111249473 B CN 111249473B
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hydroxychloroquine
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CN111249473A (en
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孙敏捷
董婧雯
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China Pharmaceutical University
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    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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    • 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
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    • A61K47/6939—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 a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Abstract

The invention discloses a delivery system of polymerized chloroquine fluorene methyl carbonyl nanogel, which comprises chloroquine fluorene methyl polymerized nanogel particles and an anti-tumor drug wrapped in the particles; the nanometer gel particles are formed by cross-linking a polysaccharide main chain modified with hydroxychloroquine and fluorenylmethyloxycarbonyl, the molecular weight of the polysaccharide main chain is 5-100 kD, the molar substitution degree of the hydroxychloroquine on the polysaccharide main chain is 5% -50%, and the molar substitution degree of the fluorenylmethyloxycarbonyl on the polysaccharide main chain is 1% -30%. The polymeric chloroquine fluorene methyl carbonyl nanogel delivery system can inhibit autophagy level in cells, block a signal path of tumor cell metastasis, and simultaneously entrap the anti-tumor drug taxol through pi-pi conjugation, so that malignant tumor metastasis and proliferation are achieved under double-tube conditions.

Description

Delivery system and preparation method of polymerized chloroquine fluorene methyl carbonyl nanogel
Technical Field
The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a polymeric chloroquine fluorene methyl carbonyl nanogel delivery system and a preparation method thereof.
Background
In China, the incidence of tumors is obviously increased in recent years, most of the patients with malignant tumors are, and the mortality of the patients is obviously improved due to the metastasis of malignant tumor cells. The traditional chemotherapeutic drug paclitaxel is combined with the tubulin in the cell to block the dissociation of the micro protein, thereby retarding the mitosis of the cell and promoting the apoptosis of the tumor cell, and bringing hope for the treatment of the tumor. However, because of the extremely low water solubility of paclitaxel, the traditional paclitaxel injection needs to use a mixed solution of polyoxyethylene castor oil and absolute ethyl alcohol as a solubilizer, but the polyoxyethylene castor oil can bring a series of allergic reactions, and patients need to be desensitized before administration, which brings pain to the patients. And because of lacking tissue specificity, the paclitaxel injection can generate serious toxic and side effects on other normal organ tissues of an organism while treating breast cancer, such as: myelosuppression, neurotoxicity, anaphylaxis, digestive tract reactions, and the like. With the development of nanotechnology and materials, the novel nanogel drug delivery system shows good clinical application potential and provides a new strategy for the efficient treatment of tumors.
The nanogel is a three-dimensional network structure system with the particle size of less than 200 nm formed by dispersing hydrophilic or amphiphilic polymer polysaccharide containing multiple functional groups in water and then performing physical or chemical crosslinking. The nanometer gel has three-dimensional network structure inside, and through ionic bond, hydrogen bond, hydrophobic force and pi-pi conjugate effect, the medicine may be encapsulated inside the nanometer gel. However, because the nanogel is mostly made of traditional gel materials and does not have the function of resisting tumor metastasis, the therapeutic effect on advanced malignant tumors is not obvious, and therefore, an innovative nanogel modification strategy is urgently sought for the advanced malignant tumors.
Disclosure of Invention
The invention aims to provide a polymeric chloroquine fluorenylmethylcarbonyl nanogel delivery system and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a delivery system of polymerized chloroquine fluorenylmethylcarbonyl nanogel comprises a nanogel particle of polymerized chloroquine fluorenylmethyl and an anti-tumor drug wrapped in the particle;
the nanometer gel particles are formed by cross-linking a polysaccharide main chain modified with hydroxychloroquine and fluorenylmethyloxycarbonyl, the molecular weight of the polysaccharide main chain is 5-200 kD, the molar substitution degree of the hydroxychloroquine on the polysaccharide main chain is 5% -50%, and the molar substitution degree of the fluorenylmethyloxycarbonyl on the polysaccharide main chain is 1% -30%.
Further, the polysaccharide backbone is selected from hydroxyethyl starch, dextrin, polyethylene glycol, hyaluronic acid, alginic acid, dextran or chitosan, preferably dextran.
Further, the antitumor drug is an aromatic ring-containing antitumor drug, such as: camptothecin, doxorubicin hydrochloride, paclitaxel, docetaxel and curcumin, preferably paclitaxel.
The preparation method of the delivery system comprises the following steps:
step 1, modification of a main chain of the poly-polysaccharide by fluorenylmethyloxycarbonyl: in a polar solvent, in the presence of a catalyst and a dehydrating agent, a fluorenylmethyloxycarbonyl donor is connected with hydroxyl on a polysaccharide main chain through esterification reaction to obtain the polysaccharide main chain with a side chain modified with fluorenylmethyloxycarbonyl;
step 2, modification of the polysaccharide skeleton by hydroxychloroquine: firstly, synthesizing hydroxychloroquine-carbonyldiimidazole in a polar solvent, and then reacting the hydroxychloroquine-carbonyldiimidazole with a polysaccharide main chain with a lateral chain modified with fluorenylmethyloxycarbonyl to obtain the polysaccharide main chain modified with hydroxychloroquine and fluorenylmethyloxycarbonyl;
step 3, preparation of nanogel delivery system: dissolving the polysaccharide main chain modified with hydroxychloroquine and fluorenylmethyloxycarbonyl and the antitumor drug in an organic solvent, dropwise adding the obtained organic solution into the aqueous solution under the stirring condition to prepare emulsion, and removing the organic solvent to obtain the nanogel delivery system.
Further, in step 1, the fluorenylmethyloxycarbonyl donor is selected from Fmoc-glycine, Fmoc-L-ornithine or N' -Fmoc-L-lysine.
Further, in step 1, the polar solvent is selected from dimethyl sulfoxide, anhydrous dimethylformamide or tetrahydrofuran, the dehydrating agent is dicyclohexylcarbodiimide, and the catalyst is dimethylaminopyridine. The reaction step is specifically that a polysaccharide main chain is dissolved in a polar solvent, a dehydrating agent, a catalyst and a fluorenylmethyloxycarbonyl donor are respectively added, the reaction is carried out in an inert atmosphere, and the product is treated to obtain the polysaccharide main chain with the side chain modified with the fluorenylmethyloxycarbonyl.
Further, in the step 2, the polar solvent is selected from propanol, acetonitrile, anhydrous dimethyl sulfoxide or anhydrous dimethylformamide, the reaction step is specifically to dissolve hydroxychloroquine and N, N '-carbonyldiimidazole in the polar solvent respectively, the hydroxychloroquine-carbonyldiimidazole is obtained by reacting the hydroxychloroquine and the N, N' -carbonyldiimidazole in an inert atmosphere, then the synthesized polysaccharide main chain of the hydroxychloroquine-carbonyldiimidazole and fluorenylmethoxycarbonyl is dissolved in the polar solvent, the reaction is carried out by stirring in the inert atmosphere, and the polysaccharide main chain of the hydroxychloroquine and the fluorenylmethoxycarbonyl is obtained after the treatment.
Further, in the step 3, the organic solvent is selected from dimethyl sulfoxide, tetrahydrofuran or dimethylformamide, the aqueous solution is selected from pure water, 5% w/v glucose solution, normal saline, phosphate buffer solution or acetate buffer solution, and the mass ratio of the polysaccharide main chain modified with hydroxychloroquine and fluorenylmethoxycarbonyl to the antitumor drug is 1-100: 1.
the nanometer gel is administered in a dosage of 1-20 mg/kg by intravenous injection or intraperitoneal injection every 2-5 days.
Common chemotherapy drugs have serious toxic and side effects, and a single drug delivery system is difficult to realize the co-delivery of multiple drugs and cannot inhibit the metastasis and proliferation of malignant tumors. Therefore, the polymeric chloroquine fluorenylmethyloxycarbonyl nanogel delivery system used by the invention can inhibit the autophagy level in cells, block a signal path of tumor cell metastasis, simultaneously encapsulate the anti-tumor drug taxol through pi-pi conjugation, and carry out metastasis and proliferation of malignant tumors under double-tube conditions. The modification amount of each component in the polymeric chloroquine fluorenylmethyl conjugate is controllable, and the formed nanoparticles have stable structure and high drug loading, and are a multifunctional delivery platform with good biological safety. By using the scheme of the invention, the further proliferation of tumor cells can be inhibited while the autophagy is inhibited and the tumor metastasis signal path is blocked, so that the malignant tumor can be effectively treated under double control.
Drawings
FIG. 1 shows the synthetic route of the polymeric chloroquine-fluorenylmethylcarbonyl glucan and the NMR spectrum of the resulting polymeric chloroquine-fluorenylmethylcarbonyl glucan in example 2.
Fig. 2 is a schematic diagram of nanogel in which the polymeric chloroquine fluorenylmethylcarbonyl dextran encapsulates the anti-tumor chemotherapeutic drug paclitaxel in example 2.
FIG. 3 shows the particle size distribution (A) and transmission electron microscope (B) of the chloroquine fluorenylmethylcarbonyl nanogel in example 2.
Fig. 4 shows the killing effect of the polymeric chloroquine fluorenylmethylcarbonyldextran nanogel skeleton-entrapped paclitaxel nanogel on tumor cells under different drug concentrations in example 2.
Fig. 5 is a result of measuring autophagy inhibition ability of the polymeric chloroquine fluorenemethylcarbonyl nanogel carrier in example 2.
Fig. 6 shows the results of the measurement of the redistribution experiment of CXCR4 of the polymeric chloroquine fluorenylmethylcarbonyl nanogel carrier in example 2.
FIG. 7 shows the results of the chloroquine fluorenylmethylcarbonyl nanogel carrier for inhibiting tumor cell invasion in example 2.
FIG. 8 shows the results of in vitro anti-tumor cell metastasis of the polymeric chloroquine fluorenylmethylcarbonyl nanogel carrier in example 2.
Detailed Description
The nanogel is a three-dimensional network structure system with the particle size of less than 200 nm formed by dispersing hydrophilic or amphiphilic polymer polysaccharide containing multiple functional groups in water and usually through physical or chemical crosslinking. The nanometer gel has three-dimensional network structure inside, and through ionic bond, hydrogen bond, hydrophobic force and pi-pi conjugate effect, the medicine may be encapsulated inside the nanometer gel. And the nanogel has a smaller particle size, so that the nanogel has a larger specific surface area and is convenient for chemical modification. Because the fluorenylmethylcarbonyl has a fluorene ring and can generate pi-pi conjugation with drugs containing aromatic rings, good drug loading effect is realized, and chemotherapeutic drugs with aromatic rings such as paclitaxel and the like are effectively loaded, the fluorenylmethylcarbonyl is selectively modified on the polysaccharide main chain and is used for loading the chemotherapeutic drugs with aromatic rings.
Metastasis and invasion are the main biological characteristics of malignant tumors, the metastasis process is complex, and the steps of separating tumor cells in primary parts from primary focuses, transporting the tumor cells to target organs along with blood or lymph fluid, continuously proliferating the tumor cells in secondary parts to form tumor cell metastases and the like are mainly adopted. In which multiple mechanisms, such as autophagy, and chemokine and its receptor, are involved. Autophagy is a process by which damaged, denatured or aged proteins and organelles within a cell are transported to lysosomes for digestive degradation. In recent years, as the research on autophagy has been advanced, the relationship between autophagy and tumor metastasis has been revealed. The paxillin degraded in the autophagy process of tumor cells can reduce the adhesion among tumor cells, and the lack of the adhesion among the cells is one of the necessary conditions for malignant metastasis to occur, so that the migration and infiltration of the tumor cells are caused. Therefore, inhibiting the autophagy pathway can reduce the degradation of pilin in the tumor cells and improve the adhesion property between the tumor cells, thereby achieving the effect of reducing the metastasis of the tumor cells. Hydroxychloroquine is a drug used to control clinical symptoms of malaria and to prevent malaria. In recent years, researches show that the classical small-molecule autophagy inhibitor hydroxychloroquine has alkalinity, can be accumulated in a lysosome to change the acidity in the lysosome, and cause the degradation of hydrolysis function, so that the degradation of paxillin is reduced, the tumor metastasis is reduced, and the method has a certain prospect in the aspect of treating malignant tumors. Chemokines, on the other hand, regulate cell migration by binding to the corresponding chemokine receptor. Chemokines are small molecule proteins with chemical chemotaxis in the cytokine superfamily, and play an important role in the development and metastasis of body tumors. The chemokine receptor CXCR4 makes a member of the chemokine receptor family widely present on the surface of tumor cells, and the chemokine SDF-1 is the only endogenous ligand of CXCR4 and is expressed at some common tumor metastasis sites, such as: lung, liver, lymph nodes and bone marrow, etc., suggesting that they are associated with tumor cell specific metastasis. These highly SDF-1 expressing organs provide fertile soil for metastasis, infiltration and further proliferation of CXCR4 highly expressing tumor cells.
Although the nano drug delivery system achieves certain achievement in the aspect of treating most tumors, the nano gel mostly adopts the traditional gel material and does not have the function of resisting tumor metastasis, so the nano drug delivery system has no remarkable treatment effect on malignant advanced tumors. Therefore, innovative nanogel modification strategies are urgently sought for advanced malignant tumors.
Hydroxychloroquine belongs to 4-aminoquinolone drugs, and is firstly applied to clinic as an antimalarial drug, and then the hydroxychloroquine is found to be capable of being used as an autophagy inhibitor to influence tumors. The hydroxychloroquine can reduce the degradation of pilin in tumor cells by inhibiting the degradation of pilin, and improve the adhesion property between tumor cells, thereby achieving the effect of reducing the metastasis of tumor cells. The composition is combined with chemotherapeutic drugs, has good curative effect on advanced malignant tumors (metastatic breast cancer, glioblastoma) and melanoma, can effectively reduce the occurrence of tumor metastasis, prolongs the life cycle of patients, and has good tolerance and safety.
Therefore, the invention provides a delivery system of a carrier encapsulated chemotherapeutic drug-containing chloroquine fluorenylmethyl nanogel with an anti-tumor cell metastasis function, which takes hydrophilic polysaccharide as a hydrophilic main chain, modifies a side chain to have a small-molecule autophagy inhibitor Hydroxychloroquine (HCQ) which can inhibit tumor cell metastasis through autophagy inhibition, and modifies the side chain to effectively inhibit proliferation and metastasis of tumor cells through pi-pi conjugated encapsulated chemotherapeutic drug paclitaxel groups fluorenylmethyloxycarbonyl, so that the dual-synergistic treatment of late malignant tumors is realized.
The hydrophilic polysaccharide main chain has hydrophilicity and is rich in hydroxyl, and substitution modification can be easily carried out. In the invention, the polysaccharide backbone is selected from one of hydroxyethyl starch, dextrin, polyethylene glycol, hyaluronic acid, alginic acid, dextran and chitosan. Dextran (Dex) is preferred because of its lower molecular weight and thus better water solubility than other high molecular weight polysaccharide backbones.
When the nanogel is formed, fluorenylmethylcarbonyl (Fmoc) and chemotherapeutics rich in aromatic rings can wrap the drugs through pi-pi conjugation to form the nanogel. In view of the hydroxyl-rich nature of the polysaccharide backbone, compounds having carboxyl groups at the tail end are selected for reaction. In the present invention, one or more selected from the group consisting of Fmoc-glycine, Fmoc-L-ornithine, and N' -Fmoc-L-lysine is used as the fluorenylmethylcarbonyl donor, preferably Fmoc-glycine.
Furthermore, the polymeric chloroquine fluorenylmethyloxycarbonyl nanogel skeleton carrier can effectively carry chemotherapeutic drugs through pi-pi conjugation, so that passive targeted delivery is realized. In order to realize better drug loading effect and avoid the self polymerization of the polymerized chloroquine and the fluorene methyl carbonyl, the molar substitution degree of the fluorene methyl carbonyl in the polysaccharide main chain is not higher than 30%.
Furthermore, as the hydrophobicity of the chloroquine is higher, in order to ensure the solubility of the polymeric chloroquine fluorene methyl carbonyl nano gel carrier in water, the substitution degree of the hydroxychloroquine on the polysaccharide main chain is not higher than 50%.
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.
Example 1
Preparation method of polymerized chloroquine-fluorenylmethylcarbonyl dextrin nanogel drug-loading system
Step 1, preparation of hydroxychloroquine: dissolving 0.6 g hydroxychloroquine sulfate in 2 mL of pure water, adding excessive 30% ammonia water under the condition of continuous stirring until the white insoluble products are not increased any more, adding dichloromethane for extraction for 4-6 times, collecting the organic phase, adding a proper amount of anhydrous sodium sulfate overnight, and evaporating the solvent under reduced pressure to obtain the final desalted product.
Step 2, synthesizing fluorenylmethylcarbonyl dextrin: dissolving 210 mg of dextrin in 2 mL of dimethylformamide, respectively adding 206mg of dicyclohexylcarbodiimide, 24 mg of 4-dimethylaminopyridine and 297 mg of Fmoc-glycine, reacting for 12-24 h under an inert gas environment, removing insoluble substances, adding reaction liquid into 10 times of anhydrous ether, centrifuging for 3 min at the rotating speed of 3000 rpm, removing supernatant to obtain a white viscous solid product, dissolving the product with ultrapure water, dialyzing, and freeze-drying to obtain fluorenylmethylcarbonyl dextrin. The feeding molar ratio is 1: 1: 0.2: 0.5-1, preferably 1: 1: 0.2: 1.
step 3, synthesizing the polymeric chloroquine-fluorenylmethylcarbonyl dextrin: respectively dissolving 200 mg of hydroxychloroquine obtained in the step 1 and 389 mg of N, N '-carbonyldiimidazole in 3 mL of dimethylformamide, dropwise adding the N, N' -carbonyldiimidazole solution under the protection of inert gas, and continuously stirring for reaction for 4-6 h. After 5 mL of dichloromethane and 2 mL of water are added to terminate the reaction, 20 mL of dichloromethane solution is added, and the reaction solution is washed by ultrapure water for 5-10 times to remove the excessive N, N' -carbonyldiimidazole, an organic phase is collected, and the solvent is evaporated under reduced pressure to obtain the hydroxychloroquine-carbonyldiimidazole. The feeding molar ratio of the hydroxyl chloroquine to the N, N' -carbonyl diimidazole is 1: 3-6, preferably 1: 4.
200 mg of hydroxychloroquine-carbonyldiimidazole and 100 mg of fluorenylmethylcarbonyl dextrin prepared in step 3 were dissolved in dimethyl sulfoxide, and the mixture was stirred under the protection of inert gas for reaction for 4 days. Adding the reaction liquid into 10 times of anhydrous ether, carrying out suction filtration, dissolving the solid with ultrapure water, dialyzing, and freeze-drying to obtain the polymeric chloroquine-fluorenylmethylcarbonyl dextrin.
Step 4, preparing the polymeric chloroquine-fluorenylmethylcarbonyl dextrin nanogel entrapping the paclitaxel: 10 mg of the prepared polymeric chloroquine-fluorenylmethylcarbonyl glucan composition and paclitaxel are dissolved in 100 mu L of organic solvent together to obtain a mixed organic solution of the polymeric chloroquine-fluorenylmethylcarbonyl glucan and the paclitaxel. The organic solvent is selected from dichloromethane, trichloromethane and tetrahydrofuran, preferably tetrahydrofuran. The feeding mass ratio of the conjugate of the polymeric chloroquine-fluorenylmethylcarbonyl glucan to the paclitaxel is 1-50: 1, preferably 5: 1.
10 mL of the aqueous solution was taken, and the mixed organic solution was added dropwise to the aqueous solution with continuous stirring. Performing ultrasonic treatment at the temperature of 20 ℃ for 3 min under the ultrasonic condition of 400W, and evaporating the organic solvent at the temperature of 37 ℃ under reduced pressure. The polymeric chloroquine-fluorenylmethylcarbonyl dextrin nanogel loaded with the chemotherapeutic drug paclitaxel is obtained, and the drug loading is 5%. The aqueous solution is selected from pure water, physiological saline, phosphate buffer solution and the like, and the phosphate buffer solution is preferred. The volume ratio of the organic mixed solution to the aqueous solution is 1: 1-100, preferably 1: 10-1: 50.
example 2
Preparation method of polymerized chloroquine-fluorenylmethylcarbonyl glucan nanogel drug-loading system
Step 1, preparation of hydroxychloroquine: dissolving 0.6 g hydroxychloroquine sulfate in 2 mL of pure water, adding excessive 30% ammonia water under the condition of continuous stirring until the white insoluble products are not increased any more, adding dichloromethane for extraction for 4-6 times, collecting the organic phase, adding a proper amount of anhydrous sodium sulfate overnight, and evaporating the solvent under reduced pressure to obtain the final desalted product.
Step 2, synthesis of fluorenylmethylcarbonyldextran (HF): dissolving 164 mg of glucan in 2 mL of dimethylformamide, respectively adding 206mg of dicyclohexylcarbodiimide, 24 mg of 4-dimethylaminopyridine and 297 mg of Fmoc-glycine, reacting for 12-24 h under an inert gas environment, removing insoluble substances, adding reaction liquid into 10 times of anhydrous ether, centrifuging for 3 min at the rotating speed of 3000 rpm, removing supernatant to obtain a white viscous solid product, dissolving the product with ultrapure water, dialyzing, and freeze-drying to obtain fluorenylmethylcarbonyl glucan (HF). The feeding molar ratio is 1: 1: 0.2: 0.5-1, preferably 1: 1: 0.2: 1.
and 3, synthesizing the polymerized chloroquine-fluorenylmethylcarbonyl dextran (CQ-HF): respectively dissolving 200 mg of hydroxychloroquine obtained in the step 1 and 389 mg of N, N '-carbonyldiimidazole in 3 mL of dimethylformamide, dropwise adding the N, N' -carbonyldiimidazole solution under the protection of inert gas, and continuously stirring for reaction for 4-6 h. After the reaction was terminated by adding 5 mL of dichloromethane and 2 mL of water, 20 mL of dichloromethane solution was added, and the excess N, N' -carbonyldiimidazole was removed by washing with ultrapure water 5 to 10 times, the organic phase was collected, and the solvent was distilled off under reduced pressure to obtain hydroxychloroquine-carbonyldiimidazole (CQ-CDI). The feeding molar ratio of the hydroxyl chloroquine to the N, N' -carbonyl diimidazole is 1: 3-6, preferably 1: 4.
200 mg of hydroxychloroquine-carbonyldiimidazole and 100 mg of fluorenylmethylcarbonyl dextran prepared in step 3 were dissolved in dimethyl sulfoxide, and the reaction was stirred under the protection of inert gas for 4 days. Adding the reaction solution into 10 times of anhydrous ether, filtering, dissolving the solid with ultrapure water, dialyzing, and lyophilizing to obtain the polymeric chloroquine-fluorenylmethylcarbonyl dextran (CQ-HF).
FIG. 1 shows the synthetic route of the polymeric chloroquine-fluorenylmethylcarbonyl glucan and the NMR spectrum of the resulting polymeric chloroquine-fluorenylmethylcarbonyl glucan, and the results demonstrate that the product prepared by carrying out the example is the polymeric chloroquine-fluorenylmethylcarbonyl glucan.
Step 4, preparation of polymeric chloroquine-fluorenylmethylcarbonyl dextran nanogel (CQ-HF/PTX) entrapping paclitaxel: 10 mg of the prepared polymeric chloroquine-fluorenylmethylcarbonyl glucan composition and paclitaxel are dissolved in 100 mu L of organic solvent together to obtain a mixed organic solution of the polymeric chloroquine-fluorenylmethylcarbonyl glucan and the paclitaxel. The organic solvent is selected from dichloromethane, trichloromethane and tetrahydrofuran, preferably tetrahydrofuran. The feeding mass ratio of the conjugate of the polymeric chloroquine-fluorenylmethylcarbonyl glucan to the paclitaxel is 1-50: 1, preferably 5: 1.
10 mL of the aqueous solution was taken, and the mixed organic solution was added dropwise to the aqueous solution with continuous stirring. Performing ultrasonic treatment at the temperature of 20 ℃ for 3 min under the ultrasonic condition of 400W, and evaporating the organic solvent at the temperature of 37 ℃ under reduced pressure. Thus obtaining the polymeric chloroquine-fluorenylmethylcarbonyl dextran nanogel (CQ-HF/PTX) loaded with the chemotherapeutic drug paclitaxel, wherein the drug loading is 7 percent. The aqueous solution is selected from pure water, physiological saline, phosphate buffer solution and the like, and the phosphate buffer solution is preferred. The volume ratio of the organic mixed solution to the aqueous solution is 1: 1-100, preferably 1: 10-1: 50.
FIG. 2 is a schematic diagram of a preparation process of the polymeric chloroquine-fluorenylmethylcarbonyl dextran nanogel.
Step 5, preparation of fluorenylmethylcarbonyldextran nanogel (HF/PTX) encapsulating paclitaxel: 10 mg of the prepared fluorenylmethylcarbonyl glucan composition and paclitaxel were dissolved together in 100. mu.L of an organic solvent to obtain a mixed organic solution of fluorenylmethylcarbonyl glucan and paclitaxel. The organic solvent is selected from dichloromethane, trichloromethane and tetrahydrofuran, preferably tetrahydrofuran. The feeding mass ratio of the conjugate of fluorenylmethylcarbonyl glucan to paclitaxel is 1-50: 1, preferably 5: 1.
10 mL of the aqueous solution was taken, and the mixed organic solution was added dropwise to the aqueous solution with continuous stirring. Performing ultrasonic treatment at the temperature of 20 ℃ for 3 min under the ultrasonic condition of 400W, and evaporating the organic solvent at the temperature of 37 ℃ under reduced pressure. Thus obtaining the polymeric fluorenylmethylcarbonyl dextran nanogel (HF/PTX) carrying the chemotherapeutic drug paclitaxel, wherein the drug loading is 7 percent. The aqueous solution is selected from pure water, physiological saline, phosphate buffer solution and the like, and the phosphate buffer solution is preferred. The volume ratio of the organic mixed solution to the aqueous solution is 1: 1-100, preferably 1: 10-1: 50.
dialyzing the obtained nanogel solution for 12 hours by using a dialysis bag with the molecular weight of 8000 kDa, replacing deionized water every 2 hours, and removing redundant paclitaxel free drugs to obtain the polymeric chloroquine-fluorenylmethylcarbonyl glucan nanogel solution.
FIG. 3 is a dynamic light scattering measurement particle size distribution of a polymeric chloroquine-fluorenylmethylcarbonyl dextran nanogel encapsulating paclitaxel and a morphology feature investigation of particles observed under a transmission electron microscope. The double-layer spherical particles with the average particle size of the prepared nanogel of 180 nm can be seen from the figure, which shows that the subsequent paclitaxel-entrapped polymeric chloroquine-fluorenylmethylcarbonyl nanogel (CQ-HF/PTX) has been successfully prepared
1. Polymeric chloroquine-fluorenylmethylcarbonyl-glucan nano-carrier and in-vitro cytotoxicity determination of paclitaxel-entrapped nanogel
Step 1, inoculating 4T1 cells of breast cancer cells into a 96-well plate, wherein the cell density is 5000 cells/well, and culturing by using a 1640 culture medium containing 10% fetal bovine serum. When the cell density is increased to 60% -70%, incubating the polymerized chloroquine-fluorenylmethylcarbonyl nanogels with different concentrations and the cells for 48 hours.
Step 2, add 20 μ L of 3- (4, 5-dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide (MTT), incubate for 4 hours, discard the well internal solution, add 200 μ L of dimethyl sulfoxide to solubilize the purple formazan produced by the live cells, measure the absorbance of each well at a wavelength of 570 nm using a microplate reader, and calculate cell viability.
FIG. 4 is an in vitro cytotoxicity assay of the polymeric chloroquine-dextran nanogel.
HF is a fluorenylmethylcarbonyl nanogel group without paclitaxel, and CQ-HF is a polymeric chloroquine-fluorenylmethylcarbonyl glucan nanogel group without paclitaxel. The left picture shows that the toxicity of the nano-carrier without paclitaxel is low, which indicates that the polymerized chloroquine-fluorenylmethylcarbonyl dextran nanogel has better safety. PTX is a commercial paclitaxel injection, HF nanogels is a fluorenylmethylcarbonyl nanogel group carrying paclitaxel, and HFH nanogels is a polymeric chloroquine-fluorenylmethyl nanogel group carrying paclitaxel. The nanogel encapsulating paclitaxel in the left panel shows significantly higher toxicity than the commercial paclitaxel injection, which may be due to the effect of chloroquine internalizing CXCR4, so that the cellular uptake capacity of the nanogel is significantly improved, and the cytotoxicity is enhanced.
2. Proton buffering capacity determination of polymerized chloroquine-fluorenylmethylcarbonyl glucan nanogel
10 mg of CQ-HF and HF, together with an equal amount of hydroxychloroquine, were dissolved in 5 mL of aqueous sodium chloride and the initial pH of the solution was adjusted to 10.3. Titration was then carried out using 0.1M hydrochloric acid solution and the change in pH of the solution was recorded for each 50. mu.L of 0.1M hydrochloric acid added until the pH had dropped to 3. HCQ is a free hydroxychloroquine group, HF is a fluorenylmethylcarbonyl nanogel group without encapsulating paclitaxel, and CQ-HF is a polymeric chloroquine-fluorenylmethyl nanogel group without encapsulating paclitaxel. FIG. 5 is a titration chart of the proton buffering capacity of the nanocarrier in vitro. The figure shows that the polymerized chloroquine-fluorenylmethylcarbonyl glucan has excellent proton buffering capacity compared with a polymer without the polymerized chloroquine, and can be used as a high-efficiency autophagy inhibitor for inhibiting the metastasis of late malignant tumors.
3. Investigation of in vitro cell autophagy inhibition function of polymerized chloroquine-fluorenylmethylcarbonyl glucan nanogel
Protein imprinting method for determination of autophagy-related protein LC 3: breast cancer cells 4T1 cells were seeded in a 6-well plate and cultured in 1640 medium containing 10% fetal bovine serum. When the cell density increased to 60% -70%, free chloroquine (HCQ), fluorenylmethylcarbonyl-dextran nanogel (HF), hydroxychloroquine-fluorenylmethylcarbonyldextran nanogel (CQ-HF) were co-incubated with the cells for 12 hours. The well solution was discarded, cell lysate was added, and after incubation for 15 minutes on ice, the supernatant was centrifuged. The supernatant was added to SDS-PAGE loading buffer, separated by SDS-PAGE gel electrophoresis, and the separated proteins were transferred to PVDF and blocked with 5% BSA at room temperature for 1 hour. After incubation of the antibody with the autophagy-related protein LC3 and a secondary antibody, color development was performed in an ultrasensitive ECL developer.
FIG. 6 shows the results of protein expression of LC3 by Western blotting. As can be seen from the figure, the ratio of LC3B/LC3A of the polymerized chloroquine-fluorenylmethylcarbonyl glucan nanogel to that of other groups is higher, which indicates that the nanogel has an obvious autophagy inhibition function, so that the effect of inhibiting cell migration can be achieved.
4. Investigation of in vitro CXCR4 antagonistic function of polymerized chloroquine-fluorenylmethylcarbonyl dextran nanogel
Human osteosarcoma cells U2OS expressing CXCR4 receptor were labeled with green fluorescent protein at 1X 10 per well5The individual cells were incubated in a confocal dish for 24 hours, incubated for 30 minutes with the various formulations, washed twice with 100. mu.L of assay (containing 2 mM L-glutamine, 1% FBS, 1% Streptomycete and 10 mM HEPES) and finally incubated for one hour with the addition of SDF-1. After fixation with 4% formaldehyde for 20 minutes, the plate was washed 4 times with PBS and observed with a confocal laser microscope.
FIG. 7 shows that the polymerized chloroquine internalizes CXCR4 on the surface of U2OS cells, so that SDF-1 cannot be combined with CXCR4 to achieve the effect of inhibiting cell migration, and therefore, the modified polymerized chloroquine has a strong function of resisting tumor cell metastasis.
5. Determination of in vitro anti-tumor cell metastasis of polymerized chloroquine-fluorenylmethylcarbonyl glucan
The Matrigel was diluted 1: 3 with a 1640 culture solution containing 1% FBS, 40. mu.L of the diluted solution was added to each well, and the mixture was left to stand at 37 ℃ for coagulation. 4T1 cells in exponential growth phase were added to a Transwell chamber containing Matrigel at 5000 cells/100. mu.L/chamber. Placing the chamber in a 24-well culture plate, adding 60 μ L of 10% FBS-containing DMEM culture solution into each well of the chamber, and culturing at 37 deg.C and 5% CO2And continuing culturing in the incubator. After 6 hours, the different preparations were incubated for 24 hours, the Transwell chamber was removed after the incubation was completed, the cells in the chamber were wiped clean with a cotton swab, washed 4 times with PBS, fixed with absolute methanol for 20 minutes, and stained with 0.2% crystal violet for 30 minutes. The cells were counted in 5 fields at random and the mean value was calculated, while observing under a 40-fold inverted microscope. And comparing the number of the cell penetrating membranes of the vector group and the control group to be used as an index for evaluating the invasion capacity of the tumor cells.
Fig. 8 is an experimental result of in vitro anti-tumor cell metastasis, and it is seen from the figure that the number of metastatic cells of the polymerized chloroquine-fluorenylmethylcarbonyl dextran nanogel (CQ-HF) is significantly reduced compared to the control group (untreated) and the chloroquine-free vector group (HF), thereby proving that the polymerized chloroquine-fluorenylmethylcarbonyl dextran nanogel has a strong anti-tumor cell metastasis function.
The above examples are merely examples for clearly illustrating the present invention, and examples of nanoparticles prepared at this time and drawings are disclosed, but not limiting the embodiments. Those skilled in the art will understand that: various alternatives, variations and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and drawings, and obvious variations or modifications derived therefrom are within the scope of the invention as herein claimed.

Claims (7)

1. A delivery system of polymerized chloroquine fluorene methyl carbonyl nanogel is characterized in that: the delivery system comprises nanogel particles of polymerized chloroquine fluorenylmethyl and an anti-tumor drug wrapped in the particles;
the nanometer gel particles are formed by cross-linking a polysaccharide main chain modified with hydroxychloroquine and fluorenylmethyloxycarbonyl, the molecular weight of the polysaccharide main chain is 5-200 kD, the molar substitution degree of the hydroxychloroquine on the polysaccharide main chain is 5% -50%, and the molar substitution degree of the fluorenylmethyloxycarbonyl on the polysaccharide main chain is 1% -30%;
the polysaccharide backbone is selected from hydroxyethyl starch, dextrin, polyethylene glycol, hyaluronic acid, alginic acid, dextran or chitosan;
the antineoplastic drug is selected from camptothecin, doxorubicin hydrochloride, paclitaxel, docetaxel or curcumin.
2. The polymeric chloroquine fluorenemethylcarbonyl nanogel delivery system of claim 1, wherein: the polysaccharide backbone is dextran.
3. The method for preparing a polymeric chloroquine fluorenemethylcarbonyl nanogel delivery system as claimed in claim 1, wherein the method comprises the following steps: the method comprises the following steps:
step 1, modification of a main chain of the poly-polysaccharide by fluorenylmethyloxycarbonyl: in a polar solvent, in the presence of a catalyst and a dehydrating agent, a fluorenylmethyloxycarbonyl donor is connected with hydroxyl on a polysaccharide main chain through esterification reaction to obtain the polysaccharide main chain with a side chain modified with fluorenylmethyloxycarbonyl;
step 2, modification of the polysaccharide skeleton by hydroxychloroquine: firstly, synthesizing hydroxychloroquine-carbonyldiimidazole in a polar solvent, and then reacting the hydroxychloroquine-carbonyldiimidazole with a polysaccharide main chain with a lateral chain modified with fluorenylmethyloxycarbonyl to obtain the polysaccharide main chain modified with hydroxychloroquine and fluorenylmethyloxycarbonyl;
step 3, preparation of nanogel delivery system: dissolving the polysaccharide main chain modified with hydroxychloroquine and fluorenylmethyloxycarbonyl and the antitumor drug in an organic solvent, dropwise adding the obtained organic solution into the aqueous solution under the stirring condition to prepare emulsion, and removing the organic solvent to obtain the nanogel delivery system.
4. The production method according to claim 3, characterized in that: in step 1, the fluorenylmethyloxycarbonyl donor is selected from Fmoc-glycine, Fmoc-L-ornithine or N' -Fmoc-L-lysine.
5. The production method according to claim 3, characterized in that: in the step 1, a polar solvent is selected from dimethyl sulfoxide, anhydrous dimethylformamide or tetrahydrofuran, a dehydrating agent is dicyclohexylcarbodiimide, and a catalyst is dimethylaminopyridine, the reaction step is specifically to dissolve a polysaccharide main chain in the polar solvent, respectively add the dehydrating agent, the catalyst and a fluorenylmethoxycarbonyl donor, react in an inert atmosphere, and obtain the polysaccharide main chain with a side chain modified with the fluorenylmethoxycarbonyl after treatment of a product.
6. The production method according to claim 3, characterized in that: in the step 2, the polar solvent is selected from propanol, acetonitrile, anhydrous dimethyl sulfoxide or anhydrous dimethylformamide, and the reaction step specifically comprises the steps of respectively dissolving hydroxychloroquine and N, N '-carbonyldiimidazole in the polar solvent, reacting the hydroxychloroquine and the N, N' -carbonyldiimidazole in an inert atmosphere to obtain hydroxychloroquine-carbonyldiimidazole, dissolving the synthesized hydroxychloroquine-carbonyldiimidazole and the polysaccharide main chain of fluorenylmethyloxycarbonyl in the polar solvent, stirring for reaction in the inert atmosphere, and treating to obtain the polysaccharide main chain of hydroxychloroquine and the fluorenylmethyloxycarbonyl.
7. The production method according to claim 3, characterized in that: in the step 3, the organic solvent is selected from dimethyl sulfoxide, tetrahydrofuran or dimethylformamide, the aqueous solution is selected from pure water, 5% w/v glucose solution, normal saline, phosphate buffer solution or acetate buffer solution, and the mass ratio of the polysaccharide main chain modified with hydroxychloroquine and fluorenylmethoxycarbonyl to the antitumor drug is 1-100: 1.
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