CN111329872A

CN111329872A - Dextrin nanogel for treating metastatic breast cancer and preparation method and application thereof

Info

Publication number: CN111329872A
Application number: CN202010163114.3A
Authority: CN
Inventors: 杨学; 边嘉怡; 孙敏捷; 王铮; 高放
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-03-10
Filing date: 2020-03-10
Publication date: 2020-06-26
Anticipated expiration: 2040-03-10
Also published as: CN111329872B

Abstract

The invention relates to the field of pharmaceutical preparations and the technical field of nano materials, in particular to dextrin nanogel for treating metastatic breast cancer and a preparation method and application thereof. dextrin-PEI-CM nanogel (DPC) is prepared by grafting Polyethyleneimine (PEI) onto a dextrin skeleton to form a dextrin-PEI polymer (DP) and connecting a CXCR4 antagonist cyclotetradecane Compound (CM) to the end of the DP. The nanogel can block CXCR4/SDF-1 axis through CM, so that the invasion and metastasis of tumors are reduced; meanwhile, the nanogel can also entrap nucleic acid such as miR-34a to reduce the expression of adhesion protein CD44 to achieve the effect of resisting metastasis, and in the invention, the DPC/miR-34a nanogel with multiple functions is prepared by a simple suspension method, and the effective treatment of tumor invasion and metastasis is realized by the synergistic effect of targeted inhibition of SDF-1/CXCR4 signal conduction and the reduction of the expression of adhesion protein CD 44.

Description

Dextrin nanogel for treating metastatic breast cancer and preparation method and application thereof

Technical Field

The invention relates to the field of pharmaceutical preparations and the technical field of nano materials, in particular to dextrin nanogel for treating metastatic breast cancer and a preparation method thereof.

Background

Metastasis is one of the leading causes of cancer death and morbidity. Metastatic Breast Cancer (MBC) is one of the most common malignancies in women, and even with increasing treatment regimens, the 5-year survival rate for patients with MBC remains low, only 5%. Tumor metastasis is a multi-stage process involving the spread of tumor cells from the primary tumor to distant metastases. The tumor cell migration chain is controlled by a variety of processes, including the destruction of tumor cell surface proteins, degradation of extracellular matrix proteins, and activation of chemokines, cytokines, and growth factors.

The tumor metastasis pathway is related to both the characteristics of the tumor cells and depends on the microenvironment of the metastatic site. Chemokines and their receptor networks play an important role in many links of tumor metastasis. First, the site to be metastasized secretes chemokines, providing a metastatic signal to direct tumor cell metastasis, consistent with the "seed and soil" theory of tumor metastasis. Both experimental and clinical evidence indicate that the chemokine receptor CXCR4 is critical in the metastatic process of breast cancer. CXCR4 belongs to a member of the family of transmembrane protein-coupled receptors and can be activated by its ligand, SDF-1, to further regulate cell proliferation, adhesion and invasion. CXCR4 has been considered as one of the most promising targets for MBC therapy in the present study, and CXCR4 antagonists such as cyclotetradecanes (Cyclam Monomer, CM) have been shown to block the CXCR4/SDF-1 axis, reducing tumor invasiveness.

Cell Adhesion Molecules (CAMs), such as CD44, are involved in cell stroma and cell-cell adhesion, and are highly expressed in malignant tumors and metastasis determinants, leading to tumor invasion and metastasis. Therefore, inhibiting the expression of CD44 in cancer cells is a potential therapeutic approach to prevent cell migration. miR-34a is a small non-coding RNA, can reduce the expression of adhesion protein CD44, and meanwhile, miR-34a can also inhibit tumor anti-apoptosis gene Bcl2 and induce tumor cell apoptosis. Therefore, the miR-34a and CM combined treatment can provide a multidimensional approach for the anti-metastasis treatment of tumors, the blocking of signal transduction and the metastasis of microenvironment.

Disclosure of Invention

The purpose of the invention can be realized by the following technical scheme:

a method of preparing a dextrin nanogel for use in the treatment of metastatic breast cancer, the method comprising the steps of:

(1) dissolving dextrin in a solvent, adding an anhydride acylating agent and a pyridine catalyst, and then stirring for reaction in an inert atmosphere to obtain a carboxylated dextrin primary solution;

preferably: the concentration of the dextrin solution is 1-250 mg/mL; further preferably: the concentration of the dextrin solution is 80-120 mg/mL;

(2) adding ether into the primary carboxylated dextrin solution obtained in the step (1) for centrifugation, collecting precipitates, dissolving the precipitates with water, putting the precipitates into a dialysis bag with the molecular weight cutoff of 5-15 kDa for dialysis, and freeze-drying to obtain carboxylated dextrin;

(3) dissolving cyclotetradecane Compound (CM) in an organic solvent, adding an anhydride acylating agent and a pyridine catalyst, and stirring the mixture for reaction to obtain a carboxylated cyclotetradecane compound crude solution;

(4) adding ether into the crude solution obtained in the step (3), centrifuging to obtain a white precipitate, redissolving the white precipitate by using an organic solvent, and removing the reaction solvent to obtain a carboxylated cyclotetradecane compound;

(5) dissolving the carboxylated dextrin obtained in the step (2), the carboxylated cyclotetradecane compound obtained in the step (4) and Polyethyleneimine (PEI) in water, adjusting the pH value to be alkaline, and reacting at room temperature in the presence of a catalyst and a condensing agent to obtain a dextrin-PEI-CM nanogel crude solution;

preferably: and (3) carboxylated dextrin: carboxylated cyclotetradecanes: the mass ratio of the polyethyleneimine is 5-200: 0.1-50: 5-400; further preferably: and (3) carboxylated dextrin: carboxylated cyclotetradecanes: the mass ratio of the polyethyleneimine is 10-50: 0.1-10: 50-100 parts;

preferably: the polyethyleneimine preferably has a molecular weight of 1.8 kDa; the condensing agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; the catalyst is selected from at least one of N-hydroxysuccinimide, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, benzotriazol-N, N, N ', N' -tetramethylurea hexafluorophosphate and benzotriazol-1-yl-oxy tripyrrolidinyl phosphorus hexafluorophosphate;

most preferably: and (3) carboxylated dextrin: carboxylated cyclotetradecanes: the mass ratio of the polyethyleneimine is 8-12: 0.5-2: 15-20; the catalyst is N-hydroxysuccinimide;

(6) and (3) putting the crude solution obtained in the step (5) into a dialysis bag with the molecular weight cutoff of 5-15 kDa for dialysis to obtain dextrin-PEI-CM nanogel (DPC).

A dextrin nanogel for use in the treatment of metastatic breast cancer, the dextrin nanogel being prepared by:

(1) dissolving dextrin in a corresponding solvent, adding an anhydride acylating agent and a pyridine catalyst, and then stirring for reaction in an inert atmosphere to obtain a carboxylated dextrin primary solution;

the polyethyleneimine preferably has a molecular weight of 1.8 kDa; the condensing agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; the catalyst is selected from at least one of N-hydroxysuccinimide, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, benzotriazol-N, N, N ', N' -tetramethylurea hexafluorophosphate and benzotriazol-1-yl-oxy tripyrrolidinyl phosphorus hexafluorophosphate;

most preferably: and (3) carboxylated dextrin: carboxylated cyclotetradecanes: the mass ratio of the polyethyleneimine is 40-60: 1-10: 75-100 parts; the catalyst is N-hydroxysuccinimide;

The technical scheme of the invention is as follows: the solvent in the step (1) comprises dimethylformamide, dimethyl sulfoxide, dimethylacetamide, pyridine, ethylene glycol, an alkaline aqueous solution and water; in the step (1) and the step (3), the anhydride acylating agent is succinic anhydride, and the pyridine catalyst is 4-dimethylamino pyridine.

The technical scheme of the invention is as follows: dextrin solution in the step (1): acid anhydride acylating agent: the mass ratio of the pyridine catalyst is as follows: 1: 0.01-10: 0.01 to 10; preferably, the dextrin solution in the step (1): acid anhydride acylating agent: the mass ratio of the pyridine catalyst is 1: 0.1-0.3: 0.08 to 0.12;

in the step (3): cyclotetradecanes: acid anhydride acylating agent: the mass ratio of the pyridine catalyst is 1: 0.01-10: 0.01 to 10; preferably: cyclotetradecanes: acid anhydride acylating agent: the mass ratio of the pyridine catalyst is 1: 0.1-0.5: 0.05 to 0.15.

The technical scheme of the invention is as follows: the organic solvent in steps (3) and (4) includes dichloromethane, methanol, ethanol, acetonitrile and acetone.

The technical scheme of the invention is as follows: the particle size of the dextrin nanogel is 50-500 nm, and preferably 180-200 nm.

Dissolving the obtained dextrin-PEI-CM nanogel in a buffer solution, adding nucleic acid, vortexing, and incubating at 37 ℃ to obtain the dextrin-PEI-CM nanogel for encapsulating the nucleic acid; wherein the nucleic acids are RNA and DNA; preferably the nucleic acid is miR-34 a; preferably: the buffer solution is HEPES buffer solution, DEPC water or TE buffer solution.

The method for encapsulating nucleic acid comprises the steps of using HEPES buffer solution with the concentration of 8-12 mM and the pH of 6.0-9.0, using DEPC water with the mass concentration of 0.05-0.2% and using TE buffer solution with the pH of 6.0-9.0 of 1 × TE.

In the above method for entrapping nucleic acids: the mass ratio of the dextrin-PEI-CM nanogel to the nucleic acid is more than 1, and preferably 3.

In the invention, the nucleic acid-loaded dextrin nanogel with the anti-metastasis effect is designed and successfully prepared, so that a CXCR antagonist CM and nucleic acids such as miR-34a are delivered at the same time, the synergistic effect between the inhibition of SDF-1/CXCR4 signaling and the down-regulation of CAMs-CD44 expression is achieved, and the effective dual-level anti-metastasis activity is realized.

Breast cancer is a highly malignant tumor in women, the metastasis and recurrence of which are the leading causes of death in breast cancer patients. Blocking the metastasis process is an important link for improving the survival quality of breast cancer patients. Therefore, in the invention, MDA-MB-231 cells (human breast cancer cells) and MDA-MB-231 tumor-bearing mice are selected as main models to be used as the evaluation objects of the anti-metastasis and anti-tumor effects of the nanogel.

The invention has the beneficial effects that:

(1) the invention provides a dextrin nanogel for treating metastatic breast cancer based on combination therapy. The traditional CXCR4 antagonist (CM) is combined with a cell adhesion molecule CD44 inhibitor, namely MiR-34a, to inhibit tumor metastasis in multiple biological paths, so that the effect of remarkably weakening the proliferation and survival of metastatic cancer cells can be achieved, and a multi-dimensional efficient path is provided for anti-metastasis treatment and signal transduction blocking of tumors.

(2) The dextrin nanogel for treating metastatic breast cancer is prepared by a suspension method, complex operation and equipment are not needed, and the preparation method is simple and convenient. Meanwhile, the used main raw material dextrin is an FDA approved safe and good pharmaceutic adjuvant, and the prepared dextrin nanogel has good biocompatibility, improves the cell uptake efficiency, reduces the carrier toxicity, and is a pharmaceutical preparation with higher production cost performance, effectiveness and low toxicity.

(3) Therefore, the invention can simply, conveniently and efficiently prepare the safe and low-toxicity dextrin nanogel with the anti-tumor and anti-metastasis effects of the combined therapy, and has potential medical prospect.

Drawings

FIG. 1 is a schematic diagram of the preparation and structure of carboxylated dextrin (Dex-COOH) (A), carboxylated cyclotetradecane compound (CM-COOH) (B) and dextrin-PEI-CM nanogel (DPC) (C).

FIG. 2 is a schematic diagram of the preparation and structure of dextrin-PEI-CM nanogel (DPC/miR-34a) for encapsulating nucleic acid.

FIG. 3 shows a nuclear magnetic hydrogen spectrum (A) of dextrin (Dex) and a nuclear magnetic hydrogen spectrum (B) of carboxylated dextrin (Dex-COOH).

FIG. 4 is a mass spectrum of a carboxylated cyclotetradecane compound.

FIG. 5 is a nuclear magnetic hydrogen spectrum of dextrin-PEI-CM nanogel.

FIG. 6 shows the particle size distribution and TEM image of dextrin-PEI-CM nanogel encapsulating nucleic acid.

FIG. 7 is a graph of particle size (A) and Zeta potential distribution (B) of dextrin-COOH (Dex-COOH), DP/miRNA and DPC/miRNA nanocomplexes for three different mass ratios w/w (PEI/miRNA) miRNA encapsulated formulations on 1.8kDa polyethyleneimine (1.8k PEI), dextrin-PEI nanogel (DP) and dextrin-PEI-CM nanogel (DPC).

Fig. 8 is the result of agarose gel electrophoresis of miRNA encapsulation tests on 1.8kDa polyethyleneimine (1.8kPEI), dextrin-PEI nanogel (DP) and dextrin-PEI-CM nanogel (DPC) according to different N/P ratios (expressed as mass ratio w/w ═ PEI/miRNA).

Fig. 9 is a graph of the effect of each preparation on Bcl2 protein expression as detected by western blot analysis.

FIG. 10 is a graph showing the effect of each preparation on the expression of CD44 protein as determined by Western blot analysis.

FIG. 11 is a microscopic image (A) of the results of various formulation groups in tumor cell invasion experiments and a histogram of the percentage of tumor cells invaded (B).

Detailed Description

The invention is further illustrated by the following examples, without limiting the scope of the invention:

the preparation process of the cyclotetradecane compound refers to the preparation method of Cyclam Monomer in the following documents.

Wang Y,Hazeldine S T,Li J,et al.Development of functional poly(amidoamine)CXCR4 antagonists with the ability to mobilize leukocytes and delivernucleic acids[J].Advanced healthcare materials,2015,4(5):729-738.

Example 1

(1) 1g of dextrin was dissolved in a mixed solution of 5mL of dimethylformamide and 5mL of dimethyl sulfoxide in a 50mL two-necked flask to obtain a dextrin solution.

(2) Adding 180mg of succinic anhydride and 85mg of 4-dimethylaminopyridine into the dextrin solution obtained in the step (1), vacuumizing a flask by installing a three-way valve, and stirring the mixture under an inert atmosphere for reaction to obtain a carboxylated dextrin primary solution.

(3) And (3) filling the carboxylated dextrin primary solution obtained in the step (2) into a 50mL centrifuge tube, adding low-temperature diethyl ether, centrifuging for 10 minutes, collecting white precipitates at the bottom of the centrifuge tube, and dissolving with 10mL of water to obtain a carboxylated dextrin crude solution.

(4) And (4) putting the crude solution obtained in the step (3) into a dialysis bag with the molecular weight cutoff of 12kDa, dialyzing for 48 hours, and freeze-drying by using a vacuum freeze dryer to obtain the carboxylated dextrin.

(5) 0.6g of Cyclotetradecanes (CM) was added to a 10mL dichloromethane solution and dissolved in a 50mL two-necked flask to obtain a cyclotetradecanes solution.

(6) 0.1g of succinic anhydride and 50mg of 4-dimethylaminopyridine were added to the flask of the solution of cyclotetradecanes of step (5), and the mixture was reacted at room temperature for 24 hours to obtain a crude solution of carboxylated cyclotetradecanes.

(7) And (3) filling the crude solution obtained in the step (6) into a 50mL centrifuge tube, adding low-temperature ether, centrifuging for 10 minutes, collecting white precipitate at the bottom of the centrifuge tube, and dissolving with 10mL dichloromethane to obtain a carboxylated cyclotetradecane compound dissolved solution.

(8) And (4) spin-drying the dissolved solution obtained in the step (7) by using a rotary evaporator to remove a reaction solvent dichloromethane to obtain the carboxylated cyclotetradecane compound.

(9) And (3) dissolving 90mg of carboxylated dextrin obtained in the step (4), 6mg of carboxylated cyclotetradecane compound obtained in the step (8) and 190mg of 1.8kDa Polyethyleneimine (PEI) in water, adding 100mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 100mg of N-hydroxysuccinimide, adjusting the pH value to be alkaline, and obtaining a crude dextrin-PEI-CM nanogel solution at room temperature.

(10) And (4) putting the crude solution obtained in the step (9) into a dialysis bag with the molecular weight cutoff of 1000Da for dialysis to obtain the dextrin-PEI-CM nanogel.

(11) And (3) dissolving the dextrin-PEI-CM nanogel obtained in the step (10) in a HEPES buffer solution (10mM HEPES, pH8.0), adding the nucleic acid miR-34a to be encapsulated, wherein the w/w (PEI/miR-34a) ratio of DPC to the nucleic acid is 1.5:1, vortexing for 1 minute, and incubating at 37 ℃ for 1 hour to obtain the dextrin-PEI-CM nanogel for encapsulating the nucleic acid.

FIG. 3 shows a nuclear magnetic hydrogen spectrum (A) of dextrin (Dex) and a nuclear magnetic hydrogen spectrum (B) of carboxylated dextrin (Dex-COOH). The figure shows the dextrin (Dex)¹The H NMR spectrum showed peaks at δ 3.35-3.95 and δ 5.30 assigned to the H2-H6 proton peak and H1 proton peak, respectively. The hydrogen spectrum of the carboxylated dextrin (Dex-COOH) shows a new proton peak at δ ═ 2.6, which is assigned to the methylene peak on succinic anhydride, demonstrating its successful synthesis.

FIG. 4 is a mass spectrum of a carboxylated cyclotetradecane compound. The exact molecular weight is 861.55, and 863.5 mass spectrum peak is shown on the magnetic mass spectrum, which proves the successful synthesis of the compound.

FIG. 5 is a nuclear magnetic hydrogen spectrum of dextrin-PEI-CM nanogel. As can be seen from the figures, the,¹the H NMR spectrum showed that the δ ═ 1.0-1.5 peaks were assigned to the Boc peak on the structure of cyclotetradecetraazane (CM 7). The peak delta-5.2-5.5 is assigned to the peak H1 on the dextrin structure. The peak delta-7.0-7.5 is classified as the H peak on the CM benzene ring structure. The peak delta-2.0-3.0 is assigned to the H peak on the PEI structure. These all demonstrate successful synthesis.

FIG. 6 shows the particle size distribution and TEM image of dextrin-PEI-CM nanogel encapsulating nucleic acid. The particle size distribution of dextrin nanogel DPC/miRNA (N/P is 3, w/w) measured by dynamic light scattering is shown in the figure, the particle size of nanogel is about 200nm, the polydispersity PDI is 0.245 +/-0.027, and the system is uniformly distributed small-particle size gel, which is beneficial to passive targeting accumulation to tumor sites through EPR effect. Meanwhile, the morphology of the nanogel is directly observed by a Transmission Electron Microscope (TEM), and the result is a uniform spherical structure.

FIG. 7 is a graph of particle size (A) and Zeta potential distribution (B) of dextrin-COOH (Dex-COOH), DP/miRNA and DPC/miRNA nanocomplexes for three different mass ratios w/w (PEI/miRNA) miRNA encapsulated formulations on 1.8kDa polyethyleneimine (1.8k PEI), dextrin-PEI nanogel (DP) and dextrin-PEI-CM nanogel (DPC). The results show that all the nanocomplexes have a particle size below 300nm, and the particle size of DPC/miRNA (w/w ═ 3) is about 180 nm. The zeta potentials of DP/miRNA and DPC/miRNA are 9.23mV and 10.25mV, respectively, while the zeta potential of dextrin-COOH (Dex-COOH) is about-18.0 mV. Therefore, due to good zeta potential and particle size, DPC/miR-34a is suitable for cellular uptake and targeting tumors via the EPR effect.

Fig. 8 is the result of agarose gel electrophoresis of miRNA encapsulation tests on 1.8kDa polyethyleneimine (1.8kPEI), dextrin-PEI nanogel (DP) and dextrin-PEI-CM nanogel (DPC) according to different N/P ratios (expressed as mass ratio w/w ═ PEI/miRNA). The results show that both DP and DPC nanogels are able to complex miRNA completely. The nucleic acid encapsulation capacity of 1.8k PEI remains after the attachment of the large dextrin and the small CM, while it can be seen that the nanogel can completely encapsulate miRNA when w/w (PEI/miRNA) is 2.5 or more.

Fig. 9 is a graph of the effect of each preparation on Bcl2 protein expression as detected by western blot analysis. Compared with a PBS group, a negative control group DPC/miNC group (DPC carries disordered miRNA) and a PEI/miR-34a group, the DPC/miR-34a nanogel has the strongest Bcl2 protein down-regulation capability, and can effectively reduce the expression level of the Bcl2 protein, thereby inducing tumor cell apoptosis.

FIG. 10 is a graph showing the effect of each preparation on the expression of CD44 protein as determined by Western blot analysis. Compared with a PBS group, a negative control group DPC/miNC group (DPC carries disordered miRNA) and a PEI/miR-34a group, the DPC/miR-34a nanogel has the strongest capacity of reducing the CD44 protein, and can effectively reduce the expression level of the CD44 protein, thereby inhibiting tumor metastasis.

FIG. 11 is a microscopic results plot (A) and a bar chart (B) of the percentage of tumor cells affected in the tumor cell invasion experiment for each formulation group, breast cancer cells MDA-MB-231 were incubated with different formulations and allowed to invade matrigel under stimulation of CXCR4 ligand SDF-1 α, resulting in microscopic counting and photographing of cell invasion conditions compared to untreated groups, Polyethyleneimine (PEI) groups, dextrin-PEI nanogel (DP) groups, free cyclotetradecane (FreeCM) groups and groups with dextrin-PEI-CM nanogel (DPC) groups, with significantly reduced cell invasion and the DPC/miR-34a group having the best effect of inhibiting tumor cell invasion, which is probably due to the dual effect of miR-34a down-regulating the CD44 and CM inhibition of the CXCR4 axis, demonstrating that miR/miR-34 a nanogels have very significant anti-metastatic effect in the in vitro metastasis model.

Example 2

(1) 1g of dextrin was dissolved in 12mL of dimethylacetamide in a 50mL two-necked flask to obtain a dextrin solution.

(2) And (2) adding 100mg of succinic anhydride and 80mg of 4-dimethylaminopyridine into the dextrin solution obtained in the step (1), vacuumizing a flask by installing a three-way valve, and stirring the mixture under an inert atmosphere to react to obtain a carboxylated dextrin primary solution.

(5) 0.5g of Cyclotetradecanes (CM) was added to a 10mL dichloromethane solution and dissolved in a 50mL two-necked flask to obtain a cyclotetradecanes solution.

(6) 0.05g of succinic anhydride and 25mg of 4-dimethylaminopyridine were added to the flask of the solution of cyclotetradecanes of step (5), and the mixture was reacted at room temperature for 24 hours to obtain a crude solution of carboxylated cyclotetradecanes.

(9) And (3) dissolving 80mg of carboxylated dextrin obtained in the step (4), 5mg of carboxylated cyclotetradecane compound obtained in the step (8) and 150mg of 1.8kDa Polyethyleneimine (PEI) in water, adding 100mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 50mg of 4-dimethylaminopyridine, adjusting the pH value to be alkaline, and reacting at room temperature to obtain a crude dextrin-PEI-CM nanogel solution.

(11) And (2) dissolving the dextrin-PEI-CM nanogel obtained in the step (10) in 0.1% DEPC water, adding nucleic acid miR-34a to be encapsulated, wherein the w/w (PEI/miR-34a) ratio of DPC to nucleic acid is 1:1, vortexing for 1 minute, and incubating for 1 hour at 37 ℃ to obtain the dextrin-PEI-CM nanogel for encapsulating nucleic acid.

Example 3

(1) 1g of dextrin was dissolved in 8.4mL of pyridine in a 50mL two-necked flask to obtain a dextrin solution.

(2) Adding 300mg succinic anhydride and 120mg 4-dimethylamino group into the dextrin solution obtained in the step (1), vacuumizing the flask by installing a three-way valve, and stirring the mixture to react under an inert atmosphere to obtain a carboxylated dextrin primary solution.

(5) Cyclotetradecanes (CM) (1 g) was added to a 10mL dichloromethane solution, and dissolved in a 50mL two-necked flask to obtain a cyclotetradecanes solution.

(6) 0.5g of succinic anhydride and 150mg of 4-dimethylaminopyridine were added to the flask of the solution of cyclotetradecanes of step (5), and the mixture was reacted at room temperature for 24 hours to obtain a crude solution of carboxylated cyclotetradecanes.

(9) And (3) dissolving 120mg of carboxylated dextrin obtained in the step (4), 20mg of carboxylated cyclotetradecane compound obtained in the step (8) and 200mg of 1.8kDa Polyethyleneimine (PEI) in water, adding 200mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 150mg of 1-hydroxybenzotriazole, adjusting the pH value to be alkaline, and reacting at room temperature to obtain a dextrin-PEI-CM nanogel crude solution.

(11) And (2) dissolving the dextrin-PEI-CM nanogel obtained in the step (10) in TE buffer solution (1 ×, pH is 8.0), adding nucleic acid miR-34a to be encapsulated, wherein the w/w (PEI/miR-34a) ratio of DPC to nucleic acid is 5:1, vortexing for 1 minute, and incubating for 1 hour at 37 ℃ to obtain the dextrin-PEI-CM nanogel for encapsulating nucleic acid.

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

1. A preparation method of dextrin nanogel for treating metastatic breast cancer is characterized by comprising the following steps: the method comprises the following steps:

(4) adding ether into the crude solution in the step (3), centrifuging to obtain a white precipitate, redissolving the white precipitate by using an organic solvent,

then removing the reaction solvent to obtain a carboxylated cyclotetradecane compound;

further preferably: and (3) carboxylated dextrin: carboxylated cyclotetradecanes: the mass ratio of the polyethyleneimine is 40-60: 1-10: 75-100 parts; the catalyst is N-hydroxysuccinimide;

2. The method of claim 1, wherein: the solvent in the step (1) comprises dimethylformamide, dimethyl sulfoxide, dimethylacetamide, pyridine, ethylene glycol, an alkaline aqueous solution and water; in the step (1) and the step (3), the anhydride acylating agent is succinic anhydride, and the pyridine catalyst is 4-dimethylamino pyridine.

3. The method of claim 1, wherein: dextrin solution in the step (1): acid anhydride acylating agent: the mass ratio of the pyridine catalyst is as follows: 1: 0.01-10: 0.01 to 10; preferably, the dextrin solution in the step (1): acid anhydride acylating agent: the mass ratio of the pyridine catalyst is 1: 0.1-0.3: 0.08 to 0.12;

4. The method of claim 1, wherein: the organic solvent in steps (3) and (4) includes dichloromethane, methanol, ethanol, acetonitrile and acetone.

5. A dextrin nanogel for use in the treatment of metastatic breast cancer, characterized in that: the dextrin nanogel is prepared by the following steps:

6. The dextrin nanogel of claim 5, wherein: the solvent in the step (1) comprises dimethylformamide, dimethyl sulfoxide, dimethylacetamide, pyridine, ethylene glycol, an alkaline aqueous solution and water; in the step (1) and the step (3), the anhydride acylating agent is succinic anhydride, and the pyridine catalyst is 4-dimethylamino pyridine;

preferably, the dextrin solution in the step (1): acid anhydride acylating agent: the mass ratio of the pyridine catalyst is as follows: 1: 0.01-10: 0.01 to 10; further preferably, the dextrin solution in the step (1): acid anhydride acylating agent: the mass ratio of the pyridine catalyst is 1: 0.1-0.3: 0.08 to 0.12;

preferably: in the step (3): cyclotetradecanes: acid anhydride acylating agent: the mass ratio of the pyridine catalyst is 1: 0.01-10: 0.01 to 10; preferably: cyclotetradecanes: acid anhydride acylating agent: the mass ratio of the pyridine catalyst is 1: 0.1-0.5: 0.05 to 0.15;

the organic solvent in steps (3) and (4) includes dichloromethane, methanol, ethanol, acetonitrile and acetone.

7. The dextrin nanogel of claim 5, wherein: the particle size of the dextrin nanogel is 50-500 nm, and preferably 180-200 nm.

8. A method for entrapping nucleic acids using the dextrin nanogel of claim 1, wherein: dissolving the obtained dextrin-PEI-CM nanogel in a buffer solution, adding nucleic acid, vortexing, and incubating at 37 ℃ to obtain the dextrin-PEI-CM nanogel for encapsulating the nucleic acid; wherein the nucleic acids are RNA and DNA; preferably the nucleic acid is miR-34 a; preferably: the buffer solution is HEPES buffer solution, DEPC water or TE buffer solution.

9. The method according to claim 8, wherein the HEPES buffer solution has a concentration of 8-12 mM and a pH of 6.0-9.0, the DEPC mass concentration in DEPC water is 0.05-0.2%, and the TE buffer solution is 1 × TE buffer solution and has a pH of 6.0-9.0.

10. The method of claim 8, wherein: the mass ratio of the dextrin-PEI-CM nanogel to the nucleic acid is more than 1, and preferably 3.