CN118976106A - An alginate-based thermogenetic CXCL12 releaser and its preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and provides an alginate-based thermogenetic CXCL12 releaser, and a preparation method and application thereof. The invention utilizes transgenic and freeze-drying technology to prepare an alginate-based thermogenetic CXCL12 releaser (ATCG) based on an optimized alternating magnetic field, the ATCG is convenient and simple to prepare, can be produced in large scale, consists of a porous magnetic bracket loaded with engineering cells, the engineering cells are stably transformed by slow viruses into a thermogenetic element of pHSP70-CXCL12, the porous magnetic bracket consists of a biological polymer material coated with magnetic particles to form a main body framework structure, and has an isotropic porous structure, and the ATCG prepared by the invention can controllably release CXCL12 chemotactic factors for DTC recruitment, activate abdominal immunity against DTC after magnetocaloric ablation, and further inhibit DTC abdominal cavity transfer.

Description

Alginate-based thermogenetic CXCL12 releaser and preparation method and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to an alginate-based thermogenetic CXCL12 releaser and a preparation method and application thereof.

Background

Peritoneal metastasis is a clinical symptom of gastric cancer and is accompanied by a high risk of death. It is characterized by the generation of a great deal of malignant ascites, abdominal pain, adhesive intestinal obstruction, nausea, vomiting and other phenomena. This is observed in more than 50% of patients who die. Gastric cancer cells (called diffuse tumor cells, DTCs) tend to metastasize to ascites due to physiological anatomical features and cellular mutations. Thus, removal of DTCs in the peritoneum can prevent metastasis. Although conventional peritoneal chemotherapy and primary cytopenia surgery are clinically accepted treatments for peritoneal metastasis, chemotherapy resistance and serious side effects still prevent widespread clinical use.

In recent years, peritoneal immunotherapy (IPIT) has been considered as a promising strategy for improving the immunocompromised environment and generating effective immunity due to the unique luminal environment and the abundance of immune cells in the peritoneal cavity. However, IPIT still faces significant challenges in immune escape due to DTC dormancy and time consuming adoptive cell therapy. Various immune cells and chemokines (CXCL 12, CCL 2) are involved in dormancy and recruitment of DTCs and accelerate the development and accurate localization of cancer. Therefore, a material is designed to promote the release of in vivo controllable chemotactic factors and induce downstream anti-DTC immunity, and has very important significance.

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provides an alginate-based thermogenetic CXCL12 releaser, and a preparation method and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of an alginate-based thermogenetic CXCL12 releaser, which comprises the following steps of:

(1) Mixing the first biopolymer material solution, the first magnetic particle solution and the cross-linking agent for cross-linking to obtain a porous magnetic scaffold;

Or mixing the second biopolymer material solution and the second magnetic particle solution, heating, and sequentially performing self-assembly and freeze drying after heating to obtain the porous magnetic stent;

(2) Adding the genetic CXCL12 engineering cells of the thermogenetics on the surface of the porous magnetic bracket, and incubating to obtain the alginate-based thermogenetics CXCL12 releaser.

Preferably, in the step (1), the first biopolymer in the first biopolymer material solution includes sodium alginate, oxidized hyaluronic acid, chitosan or silk fibroin;

the first magnetic particles in the first magnetic particle solution comprise iron-cobalt-graphite nanocapsules, ferrite particles, neodymium-iron-boron particles or alloy particles containing iron, cobalt and nickel; the mass concentration of the first magnetic particle solution is 8-12 mg/mL.

Preferably, when the first biopolymer material is sodium alginate, the method for preparing the porous magnetic stent comprises the following steps:

Mixing sodium alginate solution, first magnetic particle solution and first cross-linking agent to carry out first cross-linking, and freeze-drying after the first cross-linking is finished to obtain an intermediate substance; mixing the intermediate substance with a second crosslinking agent for second crosslinking to obtain the porous magnetic scaffold;

the mass fraction of the sodium alginate solution is 1.5-2.5%; the first cross-linking agent is calcium gluconate solution, and the mass fraction of the calcium gluconate solution is 0.5-1.0%; the second cross-linking agent is calcium chloride solution, and the concentration of the calcium chloride solution is 0.5-1.5 mol/L; the volume ratio of the sodium alginate solution to the first magnetic particle solution to the calcium gluconate solution is 1-3: 0.5 to 1.5:0.5 to 1.5;

The temperature of the first crosslinking is 20-30 ℃, and the time of the first crosslinking is 10-15 min; the freeze drying temperature is-15 to-25 ℃, and the freeze drying time is 22 to 26 hours; the temperature of the second crosslinking is 20-30 ℃, and the time of the second crosslinking is 1.5-2.5 h.

Preferably, when the first biopolymer material is oxidized hyaluronic acid, the method for preparing the porous magnetic scaffold comprises the following steps:

mixing the oxidized hyaluronic acid solution, the first magnetic particle solution and the cross-linking agent for cross-linking, and freeze-drying after the cross-linking is finished to obtain the porous magnetic scaffold;

The mass fraction of the oxidized hyaluronic acid solution is 3-7%; the cross-linking agent is branched polyethyleneimine; the volume ratio of the oxidized hyaluronic acid solution to the first magnetic particle solution is 0.5-1.5: 0.5 to 1.5; the volume ratio of the oxidized hyaluronic acid solution to the branched polyethyleneimine is 0.5-1.5: 0.5 to 1.5;

the cross-linking temperature is 20-30 ℃, and the cross-linking time is 3-7 min; the freeze drying temperature is-15 to-25 ℃, and the freeze drying time is 22 to 26 hours.

Preferably, when the first biopolymer material is chitosan, the method for preparing the porous magnetic scaffold comprises the following steps:

Mixing acetic acid solution of chitosan, first magnetic particle solution and cross-linking agent, cross-linking, and sequentially performing self-assembly and freeze-drying after cross-linking to obtain a porous magnetic bracket;

The mass fraction of chitosan in the acetic acid solution of chitosan is 5-7%; the cross-linking agent is glutaraldehyde solution, and the mass fraction of the glutaraldehyde solution is 2-4%; the volume ratio of the acetic acid solution of chitosan, the first magnetic particle solution and the glutaraldehyde solution is 0.5-1.5: 0.5 to 1.5:0.5 to 1.5;

The cross-linking temperature is 50-60 ℃, and the cross-linking time is 25-35 min; the self-assembly temperature is 20-30 ℃, and the self-assembly time is 46-50 h; the freeze drying temperature is-15 to-25 ℃, and the freeze drying time is 22 to 26 hours.

Preferably, when the first biopolymer material is silk fibroin, the method for preparing the porous magnetic scaffold comprises the following steps:

Mixing the silk fibroin solution and the first magnetic particle solution, and freeze-drying to obtain an intermediate substance after freeze-drying; mixing the intermediate substance with a cross-linking agent for cross-linking to obtain the porous magnetic scaffold;

The mass fraction of the silk fibroin solution is 8-12%; the volume ratio of the silk fibroin solution to the first magnetic particle solution is 0.5-1.5: 0.5 to 1.5; the cross-linking agent is methanol; the volume ratio of the silk fibroin solution to the methanol is 0.5-1.5: 2.5 to 3.5;

The freeze drying temperature is-15 to-25 ℃, and the freeze drying time is 22 to 26 hours; the cross-linking temperature is 20-30 ℃, and the cross-linking time is 1.5-2.5 h.

Preferably, the second biopolymer in the second biopolymer material solution in step (1) includes agarose or collagen; when the second biopolymer is agarose, the mass fraction of the second biopolymer solution is 1.5-2.5%; when the second biopolymer is collagen, the mass fraction of the second biopolymer solution is 8-12%;

the second magnetic particles in the second magnetic particle solution comprise iron-cobalt graphite nanocapsules, ferrite particles, neodymium-iron-boron particles or alloy particles containing iron, cobalt and nickel; the mass concentration of the second magnetic particle solution is 8-12 mg/mL;

The volume ratio of the second biopolymer solution to the second magnetic particle solution is 0.5-1.5: 0.5 to 1.5;

The heating temperature in the step (1) is 70-100 ℃, the time is 0.5-1.5 min, the self-assembly temperature is 20-30 ℃ and the time is 25-35 min; the freeze drying temperature is-15 to-25 ℃ and the time is 22 to 26 hours.

Preferably, the ratio of the number of the genetically engineered cells of the metagenetics CXCL12 to the volume of the porous magnetic scaffold of step (2) is 0.8x10⁵～1.2×10⁵: 80-120 mm³;

The temperature of the co-incubation is 35-40 ℃, and the time of the co-incubation is 5-15 min.

The invention also provides an alginate-based thermogenetic CXCL12 releaser prepared by the preparation method.

The invention also provides application of the alginate-based thermogenetic CXCL12 releaser in preparing anti-tumor immunity medicines or medicines for inhibiting peritoneal metastasis.

The beneficial effects of the invention are as follows:

The invention utilizes transgenic and freeze-drying technology to prepare an alginate-based thermogenetic CXCL12 releaser (ATCG) based on optimized Alternating Magnetic Field (AMF), the preparation is convenient and simple, the large-scale production can be realized, the ATCG consists of a porous magnetic bracket (IMS) loaded with engineering cells, the engineering cells are stably transformed by slow viruses into the thermogenetic element of pHSP70-CXCL12, the porous magnetic bracket consists of a main body framework structure made of a biological polymer material coated with magnetic particles, the main body framework structure has an isotropic porous structure, and the ATCG prepared by the invention can controllably release CXCL12 chemotactic factors for DTC recruitment, and activates abdominal immunity against DTC after magnetocaloric ablation, thereby inhibiting DTC abdominal cavity transfer.

Drawings

FIG. 1 is a diagram showing the basic structure of IMS containing GFP-engineered cells in example 1 (Scaffold/Cell-Scaffold/Cell, before Beforewash-AFTERWASHING-after wash, cell loading ratio-Cell loading ratio); in fig. 1, a is a laser confocal microscope characterization diagram of an IMS containing GFP-engineered cells, b is a fluorescence imaging contrast diagram of an IMS containing GFP-engineered cells and a P-IMS containing GFP-engineered cells, c is a cell load factor contrast diagram of an IMS and a P-IMS, d is a scanning electron microscope characterization diagram of an IMS containing GFP-engineered cells, and e is an elemental scanning characterization diagram of an IMS containing GFP-engineered cells;

FIG. 2 is a graph of the magnetocaloric effect characterization of IMS and ALG in example 1 (Temperature, time); in fig. 2, a is an infrared thermal imaging contrast diagram of IMS and ALG; b is a schematic diagram of the magnetocaloric conversion capability of the IMS under different alternating magnetic field intensities;

FIG. 3 is a graph depicting the thermal response capacity of the different cells of example 1 and example 2; in FIG. 3, a is a fluorescence characterization diagram of GES-1^pHSP70-GFP after thermal stimulation, b is a fluorescence characterization diagram of NIH-3T3^pHSP70-GFP after thermal stimulation, c is a WB characterization diagram of CXCL12 protein of GES-1^{pHSP70-hCXCL12} after thermal stimulation, and d is a WB characterization diagram of CXCL12 protein of NIH-3T3^{pHSP70-mCXCL12} after thermal stimulation;

FIG. 4 is a graph showing the CXCL12 release capacity after ATCG-1 magnetocaloric treatment (Concentration of hCXCL-concentration of hCCCL 12, CXCL12 Relative Expression-CXCL 12 expression level, time-Time); in fig. 4, a is a CXCL12 protein secretion ELISA data characterization diagram of ATCG-1 after magnetocaloric stimulation, b is a CXCL12 gene expression qPCR data characterization diagram of ATCG-1 after magnetocaloric stimulation, c is a CXCL12 protein secretion kinetics schematic diagram of ATCG-1 after magnetocaloric stimulation, and d is a CXCL12 gene expression kinetics schematic diagram of ATCG-1 after magnetocaloric stimulation;

FIG. 5 is a graph of the DTC chemotactic ability assessment of ATCG-1 (TRANSMIGRETED CELL-chemotactic cells, radiance-fluorescence intensity); in FIG. 5, a is a Transwell staining pattern of the ATCG-1 on MGC-803 and MFC^mCxcr4 cells after the magnetocaloric stimulation, b is a chemotactic pattern of the ATCG-1 on the MGC-803 cells after the magnetocaloric stimulation, c is a chemotactic pattern of the ATCG-1 on the MFC^mCxcr4 cells after the magnetocaloric stimulation, d is a luciferase imaging pattern of the ATCG-1 after the recruitment of DTC in the abdominal cavity of the mouse after the magnetocaloric stimulation, e is a fluorescent quantitative statistical pattern of the recruitment of DTC in the abdominal cavity of the mouse after the magnetocaloric stimulation;

FIG. 6 is a graph of evaluation of therapeutic effect of ATCG-2 in a mice model of peritoneal metastasis (Number of metastatic-metastasis number, precent-percent); in FIG. 6, a is a CT three-dimensional imaging diagram of ATCG-2 after the treatment of the peritoneal transfer mouse model, b is a statistical diagram of the number of peritoneal transfer foci of ATCG-2 after the treatment of the peritoneal transfer mouse model, and c is a statistical diagram of T cells of peritoneal cavities CD4 and CD8 of ATCG-2 after the treatment of the peritoneal transfer mouse model.

Detailed Description

The invention provides a preparation method of an alginate-based thermogenetic CXCL12 releaser, which comprises the following steps of:

(1) Mixing the first biopolymer material solution, the first magnetic particle solution and the cross-linking agent for cross-linking to obtain a porous magnetic scaffold;

Or mixing the second biopolymer material solution and the second magnetic particle solution, heating, and sequentially performing self-assembly and freeze drying after heating to obtain the porous magnetic stent;

(2) Adding the genetic CXCL12 engineering cells of the thermogenetics on the surface of the porous magnetic bracket, and incubating to obtain the alginate-based thermogenetics CXCL12 releaser.

In the present invention, the first biopolymer in the first biopolymer material solution in step (1) preferably includes sodium alginate, oxidized hyaluronic acid, chitosan or silk fibroin.

In the invention, the first magnetic particles in the first magnetic particle solution preferably comprise iron-cobalt-graphite nanocapsules, ferrite particles, neodymium-iron-boron particles or alloy particles containing iron, cobalt and nickel, and the solvent in the first magnetic particle solution is water; the mass concentration of the first magnetic particle solution is preferably 8 to 12mg/mL, more preferably 9 to 11mg/mL, and even more preferably 10 to 10.5mg/mL.

In the invention, the preparation method of the iron-cobalt graphite nanocapsules refers to a patent CN115679472A (a magnetocaloric fiber and a preparation method and application thereof); the ferrite particles are preferably compounds containing an oxygen element and an iron element, and more preferably, ferroferric oxide or ferric oxide; preparation method of NdFeB particles reference Electronic Structure ofNdFeCoB Oxide Magnetic Particles Studiedby DFT Calculations andXPS-PMC(nih.gov); iron-cobalt-nickel alloy reference Magnetic Graphitic Nanocapsules:Fabrication,Classification,and Theranostic Applications|Chemical&Biomedical Imaging(acs.org).

In the present invention, when the first biopolymer material is sodium alginate, the method for preparing the porous magnetic scaffold preferably comprises the following steps:

Mixing sodium alginate solution, first magnetic particle solution and first cross-linking agent to carry out first cross-linking, and freeze-drying after the first cross-linking is finished to obtain an intermediate substance; and mixing the intermediate substance with a second crosslinking agent for second crosslinking to obtain the porous magnetic scaffold.

In the invention, the solvent in the sodium alginate solution is water, and the mass fraction of the sodium alginate solution is preferably 1.5-2.5%, more preferably 1.7-2.3%, and even more preferably 2-2.1%; the first cross-linking agent is preferably a calcium gluconate solution, wherein the solvent in the calcium gluconate solution is water, and the mass fraction of the calcium gluconate solution is preferably 0.5-1.0%, more preferably 0.6-0.9%, and even more preferably 0.7-0.8%; the second crosslinking agent is preferably a calcium chloride solution in which the solvent is water, and the concentration of the calcium chloride solution is preferably 0.5 to 1.5mol/L, more preferably 0.7 to 1.3mol/L, and still more preferably 0.8 to 1.0mol/L; the volume ratio of the sodium alginate solution, the first magnetic particle solution and the calcium gluconate solution is preferably 1-3: 0.5 to 1.5:0.5 to 1.5, more preferably 1.5 to 2.5:0.7 to 1.3:0.7 to 1.3, more preferably 2 to 2.3:1 to 1.2:1 to 1.2; the invention does not limit the dosage of the calcium chloride solution, so long as the first biopolymer material can be ensured to complete sufficient crosslinking.

In the present invention, the temperature of the first crosslinking is preferably 20 to 30 ℃, more preferably 22 to 28 ℃, still more preferably 25 to 26 ℃; the time for the first crosslinking is preferably 10 to 15 minutes, more preferably 11 to 14 minutes, still more preferably 12 to 13 minutes; the temperature of freeze-drying is preferably-15 to-25 ℃, more preferably-17 to-23 ℃, and even more preferably-20 to-22 ℃; the time for freeze-drying is preferably 22 to 26 hours, more preferably 23 to 25 hours, and still more preferably 23.5 to 24 hours; the temperature of the second crosslinking is preferably 20 to 30 ℃, more preferably 22 to 28 ℃, still more preferably 25 to 26 ℃; the time for the second crosslinking is preferably 1.5 to 2.5 hours, more preferably 1.7 to 2.3 hours, and still more preferably 1.9 to 2 hours.

In the present invention, after the second crosslinking is completed, the obtained sample is eluted with water, whereby Ca²⁺ which is not crosslinked is sufficiently eluted to obtain a porous magnetic scaffold, which is stored at 4 ℃.

In the present invention, when the first biopolymer material is oxidized hyaluronic acid, the method for preparing a porous magnetic scaffold preferably comprises the steps of:

And mixing the oxidized hyaluronic acid solution, the first magnetic particle solution and the cross-linking agent for cross-linking, and freeze-drying after the cross-linking is finished to obtain the porous magnetic scaffold.

In the present invention, the method for producing oxidized hyaluronic acid preferably comprises the steps of:

mixing the sodium periodate solution and the hyaluronic acid solution, stirring at room temperature for preferably 1-3 hours, more preferably 1.5-2.5 hours, more preferably 2 hours, adding the ethylene glycol solution, continuously stirring at room temperature for preferably 1-3 hours, more preferably 1.5-2.5 hours, more preferably 2 hours, transferring to a dialysis bag with a molecular weight cut-off of 3kDa after stirring, dialyzing with water for preferably 2-4 days, more preferably 2.5-3.5 days, more preferably 3 days, and freeze-drying by adopting a conventional technical means in the art after dialyzing to obtain the oxidized hyaluronic acid.

In the present invention, the concentration of the sodium periodate solution is preferably 0.3 to 0.7mol/L, more preferably 0.4 to 0.6mol/L, still more preferably 0.5mol/L; the mass fraction of the hyaluronic acid solution is preferably 0.5 to 1.5%, more preferably 0.7 to 1.3%, still more preferably 0.8 to 1.0%; the volume ratio of the sodium periodate solution to the hyaluronic acid solution is preferably 3-7: 80 to 120, more preferably 4 to 6:90 to 110, more preferably 5:100.

In the present invention, the solvent in the oxidized hyaluronic acid solution is water, and the mass fraction of the oxidized hyaluronic acid solution is preferably 3 to 7%, more preferably 4 to 6%, and even more preferably 5 to 5.5%; the cross-linking agent is preferably branched polyethylenimine; the volume ratio of the oxidized hyaluronic acid solution to the first magnetic particle solution is preferably 0.5 to 1.5:0.5 to 1.5, more preferably 0.7 to 1.3:0.7 to 1.3, more preferably 1 to 1.2:1 to 1.2; the volume ratio of the oxidized hyaluronic acid solution to the branched polyethyleneimine is preferably 0.5 to 1.5:0.5 to 1.5, more preferably 0.7 to 1.3:0.7 to 1.3, more preferably 1 to 1.2:1 to 1.2.

In the present invention, the temperature of crosslinking is preferably 20 to 30 ℃, more preferably 22 to 28 ℃, still more preferably 25 to 26 ℃; the crosslinking time is preferably 3 to 7 minutes, more preferably 4 to 6 minutes, and still more preferably 4.5 to 5 minutes; the temperature of freeze-drying is preferably-15 to-25 ℃, more preferably-17 to-23 ℃, and even more preferably-20 to-22 ℃; the time for freeze-drying is preferably 22 to 26 hours, more preferably 23 to 25 hours, and still more preferably 23.5 to 24 hours; after freeze drying, a porous magnetic scaffold is obtained and stored at 4 ℃.

In the present invention, when the first biopolymer material is chitosan, the method for preparing the porous magnetic scaffold preferably comprises the steps of:

mixing acetic acid solution of chitosan, first magnetic particle solution and cross-linking agent, cross-linking, self-assembling and freeze-drying after cross-linking is finished, and obtaining the porous magnetic scaffold.

In the present invention, the mass fraction of chitosan in the acetic acid solution of chitosan is preferably 5 to 7%, more preferably 5.5 to 6.5%, and still more preferably 6 to 6.2%; the cross-linking agent is preferably glutaraldehyde solution, the solvent in the glutaraldehyde solution is water, the mass fraction of the glutaraldehyde solution is preferably 2-4%, more preferably 2.5-3.5%, and even more preferably 3-3.2%; the volume ratio of the acetic acid solution of chitosan, the first magnetic particle solution and the glutaraldehyde solution is preferably 0.5-1.5: 0.5 to 1.5:0.5 to 1.5, more preferably 0.7 to 1.3:0.7 to 1.3:0.7 to 1.3, more preferably 1 to 1.2:1 to 1.2:1 to 1.2.

In the present invention, the temperature of crosslinking is preferably 50 to 60 ℃, more preferably 52 to 58 ℃, still more preferably 55 to 56 ℃; the crosslinking time is preferably 25 to 35 minutes, more preferably 27 to 33 minutes, and still more preferably 28 to 30 minutes; the self-assembly temperature is preferably 20 to 30 ℃, more preferably 22 to 28 ℃, and even more preferably 25 to 26 ℃; the self-assembly time is preferably 46 to 50 hours, more preferably 47 to 49 hours, and even more preferably 47.5 to 48 hours; the temperature of freeze-drying is preferably-15 to-25 ℃, more preferably-17 to-23 ℃, and even more preferably-20 to-22 ℃; the time for freeze-drying is preferably 22 to 26 hours, more preferably 23 to 25 hours, and still more preferably 23.5 to 24 hours; after freeze drying, a porous magnetic scaffold is obtained and stored at 4 ℃.

In the present invention, when the first biopolymer material is silk fibroin, the method for preparing the porous magnetic scaffold preferably comprises the steps of:

Mixing the silk fibroin solution and the first magnetic particle solution, and freeze-drying to obtain an intermediate substance after freeze-drying; and mixing the intermediate substance with a crosslinking agent for crosslinking to obtain the porous magnetic scaffold.

In the invention, the solvent in the silk fibroin solution is water, and the mass fraction of the silk fibroin solution is preferably 8-12%, more preferably 9-11%, and even more preferably 10-10.5%; the volume ratio of the silk fibroin solution to the first magnetic particle solution is preferably 0.5-1.5: 0.5 to 1.5, more preferably 0.7 to 1.3:0.7 to 1.3, more preferably 1 to 1.2:1 to 1.2; the cross-linking agent is preferably methanol; the volume ratio of the silk fibroin solution to the methanol is preferably 0.5-1.5: 2.5 to 3.5, more preferably 0.7 to 1.3:2.7 to 3.3, more preferably 1 to 1.2:3 to 3.2.

In the present invention, the temperature of freeze-drying is preferably-15 to-25 ℃, more preferably-17 to-23 ℃, and still more preferably-20 to-22 ℃; the time for freeze-drying is preferably 22 to 26 hours, more preferably 23 to 25 hours, and still more preferably 23.5 to 24 hours; the crosslinking temperature is preferably 20 to 30 ℃, more preferably-17 to-23 ℃, and even more preferably-20 to-22 ℃; the crosslinking time is preferably 1.5 to 2.5 hours, more preferably 1.7 to 2.3 hours, and still more preferably 1.9 to 2 hours.

In the invention, after the crosslinking is finished, the obtained sample is eluted by water to obtain the porous magnetic stent, and the porous magnetic stent is stored at 4 ℃.

In the present invention, the second biopolymer in the second biopolymer material solution in the step (1) preferably includes agarose or collagen, and the solvent in the second biopolymer material solution is water; when the second biopolymer is agarose, the mass fraction of the second biopolymer solution is preferably 1.5 to 2.5%, more preferably 1.7 to 2.3%, still more preferably 2 to 2.1%; when the second biopolymer is collagen, the mass fraction of the second biopolymer solution is preferably 8 to 12%, more preferably 9 to 11%, still more preferably 10 to 10.5%.

In the invention, the second magnetic particles in the second magnetic particle solution preferably comprise iron-cobalt-graphite nanocapsules, ferrite particles, neodymium-iron-boron particles or alloy particles containing iron, cobalt and nickel, the source of the second magnetic particles is the same as that of the first magnetic particles, and the solvent in the second magnetic particle solution is water; the mass concentration of the second magnetic particle solution is preferably 8 to 12mg/mL, more preferably 9 to 11mg/mL, and even more preferably 10 to 10.5mg/mL.

In the present invention, the volume ratio of the second biopolymer solution to the second magnetic particle solution is preferably 0.5 to 1.5:0.5 to 1.5, more preferably 0.7 to 1.3:0.7 to 1.3, more preferably 1 to 1.2:1 to 1.2.

In the present invention, the heating temperature in the step (1) is preferably 70 to 100 ℃, more preferably 80 to 90 ℃, still more preferably 85 to 87 ℃; the time is preferably 0.5 to 1.5min, more preferably 0.7 to 1.3min, and still more preferably 0.8 to 1min; the self-assembly temperature is preferably 20 to 30 ℃, more preferably 22 to 28 ℃, and even more preferably 25 to 26 ℃; the time is preferably 25 to 35 minutes, more preferably 27 to 33 minutes, and still more preferably 28 to 30 minutes; the temperature of freeze-drying is preferably-15 to-25 ℃, more preferably-17 to-23 ℃, and even more preferably-20 to-22 ℃; the time is preferably 22 to 26 hours, more preferably 23 to 25 hours, and still more preferably 23.5 to 24 hours; after freeze drying, a porous magnetic scaffold is obtained and stored at 4 ℃.

In the present invention, the method for producing a genetically engineered CXCL12 cell in step (2) preferably comprises the steps of:

Transforming a central DNA plasmid (the promoter (marked as pHSP 70) and a thermal genetics CXCL12 gene are constructed on the central DNA plasmid), a packaging plasmid pMD2.G and a packaging plasmid psPAX into 293T cells, collecting lentivirus in a culture solution after 3 days of transfection, then incubating the lentivirus with GES-1 or NIH-3T3 for 3 days, adding puromycin after the incubation is finished for screening for 7 days, and finally obtaining the surviving cells which are the thermal genetics CXCL12 gene engineering cells.

In the present invention, the sequence of the metagenetics CXCL12 gene is obtained from ncbi database, and when the lentivirus is co-incubated with GES-1, the metagenetics CXCL12 gene is hCXCL12; when lentiviruses were co-incubated with NIH-3T3, the thermo-genetics CXCL12 gene was mCXCL12; the preparation method of the central DNA plasmid is a seamless connection technology and is a conventional technology in the field; references are Complete CHEMICAL SYNTHESIS, assemble, and Cloning of a Mycoplasma genitalium Genome |science and Enzymatic Assembly of DNA molecules up to several hundred kilobases |Nature Methods, also available from Geneart seamless cloning and GibsonThermo FISHER SCIENTIFIC-CN; packaging plasmid pMD2.G and packaging plasmid psPAX.G are purchased from Addgene (the purchase website of packaging plasmid pMD2.G is https:// www.addgene.org/12259/, and the purchase website of packaging plasmid psPAX2 is https:// www.addgene.org/12260 /); 293T cells were purchased from ATCC (purchase website https:// www.atcc.org/products/crl-3216); GES-1 was purchased from ATCC; NIH-3T3 was purchased from Procell Pronoxel (purchase website https:// www.procell.com.cn/view/8947. Html).

In the present invention, the hCCCL 12 gene sequence (SEQ ID NO. 1) is:

ATGGACGCCAAGGTCGTCGCCGTGCTGGCCCTGGTGCTGGCCGCGCTCTGCATCAGTGACGGTAAACCAGTCAGCCTGAGCTACCGATGCCCCTGCCGGTTCTTCGAGAGCCACATCGCCAGAGCCAACGTCAAGCATCTGAAAATCCTCAACACTCCAAACTGTGCCCTTCAGATTGTTGCACGGCTGAAGAACAACAACAGACAAGTGTGCATTGACCCGAAATTAAAGTGGATCCAAGAGTACCTGGAGAAAGCTTTAAACAAGAGGCTCAAGATG.

In the present invention, mCXCL gene sequence (SEQ ID NO. 2) is:

ATGAACGCCAAGGTCGTGGTCGTGCTGGTCCTCGTGCTGACCGCGCTCTGCCTCAGCGACGGGAAGCCCGTCAGCCTGAGCTACAGATGCCCATGCCGATTCTTCGAAAGCCATGTTGCCAGAGCCAACGTCAAGCATCTCAAAATTCTCAACACTCCAAACTGTGCCCTTCAGATTGTAGCCCGGCTGAAGAACAACAACAGACAAGTGTGCATTGACCCGAAGCTAAAGTGGATTCAGGAGTACCTGGAGAAAGCTTTAAACAAGAGGTTCAAGATG.

in the invention, the collection of lentiviruses in culture fluid is completed by adopting the conventional technical means in the field, specifically, after the central DNA plasmid, the packaging plasmid pMD2.G and the packaging plasmid psPAX are transformed into 293T cells, the genes on the plasmids start to express, the pMD2.G and the psPAX2 start to express protein shells of the lentiviruses, the shells are assembled with the central DNA plasmid, then the host cells are killed by the lentiviruses of the new generation, the lentiviruses are released outside the cells, and then the lentiviruses are collected by filtration and concentration; the temperature at which the lentivirus is incubated with GES-1 or NIH-3T3 is preferably 35 to 40℃and more preferably 36 to 39℃and even more preferably 37 to 38 ℃.

In the present invention, the volume ratio of the number of the genetically engineered cells of the CXCL12 of the thermogenetics in step (2) to the porous magnetic scaffold is preferably 0.8x10⁵～1.2×10⁵: 80 to 120mm³, more preferably 0.9X10⁵～1.1×10⁵: 90 to 110mm³, more preferably 1X 10⁵: 100mm³.

In the present invention, in step (2), the genetically engineered cells of the thermogenetics CXCL12 are added to the surface of the porous magnetic scaffold, specifically, the genetically engineered cells of the thermogenetics CXCL12 are dropped onto the plane of the porous magnetic scaffold (the porous magnetic scaffold is cylindrical, and the plane of the porous magnetic scaffold is the upper bottom surface or the lower bottom surface of the cylinder).

In the present invention, the temperature of the co-incubation is preferably 35 to 40 ℃, more preferably 36 to 39 ℃, and even more preferably 37 to 38 ℃; the co-incubation time is preferably 5 to 15 minutes, more preferably 7 to 13 minutes, and even more preferably 8 to 10 minutes.

The invention also provides an alginate-based thermogenetic CXCL12 releaser prepared by the preparation method.

The invention also provides application of the alginate-based thermogenetic CXCL12 releaser in preparing anti-tumor immunity medicines or medicines for inhibiting peritoneal metastasis.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Mixing a sodium alginate solution with the mass fraction of 2%, an iron-cobalt-graphite nanocapsule solution with the mass concentration of 10mg/mL and a calcium gluconate solution with the mass fraction of 0.8% (the volume ratio of the sodium alginate solution to the iron-cobalt-graphite nanocapsule solution to the calcium gluconate solution is 2:1:1), crosslinking for 12min at 25 ℃, freeze-drying for 24h at-20 ℃ after the crosslinking is finished, adding an excessive calcium chloride solution with the concentration of 1.0mol/L, crosslinking for 2h at 25 ℃, eluting the obtained sample with water after the crosslinking is finished, and fully eluting uncrosslinked Ca²⁺ to obtain a porous magnetic bracket (marked as IMS);

Transforming a central DNA plasmid (pHSP 70 and a thermogenetics hCCCL 12 gene are constructed on the central DNA plasmid), a packaging plasmid pMD2.G and a packaging plasmid psPAX into 293T cells, collecting lentivirus in a culture solution after 3 days of transfection, then incubating the lentivirus and GES-1 for 3 days at 37 ℃, adding puromycin after the incubation is finished for screening for 7 days, and obtaining cells which are finally survived, namely the thermogenetics CXCL12 genetically engineered cells (marked as GES-1^{pHSP70-hCXCL12});

10⁵ GES-1^{pHSP70-hCXCL12} were added to the surface of IMS with a volume of 100mm³ and incubated at 37℃for 10min to give the alginate-based thermogenetic CXCL12 releaser (labeled ATCG-1).

Transforming a central DNA plasmid (pHSP 70 and GFP are constructed on the central DNA plasmid, GFP is green fluorescent protein), a packaging plasmid pMD2.G and a packaging plasmid psPAX into 293T cells, collecting slow viruses in a culture solution after 3 days of transfection, then incubating the slow viruses with GES-1 for 3 days at 37 ℃, adding puromycin after the incubation is finished for screening for 7 days, and obtaining GFP engineering cells (namely GES-1^pHSP70-GFP) as final survival cells; 10⁵ GFP engineering cells are added to the surface of the IMS prepared in the embodiment with the volume of 100mm³, and incubated for 10min at 37 ℃ to obtain the IMS containing the GFP engineering cells; meanwhile, pressing the IMS prepared in the embodiment with a weight of 10g for 3 days to obtain a magnetocaloric non-porous support (marked as P-IMS); 10⁵ GFP-engineered cells were added to the surface of P-IMS with a volume of 100mm³ and incubated at 37℃for 10min to give P-IMS containing GFP-engineered cells. Characterizing the IMS containing GFP engineering cells under a laser confocal microscope, photographing the IMS containing GFP engineering cells and the P-IMS containing GFP engineering cells by using a fluorescent imaging system, respectively cleaning the IMS containing GFP engineering cells by using DPBS for 3 times after photographing, and photographing by using the fluorescent imaging system again after cleaning; And quantifying the cell load rates of IMS and P-IMS by the signal intensity of GFP; finally, fixing IMS containing GFP engineering cells on conductive adhesive, spraying metal for 90s, improving conductivity, observing the surface microstructure by a scanning electron microscope (TESCAN) under the acceleration voltage of 20kV, and performing element scanning analysis by using an X-ray energy dispersion spectrometer; the basic structure characterization of IMS containing GFP engineered cells in example 1 was obtained as shown in FIG. 1 (Scaffold/Cell-Scaffold/Cell, before Beforewash-AFTER WASHING-after wash, cell loading ratio-Cell loading ratio); In fig. 1, a is a laser confocal microscope characterization of IMS containing GFP-engineered cells, b is a fluorescence imaging contrast of IMS containing GFP-engineered cells and P-IMS containing GFP-engineered cells, c is a cell load factor contrast of IMS and P-IMS, d is a scanning electron microscope characterization of IMS containing GFP-engineered cells, and e is an elemental scanning characterization of IMS containing GFP-engineered cells. As can be seen from fig. 1, there are a large number of engineered cells within the IMS containing GFP engineered cells, which cells are effectively retained in the void structure of the IMS; the IMS containing GFP engineering cells can effectively load cells in the bracket due to the porous structure, and the cell load rate of the IMS can reach 82.2+/-16.6 percent and can reach 13.05 times of that of P-IMS; Cells are visible in IMS containing GFP engineered cells in scaffolds with elemental carbon, iron, and cobalt. Taken together, there were a large number of engineered cells and elemental carbon, iron, cobalt in the IMS containing GFP-engineered cells, indicating successful preparation of IMS containing GFP-engineered cells.

Mixing sodium alginate solution with the mass fraction of 2% and calcium gluconate solution with the mass fraction of 0.8% (the volume ratio of the sodium alginate solution to the calcium gluconate solution is 2:1), crosslinking for 12min at 25 ℃, freeze-drying for 24h at-20 ℃ after the crosslinking is finished, adding excessive calcium chloride solution with the concentration of 1.0mol/L, crosslinking for 2h at 25 ℃, eluting the obtained sample with water after the crosslinking is finished, and fully eluting uncrosslinked Ca²⁺ to obtain a bracket (marked as ALG) of an undoped magnetocaloric material; IMS and ALG are respectively exposed to a coil with the diameter of 60mm, and the whole temperature rising process is monitored by a thermal infrared imager (FOTRIC) under the conditions of the frequency of 340kHz and the magnetic field strength of 60% (corresponding to an alternating magnetic field of 27 kA/m); in addition, in order to optimize the temperature rising effect of the IMS, the magnetocaloric conversion capability of the IMS under different alternating magnetic field intensities (40-60% of magnetic field intensity, corresponding to 18-27 kA/m of alternating magnetic field) is monitored; a magnetocaloric effect characterization diagram of IMS and ALG in example 1 was obtained, as shown in fig. 2 (Temperature-Time); in fig. 2, a is an infrared thermal imaging contrast diagram of IMS and ALG; b is a schematic diagram of the magnetocaloric conversion capability of IMS under different alternating magnetic field intensities (60% refers to an alternating magnetic field of 27kA/m, 50% refers to an alternating magnetic field of 22.5kA/m, 45% refers to an alternating magnetic field of 20.25kA/m, and 40% refers to an alternating magnetic field of 18 kA/m). As can be seen from fig. 2, compared with ALG, IMS has a significant heating effect under an alternating magnetic field, the heating effect of IMS can be effectively adjusted by adjusting the intensity of the alternating magnetic field where IMS is located, the higher the intensity of the alternating magnetic field is, the faster the heating speed of IMS is, the heating speed of IMS can reach 0.1-0.3 ℃/s, the heating speed of IMS can reach about 45 ℃ under the alternating magnetic field of 22.5kA/m, the heating speed can reach about 55 ℃ under the alternating magnetic field of 27kA/m, wherein 45 ℃ can be used for thermal activation of the genetically engineered CXCL12 cells of thermogenetics, and 55 ℃ can be used for TAA (tumor associated antigen) release and peritoneal immunity activation from DTC sources. The data show that the IMS has remarkable magneto-thermal response capability, and the magneto-thermal heating effect can be adjusted by adjusting the intensity of the alternating magnetic field, so that the IMS has good magneto-thermal effect and controllability.

Example 2

The other conditions for preparing ATCG-1 in example 1 were controlled to be unchanged, and the thermogenetics hCCCL 12 gene was replaced with the thermogenetics mCXCL gene and GES-1 was replaced with NIH-3T3 to obtain NIH-3T3^{pHSP70-mCXCL12};

10⁵ NIH-3T3^{pHSP70-mCXCL12} were added to the surface of IMS with a volume of 100mm³ and incubated at 37℃for 10min to give the alginate-based thermogenetic CXCL12 releaser (labeled ATCG-2).

Transforming a central DNA plasmid (pHSP 70 and GFP are constructed on the central DNA plasmid, GFP is green fluorescent protein), a packaging plasmid pMD2.G and a packaging plasmid psPAX into 293T cells, collecting slow viruses in a culture solution after 3 days of transfection, then incubating the slow viruses with NIH-3T3 for 3 days at 37 ℃, adding puromycin after the incubation is finished for screening for 7 days, and finally obtaining cells which are obtained by survival, namely NIH-3T3^pHSP70-GFP; The GES-1^pHSP70-GFP prepared in example 1 and the NIH-3T3^pHSP70-GFP prepared in the example are used for visualizing engineering cells, the laser confocal microscope is used for characterizing the GFP expression capability of the GES-1^pHSP70-GFP and the NIH-3T3^pHSP70-GFP after 12 hours of stimulation at 45 ℃, meanwhile, the GES-1^{pHSP70-hCXCL12} prepared in the embodiment 1 and the NIH-3T3^{pHSP70-mCXCL12} prepared in the embodiment are used for protein secretion characterization of genetic CXCL12 engineering cells of the thermogenetics, and the WB detection means is used for characterizing the protein secretion capacity of CXCL12 after the GES-1^{pHSP70-hCXCL12} and the NIH-3T3^{pHSP70-mCXCL12} are stimulated for 12 hours at 45 ℃; characterization of the thermal response capacity of the different cells of example 1 and example 2 was obtained as shown in FIG. 3; In FIG. 3, a is a fluorescence characterization diagram of GES-1^pHSP70-GFP after thermal stimulation, b is a fluorescence characterization diagram of NIH-3T3^pHSP70-GFP after thermal stimulation, c is a WB characterization diagram of CXCL12 protein of GES-1^{pHSP70-hCXCL12} after thermal stimulation, d is a WB characterization of CXCL12 protein of NIH-3T3^{pHSP70-mCXCL12} after thermal stimulation. In a, GES-1 is a single human cell GES-1, GES-1^pHSP70-GFP is GES-1^pHSP70-GFP,GES-1^pHSP70-GFP +heat prepared in example 1 is GES-1^pHSP70-GFP after thermal stimulation, BF is the abbreviation of Bright Field, Meaning bright field; In b, 3T3 is a single murine cell NIH-3T3,3T3^pHSP70-GFP, and NIH-3T3^pHSP70-GFP,3T3^pHSP70-GFP + heat prepared in example 2 is NIH-3T3^pHSP70-GFP after thermal stimulation; in c, control is a Control group, namely single human cells GES-1 without any operation, pHSP 70-hCCCL 12 is GES-1^{pHSP70-hCXCL12} prepared in example 1, pHSP 70-hCCCL 12+heat is GES-1^{pHSP70-hCXCL12} after thermal stimulation, and GAPDH is an internal reference gene; In d, control is Control group, i.e. single murine cell NIH-3T3 without any manipulation, pHSP70-mCXCL is NIH-3T3^{pHSP70-mCXCL12} prepared in example 2, pHSP70-mCXCL12+heat is NIH-3T3^{pHSP70-mCXCL12} after heat stimulation, GAPDH is reference gene. As can be seen from FIG. 3, GES-1^pHSP70-GFP and NIH-3T3^pHSP70-GFP have significant GFP expression under thermal stimulation, GES-1^{pHSP70-hCXCL12} and NIH-3T3^{pHSP70-mCXCL12} successfully expressed and secreted CXCL12, Indicating that the cells effectively promote the expression of CXCL12 chemokines under thermal stimulation. The data show that the thermogenetic genetic engineering cells have obvious thermal response capability and can effectively promote the expression of GFP and CXCL12 under the stimulation of 45 ℃.

The ATCG-1 prepared in example 1 was exposed to a coil of 60mm diameter, subjected to a magnetic heat treatment under an alternating magnetic field of 22.5kA/m at a frequency of 340kHz for 10 minutes, and after the treatment was completed, the ATCG-1 was placed in a 37℃and 5% CO₂ incubator (the mass fraction of CO₂ in the medium was 5%) and cultured for 12 hours, Collecting culture medium for ELISA (enzyme-linked immunosorbent assay) to detect CXCL12 concentration; In addition, the cells in the bracket are collected to extract RNA, qPCR (real-Time fluorescence quantification) is used for detecting the expression level of CXCL12 genes, and a CXCL12 release capacity characterization diagram after ATCG-1 magneto-thermal treatment is obtained, as shown in FIG. 4 (Concentration ofhCXCL-concentration of hCCCL 12, CXCL12 Relative Expression-CXCL 12 expression level, time); in FIG. 4, a is a CXCL12 protein secretion ELISA data characterization diagram of ATCG-1 after magnetocaloric stimulation, b is a CXCL12 gene expression qPCR data characterization diagram of ATCG-1 after magnetocaloric stimulation, c is a CXCL12 protein secretion kinetics schematic diagram of ATCG-1 after magnetocaloric stimulation, and d is a CXCL12 gene expression kinetics schematic diagram of ATCG-1 after magnetocaloric stimulation. In a, 1,2, 3 and 5 in the abscissa are control groups, and 4 is an experimental group; specifically, 1 is to add 10⁵ GES-1 onto the surface of IMS with the volume of 100mm³, incubate for 10min at 37 ℃ to obtain IMS containing GES-1, and not perform magneto-thermal treatment; 2, adding 10⁵ GES-1 onto the surface of IMS with the volume of 100mm³, incubating for 10min at 37 ℃ to obtain IMS containing the GES-1, and performing magnetocaloric treatment on the IMS containing the GES-1 by adopting the same method; 3 is ATCG-1 prepared in example 1, without magneto-caloric treatment; 4 is the experimental group described above, namely, the ATCG-1 prepared in example 1 is subjected to the magneto-caloric treatment; 5 is adding hCCCL 12 protein inhibitor on the basis of carrying out magnetic heat treatment on the ATCG-1 prepared in the example 1; the abscissa in b has the same meaning as a; in c, ATCG-1+AMF is that ATCG-1 prepared in example 1 is subjected to magnetic heat treatment, and ATCG-1 is that ATCG-1 prepared in example 1 is not subjected to magnetic heat treatment; d is the same as c. As can be seen from FIG. 4, the protein secretion concentration of CXCL12 can reach 1.73+/-0.11 ng/mL 12h after thermal stimulation, which is 34.6 times that of the control group (1), the CXCL12 gene expression level is 601.09 times that of the control group (1), and furthermore, the gene expression and protein secretion of CXCL12 of the ATCG-1 are not substantially affected after inhibitor treatment; The protein concentration of ATCG-1 reaches a peak value 24h after the magnetocaloric stimulation, the gene expression reaches a peak value 8h after the thermal stimulation, and the protein secretion capacity and the gene expression capacity are reversibly restored to the pre-stimulation level after 48 h. The data show that ATCG-1 has obvious thermal response capability, and can effectively promote CXCL12 protein secretion and gene expression under magnetocaloric stimulation, and the protein can be reversibly restored to the pre-stimulation level after 48 hours.

Incubating ATCG-1 prepared in example 1 with human gastric cancer cells MGC-803 in vitro in a Transwell for 10min under an alternating magnetic field of 22.5kA/m, standing for 3 days after the end of the thermal stimulation, taking out the Transwell, washing 3 times with DPBS, staining for 5min with an aqueous solution of crystal violet with a mass fraction of 0.1%, gently wiping the upper layer cells of the Transwell with a cotton swab, observing the cells with an optical microscope, and counting the number of cells with ImageJ; the conditions are controlled to be unchanged, the ATCG-1 prepared in the example 1 is replaced by the ATCG-2 prepared in the example 2, meanwhile, the human gastric cancer cell MGC-803 is replaced by the murine gastric cancer cell MFC^mCxcr4, and finally the number of cells is counted by using imageJ; On the other hand, an experimental peritoneal transfer model is constructed in 8-week-old female nude mice, 10⁵ human-derived gastric cancer cells MGC-803 are transplanted into the abdominal cavity of the nude mice, after 3 days, ATCG-1 is transplanted into the abdominal cavity of the nude mice, the nude mice are exposed to a coil with the diameter of 60mm, the frequency of 340kHz and the alternating magnetic field of 22.5kA/m, the magnetic heat treatment is carried out for 10 minutes at intervals of one day, the magnetic heat treatment is carried out for 10 minutes under the same conditions, and after the ATCG-1 is taken out from the abdominal cavity on the 7 th day, Performing imaging characterization (7 parallel experiments) on the ATCG-1 by using a fluorescence imaging system, and then quantifying (averaging) the fluorescence intensity by using software to obtain a DTC chemotactic capacity assessment graph of the ATCG-1, as shown in FIG. 5 (TRANSMIGRETED CELL-chemotactic cells, radiance-fluorescence intensity); In FIG. 5, a is a Transwell staining pattern of MGC-803 and MFC^mCxcr4 cells by ATCG-1 after magnetocaloric stimulation, b is a chemotactic pattern of MGC-803 cells by ATCG-1 after magnetocaloric stimulation, c is a chemotactic pattern of MFC^mCxcr4 cells by ATCG-1 after magnetocaloric stimulation, d is a luciferase imaging pattern of ATCG-1 after recruitment of DTC in the abdominal cavity of a mouse after magnetocaloric stimulation, e is a fluorescent quantitative statistical plot of ATCG-1 recruiting DTCs in the abdominal cavity of mice after magnetocaloric stimulation. In a, 1 is adding 10⁵ GES-1 onto the surface of IMS with volume of 100mm³, incubating for 10min at 37 ℃ to obtain IMS containing GES-1, and not performing thermal stimulation; 2 is to add 10⁵ GES-1 onto the surface of IMS with volume of 100mm³, incubate for 10min at 37deg.C to obtain IMS containing GES-1, and heat-stimulate the IMS containing GES-1 by the same method; 3 is ATCG-1 prepared in example 1, without thermal stimulation; 4 is the thermal stimulation of ATCG-1 prepared in example 1; 5 is to add hCCCL 12 protein inhibitor based on the thermal stimulation of ATCG-1 prepared in example 1; 6 is to add 10⁵ NIH-3T3 to the surface of IMS with volume of 100mm³, incubate for 10min at 37deg.C to obtain IMS containing NIH-3T3 without thermal stimulation; 7, adding 10⁵ NIH-3T3 on the surface of IMS with the volume of 100mm³, incubating for 10min at 37 ℃ to obtain IMS containing NIH-3T3, and performing heat stimulation on the IMS containing NIH-3T3 by adopting the same method; 8 is ATCG-2 prepared in example 2 without thermal stimulation; 9 is the ATCG-2 prepared in example 2 subjected to thermal stimulation; 10 is adding hCCCL 12 protein inhibitor based on thermal stimulation of ATCG-2 prepared in example 2; b. the abscissa in c has the same meaning as the corresponding label in a; in d, adding 10⁵ GES-1 onto the surface of IMS with the volume of 100mm³, and incubating at 37 ℃ for 10min to obtain an IMS group containing the GES-1; GES-1^CXCL12 is the ATCG-1 group prepared in example 1; GES-1^CXCL12 +AMF is a set of magneto-thermal treatments based on ATCG-1 prepared in example 1; e is the same as d, with the ordinate 1 x 10⁰⁹, i.e. 1 x 10⁹,5*10⁰⁸, i.e. 5 x 10⁸. As can be obtained from fig. 5, in the in vitro Transwell co-culture system, ATCG-1 and ATCG-2 have significant chemotaxis effects on human gastric cancer cells MGC-803 and murine gastric cancer cells MFC^mCxcr4 after 3 days of thermal stimulation, and the quantitative display effect on chemotactic cells can reach 5.56-7.53 times that of the control groups (1 and 6), and significantly inhibit DTC chemotaxis level after inhibitor treatment; after in vivo magnetic heat stimulation for 7 days, the ATCG-1 has remarkable recruitment capability to MGC-803 cells and induces DTC in the abdominal cavity to fix on an ATCG-1 bracket, and the display effect after fluorescence intensity quantification can reach 14.05 times of that of a control group (GES-1). The data show that ATCG has obvious chemotactic and recruiting ability to gastric cancer cells in vitro and in vivo and induces tumor cells to colonize on the scaffold.

10⁵ Murine gastric cancer cells MFC^mCxcr4 are injected into the abdominal cavity of a mouse 3 days in advance to construct an immune-sound experimental peritoneal transfer model, then after ATCG-2 is transplanted into the abdominal cavity of the mouse, the mouse is exposed to a coil with the diameter of 60mm, the frequency of 340kHz and an alternating magnetic field of 22.5kA/m (the temperature can be raised to about 45 ℃ under the alternating magnetic field of 22.5 kA/m) on the 1 st day and the 3 rd day, The method comprises the steps of carrying out magneto-thermal treatment for 10min, inducing ATCG-2 to secrete chemokine CXCL12 to recruit DTC in abdominal cavity, exposing mice to a coil with the diameter of 60mm, the frequency of 340kHz and an alternating magnetic field of 27kA/m (the alternating magnetic field of 27kA/m can be heated to about 55 ℃) on the 5 th day and the 7 th day, carrying out magneto-thermal treatment for 10min, inducing ATCG-2 to generate high-temperature environmental damage DTC and releasing TAA. After day 14, imaging the mice by using an IVIS-CT system, collecting the peritoneal fluid of the mice, and analyzing by using the CD4 positive and CD8 positive T cells of the flow cytometry to obtain an evaluation chart of the therapeutic effect of ATCG-2 in a peritoneal transfer mouse model, as shown in FIG. 6 (Number ofmetastatic-transfer number, precent-percent); in FIG. 6, a is a CT three-dimensional imaging diagram of ATCG-2 after the treatment of the peritoneal transfer mouse model, b is a statistical diagram of the number of peritoneal transfer foci of ATCG-2 after the treatment of the peritoneal transfer mouse model, and c is a statistical diagram of T cells of peritoneal cavities CD4 and CD8 of ATCG-2 after the treatment of the peritoneal transfer mouse model. In a, control is that only 10⁵ murine gastric cancer cells MFC^mCxcr4 were injected into the abdominal cavity of mice without any treatment group; 3T3 is prepared by adding 10⁵ NIH-3T3 onto the surface of IMS with volume of 100mm³, incubating at 37deg.C for 10min to obtain IMS containing NIH-3T3, injecting MFC^mCxcr4 into abdominal cavity of mouse, transplanting an IMS containing NIH-3T3 into the mice peritoneal group; 3T3¹² is the implantation of ATCG-2 into the group of mouse abdominal cavities after injecting MFC^mCxcr4 into the mouse abdominal cavities; 3T3¹² AMF is prepared by injecting MFC^mCxcr4 into the abdominal cavity of a mouse, transplanting ATCG-2 into the abdominal cavity of the mouse, exposing the mouse to a coil with 60mm diameter, a frequency of 340kHz and an alternating magnetic field of 22.5kA/m on days 1 and3, performing magnetocaloric treatment for 10min, and inducing ATCG-2 to secrete chemokine CXCL12 to recruit intraperitoneal DTC; 3T3¹² AMF is prepared by injecting MFC^mCxcr4 into the abdominal cavity of a mouse, transplanting ATCG-2 into the abdominal cavity of the mouse, exposing the mouse to 60mm diameter coil, 340kHz frequency, 27kA/m alternating magnetic field on day 5 and day 7, magnetically heating for 10min, inducing ATCG-2 to generate high temperature environmental injury DTC, And releasing the TAA group; 3T3¹² AMF55AMF is the experimental group initially described above; b. and c is as defined in a. As can be seen from fig. 6, after ATCG-2 treatment, the IVIS-CT imaging result shows that ATCG-2 can effectively reduce the number of peritoneal metastases, and the quantification result shows that the number of peritoneal metastases can be reduced by 6.09 times; the number of CD4 positive cells in the abdominal cavity changed less, while CD8 positive T cells were significantly elevated 1.8-fold in ATCG-2 treated group. the above data show that ATCG-2 is effective in attenuating peritoneal metastasis and in activating the peritoneal immune levels, resulting in intraperitoneal immunotherapy against DTCs.

From the above examples, the invention provides an alginate-based thermogenetic CXCL12 releaser (ATCG) based on an optimized Alternating Magnetic Field (AMF) prepared by using transgenic and freeze-drying technologies, the preparation is convenient and simple, the large-scale production can be realized, the ATCG consists of a porous magnetic scaffold (IMS) loaded with engineering cells, the engineering cells are stably transformed by slow viruses into pHSP70-CXCL12, the porous magnetic scaffold consists of a main framework structure made of a biological polymer material coated with magnetic particles, the porous magnetic scaffold has an isotropic porous structure, and the ATCG prepared by the invention can controllably release CXCL12 chemotactic factors for DTC recruitment and activate the abdominal immunity against DTC after magnetocaloric ablation, thereby inhibiting DTC abdominal cavity transfer.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

Translated fromChinese

1.一种藻酸盐基热遗传学CXCL12释放器的制备方法，其特征在于，包含下列步骤：1. A method for preparing an alginate-based thermogenetic CXCL12 releaser, comprising the following steps:(1)将第一生物高分子材料溶液、第一磁性颗粒溶液和交联剂混合进行交联，得到多孔磁性支架；(1) mixing a first biopolymer material solution, a first magnetic particle solution and a cross-linking agent to perform cross-linking to obtain a porous magnetic scaffold;或者将第二生物高分子材料溶液和第二磁性颗粒溶液混合进行加热，加热结束后顺次进行自组装和冷冻干燥，得到多孔磁性支架；Alternatively, the second biopolymer material solution and the second magnetic particle solution are mixed and heated, and after the heating is completed, self-assembly and freeze-drying are performed in sequence to obtain a porous magnetic scaffold;(2)将热遗传学CXCL12基因工程细胞加入到多孔磁性支架的表面上，进行共孵育，得到所述的藻酸盐基热遗传学CXCL12释放器。(2) Adding thermogenetic CXCL12 genetically engineered cells to the surface of the porous magnetic support for co-incubation to obtain the alginate-based thermogenetic CXCL12 releaser.2.如权利要求1所述的藻酸盐基热遗传学CXCL12释放器的制备方法，其特征在于，步骤(1)所述第一生物高分子材料溶液中第一生物高分子包括海藻酸钠、氧化透明质酸、壳聚糖或丝素蛋白；2. The method for preparing an alginate-based thermogenetic CXCL12 releaser according to claim 1, characterized in that the first biopolymer in the first biopolymer material solution in step (1) comprises sodium alginate, oxidized hyaluronic acid, chitosan or silk fibroin;所述第一磁性颗粒溶液中第一磁性颗粒包含铁钴石墨纳米囊、铁氧体颗粒、钕铁硼颗粒或含铁、钴、镍的合金颗粒；所述第一磁性颗粒溶液的质量浓度为8～12mg/mL。The first magnetic particles in the first magnetic particle solution include iron-cobalt graphite nanocapsules, ferrite particles, neodymium-iron-boron particles or alloy particles containing iron, cobalt and nickel; the mass concentration of the first magnetic particle solution is 8-12 mg/mL.3.如权利要求2所述的藻酸盐基热遗传学CXCL12释放器的制备方法，其特征在于，当第一生物高分子材料为海藻酸钠时，制备多孔磁性支架的方法包含下列步骤：3. The method for preparing the alginate-based thermogenetic CXCL12 releaser according to claim 2, characterized in that when the first biopolymer material is sodium alginate, the method for preparing the porous magnetic scaffold comprises the following steps:将海藻酸钠溶液、第一磁性颗粒溶液和第一交联剂混合进行第一交联，第一交联结束后进行冷冻干燥，得到中间物质；将中间物质和第二交联剂混合进行第二交联，得到多孔磁性支架；The sodium alginate solution, the first magnetic particle solution and the first cross-linking agent are mixed to perform a first cross-linking, and after the first cross-linking is completed, freeze-dried to obtain an intermediate substance; the intermediate substance is mixed with a second cross-linking agent to perform a second cross-linking to obtain a porous magnetic scaffold;所述海藻酸钠溶液的质量分数为1.5～2.5％；第一交联剂为葡萄糖酸钙溶液，葡萄糖酸钙溶液的质量分数为0.5～1.0％；第二交联剂为氯化钙溶液，氯化钙溶液的浓度为0.5～1.5mol/L；海藻酸钠溶液、第一磁性颗粒溶液和葡萄糖酸钙溶液的体积比为1～3：0.5～1.5：0.5～1.5；The mass fraction of the sodium alginate solution is 1.5-2.5%; the first cross-linking agent is a calcium gluconate solution, the mass fraction of the calcium gluconate solution is 0.5-1.0%; the second cross-linking agent is a calcium chloride solution, the concentration of the calcium chloride solution is 0.5-1.5 mol/L; the volume ratio of the sodium alginate solution, the first magnetic particle solution and the calcium gluconate solution is 1-3:0.5-1.5:0.5-1.5;第一交联的温度为20～30℃，第一交联的时间为10～15min；冷冻干燥的温度为-15～-25℃，冷冻干燥的时间为22～26h；第二交联的温度为20～30℃，第二交联的时间为1.5～2.5h。The temperature of the first cross-linking is 20-30°C, and the time of the first cross-linking is 10-15 minutes; the temperature of freeze-drying is -15--25°C, and the time of freeze-drying is 22-26 hours; the temperature of the second cross-linking is 20-30°C, and the time of the second cross-linking is 1.5-2.5 hours.4.如权利要求2所述的藻酸盐基热遗传学CXCL12释放器的制备方法，其特征在于，当第一生物高分子材料为氧化透明质酸时，制备多孔磁性支架的方法包含下列步骤：4. The method for preparing the alginate-based thermogenetic CXCL12 releaser according to claim 2, characterized in that when the first biopolymer material is oxidized hyaluronic acid, the method for preparing the porous magnetic scaffold comprises the following steps:将氧化透明质酸溶液、第一磁性颗粒溶液和交联剂混合进行交联，交联结束后进行冷冻干燥，得到多孔磁性支架；The oxidized hyaluronic acid solution, the first magnetic particle solution and the cross-linking agent are mixed for cross-linking, and freeze-dried after the cross-linking is completed to obtain a porous magnetic scaffold;所述氧化透明质酸溶液的质量分数为3～7％；交联剂为支化聚乙烯亚胺；氧化透明质酸溶液和第一磁性颗粒溶液的体积比为0.5～1.5：0.5～1.5；氧化透明质酸溶液和支化聚乙烯亚胺的体积比为0.5～1.5：0.5～1.5；The mass fraction of the oxidized hyaluronic acid solution is 3-7%; the cross-linking agent is branched polyethyleneimine; the volume ratio of the oxidized hyaluronic acid solution to the first magnetic particle solution is 0.5-1.5:0.5-1.5; the volume ratio of the oxidized hyaluronic acid solution to the branched polyethyleneimine is 0.5-1.5:0.5-1.5;交联的温度为20～30℃，交联的时间为3～7min；冷冻干燥的温度为-15～-25℃，冷冻干燥的时间为22～26h。The cross-linking temperature is 20 to 30° C., and the cross-linking time is 3 to 7 minutes; the freeze-drying temperature is -15 to -25° C., and the freeze-drying time is 22 to 26 hours.5.如权利要求2所述的藻酸盐基热遗传学CXCL12释放器的制备方法，其特征在于，当第一生物高分子材料为壳聚糖时，制备多孔磁性支架的方法包含下列步骤：5. The method for preparing the alginate-based thermogenetic CXCL12 releaser according to claim 2, characterized in that when the first biopolymer material is chitosan, the method for preparing the porous magnetic scaffold comprises the following steps:将壳聚糖的醋酸溶液、第一磁性颗粒溶液和交联剂混合，进行交联，交联结束后顺次进行自组装和冷冻干燥，得到多孔磁性支架；The acetic acid solution of chitosan, the first magnetic particle solution and the cross-linking agent are mixed to perform cross-linking, and after the cross-linking is completed, self-assembly and freeze-drying are performed in sequence to obtain a porous magnetic scaffold;所述壳聚糖的醋酸溶液中壳聚糖的质量分数为5～7％；交联剂为戊二醛溶液，戊二醛溶液的质量分数为2～4％；壳聚糖的醋酸溶液、第一磁性颗粒溶液和戊二醛溶液的体积比为0.5～1.5：0.5～1.5：0.5～1.5；The mass fraction of chitosan in the chitosan acetic acid solution is 5-7%; the cross-linking agent is glutaraldehyde solution, and the mass fraction of the glutaraldehyde solution is 2-4%; the volume ratio of the chitosan acetic acid solution, the first magnetic particle solution and the glutaraldehyde solution is 0.5-1.5:0.5-1.5:0.5-1.5;交联的温度为50～60℃，交联的时间为25～35min；自组装的温度为20～30℃，自组装的时间为46～50h；冷冻干燥的温度为-15～-25℃，冷冻干燥的时间为22～26h。The cross-linking temperature is 50-60°C, and the cross-linking time is 25-35 minutes; the self-assembly temperature is 20-30°C, and the self-assembly time is 46-50 hours; the freeze-drying temperature is -15--25°C, and the freeze-drying time is 22-26 hours.6.如权利要求2所述的藻酸盐基热遗传学CXCL12释放器的制备方法，其特征在于，当第一生物高分子材料为丝素蛋白时，制备多孔磁性支架的方法包含下列步骤：6. The method for preparing the alginate-based thermogenetic CXCL12 releaser according to claim 2, characterized in that when the first biopolymer material is silk fibroin, the method for preparing the porous magnetic scaffold comprises the following steps:将丝素蛋白溶液和第一磁性颗粒溶液混合进行冷冻干燥，冷冻干燥结束后得到中间物质；将中间物质和交联剂混合进行交联，得到多孔磁性支架；The silk fibroin solution and the first magnetic particle solution are mixed and freeze-dried to obtain an intermediate substance after the freeze-drying is completed; the intermediate substance and a cross-linking agent are mixed and cross-linked to obtain a porous magnetic scaffold;所述丝素蛋白溶液的质量分数为8～12％；丝素蛋白溶液和第一磁性颗粒溶液的体积比为0.5～1.5：0.5～1.5；交联剂为甲醇；丝素蛋白溶液与甲醇的体积比为0.5～1.5：2.5～3.5；The mass fraction of the silk fibroin solution is 8-12%; the volume ratio of the silk fibroin solution to the first magnetic particle solution is 0.5-1.5:0.5-1.5; the crosslinking agent is methanol; the volume ratio of the silk fibroin solution to methanol is 0.5-1.5:2.5-3.5;冷冻干燥的温度为-15～-25℃，冷冻干燥的时间为22～26h；交联的温度为20～30℃，交联的时间为1.5～2.5h。The freeze-drying temperature is -15 to -25°C, and the freeze-drying time is 22 to 26 hours; the cross-linking temperature is 20 to 30°C, and the cross-linking time is 1.5 to 2.5 hours.7.如权利要求1所述的藻酸盐基热遗传学CXCL12释放器的制备方法，其特征在于，步骤(1)所述第二生物高分子材料溶液中第二生物高分子包括琼脂糖或胶原蛋白；当第二生物高分子为琼脂糖时，第二生物高分子溶液的质量分数为1.5～2.5％；当第二生物高分子为胶原蛋白时，第二生物高分子溶液的质量分数为8～12％；7. The method for preparing an alginate-based thermogenetic CXCL12 releaser according to claim 1, characterized in that, in step (1), the second biopolymer in the second biopolymer material solution comprises agarose or collagen; when the second biopolymer is agarose, the mass fraction of the second biopolymer solution is 1.5-2.5%; when the second biopolymer is collagen, the mass fraction of the second biopolymer solution is 8-12%;所述第二磁性颗粒溶液中第二磁性颗粒包含铁钴石墨纳米囊、铁氧体颗粒、钕铁硼颗粒或含铁、钴、镍的合金颗粒；所述第二磁性颗粒溶液的质量浓度为8～12mg/mL；The second magnetic particles in the second magnetic particle solution include iron-cobalt graphite nanocapsules, ferrite particles, neodymium-iron-boron particles, or alloy particles containing iron, cobalt, and nickel; the mass concentration of the second magnetic particle solution is 8 to 12 mg/mL;第二生物高分子溶液和第二磁性颗粒溶液的体积比为0.5～1.5：0.5～1.5；The volume ratio of the second biopolymer solution to the second magnetic particle solution is 0.5-1.5:0.5-1.5;步骤(1)所述加热的温度为70～100℃，时间为0.5～1.5min，自组装的温度为20～30℃，时间为25～35min；冷冻干燥的温度为-15～-25℃，时间为22～26h。In step (1), the heating temperature is 70-100° C. for 0.5-1.5 min, the self-assembly temperature is 20-30° C. for 25-35 min, and the freeze-drying temperature is -15--25° C. for 22-26 h.8.如权利要求7所述的藻酸盐基热遗传学CXCL12释放器的制备方法，其特征在于，步骤(2)所述热遗传学CXCL12基因工程细胞的数量与多孔磁性支架的体积比为0.8×10⁵～1.2×10⁵个：80～120mm³；8. The method for preparing the alginate-based thermogenetic CXCL12 releaser according to claim 7, characterized in that the volume ratio of the thermogenetic CXCL12 genetically engineered cells in step (2) to the porous magnetic scaffold is 0.8×10⁵ to 1.2×10⁵ cells: 80 to 120 mm³ ;所述共孵育的温度为35～40℃，共孵育的时间为5～15min。The co-incubation temperature is 35-40° C., and the co-incubation time is 5-15 min.9.权利要求1～8任意一项所述制备方法制备得到的藻酸盐基热遗传学CXCL12释放器。9. The alginate-based thermogenetic CXCL12 releaser prepared by the preparation method according to any one of claims 1 to 8.10.权利要求9所述的藻酸盐基热遗传学CXCL12释放器在制备抗肿瘤免疫的药物或制备抑制腹腔转移的药物中的应用。10. Use of the alginate-based thermogenetic CXCL12 releaser according to claim 9 in the preparation of anti-tumor immune drugs or drugs for inhibiting peritoneal metastasis.