A tumor microenvironment regulation composite gene vector and its preparation method and application

Abstract

A tumor microenvironment regulation composite gene vector and its preparation method and application belong to the technical field of biomedical organic polymer materials. In order to solve the technical problems of low transfection efficiency and target cell selectivity of non-viral gene vectors in the prior art, the present invention provides a tumor microenvironment regulation composite gene vector, the composite gene vector includes the following components: macrophage polarizer BLZ945, silk fibroin, polyethyleneimine and pDNA expressing tumor suppressor gene; the packaging form of the composite gene vector is mixed packaging or layer packaging. The composite gene vector provided by the present invention has the function of converting the tumor-promoting phenotype of tumor-associated macrophages into an anti-tumor phenotype, reshaping the immunosuppressive tumor microenvironment, regulating the tumor microenvironment, and improving the immune anti-tumor effect; the composite gene vector can transfer anti-tumor genes into tumor cells, inhibit the proliferation and apoptosis of tumor cells, and can be used to prepare anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical organic polymer materials, and in particular relates to a tumor microenvironment regulation composite gene carrier and a preparation method and application thereof. Background Art

[0002] Gene therapy has become an important direction for exploring new and efficient treatment strategies with its precision and personalized characteristics for the mechanism of tumorigenesis. However, gene therapy for clinical solid tumors in existing technologies has not achieved the expected ideal effect. The main reason is that the tumor microenvironment is dysfunctional during the treatment process, and is even domesticated into a tumor-promoting accomplice, which greatly weakens the anti-solid tumor efficacy. Specifically, macrophages in the tumor microenvironment recruit peripheral blood monocytes to solid tumors and differentiate into tumor-associated macrophages (TAMs). TAMs are highly plastic and will switch from one phenotype to another (M1↔M2) according to changes in microenvironment signals. Among them, M1-like phenotype macrophages have anti-tumor effects, but they will be "educated" by Th2 cells in the tumor microenvironment into a tumor-promoting M2-like phenotype; M2 not only has the function of promoting tumor angiogenesis and maintaining tumor stemness, but is also the main accomplice in the formation of an immunosuppressive microenvironment, promoting tumor invasion and metastasis.

[0003] Gene therapy relies on gene vectors, and compared with viral gene vectors, non-viral gene vectors have the characteristics of lower immunogenicity, higher gene loading capacity, higher safety, and simpler preparation methods. However, the naked plasmid (pDNA) in non-viral gene vectors is easily degraded by nucleases in the extracellular matrix, has poor pharmacokinetic properties, and is difficult to pass through the cell membrane. Therefore, a vector system is needed to deliver pDNA to target cells. At present, the non-viral vectors that have been reported (including: liposomes, polyethyleneimine, zein, chitosan, and silicates, etc.) have problems with low transfection efficiency and target cell selectivity, resulting in unsatisfactory gene therapy effects for clinical solid tumors. Summary of the invention

[0004] The present invention aims to solve the technical problems of low transfection efficiency and target cell selectivity of non-viral gene vectors in the prior art, and provides a tumor microenvironment regulation composite gene vector and a preparation method and application thereof.

[0005] One of the purposes of the present invention is to provide a tumor microenvironment regulation composite gene vector, which comprises the following components: macrophage polarizer BLZ945, silk fibroin, polyethyleneimine and pDNA expressing tumor suppressor gene.

[0006] In a preferred embodiment of the present invention, the tumor suppressor gene expressed by the pDNA is one or two of the p53 gene, growth inhibitory factor 4, interleukin family gene, tumor necrosis factor-α, multiple tumor suppressor gene p16, adenomatous polyposis gene or retinoblastoma gene RB.

[0007] In a preferred embodiment of the present invention, the silk fibroin includes Bombyx mori silk fibroin or Tussah silk fibroin, and the molecular weight of the polyethyleneimine is less than 2500 Da.

[0008] In a preferred embodiment of the present invention, the composite gene carrier is packaged in the form of mixed packaging or layer-by-layer packaging.

[0009] In a preferred embodiment of the present invention, the method for preparing the composite gene carrier in the mixed packaging form comprises the following steps:

[0010] S1: Dissolve the macrophage polarizing agent BLZ945 in anhydrous dimethylformamide, add the silk fibroin solution, place in an ice bath and stir for 12-20 h, centrifuge and ultrafilter at 0-4°C and 8000-10000 rpm for 30-45 min to obtain the silk fibroin solution modified with the polarizing agent;

[0011] S2: The polarizer-modified silk fibroin solution obtained in S1 was mixed with the polyethyleneimine solution, vortexed for 5-10 min, and then filtered through a 0.22 μm filter to obtain a mixture of silk fibroin and polyethyleneimine; pDNA solution was added to the above mixture, vortexed for 60 s, and allowed to stand at 0-4°C for 30-45 min to obtain a pDNA composite gene vector in a co-packaged form.

[0012] In a preferred embodiment of the present invention, the method for preparing the composite gene carrier in the form of layer-by-layer packaging comprises the following steps:

[0013] S11: dissolving the macrophage polarizing agent BLZ945 in anhydrous dimethylformamide, adding the silk fibroin solution, placing in an ice bath and stirring for 12-20 h, centrifuging and ultrafiltration at 0-4°C and 8000-10000 rpm for 30-45 min to obtain the silk fibroin solution modified with the polarizing agent;

[0014] S22: Mix the polyethyleneimine solution and the pDNA solution evenly, vortex for 60 seconds, and let stand at 0-4°C for 30-45 minutes to obtain a polyethyleneimine and pDNA complex; add the polarizer-modified silk fibroin solution obtained in S11 to the above-obtained complex, vortex for 5-10 minutes, and let stand at 0-4°C for 30-45 minutes to obtain a pDNA composite gene vector in a layered packaging form.

[0015] In a preferred embodiment of the present invention, the concentration of the macrophage polarizer BLZ945 in S1 and S11 is 0.1-4 mg / mL, the concentration of the silk fibroin solution is 10-40 mg / mL, the mixing mass ratio of the macrophage polarizer BLZ945 to the silk fibroin solution is 1:(10-100), and the molecular weight cutoff of the ultrafiltration tube in the ultrafiltration treatment is 3000 Da.

[0016] In a preferred embodiment of the present invention, the concentration of the polarizer-modified silk fibroin solution in S2 and S22 is 1-10 mg / mL, the concentration of the polyethyleneimine solution is 0.1-4 mg / mL, and the concentration of the pDNA solution is 20-200 μg / mL.

[0017] In a preferred embodiment of the present invention, the mass ratio of the polarizer-modified silk fibroin, polyethyleneimine and pDNA in the composite gene carriers in S2 and S22 is (10-100): (2-20): 1.

[0018] The second purpose of the present invention is to provide the use of the above-mentioned tumor microenvironment regulating composite gene vector in the preparation of anti-tumor drugs.

[0019] The beneficial effects of the present invention are as follows: silk fibroin has good biocompatibility and biodegradability, low immunogenicity, safety and non-toxicity, and the tussah silk fibroin is rich in arginine-glycine-aspartic acid (RGD) tripeptide sequence, which can specifically recognize and bind to αvβ3 and αvβ5 integrins expressed in large quantities during tumor angiogenesis, thereby enhancing the adhesion of gene carriers to tumor cells. The silk fibroin used in the present invention includes silk fibroin from Bombyx mori or tussah silk fibroin, and the carboxyl groups of aspartic acid and glutamic acid contained in the silk fibroin react with the hydroxyl groups in the macrophage polarizer BLZ945 to chemically bind the polarization activator to the side chain of the silk fibroin; at the same time, the polarizer BLZ945 is fully loaded by physical forces such as hydrophilic and hydrophobic effects and hydrogen bonds; the present invention uses the biocompatible and biodegradable silk fibroin as a gene carrier material, which can not only protect the gene from being degraded by nucleases and improve the efficiency of transgenic, but also the carrier can effectively release the gene when entering the tumor cell, and induce tumor cell apoptosis.

[0020] The molecular weight of polyethyleneimine used in the present invention is less than 2500 Da. Low molecular weight polyethyleneimine contains a large number of amino groups and imino groups, resulting in a large positive charge density on its surface, and the cytotoxicity is much lower than that of high molecular weight polyethyleneimine. At the same time, the present invention can effectively compress pDNA when the silk protein reacted with the macrophage polarizer BLZ945 is mixed or layered with low molecular weight polyethyleneimine to express the pDNA of the tumor suppressor gene, forming a nano-carrier with a smaller size, which can effectively stabilize the gene carrier structure and nano-size; and it is easy to combine with the silk protein carrying negative charge on the surface through electrostatic action, protecting pDNA from penetrating the cell membrane and improving the transgenic efficiency.

[0021] In the prior art, although silk fibroin is also used to prepare carriers, it is often prepared in combination with potassium permanganate and polyacrylamine hydrochloride; or the carrier is prepared only by combining sodium heparin, polyethyleneimine, and polyethyleneimine-transmembrane peptide, and the molecular weight of the polyethyleneimine used is relatively high; the silk fibroin used is also not treated with the macrophage polarizing agent BLZ945, and the substances interacting with the silk fibroin are different; in the prior art, the molecular weight of the polyethyleneimine used is relatively high, which is different from the polyethyleneimine standard selected in the present invention, and the packaging form is different from the blended packaging or layered packaging form used in the present invention, and there is a substantial difference.

[0022] The tumor microenvironment regulation composite gene vector provided by the present invention, in an acidic tumor microenvironment, anchors the CSF1R overexpressed in M2 macrophages through physical desorption and chemical bond breaking and sustained release of polarizing agents, polarizes and reprograms tumor-promoting M2-like phenotype macrophages into anti-tumor M1-like phenotype macrophages, transforms them from a tumor-promoting phenotype to an anti-tumor phenotype, and transforms tumor "accomplices in crime" into "attack force"; the tumor microenvironment regulation composite gene vector has the function of reshaping the immunosuppressive tumor microenvironment, regulating the tumor microenvironment, and improving the immune anti-tumor effect. The tumor microenvironment regulation composite gene vector provided by the present invention can transfer anti-tumor genes into tumor cells, inhibit tumor cell proliferation and induce tumor cell apoptosis, realize dual-channel improvement of anti-tumor treatment effects, and can be used to prepare anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the construction of the mixed and packaged tumor microenvironment regulating composite gene vector in Example 1;

[0024] Figure 2 This is a schematic diagram of the construction of the layer-by-layer packaged tumor microenvironment regulation composite gene vector in Example 2;

[0025] Figure 3: are scanning electron micrographs of the composite gene vectors obtained in Example 1 and Example 2; A is a scanning electron micrograph of the composite gene vectors in mixed packaging, and B is a scanning electron micrograph of the composite gene vectors in layer-by-layer packaging;

[0026] Figure 4 The flow cytometry spectra of M1 and M2 cells before and after activation of the composite gene vector in Example 3;

[0027] Figure 5 : are laser confocal images of lung tumor cells H460 expressing green fluorescent protein in Example 4; A is a laser confocal image of lung tumor cells H460 transfected with pure plasmid pDNA, B is a laser confocal image of lung tumor cells H460 transfected with a co-packaged composite gene vector, and C is a laser confocal image of lung tumor cells H460 transfected with a layer-by-layer packaged composite gene vector;

[0028] Figure 6 A is a flow cytometric graph of the positive cell ratio of lung tumor cells H460 expressing green fluorescent protein in Example 4; A is a flow cytometric graph of the positive cell ratio of lung tumor cells H460 after transfection with pure plasmid pDNA, B is a flow cytometric graph of the positive cell ratio of lung tumor cells H460 after transfection with the mixed packaged composite gene vector, and C is a flow cytometric graph of the positive cell ratio of lung tumor cells H460 after transfection with the layer-by-layer packaged composite gene vector;

[0029] Figure 7 The cell survival rate statistics of lung tumor cells H460 transfected with the gene vector in Example 4 at 1, 3, 5, and 7 days of culture. DETAILED DESCRIPTION

[0030] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0031] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments and the accompanying drawings of the specification. The test methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0032] Example 1: Preparation of a co-packaged tumor microenvironment regulating composite gene vector

[0033] S1: Take 100 g of raw silkworm silk and place it in 5 L of NaHCO3 aqueous solution with a mass concentration of 5‰, boil it three times, each boiling time is 30 min, and wash it three times with deionized water; place the washed silkworm silk in a 60℃ oven to dry and loosen it; take 100 mL of 9.3 M lithium bromide solution and heat it to 60℃ for use, take 15 g of the above dried and loosened degummed silkworm silk and add it to the above lithium bromide solution in batches to dissolve until there is no floc, the dissolution time is 2 h; after cooling, transfer it to a dialysis bag (molecular weight cutoff is 10 kDa), dialyze it in deionized water at 4℃ for 4 d, change the deionized water every 2 h, and obtain the purified silk fibroin solution after filtration; the concentration of the above silk fibroin solution is 42 mg / mL measured by weighing method;

[0034] S2: Use triple distilled water to dilute the silk fibroin solution obtained in S1 to a silk fibroin solution with a concentration of 10 mg / mL, dissolve the macrophage polarizer BLZ945 in anhydrous dimethylformamide (DMF) to prepare a macrophage polarizer BLZ945 solution with a concentration of 0.1 mg / mL; then add an equal volume of diluted silk fibroin solution to the diluted macrophage polarizer BLZ945 and mix evenly, place in an ice bath and stir for 12 h, centrifuge and ultrafilter at 4°C and 8000 rpm for 30 min, the molecular weight cutoff of the ultrafiltration tube is 3000 Da, remove unreacted free BLZ945, and obtain a silk fibroin solution modified with a polarizer; the concentration of the silk fibroin solution modified with a polarizer is 7.6 mg / mL measured by weighing method, and then diluted to 2 mg / mL;

[0035] S3: Use deionized water to prepare polyethyleneimine with a molecular weight of 2.0 kDa into a polyethyleneimine aqueous solution with a concentration of 0.2 mg / mL, and evenly blend the polyethyleneimine aqueous solution with an equal volume of the diluted silk fibroin solution modified with the polarizer obtained in S2. The polyethyleneimine aqueous solution needs to be added dropwise to the diluted silk fibroin solution modified with the polarizer in an ice bath, stirred evenly, vortexed for 10 min, and then filtered through a 0.22 μm filter to obtain a blended solution;

[0036] S4: Use sterile triple-distilled water to prepare the human growth factor 4 and interleukin-24 co-expression plasmid into a pDNA solution with a concentration of 20 μg / mL. The mixed solution obtained in S3 is evenly mixed with an equal volume of the above pDNA solution, vortexed for 60 s, and allowed to stand in a 0°C environment for 30 min to obtain a tumor microenvironment regulation composite gene vector co-packaged with silk fibroin and polyethyleneimine. The mass ratio of silk fibroin, polyethyleneimine and pDNA in the co-packaged tumor microenvironment regulation composite gene vector is 50:5:1.

[0037] The schematic diagram of the construction of the co-packaged tumor microenvironment regulation composite gene vector prepared in this example is as follows: Figure 1 As shown; the above obtained mixed packaged tumor microenvironment regulation composite gene vector was subjected to scanning electron microscopy, and the results were as follows Figure 3 As shown in A, it can be seen that the co-packaged tumor microenvironment regulation composite gene carrier is in the form of nanoparticles, evenly dispersed, and has a particle size between 103-168 nm.

[0038] Example 2: Preparation of a layer-by-layer packaged tumor microenvironment regulation composite gene vector

[0039] S1: Take 100 g of raw silkworm silk and place it in 5 L of NaHCO3 aqueous solution with a mass concentration of 5‰, boil it three times, each boiling time is 30 min, and wash it three times with deionized water; place the washed silkworm silk in a 60℃ oven to dry and loosen it; take 100 mL of 9.3 M lithium bromide solution and heat it to 60℃ for use, take 15 g of the above dried and loosened degummed silkworm silk and add it to the lithium bromide solution in batches to dissolve until there is no floc, the dissolution time is 2 h; after cooling, transfer it to a dialysis bag (molecular weight cutoff is 10 kDa), dialyze it in deionized water at 4℃ for 4 d, change the deionized water every 2 h, and obtain the purified silk fibroin solution after filtration; the concentration of the above silk fibroin solution is 42 mg / mL measured by weighing method;

[0040] S2: Use triple distilled water to dilute the silk fibroin solution obtained in S1 to a silk fibroin solution with a concentration of 40 mg / mL, dissolve the macrophage polarizer BLZ945 in anhydrous dimethylformamide (DMF) to prepare a macrophage polarizer BLZ945 solution with a concentration of 0.1 mg / mL; then add an equal volume of the diluted silk fibroin solution to the above macrophage polarizer BLZ945 solution and mix evenly, place in an ice bath and stir for 12 h, centrifuge and ultrafilter at 4°C and 8000 rpm for 30 min, the molecular weight cutoff of the ultrafiltration tube is 3000 Da, remove the unreacted free BLZ945, and obtain the silk fibroin solution modified by the polarizer; the concentration of the silk fibroin solution modified by the polarizer is measured by weighing method to be 28.6 mg / mL, and it is diluted to 10 mg / mL;

[0041] S3: Use deionized water to prepare polyethyleneimine with a molecular weight of 2.0 kDa into a polyethyleneimine aqueous solution with a concentration of 4 mg / mL, use sterile triple-distilled water to prepare a pDNA solution with a concentration of 200 μg / mL for the co-expression plasmid of human growth factor 4 and interleukin-24, mix the polyethyleneimine aqueous solution with an equal volume of pDNA solution, vortex for 60 s, and stand at 0°C for 30 min to obtain a polyethyleneimine and pDNA complex;

[0042] S4: The polarizer-modified silk fibroin solution obtained in S2 was evenly mixed with an equal volume of the polyethyleneimine and pDNA complex obtained in S3, vortexed for 10 s, and allowed to stand in a 0°C environment for 30 min to obtain a tumor microenvironment regulating composite gene vector packaged with silk fibroin and polyethyleneimine. The mass ratio of silk fibroin, polyethyleneimine and pDNA in the layer-by-layer packaged tumor microenvironment regulating composite gene vector was 100:20:1.

[0043] The schematic diagram of the construction of the layer-by-layer packaged tumor microenvironment regulation composite gene vector prepared in this example is as follows: Figure 2 As shown, the above-obtained layer-by-layer packaged tumor microenvironment regulation composite gene vector was subjected to scanning electron microscopy, and the results were as follows Figure 3 As shown in B, it can be seen that the layer-by-layer packaged tumor microenvironment regulation composite gene carrier is in the form of nanoparticles, evenly dispersed, and the particle size is between 132-189 nm.

[0044] Example 3: Application of layer-by-layer packaged tumor microenvironment regulation composite gene vector

[0045] S1: Take 100 g of raw tussah silk and place it in 5 L of NaHCO3 aqueous solution with a mass concentration of 2.5‰, boil it three times, each boiling time is 45 min, and wash it three times with deionized water; place the washed tussah silk in a 60℃ oven to dry and loosen it; take 150 mL of calcium nitrate tetrahydrate, heat and melt it, filter it, and keep the temperature at 105℃, take 15 g of the above-mentioned dried and loosened degummed tussah silk and add it to the above-mentioned calcium nitrate tetrahydrate in batches to dissolve until there is no floc, and the dissolution time is 5 h; after cooling, transfer it to a dialysis bag (molecular weight cutoff is 12 kDa), dialyze it in deionized water at 0℃ for 48 d, change the deionized water every 1 h, and obtain the purified tussah silk fibroin solution after filtration; the concentration of the above-mentioned tussah silk fibroin solution is 45 mg / mL measured by weighing method;

[0046] S2: Use triple distilled water to dilute the tussah silk fibroin solution obtained in S1 to a concentration of 20 mg / mL tussah silk fibroin solution, dissolve the macrophage polarizer BLZ945 in anhydrous dimethylformamide (DMF) to prepare a macrophage polarizer BLZ945 solution with a concentration of 4 mg / mL; then add an equal volume of the diluted tussah silk fibroin solution to the above macrophage polarizer BLZ945 solution and mix evenly, place in an ice bath and stir for 20 h, centrifuge and ultrafilter at 0°C and 10000 rpm for 45 min, the molecular weight cutoff of the ultrafiltration tube is 3000 Da, remove the unreacted free BLZ945, and obtain the polarizer-modified tussah silk fibroin solution; the concentration of the polarizer-modified tussah silk fibroin solution is 17.5 mg / mL by weighing method, and then dilute it to 10 mg / mL;

[0047] S3: Use deionized water to prepare polyethyleneimine with a molecular weight of 2.0 kDa into a polyethyleneimine aqueous solution with a concentration of 2 mg / mL, use sterile triple-distilled water to prepare a pDNA solution with a concentration of 200 μg / mL for the co-expression plasmid of human growth factor 4 and interleukin-24, mix the polyethyleneimine aqueous solution with an equal volume of pDNA solution, vortex for 60 s, and stand at 0°C for 45 min to obtain a polyethyleneimine and pDNA complex;

[0048] S4: The polarizer-modified diluted tussah fibroin solution obtained in S2 was evenly mixed with an equal volume of the polyethyleneimine and pDNA complex obtained in S3, vortexed for 5 min, and allowed to stand in a 0°C environment for 45 min to obtain a tumor microenvironment regulating composite gene vector packaged with tussah fibroin protein and polyethyleneimine. The mass ratio of tussah fibroin protein, polyethyleneimine and pDNA in the layer-by-layer packaged tumor microenvironment regulating composite gene vector was 100:10:1.

[0049] Effect experiment:

[0050] In this example, mouse bone marrow-derived cells were induced to obtain M0 phenotype macrophages, and M2 phenotype macrophages were obtained by incubation for 24 h after IL-4 stimulation. The M2 cells were activated by the layer-by-layer packaged tumor microenvironment regulation composite gene vector obtained in S4 above, and polarized into M1 phenotype macrophages. In this example, the above M1 and M2 cells were detected by flow cytometry. The structure is as follows Figure 4As shown, it can be seen that the macrophage phenotype when activated by the tumor microenvironment regulation composite gene carrier that has not been packaged layer by layer is mainly M2 type, accounting for 62%; the proportion of M2 type cells after activation by the tumor microenvironment regulation composite gene carrier that has been packaged layer by layer is 28%, which is significantly reduced compared to the proportion of M2 cells before activation, while the proportion of M1 type cells has increased significantly from 25% to 57%. This shows that the tumor microenvironment regulation composite gene carrier packaged layer by layer provided in this embodiment has the ability to significantly induce macrophage phenotypic transformation and can provide a favorable immunosuppressive microenvironment for tumor treatment.

[0051] Example 4: Application of composite gene vector for tumor microenvironment regulation

[0052] S1: Take 100 g of raw silk from Bombyx mori and place it in 5 L of NaHCO3 aqueous solution with a mass concentration of 2.5‰, boil it three times, and boil it for 45 min each time, and wash it three times with deionized water; dry the washed tussah silk in a 60℃ oven and loosen it; heat and melt 150 mL of calcium nitrate tetrahydrate, filter it, and keep the temperature at 105℃, take 15 g of the dried and loosened degummed tussah silk and add it to the above calcium nitrate tetrahydrate in batches to dissolve until there is no floc, and the dissolution time is 5 h; after cooling, transfer it to a dialysis bag (molecular weight cutoff is 12 kDa), dialyze it in deionized water at 0℃ for 48 d, change the deionized water every 1 h, and obtain the purified tussah fibroin solution after filtration; the concentration of the above tussah fibroin solution is 45 mg / mL measured by weighing method;

[0053] S2: Use triple distilled water to dilute the tussah fibroin solution obtained in S1 to a concentration of 20 mg / mL tussah fibroin solution, dissolve the macrophage polarizer BLZ945 in anhydrous dimethylformamide (DMF) to prepare a macrophage polarizer BLZ945 solution with a concentration of 4 mg / mL; then add an equal volume of the diluted tussah fibroin solution to the above macrophage polarizer BLZ945 solution and mix evenly, place in an ice bath and stir for 20 h, centrifuge and ultrafilter at 0°C and 10000 rpm for 30 min, the molecular weight cutoff of the ultrafiltration tube is 3000 Da, remove the unreacted free BLZ945, and obtain the polarizer-modified tussah fibroin solution; the concentration of the polarizer-modified tussah fibroin solution is 17.5 mg / mL by weighing method, and then dilute it to 10 mg / mL;

[0054] S3: Use deionized water to prepare polyethyleneimine with a molecular weight of 2.0 kDa into a polyethyleneimine aqueous solution with a concentration of 1 mg / mL, and evenly blend the polyethyleneimine aqueous solution with an equal volume of the diluted tussah fibroin solution modified with the polarizer obtained in S2. The polyethyleneimine aqueous solution needs to be added dropwise to the tussah fibroin solution modified with the polarizer in an ice bath, stirred evenly, vortexed for 5 min, and then filtered through a 0.22 μm filter to obtain a blended solution;

[0055] S4: The human growth factor 4 and interleukin-24 co-expression plasmids were prepared into a pDNA solution with a concentration of 50 μg / mL using sterile triple distilled water, and the mixed solution obtained in S3 was evenly mixed with the above-mentioned equal volume of pDNA solution, vortexed for 60 seconds, and allowed to stand at 0°C for 45 minutes to obtain a tumor microenvironment regulation composite gene vector co-packaged with tussah fibroin protein and polyethyleneimine, wherein the mass ratio of tussah fibroin protein, polyethyleneimine and pDNA in the co-packaged tumor microenvironment regulation composite gene vector was 100:10:1;

[0056] S5: Use deionized water to prepare polyethyleneimine with a molecular weight of 2.0 kDa into a polyethyleneimine aqueous solution with a concentration of 2 mg / mL, use sterile triple-distilled water to prepare a pDNA solution with a concentration of 200 μg / mL for the co-expression plasmid of human growth factor 4 and interleukin-24, mix the polyethyleneimine aqueous solution with an equal volume of pDNA solution, vortex for 60 s, and stand at 0°C for 45 min to obtain a polyethyleneimine and pDNA complex;

[0057] S6: The polarizer-modified diluted tussah fibroin solution obtained in S2 was evenly mixed with an equal volume of the polyethyleneimine and pDNA complex obtained in S5, vortexed for 5 min, and allowed to stand in a 0°C environment for 45 min to obtain a tumor microenvironment regulating composite gene vector packaged with tussah fibroin protein and polyethyleneimine. The mass ratio of tussah fibroin protein, polyethyleneimine and pDNA in the layer-by-layer packaged tumor microenvironment regulating composite gene vector was 100:10:1.

[0058] Effect experiment:

[0059] (1) In this example, the mixed packaged tumor microenvironment regulation composite gene vector obtained in S4 and the layer-by-layer packaged tumor microenvironment regulation composite gene vector obtained in S6 were transfected into lung tumor cells H460, and the expression of green fluorescent protein after pDNA was transferred into cells was observed using a laser confocal microscope. The results are as follows: Figure 5As shown. It can be seen that only a few lung tumor cells H460 in the lung tumor cells transfected with pure plasmid pDNA expressed green fluorescence, indicating that pure plasmid pDNA is difficult to be transferred into lung tumor cells H460; while in the lung tumor cells H460 transfected with the mixed packaging or layer-by-layer packaging tumor microenvironment regulation composite gene vector provided by the present invention, the number of lung tumor cells H460 expressing green fluorescence was significantly higher than that of lung tumor cells H460 transfected with pure plasmid pDNA; and there was no significant difference in the number of cells expressing green fluorescence and the fluorescence intensity after the above-mentioned mixed packaging and layer-by-layer packaging tumor microenvironment regulation composite gene vectors were transfected into lung tumor cells H460, indicating that the mixed packaging or layer-by-layer packaging tumor microenvironment regulation composite gene vectors have the ability to efficiently transfect H460 cells.

[0060] (2) In this example, flow cytometry was used to detect lung tumor cells H460 transfected with the above-mentioned pure plasmid pDNA, co-packaged and layer-packaged tumor microenvironment regulation composite gene vectors. The proportion of positive cells expressing green fluorescence was the transfection efficiency. The results are as follows: Figure 6 The transfection efficiency of pure plasmid pDNA was 6.75%, the transfection efficiency of co-packaged composite gene vector was 28.49%, and the transfection efficiency of layer-by-layer packaged composite gene vector was 30.26%. It can be seen that the transfection efficiency of co-packaged and layer-by-layer packaged composite gene vectors for tumor microenvironment regulation was significantly higher than that of pure plasmid pDNA, indicating that co-packaged and layer-by-layer packaged composite gene vectors for tumor microenvironment regulation have the ability to efficiently transduce target genes into tumor cells and express them.

[0061] (3) Lung tumor cells H460 were expressed at 4×10 3 The cells / well were inoculated at a density of 100 μL / well in a 96-well plate, with 5 replicate wells. They were cultured in a DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2 for 24 h. After the medium was removed, 100 μL of pure plasmid pDNA, the mixed-packaged tumor microenvironment regulation composite gene vector obtained in S4, and the layer-packaged tumor microenvironment regulation composite gene vector obtained in S6 were added to the wells, and the above lung tumor cells H460 were transfected 24 h later. The pure plasmid and composite vector solution were removed, and 200 μL of serum-containing medium was added to each well. The culture was continued for 1, 3, 5 and 7 days, and the serum-containing medium was used as the blank control group. The absorbance (OD) value of each well at 450 nm was determined by CCK-8 method at 4 time points, and the cell survival rate was:

[0062] Cell survival rate (%) = (OD value of experimental group / OD value of blank group) × 100%

[0063] The results are as follows Figure 7As shown, after pure plasmid pDNA was transfected into lung tumor cells H460, due to the very low transfection efficiency, it had no significant effect on the proliferation of lung tumor cells H460, and the cell survival rate was close to 100%; when the lung tumor cells H460 were transfected with the tumor microenvironment regulation composite gene vectors packaged in a mixed manner and packaged in layers, the survival rate of the lung tumor cells H460 was significantly reduced when they were cultured to the 5th and 7th days, and the cell survival rate was less than 80%, indicating that the proliferation of lung tumor cells H460 was significantly inhibited, and the tumor microenvironment regulation composite gene vectors packaged in a mixed manner and packaged in layers provided by the present invention had obvious anti-tumor activity.

[0064] The contents not described in detail in the specification of the present invention are well-known technologies for those skilled in the art. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.

Claims

1. A tumor microenvironment regulation composite gene vector, characterized in that: The tumor microenvironment regulation composite gene vector comprises the following components: macrophage polarizing agent BLZ945, silk protein, polyethyleneimine and pDNA expressing tumor suppressor gene; The tumor suppressor gene expressed by the pDNA is one or two of the p53 gene, growth inhibitory factor 4, interleukin family gene, tumor necrosis factor-α, multiple tumor suppressor gene p16, adenomatous polyposis gene or retinoblastoma gene RB; The silk fibroin includes silk fibroin from Bombyx mori or Tussah silk fibroin; The molecular weight of the polyethyleneimine is less than 2500Da; The packaging form of the composite gene carrier is mixed packaging; The preparation method of the composite gene carrier in the mixed packaging form comprises the following steps: S1: Dissolve the macrophage polarizing agent BLZ945 in anhydrous dimethylformamide, add the silk fibroin solution, place in an ice bath and stir for 12-20 hours, centrifuge and ultrafilter at 0-4°C and 8000-10000 rpm for 30-45 minutes to obtain the silk fibroin solution modified with the polarizing agent; S2: The polarizer-modified silk fibroin solution obtained in S1 was mixed with the polyethyleneimine solution, vortexed for 5-10 minutes, and then filtered through a 0.22 μm filter to obtain a mixture of silk fibroin and polyethyleneimine; the pDNA solution was added to the above mixture, vortexed for 60 seconds, and allowed to stand at 0-4°C for 30-45 minutes to obtain a pDNA composite gene vector in a mixed packaging form.

2. A tumor microenvironment regulation composite gene vector, characterized in that: The tumor microenvironment regulation composite gene vector comprises the following components: macrophage polarizing agent BLZ945, silk protein, polyethyleneimine and pDNA expressing tumor suppressor gene; The tumor suppressor gene expressed by the pDNA is one or two of the p53 gene, growth inhibitory factor 4, interleukin family gene, tumor necrosis factor-α, multiple tumor suppressor gene p16, adenomatous polyposis gene or retinoblastoma gene RB; The silk fibroin includes silk fibroin from Bombyx mori or Tussah silk fibroin; The molecular weight of the polyethyleneimine is less than 2500Da; The packaging form of the composite gene carrier is layer-by-layer packaging; The method for preparing the composite gene carrier in the form of layer-by-layer packaging comprises the following steps: S11: dissolving giant cell polarizing agent BLZ945 in anhydrous dimethylformamide, adding silk fibroin solution, placing in an ice bath and stirring for 12-20 hours, centrifuging and ultrafiltration at 0-4°C and 8000-10000 rpm for 30-45 minutes to obtain a silk fibroin solution modified with polarizing agent; S22: Mix the polyethyleneimine solution and the pDNA solution evenly, vortex for 60 seconds, and let stand at 0-4°C for 30-45 minutes to obtain a polyethyleneimine and pDNA complex; add the polarizer-modified silk fibroin solution obtained in S11 to the above-obtained complex, vortex for 5-10 minutes, and let stand at 0-4°C for 30-45 minutes to obtain a pDNA composite gene vector in a layered packaging form.

3. The composite gene carrier according to any one of claim 1 or claim 2, characterized in that: The concentration of the macrophage polarizer BLZ945 is 0.1-4 mg / mL, the concentration of the silk fibroin solution is 10-40 mg / mL, the mixing mass ratio of the macrophage polarizer BLZ945 to the silk fibroin solution is 1:(10-100), and the molecular weight cutoff of the ultrafiltration tube in the ultrafiltration treatment is 3000 Da.

4. The composite gene carrier according to any one of claim 1 or claim 2, characterized in that: The concentration of the polarizer-modified silk fibroin solution is 1-10 mg / mL, the concentration of the polyethyleneimine solution is 0.1-4 mg / mL, and the concentration of the pDNA solution is 20-200 g / ml.

5. The composite gene carrier according to any one of claim 1 or claim 2, characterized in that: The mass ratio of the polarizer-modified silk fibroin, polyethyleneimine and pDNA in the composite gene carrier is (10-100): (2-20):

1.

6. Use of the tumor microenvironment regulating composite gene vector according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.

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