Preparation method and application of temperature-controlled gene expression nanomaterial

Abstract

The invention relates to a preparation method and application of a temperature-controlled gene expression nanomaterial, which can effectively solve the problem of avoiding damage to transfected cells and surrounding tissues caused by excessive heat through precise temperature control and realizing non-destructive photothermal activation of genetic engineering. The preparation method is as follows: chitosan is dissolved in an acetic acid deionized water solution to prepare a solution A; EDC and p-anisic acid are added to DCM, NHS is added, stirred under argon at room temperature, dried, and then thiol-polyethylene glycol-carboxyl is added to react under N2 protection to synthesize AEAA-PEG-COOH; NHS and EDC are added to the solution A, stirred for 1 hour, and then AEAA-PEG-COOH is added, stirred overnight, dialyzed, and freeze-dried to obtain a powder B; a mixture of thermosensitive green, beta-naphthol and polyethylene glycol is infrared-heated and dissolved in acetone to obtain a solution C; the solution C is dripped into the powder B, stirred evenly, centrifuged, and impurities are removed to obtain a nanomaterial D; the thermosensitive green in the nanomaterial D is reacted with a plasmid, centrifuged, and the unadsorbed plasmid is removed to obtain the temperature-controlled gene expression nanomaterial.

Description

Technical Field

[0001] The present invention relates to the medical field, and in particular to a preparation method and application of a temperature-controlled gene expression nano material. Background Art

[0002] Transgenic systems are nucleic acids that carry genetic material or gene editing devices into cells to continuously produce therapeutic proteins or correct cellular gene errors, providing useful tools for regulating cell behavior and potentially leading to innovative therapies. In recent years, gene therapy, which delivers exogenous nucleic acids into specific cells via viral or non-viral vectors, has become a promising treatment for genetic diseases and cancer. However, expression in non-specific sites not only reduces the efficiency of treatment but may also cause irreversible side effects, thus limiting their clinical application.

[0003] In gene therapy, encoding specific promoters is a feasible solution, in which light is an ideal inducer of gene expression due to its low toxicity, easy regulation and high spatiotemporal resolution. Among them, photothermal conversion materials can generate heat under laser irradiation, stimulating the transformation of heat shock factor (HSF) from monomer to trimer, and then HSF trimer translocation to the nucleus, binding to heat shock element (HSE) in HSP70 and activating transcription, which can achieve precise regulation of gene expression at a specified location and time. In addition, heat can increase the permeability of cell membranes to improve cell uptake and gene transfection efficiency. In recent years, inorganic materials and near-infrared dyes have been applied to hot-start gene therapy. However, during laser irradiation, temperature is difficult to be precisely controlled. Excessive temperature inevitably damages transfected cells, affects gene expression efficiency, and causes irreversible damage to surrounding normal tissues. Therefore, it is urgent to develop a precise temperature-controlled gene expression system to achieve non-destructive photothermal activation of gene expression to improve the efficiency and safety of gene therapy. At present, there is no report on a method for achieving safe and non-destructive initiation of gene expression through a mild and constant temperature transgenic system. Summary of the invention

[0004] In view of the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide a method for preparing temperature-controlled gene expression nanomaterials and their application, which can effectively solve the problem of non-destructive photothermal activation of genetic engineering by precisely controlling the temperature to avoid damage to transfected cells and surrounding tissues caused by excessive heat.

[0005] The technical solution provided by the present invention is a method for preparing a temperature-controlled gene expression nanomaterial, comprising the following steps:

[0006] 1) Dissolve chitosan (CS) in 20 mL of 1% acetic acid deionized water solution to a concentration of 5-10 mg / mL, and stir at 300 rpm for 8-12 hours overnight to prepare solution A;

[0007] 2) Add 1.6-2.2 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.8-1.2 g of p-anisic acid to 250 mL of dichloromethane (DCM), then add 0.8-1.3 g of N-hydroxysuccinimide (NHS), mix well, stir at room temperature under argon for 42 h, and dry to obtain NHS-activated aminoethylanisamide (AEAA-NHS);

[0008] 1.8-2.2 mL of NHS-activated aminoethyl anisamide (AEAA-NHS) with a concentration of 0.034 mmol and 90-110 mg of thiol-polyethylene glycol-carboxyl (SH-PEG-COOH) were reacted under N2 protection for 48 h to synthesize carboxylated polyethylene glycol-grafted aminoethyl anisamide (AEAA-PEG-COOH);

[0009] 5.5-6.0 mg of N-hydroxysuccinimide (NHS) and 19.0-19.3 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were added to solution A, stirred for 1 hour, and then carboxylated polyethylene glycol-grafted aminoethyl anisamide (AEAA-PEG-COOH) was added, and the reaction mixture was stirred at room temperature for 8-12 hours overnight; the mixture solution was collected, dialyzed in deionized water for 46-50 hours using a dialysis bag with MWCO=8000-14000, and the dialyzate was freeze-dried for 46-50 hours to obtain powder B;

[0010] 3) Heat 320-340 mg of a mixture of thermal green, β-naphthol and polyethylene glycol in an infrared heating furnace at 90-110 W for 4-6 minutes to obtain a transparent melt, and then add acetone to dissolve it to obtain solution C;

[0011] The mass ratio of the thermal green, β-naphthol and polyethylene glycol is 1:5:60;

[0012] 4) Add solution C dropwise into powder B with stirring and stir for 2 minutes to form a uniform green solution. Centrifuge through a 50kDa ultrafiltration tube to remove impurities to obtain nanomaterial D. React the thermosensitive green in nanomaterial D with a plasmid expressing co-stimulatory molecules CD86 or CD80 at a mass ratio of 1 to 10:1 for 30 minutes, centrifuge and remove the unadsorbed plasmid to obtain a temperature-controlled gene expression nanomaterial with a particle size of 100 to 150 nm.

[0013] The molecular weight of the chitosan is one of 5kDa, 15kDa, 25kDa and 100kDa or a mixture of two or more thereof.

[0014] The molecular weight of the polyethylene glycol is one of 1000, 1500, 2000, 4000 or a mixture of two or more thereof.

[0015] The temperature-controlled gene expression nanomaterial prepared by the method is used in the preparation of gene therapy drugs, and the gene therapy drugs are DNA drugs and RNA drugs.

[0016] This invention will take tumor-associated fibroblasts (CAFs) as an example. CAFs are one of the most abundant matrix components in the microenvironment of solid tumors. Accumulated evidence shows that CAFs can directly disable CD8 cytotoxic T lymphocytes. However, current attempts to combat CAFs-mediated immunosuppression are mainly focused on the removal of targeted CAFs, and their clinical translation may be hindered by factors such as limited removal efficiency and potential risk of tumor metastasis. Considering the dual role of CAFs in cancer progression (immunosuppression and maintenance of tumor tissue homeostasis), this invention uses precise temperature-controlled gene expression nanomaterials to safely reprogram immunosuppressive CAFs into immune-activated antigen-presenting cells in situ. Unlike current CAFs elimination technology, this invention both amplifies immunotherapy and avoids the risk of metastasis caused by the destruction of tumor tissue homeostasis.

[0017] The precise temperature-controlled gene expression nanomaterial prepared by the present invention can precisely start the gene expression of a variety of transfected cells, and the transfected cells include CAF cells, 4T1 cells, B16F10 cells, NIH3T3 cells, L929 cells, and 293 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the potential and particle size analysis of the temperature-controlled gene expression nanomaterial of the present invention;

[0019] Figure 2 This is a transmission electron microscope image of the temperature-controlled gene expression nanomaterial of the present invention;

[0020] Figure 3 This is a photothermal effect diagram of the temperature-controlled gene expression nanomaterial of the present invention;

[0021] Figure 4 This is a diagram of the photothermal conversion stability of the temperature-controlled gene expression nanomaterial of the present invention after being irradiated by cyclic laser;

[0022] Figure 5 The agarose gel electrophoresis diagram of the temperature-controlled gene expression nanomaterial of the present invention at different thermosensitive green / plasmid weight ratios (T is the representative of the thermosensitive green in TNP@CS-A);

[0023] Figure 6 This is an agarose gel shift diagram of the temperature-controlled gene expression nanomaterial of the present invention protecting DNA from DNase I degradation;

[0024] Figure 7 This is a stability diagram of the temperature-controlled gene expression nanomaterial of the present invention in serum;

[0025] Figure 8 This is a graph showing the effects of different concentrations of the temperature-controlled gene expression nanomaterials of the present invention on CAFs cell viability;

[0026] Fig. 9 The figure is a graph showing the effect of the temperature-controlled gene expression nanomaterial of the present invention on the viability of CAFs cells with or without laser irradiation (laser (+) or without laser irradiation (−));

[0027] Fig.10 This is a diagram showing the effect of the temperature-controlled gene expression nanomaterial of the present invention on the viability of CAFs cells in a live / dead cell staining experiment;

[0028] Fig.11 This is a diagram of gene expression after CAFs are transfected with the temperature-controlled gene expression nanomaterial of the present invention. DETAILED DESCRIPTION

[0029] The specific implementation of the present invention is described in detail below with reference to the accompanying drawings and examples.

[0030] The present invention can be specifically implemented by the following examples:

[0031] Example 1

[0032] The present invention provides a method for preparing a temperature-controlled gene expression nanomaterial, characterized by comprising the following steps:

[0033] 1) Dissolve 100 mg of chitosan in 20 mL of 1% acetic acid deionized water solution, and stir at 300 rpm for 8-12 hours overnight to prepare solution A;

[0034] 2) Add 1.9 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1.0 g of p-anisic acid to 250 mL of dichloromethane (DCM), then add 1.1 g of N-hydroxysuccinimide (NHS), mix well, stir at room temperature under argon for 42 h, and dry to obtain NHS-activated aminoethylanisamide (AEAA-NHS);

[0035] 2 mL of AEAA-NHS with a concentration of 0.034 mmol was reacted with 100 mg of thiol-polyethylene glycol-carboxyl (SH-PEG-COOH) under N2 protection for 48 h to synthesize carboxylated polyethylene glycol-grafted aminoethyl anisamide (AEAA-PEG-COOH);

[0036] 5.75 mg of NHS and 19.17 mg of EDC were added to solution A, stirred for 1 hour, and then carboxylated polyethylene glycol-grafted aminoethyl anisamide (AEAA-PEG-COOH) was added, and the reaction mixture was stirred at room temperature for 10 hours overnight; the mixture solution was collected, dialyzed in deionized water for 48 hours using a dialysis bag with MWCO=10000, and the dialyzate was freeze-dried for 48 hours to obtain powder B;

[0037] 3) Heat 330 mg of a mixture of thermal green, β-naphthol and polyethylene glycol in an infrared heating furnace at 100 W for 5 min to obtain a transparent melt, then add acetone to dissolve to obtain solution C;

[0038] 4) Add solution C dropwise into powder B with stirring and stir for 2 minutes to form a uniform green solution. Centrifuge through a 50kDa ultrafiltration tube to remove impurities to obtain nanomaterial D. React the thermosensitive green in nanomaterial D with a plasmid expressing co-stimulatory molecules CD86 or CD80 at a mass ratio of 2.5:1 for 30 minutes. Centrifuge and remove the unadsorbed plasmid to obtain a temperature-controlled gene expression nanomaterial with a particle size of 100-150 nm.

[0039] Example 2

[0040] The present invention provides a method for preparing a temperature-controlled gene expression nanomaterial, characterized by comprising the following steps:

[0041] 1) Dissolve 160 mg of chitosan in 20 mL of 1% acetic acid deionized water solution, and stir at 300 rpm for 8-12 hours overnight to prepare solution A;

[0042] 2) Add 1.7 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.9 g of p-anisic acid to 250 mL of dichloromethane (DCM), then add 0.9 g of N-hydroxysuccinimide (NHS), mix well, stir under argon at room temperature for 42 h, and dry to obtain NHS-activated aminoethylanisamide (AEAA-NHS);

[0043] 1.9 mL of NHS-activated aminoethyl anisamide (AEAA-NHS) with a concentration of 0.034 mmol was reacted with 95 mg of thiol-polyethylene glycol-carboxyl (SH-PEG-COOH) under N2 protection for 48 h to synthesize carboxylated polyethylene glycol-grafted aminoethyl anisamide (AEAA-PEG-COOH);

[0044] 5.6 mg of N-hydroxysuccinimide (NHS) and 19.0 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were added to solution A and stirred for 1 hour. Then, aminoethyl anisamide grafted with carboxylated polyethylene glycol (AEAA-PEG-COOH) was added, and the reaction mixture was stirred at room temperature for 9 hours overnight. The mixture solution was collected and dialyzed in deionized water for 49 hours using a dialysis bag with MWCO=9000. The dialyzate was freeze-dried for 49 hours to obtain powder B.

[0045] 3) Heat 310 mg of a mixture of thermal green, β-naphthol and polyethylene glycol in an infrared heating furnace at 90 W for 6 min to obtain a transparent melt, then add acetone to dissolve to obtain solution C;

[0046] 4) Add solution C dropwise into powder B with stirring and stir for 2 minutes to form a uniform green solution. Centrifuge through a 50kDa ultrafiltration tube to remove impurities to obtain nanomaterial D. React the thermosensitive green in nanomaterial D with a plasmid expressing the co-stimulatory molecule CD86 at a mass ratio of 5:1 for 30 minutes, centrifuge and remove the unadsorbed plasmid to obtain a temperature-controlled gene expression nanomaterial with a particle size of 100-150 nm.

[0047] Example 3

[0048] The present invention provides a method for preparing a temperature-controlled gene expression nanomaterial, characterized by comprising the following steps:

[0049] 1) Dissolve 120 mg of chitosan in 20 mL of 1% acetic acid deionized water solution, and stir at 300 rpm for 8-12 hours overnight to prepare solution A;

[0050] 2) Add 2.1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1.1 g of p-anisic acid to 250 mL of dichloromethane (DCM), then add 1.2 g of N-hydroxysuccinimide (NHS), mix well, stir at room temperature under argon for 42 h, and dry to obtain NHS-activated aminoethylanisamide (AEAA-NHS);

[0051] 2.1 mL of NHS-activated aminoethyl anisamide (AEAA-NHS) with a concentration of 0.034 mmol was reacted with 110 mg of thiol-polyethylene glycol-carboxyl (SH-PEG-COOH) under N2 protection for 48 h to synthesize carboxylated polyethylene glycol-grafted aminoethyl anisamide (AEAA-PEG-COOH);

[0052] 5.9 mg of N-hydroxysuccinimide (NHS) and 19.3 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were added to solution A and stirred for 1 hour. Then, aminoethyl anisamide grafted with carboxylated polyethylene glycol (AEAA-PEG-COOH) was added, and the reaction mixture was stirred at room temperature for 11 hours overnight. The mixture solution was collected and dialyzed in deionized water for 47 hours using a dialysis bag with MWCO=13000. The dialyzate was freeze-dried for 47 hours to obtain powder B.

[0053] 3) Heat 340 mg of the mixture of thermal green, β-naphthol and polyethylene glycol in an infrared heating furnace at 140 W for 4 min to obtain a transparent melt, and then add acetone to dissolve it to obtain solution C;

[0054] 4) Add solution C dropwise into powder B under stirring and stir for 2 minutes to form a uniform green solution. Centrifuge through a 50 kDa ultrafiltration tube to remove impurities to obtain nanomaterial D. React the thermosensitive green in nanomaterial D with a plasmid expressing co-stimulatory molecules CD86 or CD80 at a mass ratio of 8:1 for 30 minutes, centrifuge and remove the unadsorbed plasmid to obtain a temperature-controlled gene expression nanomaterial with a particle size of 100 to 150 nm.

[0055] It should be pointed out that the above is only an embodiment of the present invention, which is used to illustrate the specific implementation of the present invention, and is not intended to limit the scope of protection of the present invention. Any equivalent or equivalent modifications and substitutions made within the spirit and principles of the present invention are essentially the same as the technical solution of the present invention and are included in the scope of protection of the present invention.

[0056] The method for preparing the temperature-controlled gene expression nanomaterial prepared by the present invention is easy to operate, the raw materials are abundant and easily available, the product quality is good, it has gene-targeted therapeutic activity, and has achieved very good beneficial technical effects through experiments. The relevant experimental data are as follows (taking Example 1 as an example):

[0057] Temperature-controlled gene expression nanomaterials for precise and safe reprogramming of cancer-associated fibroblasts (CAFs)

[0058] CAFs cells (4 × 10 4 cells / well) were cultured in 300 μL of culture medium (24-well plate) for 24 h.

[0059] Detection and analysis

[0060] 1) The complex of temperature-controlled gene expression nanomaterial and plasmid was subjected to fluid dynamics analysis of potential level and particle size distribution. The results are as follows Figure 1 shown.

[0061] from Figure 1From the analysis, it can be seen that the potential of the temperature-controlled gene expression nanomaterial (TNP@CS-A) is +39 mV, and the potential of the complex of the precise temperature-controlled gene expression nanomaterial and the plasmid (TNP@CS-A / pDNA) formed after electrostatic adsorption of the plasmid is +2 mV. The hydrodynamic size of TNP@CS-A is about 124 nm, and the hydrodynamic size of TNP@CS-A / pDNA increases to about 137nm. Among them, TNP@CS-A represents the precise temperature-controlled gene expression nanomaterial without plasmid, and TNP@CS-A / pDNA represents the complex of the precise temperature-controlled gene expression nanomaterial and the plasmid.

[0062] 2) The complex of temperature-controlled gene expression nanomaterial and plasmid was analyzed by transmission electron microscopy. The scale bar is 100 nm. The results are as follows Figure 2 shown.

[0063] from Figure 2 Observation and analysis show that the complex of precise temperature-controlled gene expression nanomaterials and plasmid (TNP@CS-A / pDNA) has a spherical structure.

[0064] 3) Experimental study on the photothermal performance of temperature-controlled gene expression nanomaterials

[0065] The specific operation is as follows: In order to study the potential of temperature-controlled gene expression nanomaterials (TNP@CS-A) in precise temperature control, different concentrations of TNP@CS-A solutions (the dosage of thermal green is 20 μg / mL, 40 μg / mL, 80 μg / mL) were exposed to 660 nm laser for 30 min (2 W / cm 2 ). The temperature of the TNP@CS-A solution at different time points after laser irradiation was measured using an infrared imager.

[0066] The results show that all TNP@CS-A samples of the set concentrations show a rapid initial temperature rise, and then stabilize after reaching the preset temperature (42°C), and continue to maintain the preset temperature. The speed of temperature change is related to the concentration. The higher the concentration of the sample, the shorter the time to reach the temperature peak ( Figure 3 ).

[0067] 4) Experimental study on the photothermal stability of temperature-controlled gene expression nanomaterials

[0068] The specific operation is as follows: In order to study the photothermal stability of temperature-controlled gene expression nanomaterials (TNP@CS-A), a 40 μg / mL TNP@CS-A solution was exposed to 660 nm laser for 30 min (2 W / cm 2), and then naturally cooled, and five cycles were performed in sequence. The UV-visible spectra of the solution after heating or cooling were measured using a UV-visible spectrometer (Japan UV2550). The results are as follows Figure 4 shown.

[0069] From the analysis of the figure, it can be seen that the TNP@CS-A solution changes from green to colorless after 30 minutes of laser irradiation, and the ultraviolet absorption at 620 nm disappears. After cooling to 25°C, the solution returns to green and the ultraviolet-visible absorption ( Figure 4 A). These results indicate that when the solution turns colorless, laser absorption stops, constant temperature control of the preset temperature is expected to be achieved, and the nanomaterial has good reversibility of photothermal conversion. In addition, after 5 cycles of laser heating and cooling, the final temperature of TNP@CS-A still reaches the same value as the first irradiation, showing good photothermal conversion stability ( Figure 4 B) provides sufficient power to hot-start gene expression.

[0070] 5) Experimental study on the plasmid loading capacity of temperature-controlled gene expression nanomaterials

[0071] The specific operation is as follows: the present invention uses gel retardation method to detect the ability of temperature-controlled gene expression nanomaterials (TNP@CS-A) to load plasmid (pDNA). First, TNP@CS-A solution and plasmid are mixed at mass ratios of 10:1, 5:1, 2.5:1, and 1:1, respectively (T is the representative of thermosensitive green in TNP@CS-A), and the mixture is vortexed for 30 seconds; then, incubated at room temperature for 30 minutes, and the migration of the mixture on the gel is analyzed using 2% agarose gel to evaluate the ability of TNP@CS-A to load plasmid DNA. The results are as follows: Figure 5 shown.

[0072] The results showed that the migration of pDNA was completely retarded when the weight ratio of TNP@CS-A to pDNA was 2.5 or higher, indicating that TNP@CS-A was fully loaded with plasmid at a mass ratio of 2.5:1.

[0073] 6) Experimental study on the protective performance of temperature-controlled gene expression nanomaterials on plasmids

[0074] The specific operation is as follows: the present invention uses gel retardation method to detect the protective performance of temperature-controlled gene expression nanomaterials (TNP@CS-A) on plasmids (pDNA). First, TNP@CS-A solution and plasmids are mixed at mass ratios of 5:1, 2.5:1, 1:1, and 0:1, respectively (T is the representative of thermosensitive green in TNP@CS-A), and the mixture is vortexed for 30 seconds; then, incubated at room temperature for 30 minutes, and the migration of the mixture on the gel is analyzed using 2% agarose gel to evaluate the protective performance of TNP@CS-A on plasmids. The results are as follows: Figure 6 shown.

[0075] The results showed that after exposure to DNase I, pDNA was completely degraded in the group without TNP@CS-A, while pDNA remained intact in the TNP@CS-A / pDNA complex fully loaded with plasmid, indicating that TNP@CS-A can effectively protect DNA from degradation by DNase I.

[0076] 7) Experiment on the stability of the complex of temperature-controlled gene expression nanomaterials and plasmids in serum

[0077] The specific operation is: the stability of the complex of temperature-controlled gene expression nanomaterials and plasmids (TNP@CS-A / pDNA) in serum is detected by gel retardation method. In short, TNP@CS-A solution and plasmid are mixed at a mass ratio of 2.5:1 (T is the representative of thermosensitive green in TNP@CS-A), and the mixture is vortexed for 30 seconds. Then incubate at room temperature for 30 minutes to form TNP@CS-A / pDNA complex, dissolve fetal bovine serum, and analyze the stability of TNP@CS-A / pDNA complex in serum by 2% gel retardation method. The results are shown in Figure 2. Figure 7 shown.

[0078] The results showed that after incubation in serum for 24 h, the TNP@CS-A / pDNA complex exhibited good stability, indicating that it has the function of gene delivery.

[0079] 8) Photothermal toxicity experiment of nanomaterials for precise temperature-controlled gene expression

[0080] Tumor-associated fibroblasts (CAFs) were used to conduct cytotoxicity experiments on the precise temperature-controlled gene expression nanomaterial (TNP@CS-A). CAFs were incubated at 10 4 The cells were seeded at a density of 100 μL in a 96-well plate and cultured for 12 h. Different concentrations of TNP@CS-A were co-incubated with CAFs for 24 h. The old culture medium was replaced with 100 μL of fresh culture medium, and the cell viability was determined according to the MTT protocol.

[0081] In order to evaluate the effect of mild photothermal treatment induced by TNP@CS-A on the activity of CAFs, CAFs were plated at 10 4 The cells were seeded at a density of 100 cells / mL in a 96-well plate and cultured overnight. Secondly, TNP@CS-A (40 μg / mL) was added and incubated with CAFs for 6 h. Then, the cell culture supernatant was removed and replaced with fresh culture medium for 2 h. Finally, the cells were irradiated with a 660 nm laser for 30 min, and the cell viability was evaluated by the MTT method.

[0082] Live / dead cell staining assay to assess cell viability: CAFs were seeded in 24-well plates (8 × 10 4 Then, CAFs were incubated with TNP@CS-A (40 μg / mL) and PBS for 6 h. After stabilization for 2 h, the cells were irradiated with 660 nm laser for 30 min (2 W / cm 2 Calcein AM (1 μM) and propidium iodide (1 μM) were added to a 24-well plate and incubated for 20 min. The results were detected by laser confocal microscopy.

[0083] The results showed that even when CAFs were co-incubated with TNP@CS-A at a concentration of up to 80 µg / mL, the cytotoxicity was negligible ( Figure 8 ), indicating that TNP@CS-A has significant biocompatibility. Subsequently, the MTT method was used to detect the non-destructive photothermal effect of TNP@CS-A on CAFs. The results showed that the viability of CAFs was not affected after co-incubation with TNP@CS-A under laser irradiation ( Fig. 9 The live / dead cell staining analysis image is similar to the MTT assay result ( Fig.10 ), which means that the gene expression nanomaterials with precise temperature control prepared by the present invention have no effect on cell viability under laser irradiation, which provides strong support for non-destructive light-activated reprogramming of cells.

[0084] 9) Using temperature-controlled gene expression nanomaterials for in vitro optogenetic activation experiments

[0085] In the present invention, tumor-associated fibroblasts (CAFs) were used for transfection research. CAFs were inoculated into 24-well plates (8×10 4 cells / well) and cultured for 24 hours. The complex of temperature-controlled gene expression nanomaterials and plasmids (TNP@CS-A / pDNA) dissolved in Opti-MEM medium (weight ratio of 2.5:1) was added to the cells; after 6 hours, the Opti-MEM medium was removed, and fresh medium containing 10% fetal bovine serum (500 µL per well) was added to the 24-well plate; the 24-well plate was placed in a cell culture incubator and incubated for another 2 hours; then, the 24-well plate was continuously exposed to 660 nm (2 W / cm 2 ) laser for 30 min and the cells were further incubated under the same conditions. After 48 h, the cells were collected and the expression of co-stimulatory molecules (CD86) was characterized by flow cytometry.

[0086] The results are as follows Fig.11As shown, the expression of plasmid (pDNA) was activated under 660 nm laser irradiation, and flow cytometry results showed that the TNP@CS-A / pDNA complex expressed CD86 on the cell membrane of CAFs. In contrast, the expression of co-stimulatory factors in the control group and blank group without laser treatment was negligible. Therefore, the TNP@CS-A / pDNA-mediated optogenetic platform can precisely and remotely control gene expression to reprogram CAFs.

[0087] 10) Experiment on the effect of reprogrammed tumor-associated fibroblasts on T cell proliferation

[0088] Extraction of 4T1 cell antigens: First, disperse 4T1 cells in PBS, sonicate in an ice-water bath at 440W for 30 min, centrifuge at 3000rpm / 10min, and take the supernatant for later use.

[0089] Transfection of tumor-associated fibroblasts with different preparations: Tumor-associated fibroblasts (CAFs) (4 × 10 4 cells, 48-well plates) were cultured in 300 μL culture medium for 24 hours, wherein the basal medium was a medium containing 10% FBS. Control group: blank culture medium; Experimental group 1: blank culture medium + TNP@CS-A (-) (temperature-controlled nanoparticles without plasmid, without laser irradiation); Experimental group 2: blank culture medium + TNP@CS-A (+) (temperature-controlled nanoparticles without plasmid, with laser irradiation); Experimental group 3: blank culture medium + TNP@CS-A / pDNA (-) (temperature-controlled nanoparticles with plasmid, without laser irradiation); Experimental group 4: blank culture medium + TNP@CS-A / pDNA (+) (temperature-controlled nanoparticles with plasmid, with laser irradiation). The cells were treated according to the groups and incubated for 48 hours.

[0090] Evaluation of T cell proliferation: 1.5 million T cells extracted from mouse spleen were added to the above transfected CAFs, and the culture medium was 10% inactivated BI serum culture medium. The experimental groups were: control group: blank culture medium + 4T1 antigen; experimental group 1: blank culture medium + 4T1 antigen + fibroblasts transfected with TNP@CS-A (-) (temperature-controlled nanoparticles without plasmids, no laser irradiation); experimental group 2: blank culture medium + 4T1 antigen + CAFs transfected with TNP@CS-A (+) (temperature-controlled nanoparticles without plasmids Experimental group 3: blank medium + 4T1 antigen + CAFs transfected with TNP@CS-A / pDNA (-) (temperature-controlled nanoparticles containing plasmids, without laser irradiation); Experimental group 4: blank medium + 4T1 antigen + CAFs transfected with TNP@CS-A / pDNA (+) (temperature-controlled nanoparticles containing plasmids, with laser irradiation); Experimental group 5: blank medium + CAFs transfected with TNP@CS-A / pDNA (+) (temperature-controlled nanoparticles containing plasmids, with laser irradiation). After 48 hours, the proliferation of CD8+ T cells was detected by flow cytometry.

[0091] The number of CD3+CD8+ T cells increased from 14.3% in Experiment 3 to 20.4% in Experiment 4, while the TNP@CS-A / p1(+) (antigen-free) group had a negligible effect on the level of CD3+CD8+ T cells. These results indicate that CAFs can be reprogrammed by remote control, and that reprogrammed CAFs expressing co-stimulatory factors can significantly promote the proliferation of CD3+CD8+ T in an antigen-dependent manner.

[0092] The temperature-controlled gene expression nanomaterial (TNP@CS-A) of the present invention provides a way to initiate cell reprogramming by photothermal induction without damage. The present invention takes tumor-associated fibroblasts as an example. Tumor-associated fibroblasts (CAFs) are one of the most abundant matrix components in the microenvironment of solid tumors. Accumulated evidence shows that CAFs can directly disable CD8 cytotoxic T lymphocytes. However, current attempts to resist CAFs-mediated immunosuppression are mainly focused on the removal of targeted CAFs, and its clinical transformation may be hindered by factors such as limited removal efficiency and potential tumor metastasis risks. Considering the dual role of CAFs in cancer progression (immunosuppression and maintenance of tumor tissue homeostasis), the present invention uses a precise temperature-controlled gene expression system to safely reprogram immunosuppressive CAFs into immune-activated antigen-presenting cells in situ. Unlike the current CAFs elimination strategy, the present invention not only amplifies immunotherapy, but also avoids the risk of metastasis caused by the destruction of tumor tissue homeostasis. In the actual preparation and research process, it was also found that the preset point temperature can be accurately adjusted by changing the molecular weight and feed ratio of polyethylene glycol, providing strong support for accurate and safe cell reprogramming.

[0093] While the above experiment was conducted on Example 1, experiments were also conducted on other examples, all of which obtained the same or similar results, which will not be listed here one by one.

[0094] Experiments show that the method for preparing the temperature-controlled gene expression nanomaterial prepared by the invention is easy to operate, the raw materials are abundant and easy to obtain, the product quality is good, and it has gene-targeted therapeutic activity. Compared with the prior art, it has the following outstanding beneficial technical effects:

[0095] 1) The precise temperature-controlled gene expression nanomaterials of the present invention have abundant sources of synthetic materials, are easy to obtain, and have low costs; they are simple to synthesize, have a short time cycle, and are easy to clinically transform;

[0096] 2) The precise temperature-controlled gene expression nanomaterial provided by the present invention can be completely loaded with plasmids when the mass ratio of thermosensitive green to plasmid is greater than 2.5, has high loading efficiency, can effectively protect DNA from degradation by DNase I, and has good stability in fetal bovine serum, providing support for in vivo delivery.

[0097] 3) The precise temperature-controlled transgenic nanosystem provided by the present invention is dark green at room temperature, can absorb light and effectively convert it into heat. Once the temperature reaches the preset point, the nanoparticles will switch to a colorless state, which can keep the temperature at a preset level. The preset point temperature of the nanosystem with thermochromic properties can be precisely controlled by changing the feed ratio or molecular weight of polyethylene glycol, providing a strong guarantee for safe and effective cell reprogramming.

[0098] 4) The present invention utilizes precise temperature-controlled gene expression nanomaterials to safely reprogram immunosuppressive CAFs into immune-activated antigen-presenting cells in situ, which not only amplifies immunotherapy but also avoids the risk of metastasis caused by the destruction of tumor tissue homeostasis. It has the value of promotion and application and has huge economic and social benefits.

Claims

1. A method for preparing a temperature-controlled gene expression nanomaterial, characterized in that: The following steps are involved: 1) Dissolve chitosan in 20 mL of 1% acetic acid deionized water solution to a chitosan concentration of 5-10 mg / mL, and stir at 300 rpm for 8-12 hours overnight to prepare solution A; 2) Add 1.6-2.2 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.8-1.2 g of p-anisic acid to 250 mL of dichloromethane, then add 0.8-1.3 g of N-hydroxysuccinimide, mix well, stir under argon at room temperature for 42 hours, and dry to obtain N-hydroxysuccinimide-activated aminoethylanisamide; 1.8-2.2 mL of aminoethyl anisamide activated by N-hydroxysuccinimide at a concentration of 0.034 mmol was reacted with 90-110 mg of thiol-polyethylene glycol-carboxyl under N2 protection for 48 h to synthesize carboxylated polyethylene glycol-grafted aminoethyl anisamide; 5.5-6.0 mg of N-hydroxysuccinimide and 19.0-19.3 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to solution A, stirred for 1 hour, and then aminoethyl anisamide grafted with carboxylated polyethylene glycol was added, and the reaction mixture was stirred at room temperature for 8-12 hours overnight; the mixture solution was collected, dialyzed in deionized water for 46-50 hours using a dialysis bag with MWCO=8000-14000, and the dialyzate was freeze-dried for 46-50 hours to obtain powder B; 3) Heat 320-340 mg of a mixture of thermal green, β-naphthol and polyethylene glycol in an infrared heating furnace at 90-110 W for 4-6 minutes to obtain a transparent melt, and then add acetone to dissolve it to obtain solution C; The mass ratio of the thermal green, β-naphthol and polyethylene glycol is 1:5:60; 4) Add solution C dropwise into powder B with stirring and stir for 2 minutes to form a uniform green solution. Centrifuge through a 50kDa ultrafiltration tube to remove impurities to obtain nanomaterial D. React the thermosensitive green in nanomaterial D with a plasmid expressing co-stimulatory molecules CD86 or CD80 at a mass ratio of 1 to 10:1 for 30 minutes, centrifuge and remove the unadsorbed plasmid to obtain a temperature-controlled gene expression nanomaterial with a particle size of 100 to 150 nm.

2. The method for preparing the temperature-controlled gene expression nanomaterial according to claim 1, characterized in that: The molecular weight of the chitosan is one of 5kDa, 15kDa, 25kDa and 100kDa or a mixture of two or more thereof.

3. The method for preparing the temperature-controlled gene expression nanomaterial according to claim 1, characterized in that: The molecular weight of the polyethylene glycol is one of 1000, 1500, 2000, 4000 or a mixture of two or more thereof.

4. The method for preparing the temperature-controlled gene expression nanomaterial according to claim 1, characterized in that: The following steps are involved: 1) Dissolve 100 mg of chitosan in 20 mL of 1% acetic acid deionized water solution, and stir at 300 rpm for 8-12 hours overnight to prepare solution A; 2) Add 1.9 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.0 g of p-anisic acid to 250 mL of dichloromethane, then add 1.1 g of N-hydroxysuccinimide, mix well, stir under argon at room temperature for 42 h, and dry to obtain N-hydroxysuccinimide-activated aminoethylanisamide; 2 mL of aminoethyl anisamide activated by N-hydroxysuccinimide at a concentration of 0.034 mmol was reacted with 100 mg of thiol-polyethylene glycol-carboxyl under N2 protection for 48 h to synthesize carboxylated polyethylene glycol-grafted aminoethyl anisamide; 5.75 mg of N-hydroxysuccinimide and 19.17 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to solution A, and the mixture was stirred for 1 hour. Then, aminoethyl anisamide grafted with carboxylated polyethylene glycol was added, and the reaction mixture was stirred at room temperature for 10 hours overnight. The mixture solution was collected and dialyzed in deionized water for 48 hours using a dialysis bag with MWCO=10000. The dialyzate was freeze-dried for 48 hours to obtain powder B. 3) Heat 330 mg of a mixture of thermal green, β-naphthol and polyethylene glycol in an infrared heating furnace at 100 W for 5 min to obtain a transparent melt, then add acetone to dissolve to obtain solution C; 4) Add solution C dropwise into powder B with stirring and stir for 2 minutes to form a uniform green solution. Centrifuge through a 50kDa ultrafiltration tube to remove impurities to obtain nanomaterial D. React the thermosensitive green in nanomaterial D with a plasmid expressing co-stimulatory molecules CD86 or CD80 at a mass ratio of 2.5:1 for 30 minutes. Centrifuge and remove the unadsorbed plasmid to obtain a temperature-controlled gene expression nanomaterial with a particle size of 100-150 nm.

5. The method for preparing the temperature-controlled gene expression nanomaterial according to claim 1, characterized in that: The following steps are involved: 1) Dissolve 160 mg of chitosan in 20 mL of 1% acetic acid deionized water solution, and stir at 300 rpm for 8-12 hours overnight to prepare solution A; 2) Add 1.7 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.9 g of p-anisic acid to 250 mL of dichloromethane, then add 0.9 g of N-hydroxysuccinimide, mix well, stir under argon at room temperature for 42 h, and dry to obtain N-hydroxysuccinimide-activated aminoethylanisamide; 1.9 mL of aminoethyl anisamide activated by N-hydroxysuccinimide with a concentration of 0.034 mmol was reacted with 95 mg of thiol-polyethylene glycol-carboxyl under N2 protection for 48 h to synthesize carboxylated polyethylene glycol-grafted aminoethyl anisamide; 5.6 mg of N-hydroxysuccinimide and 19.0 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to solution A, and the mixture was stirred for 1 hour. Then, aminoethyl anisamide grafted with carboxylated polyethylene glycol was added, and the reaction mixture was stirred at room temperature for 9 hours overnight. The mixture solution was collected and dialyzed in deionized water for 49 hours using a dialysis bag with MWCO=9000, and the dialyzate was freeze-dried for 49 hours to obtain powder B. 3) Heat 310 mg of a mixture of thermal green, β-naphthol and polyethylene glycol in an infrared heating furnace at 90 W for 6 min to obtain a transparent melt, then add acetone to dissolve to obtain solution C; 4) Add solution C dropwise into powder B with stirring and stir for 2 minutes to form a uniform green solution. Centrifuge through a 50kDa ultrafiltration tube to remove impurities to obtain nanomaterial D. React the thermosensitive green in nanomaterial D with a plasmid expressing the co-stimulatory molecule CD86 at a mass ratio of 5:1 for 30 minutes, centrifuge and remove the unadsorbed plasmid to obtain a temperature-controlled gene expression nanomaterial with a particle size of 100-150 nm.

6. The method for preparing the temperature-controlled gene expression nanomaterial according to claim 1, characterized in that: The following steps are involved: 1) Dissolve 120 mg of chitosan in 20 mL of 1% acetic acid deionized water solution, and stir at 300 rpm for 8-12 hours overnight to prepare solution A; 2) Add 2.1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.1 g of p-anisic acid to 250 mL of dichloromethane, then add 1.2 g of N-hydroxysuccinimide, mix well, stir under argon at room temperature for 42 h, and dry to obtain N-hydroxysuccinimide-activated aminoethylanisamide; 2.1 mL of aminoethyl anisamide activated by N-hydroxysuccinimide with a concentration of 0.034 mmol was reacted with 110 mg of thiol-polyethylene glycol-carboxyl under N2 protection for 48 h to synthesize carboxylated polyethylene glycol-grafted aminoethyl anisamide; 5.9 mg of N-hydroxysuccinimide and 19.3 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to solution A, and stirred for 1 hour. Then, aminoethyl anisamide grafted with carboxylated polyethylene glycol was added, and the reaction mixture was stirred at room temperature for 11 hours overnight. The mixture solution was collected and dialyzed in deionized water for 47 hours using a dialysis bag with MWCO=13000, and the dialyzate was freeze-dried for 47 hours to obtain powder B. 3) Heat 340 mg of the mixture of thermal green, β-naphthol and polyethylene glycol in an infrared heating furnace at 140 W for 4 min to obtain a transparent melt, and then add acetone to dissolve it to obtain solution C; 4) Add solution C dropwise into powder B under stirring and stir for 2 minutes to form a uniform green solution. Centrifuge through a 50 kDa ultrafiltration tube to remove impurities to obtain nanomaterial D. React the thermosensitive green in nanomaterial D with a plasmid expressing co-stimulatory molecules CD86 or CD80 at a mass ratio of 8:1 for 30 minutes, centrifuge and remove the unadsorbed plasmid to obtain a temperature-controlled gene expression nanomaterial with a particle size of 100 to 150 nm.

7. Use of the temperature-controlled gene expression nanomaterial prepared by the method according to any one of claims 1 to 6 in the preparation of gene therapy drugs, wherein the gene therapy drugs are DNA drugs and RNA drugs.

8. Use of the temperature-controlled gene expression nanomaterial prepared by the method of claim 7 in the preparation of gene therapy drugs, characterized in that: The gene-transfected cells include tumor-associated fibroblasts, 4T1 cells, B16F10 cells, NIH3T3 cells, L929 cells, and 293 cells.

Images