CN115887653B - Preparation method and application of temperature-controlled gene expression nanomaterial - Google Patents
Preparation method and application of temperature-controlled gene expression nanomaterial Download PDFInfo
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Abstract
本发明涉及温控基因表达纳米材料的制备方法及其应用，可有效解决通过精准控温，避免过高热对被转染细胞及周围组织造成的损伤，实现基因工程的非破坏性光热激活的问题，制备方法是，壳聚糖溶于醋酸去离子水溶液中，制成溶液A；将EDC和对茴香酸加入到DCM中，加入NHS，在室温氩气下搅拌，干燥，再加入巯基‑聚乙二醇‑羧基在N2保护下反应，合成AEAA‑PEG‑COOH；将NHS和EDC加入溶液A中，搅拌1h，再加入AEAA‑PEG‑COOH，搅拌过夜，透析，冻干，得粉末B；将热敏绿、β‑萘酚、聚乙二醇的混合物红外加热，丙酮溶解，成溶液C；将溶液C滴加到粉末B中，搅拌均匀，离心，除杂，得纳米材料D，将纳米材料D中的热敏绿与质粒反应，离心，除去未吸附上的质粒，得温控基因表达纳米材料。
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
The invention relates to the field of medicines, in particular to a preparation method and application of a temperature-controlled gene expression nanomaterial.
Background
Transgenic systems are nucleic acids that bring genetic material or gene editing devices into cells to continue the production of therapeutic proteins or correct cellular error genes, provide a useful tool for regulating cellular behavior, and potentially lead to innovative therapies. In recent years, gene therapy, in which exogenous nucleic acid is delivered into specific cells by viral or non-viral vectors, has become a promising therapeutic approach to treat genetic diseases and cancers. However, expression at non-specific sites not only reduces therapeutic efficiency but may also lead to irreversible side effects, thereby limiting their clinical use.
In gene therapy, coding for specific promoters is a viable solution, where light is an ideal inducer of gene expression due to its low toxicity, ease of regulation, and high spatial-temporal resolution. The photothermal conversion material can generate heat under the irradiation of laser, stimulates the transformation of a thermal shock factor (HSF) from a monomer to a trimer, then the HSF trimer is translocated to a cell nucleus, is combined with a Heat Shock Element (HSE) in HSP70 and activates transcription, and can realize accurate regulation and control of gene expression at a designated position and time. In addition, heat can increase the permeability of cell membranes to increase cellular 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, the temperature is difficult to be precisely controlled. Excessive temperatures inevitably damage transfected cells, affect gene expression efficiency, and cause irreversible damage to surrounding normal tissues. Therefore, there is an urgent need to develop a precise temperature-controlled gene expression system to achieve intact photothermal activation of gene expression to improve gene therapy efficiency and safety. At present, no report is made on a method for realizing safe and lossless gene expression by a gentle and isothermal transgenic system.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide 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 and realizing nondestructive photo-thermal activation of genetic engineering by accurately controlling temperature.
The technical scheme of the invention is that the preparation method of the temperature-controlled gene expression nanomaterial comprises the following steps:
1) Dissolving Chitosan (CS) in 20mL of acetic acid deionized water solution with volume concentration of 1%, enabling the concentration of chitosan to be 5-10 mg/mL, and stirring at 300rpm for 8-12h overnight to prepare solution A;
2) 1.6 to 2.2g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.8 to 1.2g of p-anisic acid are added into Dichloromethane (DCM) 250 mL, then N-hydroxysuccinimide (NHS) 0.8 to 1.3 g are added, uniformly mixed, stirred for 42h under room temperature argon, and dried to obtain NHS activated amino ethyl anisoamide (AEAA-NHS);
Reacting 1.8-2.2mL of NHS activated amino ethyl anisoamide (AEAA-NHS) with 90-110mg of mercapto-polyethylene glycol-carboxyl (SH-PEG-COOH) under N 2 protection for 48h to synthesize carboxylated polyethylene glycol grafted amino ethyl anisoamide (AEAA-PEG-COOH) at a concentration of 0.034 mmol;
adding 5.5-6.0mg of N-hydroxysuccinimide (NHS) and 19.0-19.3mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) into the solution A, stirring for 1h, adding carboxylated polyethylene glycol grafted amino ethyl anisoamide (AEAA-PEG-COOH), stirring the reaction mixture at room temperature for 8-12 h overnight, collecting the mixture solution, dialyzing in deionized water for 46-50 h by using a dialysis bag with MWCO=8000-14000, and freeze-drying the dialyzate for 46-50 h to obtain powder B;
3) Heating 320-340mg of a mixture of thermosensitive green, beta-naphthol and polyethylene glycol for 4-6 min by an infrared heating furnace at 90-110W to obtain transparent melt, and then adding acetone for dissolution to obtain a solution C;
the thermosensitive green, beta-naphthol mass of polyethylene glycol the ratio was 1. 5.60;
4) Dropwise adding the solution C into the powder B under stirring, stirring for 2min to form a uniform green solution, centrifuging by a 50kDa ultrafiltration tube, removing impurities to obtain a nanomaterial D, reacting thermosensitive green in the nanomaterial D with plasmids expressing co-stimulatory molecules CD86 or CD80 in a mass ratio of 1-10..1 for 30min, centrifuging, and removing the plasmids which are not adsorbed to obtain the temperature-controlled gene expression nanomaterial, wherein the particle size is 100-150 nm.
The molecular weight of the chitosan is one or a mixture of more than two of 5kDa,15kDa,25kDa and 100 kDa.
The molecular weight of the polyethylene glycol is one or more than two of 1000, 1500, 2000 and 4000.
The temperature-controlled gene expression nanomaterial prepared by the method is applied to the preparation of gene therapy drugs, wherein the gene therapy drugs are DNA drugs and RNA drugs.
In the invention, tumor-related fibroblasts are taken as an example (CAFs), CAFs is one of the most abundant matrix components in the microenvironment of solid tumors, and accumulated evidence shows that CAFs can directly disable CD8 cytotoxic T lymphocytes. However, current attempts at anti-CAFs mediated immunosuppression have focused mainly on targeting CAFs clearance, whose clinical transformation may be hampered by factors such as limited clearance efficiency and potential risk of tumor metastasis. In view of the dual role of CAFs in cancer progression (immunosuppression and maintenance of tumor tissue homeostasis), the present invention utilizes accurate temperature-controlled gene expression nanomaterials to safely reprogram immunosuppressed CAFs into immunocompetent antigen presenting cells in situ, unlike current CAFs elimination techniques, which both amplifies immunotherapy and avoids the risk of metastasis due to disruption of tumor tissue homeostasis.
The accurate temperature control gene expression nanomaterial prepared by the invention can accurately start gene expression of various transfected cells, wherein the transfected cells comprise CAF cells, 4T1 cells, B16F10 cells, NIH3T3 cells, L929 cells and 293 cells.
Drawings
FIG. 1 is a graph showing the potential and particle size analysis of the temperature-controlled gene expression nanomaterial of the present invention;
FIG. 2 is a transmission electron microscope image of the temperature-controlled gene expression nanomaterial of the present invention;
FIG. 3 is a photo-thermal effect diagram of the temperature-controlled gene expression nanomaterial of the present invention;
FIG. 4 is a graph showing the photo-thermal conversion stability of the temperature-controlled gene expression nanomaterial of the present invention after being irradiated with a circulating laser;
FIG. 5 is an agarose gel electrophoresis chart of the temperature-controlled gene expression nanomaterial of the present invention at different thermosensitive green/plasmid weight ratios (T is representative of thermosensitive green in TNP@CS-A);
FIG. 6 is a agarose gel displacement diagram of the temperature controlled gene expression nanomaterial of the present invention to protect DNA from DNase I degradation;
FIG. 7 is a graph showing the stability of the temperature-controlled gene expression nanomaterial of the present invention in serum;
FIG. 8 is a graph showing the effect of different concentrations of the temperature-controlled gene expression nanomaterial on CAFs cell viability;
FIG. 9 is a graph showing the effect of the temperature-controlled gene expression nanomaterial of the present invention on CAFs cell viability with or without laser irradiation (laser (+) or no laser irradiation (-));
FIG. 10 is a graph showing the effect of the temperature-controlled gene expression nanomaterial of the present invention on CAFs cell viability in a live/dead cell staining experiment;
FIG. 11 is a diagram showing the expression of the gene after the temperature-controlled gene expression nanomaterial of the present invention is transfected CAFs.
Detailed Description
The following describes in detail the embodiments of the present invention with reference to the drawings and examples.
The invention can be embodied by the following examples
Example 1
The invention relates to a preparation method of a temperature-controlled gene expression nanomaterial, which is characterized by comprising the following steps of:
1) 100mg of chitosan is dissolved in 20mL of acetic acid deionized water solution with volume concentration of 1%, and stirred at 300rpm for 8-12h overnight to prepare solution A;
2) 1.9g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 1.0g of p-anisic acid are added into 250mL of Dichloromethane (DCM), 1.1g of N-hydroxysuccinimide (NHS) is then added, the mixture is uniformly mixed, stirred for 42h under argon at room temperature and dried to obtain NHS activated amino ethyl anisoamide (AEAA-NHS);
2mL of AEAA-NHS with the concentration of 0.034mmol is reacted with 100mg of sulfhydryl-polyethylene glycol-carboxyl (SH-PEG-COOH) under the protection of N 2 for 48h to synthesize carboxylated polyethylene glycol grafted amino ethyl anisoamide (AEAA-PEG-COOH);
Adding NHS 5.75mg and EDC 19.17mg into solution A, stirring for 1h, adding carboxylated polyethylene glycol grafted amino ethyl anisoamide (AEAA-PEG-COOH), stirring the reaction mixture at room temperature for 10h overnight, collecting the mixture solution, dialyzing in deionized water for 48h with a dialysis bag with MWCO=10000, and freeze-drying the dialysate for 48h to obtain powder B;
3) Heating 330mg of a mixture of thermosensitive green, beta-naphthol and polyethylene glycol for 5min by using an infrared heating furnace at 100W to obtain a transparent melt, and then adding acetone for dissolution to obtain a solution C;
4) Dropwise adding the solution C into the powder B under stirring, stirring for 2min to form a uniform green solution, centrifuging by a 50kDa ultrafiltration tube, removing impurities to obtain a nanomaterial D, reacting thermosensitive green in the nanomaterial D with plasmids expressing co-stimulatory molecules CD86 or CD80 in a mass ratio of 2.5..1 for 30min, centrifuging, and removing the plasmids which are not adsorbed to obtain the temperature-controlled gene expression nanomaterial, wherein the particle size is 100-150 nm.
Example 2
The invention relates to a preparation method of a temperature-controlled gene expression nanomaterial, which is characterized by comprising the following steps of:
1) 160mg of chitosan is dissolved in 20mL of acetic acid deionized water solution with volume concentration of 1%, and stirred at 300rpm for 8-12h overnight to prepare solution A;
2) 1.7g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.9g of p-anisic acid are added into Dichloromethane (DCM) 250mL g, then 0.9g of N-hydroxysuccinimide (NHS) is added, uniformly mixed, stirred for 42h under room temperature argon and dried to obtain NHS activated amino ethyl anisoamide (AEAA-NHS);
NHS activated amino ethyl anisoamide (AEAA-NHS) 1.9mL at a concentration of 0.034mmol was reacted with thiol-polyethylene glycol-carboxyl (SH-PEG-COOH) 95mg under N 2 protection for 48h to synthesize carboxylated polyethylene glycol grafted amino ethyl anisoamide (AEAA-PEG-COOH);
5.6mg of N-hydroxysuccinimide (NHS) and 19.0mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were added to solution A, stirred for 1h, and then carboxylated polyethylene glycol-grafted aminoethyl anisoamide (AEAA-PEG-COOH) was added, and the reaction mixture was stirred at room temperature for 9h overnight;
3) Heating 310mg of a mixture of thermosensitive green, beta-naphthol and polyethylene glycol for 6min by an infrared heating furnace 90W to obtain a transparent melt, and then adding acetone for dissolution to obtain a solution C;
4) Dropwise adding the solution C into the powder B under stirring, stirring for 2min to form a uniform green solution, centrifuging by a 50kDa ultrafiltration tube, removing impurities to obtain a nanomaterial D, reacting thermosensitive green in the nanomaterial D with plasmids expressing co-stimulatory molecules CD86 in a mass ratio of 5..1 for 30min, centrifuging, and removing the plasmids which are not adsorbed to obtain the temperature-controlled gene expression nanomaterial, wherein the particle size is 100-150 nm.
Example 3
The invention relates to a preparation method of a temperature-controlled gene expression nanomaterial, which is characterized by comprising the following steps of:
1) 120mg of chitosan is dissolved in 20mL of acetic acid deionized water solution with volume concentration of 1%, and stirred at 300rpm for 8-12h overnight to prepare solution A;
2) 2.1g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 1.1g of p-anisic acid are added into Dichloromethane (DCM) 250mL, 1.2g of N-hydroxysuccinimide (NHS) is then added, the mixture is uniformly mixed, stirred for 42h under argon at room temperature and dried to obtain NHS activated amino ethyl anisoamide (AEAA-NHS);
2.1mL of NHS-activated amino ethyl anisoamide (AEAA-NHS) with a concentration of 0.034mmol was reacted with 110mg of mercapto-polyethylene glycol-carboxyl (SH-PEG-COOH) under N 2 protection for 48h to synthesize carboxylated polyethylene glycol grafted amino ethyl anisoamide (AEAA-PEG-COOH);
5.9mg of N-hydroxysuccinimide (NHS) and 19.3mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were added to solution A, stirred for 1h, and then carboxylated polyethylene glycol-grafted aminoethyl anisoamide (AEAA-PEG-COOH) was added, and the reaction mixture was stirred at room temperature for 11h overnight;
3) Heating 340mg of a mixture of thermosensitive green, beta-naphthol and polyethylene glycol for 4min through an infrared heating furnace 140W to obtain a transparent melt, and then adding acetone for dissolution to obtain a solution C;
4) Dropwise adding the solution C into the powder B under stirring, stirring for 2min to form a uniform green solution, centrifuging by a 50kDa ultrafiltration tube, removing impurities to obtain a nanomaterial D, reacting thermosensitive green in the nanomaterial D with plasmids expressing co-stimulatory molecules CD86 or CD80 in a mass ratio of 8..1 for 30min, centrifuging, and removing the plasmids which are not adsorbed to obtain the temperature-controlled gene expression nanomaterial, wherein the particle size is 100-150 nm.
It should be noted that the foregoing embodiments of the present invention are merely illustrative of specific embodiments of the present invention, and are not intended to limit the scope of the invention, and any equivalent modifications and substitutions made within the spirit and principles of the present invention are essentially the same as the technical solutions of the present invention and are included in the scope of the present invention.
The preparation method of the temperature-control gene expression nanomaterial prepared by the invention is easy to operate, the raw materials are rich and easy to obtain, the product quality is good, the temperature-control gene expression nanomaterial has gene targeting therapeutic activity, and the experiment has very good beneficial technical effects, and the related experimental data are as follows (taking example 1):
The temperature-controlled gene expression nano material is used for accurate and safe tumor-related fibroblast (CAFs) reprogramming experiment
CAFs cells (4X 10 4 cells/well) were cultured in 300. Mu.L of medium (24 well plate) for 24 hours.
Detection and analysis
1) The composite of the temperature-controlled gene expression nanomaterial and the plasmid is subjected to hydrodynamic analysis on potential level and particle size distribution, and the result is shown in figure 1.
As can be seen from the analysis in FIG. 1, the potential of the temperature-controlled gene expression nanomaterial (TNP@CS-A) is + mV, and the potential of the complex (TNP@CS-A/pDNA) of the accurate temperature-controlled gene expression nanomaterial formed by electrostatic adsorption of plasmids and plasmids is +2 mV. The hydrodynamic size of TNP@CS-A was about 124 nm and the hydrodynamic size of TNP@CS-A/pDNA increased to about 137 nm. Wherein TNP@CS-A represents A precise temperature control gene expression nanomaterial not carrying plasmids, and TNP@CS-A/pDNA represents A complex of the precise temperature control gene expression nanomaterial and plasmids.
2) The composite of the temperature-controlled gene expression nanomaterial and the plasmid was subjected to transmission electron microscope analysis at a scale of 100 nm, and the result is shown in fig. 2.
From the observation analysis in FIG. 2, it is known that the complex of the accurate temperature-controlled gene expression nanomaterial and plasmid (TNP@CS-A/pDNA) has A spherical structure.
3) Investigation experiment of photo-thermal performance of temperature control gene expression nano material
Specific manipulations to investigate the potential of temperature-controlled gene-expressing nanomaterials (TNP@CS-A) in precisely controlled temperatures, TNP@CS-A solutions of different concentrations (doses of thermosensitive green 20. Mu.g/m, 40. Mu.g/m, 80. Mu.g/m) were exposed to 660 nm lasers for 30min (2W/cm 2). The temperature of the TNP@CS-A solution at various time points after the TNP@CS-A solution was irradiated with laser was measured by an infrared imager.
The results show that the TNP@CS-A samples at the set concentrations all showed A rapid initial temperature rise, followed by steady state after reaching the preset point temperature (42 ℃) and continued to maintain the preset point temperature, with the rate of temperature change being dependent on the concentration, the higher the concentration of the sample, the shorter the time to reach the temperature peak (FIG. 3).
4) Light-heat stability investigation experiment is carried out on temperature control gene expression nano material
Specifically, in order to study the photo-thermal stability of the temperature-controlled gene expression nanomaterial (TNP@CS-A), 40 μg/m TNP@CS-A solution was exposed to 660 nm laser light for continuous irradiation of 30min (2W/cm 2), then naturally cooled, and sequentially subjected to 5 cycles. And ultraviolet-visible spectrum after the solution was heated or cooled was measured using an ultraviolet-visible spectrometer (japanese UV 2550), and the result is shown in fig. 4.
From the analysis in the figure, it is seen that the TNP@CS-A solution turns from green to colorless upon irradiation with 30 min laser light, and the ultraviolet absorption disappears at 620 nm. After cooling to 25 ℃, the solution recovered green while uv-visible absorption was recovered (fig. 4A). These results indicate that when the solution turns colorless, stopping the laser absorption is expected to achieve the constant temperature control of the preset temperature, and shows that the nanomaterial has good photo-thermal conversion reversibility. In addition, TNP@CS-A reached the same value as the first irradiation after 5 cycles of laser heating and cooling, showing good photo-thermal conversion stability (FIG. 4B), providing sufficient power for hot-start gene expression.
5) Investigation experiment of plasmid loading capability of temperature control gene expression nano material
Specifically, the present invention examined the ability of A temperature controlled gene expression nanomaterial (TNP@CS-A) to load plasmid (pDNA) by gel blocking, first, TNP@CS-A solution was mixed with plasmid at mass ratios of 10:1, 5:1, 2.5:1, 1:1, respectively (T is representative of thermal green in TNP@CS-A), the mixture was vortexed for 30 seconds, then 30min was incubated at room temperature, and migration of the mixture on gel was analyzed by 2% agarose gel to assess the ability of TNP@CS-A to load plasmid DNA, and the results are shown in FIG. 5.
The results showed that when the weight ratio of TNP@CS-A and pDNA was 2.5 or higher, migration of pDNA was completely delayed, indicating that the plasmid was completely loaded when the mass ratio of TNP@CS-A was 2.5:1.
6) Investigation experiment of protective performance of temperature control gene expression nano material on plasmid
Specifically, the present invention examined the protective properties of the temperature controlled gene expression nanomaterial (TNP@CS-A) against plasmid (pDNA) by gel blocking, first, TNP@CS-A solution was mixed with plasmid in mass ratios of 5:1, 2.5:1, 1:1, 0:1, respectively (T is representative of thermal green in TNP@CS-A), the mixture was vortexed for 30 seconds, then 30min was incubated at room temperature, and migration of the mixture on gel was analyzed by 2% agarose gel to assess the protective properties of TNP@CS-A against plasmid, and the results are shown in FIG. 6.
The results showed that pDNA was completely degraded in TNP@CS-A free groups after exposure to DNase I, while the pDNA remained intact in the TNP@CS-A/pDNA complex completely loaded with the plasmid, indicating that TNP@CS-A can effectively protect DNA from DNase I degradation.
7) Stability investigation experiment of temperature-controlled gene expression nanomaterial and plasmid composite in serum
The specific operation is that A gel blocking method is adopted to detect the stability of A composite (TNP@CS-A/pDNA) of A temperature controlled gene expression nano material and A plasmid in serum. Briefly, TNP@CS-A solution was mixed with plasmid at A mass ratio of 2.5:1 (T being representative of the thermal green in TNP@CS-A), and the mixture was vortexed for 30 seconds. Then incubated at room temperature for 30min to form TNP@CS-A/pDNA complex, the fetal bovine serum was solubilized, and the TNP@CS-A/pDNA complex was analyzed for stability in serum by A2% gel blocking method, and the results are shown in FIG. 7.
The results showed that the TNP@CS-A/pDNA complex showed good stability by incubation in serum for 24h, indicating that it has the function of gene transfer.
8) Photo-thermal toxicity experiment of accurate temperature control gene expression nano material
And carrying out cytotoxicity experiments on the accurate temperature control gene expression nano material (TNP@CS-A) by adopting tumor related fibroblasts (CAFs). CAFs cells were seeded into 96-well plates at A density of 10 4 cells per well and incubated for 12 hours, TNP@CS-A at different concentrations was incubated with CAFs for 24h, and the old medium was replaced with 100. Mu. of fresh medium and cell viability was determined according to the MTT protocol.
To evaluate the effect of TNP@CS-A on CAFs activity induced by gentle light and heat under laser, CAFs was seeded in 96-well plates at A density of 10 4 cells per well and incubated overnight, next TNP@CS-A (40 μg/m) was added and incubated with CAFs for 6h, then cell culture supernatant was removed and replaced with fresh medium for stabilization of 2h, finally cells were continuously irradiated with 660 nm laser for 30 min and cell viability was assessed by MTT method.
Live/dead cell staining experiments cell viability was assessed by inoculating CAFs in 24 well plates (8 x 10 4 cells per well) and culturing for 12 hours. Then CAFs was incubated with TNP@CS-A (40. Mu.g/m) and PBS, respectively, for 6 h. After stabilization of 2h, the cells 30min (2W/cm 2) were irradiated with 660 nm laser. Calcein AM (1 μm) and propidium iodide (1 μm) were added to 24 well plates, incubated 20 min, and the results were examined by confocal laser.
The results showed that even CAFs incubated with TNP@CS-A up to 80. Mu.g/m, cytotoxicity was negligible (FIG. 8), indicating that TNP@CS-A had significant biocompatibility. Subsequently, the non-destructive photothermal effect of TNP@CS-A on CAF was examined by MTT method, and the results showed that the viability of CAF after co-incubation with TNP@CS-A was not affected under laser irradiation (FIG. 9). The analysis image of the live/dead cell staining is similar to the detection result of the MTT method (figure 10), which means that the gene expression nanomaterial with accurate temperature control prepared by the invention has no influence on the cell viability under the irradiation of laser, and provides strong support for nondestructively photo-activated reprogrammed cells.
9) Temperature-controlled gene expression nanomaterial and in-vitro optogenetic activation experiment
The invention uses tumor-associated fibroblasts (CAFs) for transfection studies, CAFs was inoculated into 24 well plates (8×10 4 cells/well) and cultured for 24 hours. The composite of temperature controlled gene expression nanomaterial dissolved in Opti-MEM medium and plasmid (TNP@CS-A/pDNA) (weight ratio of 2.5:1) was added to cells, after 6h, opti-MEM medium was removed, fresh medium containing 10% fetal bovine serum (500. Mu. per well) was added to 24 well plates, 24 well plates were placed in A cell incubator for 2 hours, then the 24 well plates were continuously exposed to 660: 660 nm (2W/cm 2) laser irradiation for 30: 30 min, cells were further incubated under the same conditions, after 48: 48 h, the cells were collected and expression of co-stimulatory molecules (CD 86) was characterized by flow cytometry.
As A result, as shown in FIG. 11, expression of plasmid (pDNA) was activated under 660 nm laser irradiation, and the results of flow cytometry showed that TNP@CS-A/pDNA complex expressed CD86 on CAFs cell membrane. In contrast, the expression of co-stimulatory factors was negligible in the control and blank groups without laser treatment. Thus, the TNP@CS-A/pDNA mediated optogenetic platform can precisely and remotely control gene expression to reprogram CAFs.
10 Experiment of the effect of reprogrammed tumor-associated fibroblasts on T cell proliferation
4T1 cell antigen extraction, namely dispersing 4T1 cells in PBS, performing ultrasonic treatment in 440W ice water bath for 30min, centrifuging at 3000 rpm/10min, and taking supernatant for later use.
Tumor-associated fibroblasts were transfected with different formulations by culturing tumor-associated fibroblasts (CAFs) (4×10× 10 4 cells per well, 48 well plate) in 300 μl of medium for 24 hours, wherein the basal medium was 10% FBS medium, control group, blank medium, experiment 1 group, blank medium+tnp@cs-A (-) (no plasmid-containing temperature-controlled nanoparticles, no laser irradiation), experiment 2 group, blank medium+tnp@cs-A (+) (no plasmid-containing temperature-controlled nanoparticles, laser irradiation), experiment 3 group, blank medium+tnp@cs-A/pdnA (-) (plasmid-containing temperature-controlled nanoparticles, no laser irradiation), experiment 4 group, blank medium+tnp@cs-A/pdnA (+) (plasmid-containing temperature-controlled nanoparticles, laser irradiation), treating cells according to groups, and incubating for 48 hours, respectively.
The proliferation of T cells was evaluated by adding 150 ten thousand T cells extracted from the spleen of the mice to the above-mentioned transfected CAFs, wherein the culture medium was 10% inactivated BI serum culture medium, the experimental group, control group, blank medium +4T1 antigen, experimental group, blank medium +4T1 antigen +TNP@CS-A (-) transfected fibroblasts (temperature control nanoparticle without plasmid, no laser irradiation), experimental group 2, blank medium +4T1 antigen + CAFs transfected by TNP@CS-A (+) (temperature control nanoparticle without plasmid, laser irradiation), experimental group 3, blank medium +4T1 antigen + CAFs transfected by TNP@CS-A/pDNA () (temperature control nanoparticle with plasmid, no laser irradiation), experimental group 4, blank medium +4T1 antigen +TNP@CS-A/pDNA (+) (temperature control nanoparticle with plasmid, no laser irradiation), experimental group 2, blank medium +4T1 antigen +TNP@CS-A/pDNA (+) transfected fibroblasts (temperature control nanoparticle with laser irradiation), and laser irradiation). 48 After h, proliferation of cd8+ T cells was detected by flow cytometry.
The number of CD3+CD8+ T cells increased from 14.3% in experiment 3 to 20.4% in experiment 4, while TNP@CS-A/p1 (+) (no antigen) had A negligible effect on CD3+CD8+ T cell levels. These results indicate that CAFs can be reprogrammed via remote control, and that CAFs, which expresses co-stimulators after reprogramming, can significantly promote proliferation of cd3+cd8+t in an antigen dependent manner.
The temperature-controlled gene expression nanomaterial (TNP@CS-A) provides A nondestructive photo-thermal initiation cell reprogramming mode. In the invention, by taking tumor-related fibroblasts as an example, the tumor-related fibroblasts (CAFs) are one of the most abundant matrix components in the microenvironment of a solid tumor, and accumulated evidence shows that CAFs can directly disable CD8 cytotoxic T lymphocytes. However, current attempts at anti-CAFs mediated immunosuppression have focused mainly on targeting CAFs clearance, whose clinical transformation may be hampered by factors such as limited clearance efficiency and potential risk of tumor metastasis. Given the dual role of CAFs in cancer progression (immunosuppression and maintenance of tumor tissue homeostasis), the present invention utilizes a precise temperature-controlled gene expression system to safely reprogram immunosuppressed CAFs into immunocompetent antigen presenting cells in situ. Unlike current CAFs elimination strategies, the present invention both amplifies immunotherapy and avoids the risk of metastasis due to disruption of tumor tissue homeostasis. In the actual preparation and research process, the temperature of the preset point can be accurately regulated by changing the molecular weight and the feeding ratio of polyethylene glycol, and a powerful support is provided for accurately and safely reprogramming cells.
The experiments described above for example 1 and other examples were performed to obtain the same or similar results, and are not listed here.
Experiments show that the preparation method of the temperature-controlled gene expression nanomaterial prepared by the invention is easy to operate, the raw materials are rich and easy to obtain, the product quality is good, and the temperature-controlled gene expression nanomaterial has gene targeting therapeutic activity, and compared with the prior art, the temperature-controlled gene expression nanomaterial has the following outstanding beneficial technical effects:
1) The accurate temperature control gene expression nanomaterial is rich in synthetic material sources, convenient to obtain, low in cost, simple to synthesize, short in time period and convenient for clinical transformation;
2) The accurate temperature control gene expression nanomaterial provided by the invention can be used for completely loading plasmids when the mass ratio of thermosensitive green to plasmids is more than 2.5, has high loading efficiency, can effectively protect DNA from being degraded by DNase I, has good stability in fetal bovine serum, and provides support for in vivo transmission.
3) The precise temperature control transgenic nano system provided by the invention is dark green at room temperature, can absorb light and effectively convert the light into heat, and once the temperature reaches a preset point, the nano particles can be switched to a colorless state, so that the temperature can be kept at a preset level, the temperature of the preset point of the nano system with thermochromic property can be precisely controlled by changing the feeding ratio or molecular weight of polyethylene glycol, and a powerful guarantee is provided for safely and effectively reprogramming cells.
4) The invention utilizes the accurate temperature control gene expression nano material to safely reprogram the immunosuppressed CAFs into the immune activated antigen presenting cell in situ, thereby not only amplifying the immunotherapy, but also avoiding the transfer risk caused by the damage of tumor tissue steady state, having popularization and application values and great economic and social benefits.
Claims (8)
1. The preparation method of the temperature-controlled gene expression nanomaterial is characterized by comprising the following steps of:
1) Dissolving chitosan in 20mL of acetic acid deionized water solution with volume concentration of 1%, enabling the concentration of chitosan to be 5-10 mg/mL, and stirring at 300rpm for 8-12h overnight to prepare solution A;
2) Adding 1.6-2.2g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 0.8-1.2g of p-anisic acid into 250mL of methylene dichloride, then adding 0.8-1.3g of N-hydroxysuccinimide, uniformly mixing, stirring for 42h under room temperature argon, and drying to obtain N-hydroxysuccinimide activated amino ethyl anisamide;
1.8-2.2mL of N-hydroxysuccinimide activated amino ethyl anisoamide with the concentration of 0.034mmol is reacted with 90-110mg of mercapto-polyethylene glycol-carboxyl under the protection of N 2 for 48h to synthesize carboxylated polyethylene glycol grafted amino ethyl anisoamide;
Adding 5.5-6.0mg of N-hydroxysuccinimide and 19.0-19.3mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride into the solution A, stirring for 1h, adding carboxylated polyethylene glycol grafted aminoethyl anisoamide, stirring the reaction mixture at room temperature for 8-12 h overnight, collecting the mixture solution, dialyzing in deionized water for 46-50 h by using a dialysis bag with MWCO=8000-14000, and freeze-drying the dialyzate liquid for 46-50 h to obtain powder B;
3) Heating 320-340mg of a mixture of thermosensitive green, beta-naphthol and polyethylene glycol for 4-6 min by an infrared heating furnace at 90-110W to obtain transparent melt, and then adding acetone for dissolution to obtain a solution C;
the thermosensitive green, beta-naphthol mass of polyethylene glycol the ratio was 1. 5.60;
4) Dropwise adding the solution C into the powder B under stirring, stirring for 2min to form a uniform green solution, centrifuging by a 50kDa ultrafiltration tube, removing impurities to obtain a nanomaterial D, reacting thermosensitive green in the nanomaterial D with plasmids expressing co-stimulatory molecules CD86 or CD80 in a mass ratio of 1-10..1 for 30min, centrifuging, and removing the plasmids which are not adsorbed to obtain the temperature-controlled gene expression nanomaterial, wherein the particle size is 100-150 nm.
2. The method for preparing a temperature-controlled gene expression nanomaterial of claim 1, wherein the chitosan has a molecular weight of one or more of 5kda,15kda,25kda, and 100 kda.
3. The method for preparing temperature-controlled gene expression nanomaterial of claim 1, wherein the polyethylene glycol has a molecular weight of one or more of 1000, 1500, 2000, 4000.
4. The method for preparing a temperature-controlled gene expression nanomaterial according to claim 1, comprising the steps of:
1) 100mg of chitosan is dissolved in 20mL of acetic acid deionized water solution with volume concentration of 1%, and stirred at 300rpm for 8-12h overnight to prepare solution A;
2) 1.9g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1.0g of p-anisic acid are added into 250mL of methylene dichloride, then 1.1g of N-hydroxysuccinimide is added, the mixture is uniformly mixed, stirred for 42h under room temperature argon, and dried to obtain N-hydroxysuccinimide activated amino ethyl anisoamide;
2mL of N-hydroxysuccinimide activated amino ethyl anisoamide with the concentration of 0.034mmol is reacted with 100mg of mercapto-polyethylene glycol-carboxyl under the protection of N 2 for 48h to synthesize carboxylated polyethylene glycol grafted amino ethyl anisoamide;
Adding 5.75mg of N-hydroxysuccinimide and 19.17mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride to the solution A, stirring for 1h, adding carboxylated polyethylene glycol grafted aminoethyl anisoamide, stirring the reaction mixture at room temperature for 10h overnight, collecting the mixture solution, dialyzing in deionized water for 48h with a dialysis bag with MWCO=10000, and freeze-drying the dialysate for 48h to obtain powder B;
3) Heating 330mg of a mixture of thermosensitive green, beta-naphthol and polyethylene glycol for 5min by using an infrared heating furnace at 100W to obtain a transparent melt, and then adding acetone for dissolution to obtain a solution C;
4) Dropwise adding the solution C into the powder B under stirring, stirring for 2min to form a uniform green solution, centrifuging by a 50kDa ultrafiltration tube, removing impurities to obtain a nanomaterial D, reacting thermosensitive green in the nanomaterial D with plasmids expressing co-stimulatory molecules CD86 or CD80 in a mass ratio of 2.5..1 for 30min, centrifuging, and removing the plasmids which are not adsorbed to obtain the temperature-controlled gene expression nanomaterial, wherein the particle size is 100-150 nm.
5. The method for preparing a temperature-controlled gene expression nanomaterial according to claim 1, comprising the steps of:
1) 160mg of chitosan is dissolved in 20mL of acetic acid deionized water solution with volume concentration of 1%, and stirred at 300rpm for 8-12h overnight to prepare solution A;
2) 1.7g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 0.9g of p-anisic acid are added into 250mL of methylene dichloride, then 0.9g of N-hydroxysuccinimide is added, the mixture is uniformly mixed, stirred for 42h under room temperature argon, and dried to obtain N-hydroxysuccinimide activated amino ethyl anisoamide;
1.9mL of N-hydroxysuccinimide activated amino ethyl anisoamide with the concentration of 0.034mmol is reacted with 95mg of mercapto-polyethylene glycol-carboxyl under the protection of N 2 for 48 hours to synthesize carboxylated polyethylene glycol grafted amino ethyl anisoamide;
Adding 5.6mg of N-hydroxysuccinimide and 19.0mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride to the solution A, stirring for 1h, adding carboxylated polyethylene glycol grafted aminoethyl anisoamide, stirring the reaction mixture at room temperature for 9h overnight;
3) Heating 310mg of a mixture of thermosensitive green, beta-naphthol and polyethylene glycol for 6min by an infrared heating furnace 90W to obtain a transparent melt, and then adding acetone for dissolution to obtain a solution C;
4) Dropwise adding the solution C into the powder B under stirring, stirring for 2min to form a uniform green solution, centrifuging by a 50kDa ultrafiltration tube, removing impurities to obtain a nanomaterial D, reacting thermosensitive green in the nanomaterial D with plasmids expressing co-stimulatory molecules CD86 in a mass ratio of 5..1 for 30min, centrifuging, and removing the plasmids which are not adsorbed to obtain the temperature-controlled gene expression nanomaterial, wherein the particle size is 100-150 nm.
6. The method for preparing a temperature-controlled gene expression nanomaterial according to claim 1, comprising the steps of:
1) 120mg of chitosan is dissolved in 20mL of acetic acid deionized water solution with volume concentration of 1%, and stirred at 300rpm for 8-12h overnight to prepare solution A;
2) Adding 2.1g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1.1g of p-anisic acid into 250mL of dichloromethane, then adding 1.2g of N-hydroxysuccinimide, uniformly mixing, stirring for 42h under room temperature argon, and drying to obtain N-hydroxysuccinimide activated amino ethyl anisoamide;
2.1mL of N-hydroxysuccinimide activated amino ethyl anisoamide with the concentration of 0.034mmol is reacted with 110mg of mercapto-polyethylene glycol-carboxyl under the protection of N 2 for 48h to synthesize carboxylated polyethylene glycol grafted amino ethyl anisoamide;
Adding 5.9mg of N-hydroxysuccinimide and 19.3mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride to the solution A, stirring for 1h, adding carboxylated polyethylene glycol grafted aminoethyl anisoamide, stirring the reaction mixture at room temperature for 11h overnight, collecting the mixture solution, dialyzing in deionized water for 47h with a dialysis bag with MWCO=13000, and freeze-drying the dialysate for 47h to obtain powder B;
3) Heating 340mg of a mixture of thermosensitive green, beta-naphthol and polyethylene glycol for 4min through an infrared heating furnace 140W to obtain a transparent melt, and then adding acetone for dissolution to obtain a solution C;
4) Dropwise adding the solution C into the powder B under stirring, stirring for 2min to form a uniform green solution, centrifuging by a 50kDa ultrafiltration tube, removing impurities to obtain a nanomaterial D, reacting thermosensitive green in the nanomaterial D with plasmids expressing co-stimulatory molecules CD86 or CD80 in a mass ratio of 8..1 for 30min, centrifuging, and removing the plasmids which are not adsorbed to obtain the temperature-controlled gene expression nanomaterial, wherein the particle size is 100-150 nm.
7. The use of the temperature-controlled gene expression nanomaterial prepared by the method of any one of claims 1 to 6 in the preparation of a gene therapy drug, wherein the gene therapy drug is a DNA drug or an RNA drug.
8. The application of the temperature-controlled gene expression nanomaterial prepared by the method of claim 7 in preparing a gene therapy drug, wherein transfected cells of the gene comprise tumor-associated fibroblasts, 4T1 cells, B16F10 cells, NIH3T3 cells, L929 cells and 293 cells.
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