CN110115764B - Voice-activated tumor efficient synergistic immunotherapy visualization micro-nano carrier system and preparation method and application thereof - Google Patents
Voice-activated tumor efficient synergistic immunotherapy visualization micro-nano carrier system and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a visual micro-nano carrier system for sound-control efficient synergistic immunotherapy of tumors, and a preparation method and application thereof. The system comprises an intelligent module consisting of pH/reduction double sensitive polymers; the voice control module consists of a voice sensitive protein MscL gene; a targeting module consisting of T cell and DC cell antibodies; a visualization module consisting of a microbubble-loaded ultrasound imaging agent. The system is expected to realize the synergy of sound control immune response enhancement, immune escape relief and immune process regulation and control at the same time through efficient gene delivery, sound control accurate starting and immune synergy integration, further realize the synergy of in-situ and systemic immune response, and generate immune memory, thereby realizing in-situ treatment, metastasis inhibition and relapse prevention of tumors to the maximum extent.
Description
Technical Field
The invention relates to the technical field of biotechnology and medicine, in particular to a visual micro-nano carrier system for sound-control tumor efficient synergistic immunotherapy and a preparation method and application thereof.
Background
The occurrence, metastasis and recurrence of tumors are a serious threat to the health and life of humans. In recent years, immunotherapy is becoming one of the main means for overcoming tumors, and has been successfully applied to the treatment of various tumors, such as liver cancer, non-small cell lung cancer, kidney cancer, prostate cancer, melanoma and the like. A variety of tumor immunotherapeutic drugs have been FDA approved for clinical research. Recent researches show that immune response enhancement, immune escape relief and immune process regulation are three important factors for effectively improving the tumor immunotherapy effect.
At present, the immune response enhancement mode mainly comprises tumor vaccine, therapeutic antibody, CAR-T cell therapy and the like, and can improve the killing capability of the immune system to the tumor. The mode of immune escape relieving mainly comprises an immune checkpoint inhibitor, a small molecule inhibitor and the like, and can relieve the inhibition of a tumor microenvironment on an immune system and enhance the recognition of the immune system on tumor cells. The immune process regulation and control mode mainly focuses on regulating and controlling respective sensitive molecules by means of light, magnetism and the like, and further realizes regulation and control of immune cells. However, due to the heterogeneity and genetic instability of tumors, the effect of enhancing immune response varies from person to person, the mechanisms of escaping body immunity are different, and the means for regulating and controlling immune processes are also extremely limited, so that the effect of tumor therapy with a single immune effect is still unsatisfactory. Therefore, if the synergy of immune response enhancement, immune escape relief and immune process regulation can be realized at the same time, the tumor immunotherapy effect can be expected to be improved.
Recent studies show that scientific construction of a functionalized vector system and efficient delivery of an immunomodulator with a therapeutic effect to an immune organ or immune cells are key to effective realization of synergy of three immune effects. Currently, various carrier systems have been developed in the field of tumor immunotherapy, including polymer-based nanoparticles and micelles, lipid-based liposomes, nanoemulsions, and inorganic-based iron oxide nanoparticles. By utilizing the special physicochemical properties of the carrier system, such as structure, particle size, morphology, surface charge, degradability and the like, the carrier system can be easily phagocytized by immune cells, and the immunotherapy medicament can be efficiently delivered. In addition, according to different treatment requirements, the physicochemical properties of the carrier system can be properly adjusted, and the surface of the nano-particles can be properly modified, so that an ideal multifunctional carrier system is obtained, and finally, the immune response of an organism is effectively induced, and the immunotherapy of tumors is realized.
However, the existing vector system has the defects of low delivery efficiency, poor safety and accuracy, single immune effect and the like. Therefore, only by creating a new method and constructing a carrier with high-efficiency delivery, accurate starting and cooperative integration functions, the high-efficiency cooperation of immune response enhancement, immune escape relief and immune process regulation can be expected to be realized, the cooperation of tumor in situ and whole body immune response is further realized, and immunological memory is generated, so that the in situ treatment, metastasis inhibition and relapse prevention effects of the tumor are effectively improved.
Disclosure of Invention
The invention aims to solve the technical problem of constructing a visual micro-nano carrier system for the sound-control efficient synergistic immunotherapy of tumors, and realizing the in-situ therapy, the metastasis inhibition and the relapse prevention of tumors through efficient gene delivery, accurate sound-control starting and immune synergistic integration. The invention discloses a reverse attack 'adoptive T cell therapy', which is characterized in that tumor infiltrating T cells are not extracted out of a body, the concentrations of calcium ions and potassium ions inside and outside T cells and DC cells in a tumor microenvironment are regulated and controlled through the response of ultrasonic sensitive ion channel proteins to ultrasonic waves, ion channels are opened, calcium ion inflow is initiated, downstream calcineurin is activated, activated cell nuclear factors are phosphorylated and transferred into nuclei, then related gene expression of immune activation is triggered, and T cells in a 'stem cell state' are activated in situ in tumors.
The invention also aims to solve another technical problem of a preparation method of the micro-nano carrier system.
The invention also aims to provide application of the micro-nano carrier system in tumor immunotherapy.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a visual micro-nano carrier system for efficient synergistic immunotherapy of acoustic control tumors is characterized in that an intelligent module is composed of pH/reduction double-sensitive polymers, an acoustic control module is composed of acoustic-sensitive protein MscL genes, a targeting module is composed of T cells and DC cell antibodies, a visual module is composed of microbubble-loaded ultrasonic imaging agents C4F10, and finally a structure that microbubbles wrap gene-loaded nanoparticles is formed.
Preferably, the micro-nano carrier system has the microbubble particle size of 1-5 microns and the gene-loaded nano particle size of 20-100 nm.
The second technical scheme of the invention is a preparation method of the micro-nano carrier system, which comprises the following steps:
1) preparation of polymer poly (dipiperidinopropane-quaternary ammonium dithioate):
(1) preparing 0.1M dimethylaminopropionic acid solution by using anhydrous dichloromethane, adding 2-3g of EDC hydrochloride and 100-120mg of dimethylaminopyridine, performing argon protection, adding 4-6g of dithiodiethanol, reacting at room temperature overnight, and performing silica gel chromatography purification on a product to obtain a reduction sensitive monomer dithio-quaternary ammonium salt molecule;
(2) weighing 2-6g of monomer dithio quaternary ammonium salt molecules and 0.5-1.5g of 2-azido ethyl acrylate, adding 4-6mL of dipiperidine propane (TDP), carrying out argon protection reaction, and carrying out dialysis bag dialysis purification on a product to obtain a cationic polymer poly (dipiperidine propane-dithio quaternary ammonium salt) (P (TDP-ANSAN)) with the molecular weight of 5000-15000;
2) preparing gene-loaded nanoparticles:
(1) mixing and dissolving a polymer P (TDP-ANSSAN) and a voice sensitive protein MscL gene in deionized water, wherein the nitrogen-phosphorus ratio of the polymer P and the voice sensitive protein MscL gene is 5:1-50:1, and self-assembling to form nanoparticles;
(2) adding 50-100 mu g T cell antibody to obtain gene-loaded nanoparticles of the target T cells;
(3) adding 50-100 mug of DC cell antibody to obtain the gene-loaded nano-particles of the targeted DC cells;
3) constructing a visual micro-nano carrier system for the sound-control efficient synergistic immunotherapy of tumors: preparing liposome microvesicle loaded with ultrasonic imaging agent by a double emulsification-solvent volatilization method, and adding 5-15mL of the gene-loaded nanoparticle solution to obtain the ultrasonic imaging microvesicle coated with the gene-loaded nanoparticles.
The third technical scheme of the invention is the application of the visual micro-nano carrier system for the acoustic control tumor high-efficiency synergetic immunotherapy, which comprises the following steps:
1) the micro-nano vector system delivers the MscL gene of the voice sensitive protein to T cells and DC cells in a targeted manner, and the expression lasts for 24-48 h;
2) carrying out ultrasonic treatment on the T cells and the DC cells, wherein the ultrasonic parameters are as follows: the frequency is 2-12MHz, the power is 1-15W, and the time is 0.5-5 min;
3) 2-24h T cells and DC cells were activated after sonication, producing an immune effect.
The micro-nano carrier system can realize the high-efficiency gene delivery with intellectualization, precision and visualization, activate immune cells by using an acoustic genetic technology, and realize the sound-control precision start of an immune effect by ultrasonic waves.
The micro-nano carrier system can simultaneously realize the coordination of immune response enhancement, immune escape relief and immune process regulation, further realize the coordination of in-situ and systemic immune response, and generate immune memory, thereby realizing in-situ treatment, metastasis inhibition and relapse prevention of tumors.
The structure of the invention has the following beneficial effects:
the visual micro-nano carrier system for the sound-control efficient synergistic immunotherapy of the tumors is an ultrasonic imaging microbubble wrapping gene-loaded nanoparticles, is uniform in shape and good in dispersity, can be used for carrying out ultrasonic imaging on tumor parts, and meanwhile releases the gene-loaded nanoparticles, so that the sound-control efficient synergistic immunotherapy of the tumors is realized by utilizing ultrasound.
Intelligent, precise and visual gene efficient delivery; the acoustic genetic technology is utilized to activate immune cells, so that the acoustic control accurate starting of the immune effect is realized; realizing the coordination of immune response enhancement, immune escape relief and immune process regulation and further realizing the coordination of in-situ and systemic immune response.
Drawings
FIG. 1: and (5) a schematic structure diagram of a micro-nano carrier system.
Detailed Description
To further illustrate the present invention, the present invention will now be described in detail by way of specific embodiments.
The invention relates to a visual micro-nano carrier system for efficient synergetic immunotherapy of acoustic control tumors, which is characterized in that an intelligent module is composed of pH/reduction double-sensitive polymers, an acoustic control module is composed of acoustic sensitive protein MscL genes, a targeting module is composed of T cells and DC cell antibodies, and a visual module is composed of microbubble-loaded ultrasonic imaging agents;
finally, the structure of the microbubble-encapsulated gene-loaded nanoparticle is formed, as shown in figure 1.
The preparation method of the visual micro-nano carrier system for the acoustic control tumor high-efficiency synergetic immunotherapy comprises the following steps: the details are as follows
(1) Preparation of polymer poly (dipiperidinopropane-quaternary ammonium dithioate): preparing 0.1M dimethylaminopropionic acid solution by using anhydrous dichloromethane, adding 2-3g of EDC hydrochloride and 100-120mg of dimethylaminopyridine, adding 4-6g of dithiodiethanol under the protection of argon, and reacting at room temperature overnight. And purifying by silica gel chromatography to obtain the reduction sensitive monomer dithio-quaternary ammonium salt molecule. 2-6g of monomer dithio quaternary ammonium salt molecule, 4-6mL of dipiperidine propane (TDP) and 0.5-1.5g of 2-azido ethyl acrylate are weighed and added into a Schlenk tube, the reaction is carried out under the protection of argon, and the product is dialyzed and purified by a dialysis bag to obtain the cationic polymer poly (dipiperidine propane-dithio quaternary ammonium salt) (P (TDP-ANSAN)) with the molecular weight of 5000-15000.
(2) Preparing gene-loaded nanoparticles: the polymer P (TDP-ANSSAN) and the sound sensitive protein MscL gene are mixed and dissolved in deionized water, the nitrogen-phosphorus ratio of the polymer P and the sound sensitive protein MscL gene is 5:1-50:1, and the polymer P and the sound sensitive protein MscL gene are self-assembled to form the nano-particles. Then 50-100 mu g T cell or DC cell antibody is added respectively to obtain the gene-loaded nano-particles which can target tumor tissue T cells or DC cells respectively.
(3) Constructing a visual micro-nano carrier system for the sound-control efficient synergistic immunotherapy of tumors: preparing liposome microvesicle loaded with ultrasonic imaging agent by a double emulsification-solvent volatilization method, and adding 5-15mL of the gene-loaded nanoparticle solution to obtain the ultrasonic imaging microvesicle coated with the gene-loaded nanoparticles.
The functional experimental technology for evaluating the MscL plasmid of the voice sensitive protein prepared by the invention is as follows:
and transfecting the constructed MscL plasmid into T cells and DC cells, and verifying the expression effect of the MscL plasmid on cell membranes by using a fluorescence microscope and Western blot. Carrying out ultrasonic treatment on the two cells at the frequency of 2-12MHz and the power of 1-15W for 0.5-5min, observing the inflow condition of calcium ions by a confocal microscope by using a calcium indicator, detecting the expression content of IL-2 and IFN-gamma of the T cell by using a kit, detecting the expression level of CD80 and CD86 proteins of the DC cell by using flow cytometry, and verifying the activation condition of the T cell and the DC cell.
The experimental technology for evaluating the in-vitro immune cell activation function of the micro-nano carrier system prepared by the invention comprises the following steps:
co-culturing the optimally prepared nano gene carrier and a T cell or a DC cell, and performing qualitative and quantitative analysis on the endocytosis effect of the nano carrier in the T cell or the DC cell by using a fluorescence microscope and a flow cytometer. After culturing for a certain time, observing the distribution of the nano gene carrier in the cell by using a confocal microscope, observing and detecting the expression of the target protein by using methods such as a fluorescence microscope, a confocal microscope, flow cytometry and the like through a Green Fluorescent Protein (GFP) label contained in the target gene, and further analyzing the content of the intracellular precursor protein by using WesternBlot. Observing the calcium ion inflow condition after ultrasonic treatment with the frequency of 2-12MHz, the power of 1-15W and the time of 0.5-5min by a confocal microscope, detecting the IL-2 and IFN-gamma expression content of T cells by a kit, and detecting the expression levels of CD80 and CD86 proteins of DC cells by a flow cytometer.
The experimental technology for evaluating the in-vivo tumor immunotherapy function of the micro-nano carrier system prepared by the invention is as follows:
constructing a mouse in-situ tumor model, observing the size and shape of the in-situ tumor through ultrasonic imaging, injecting a micro-nano carrier system into the tumor at multiple points, and simultaneously starting tumor immunotherapy through ultrasonic treatment with the frequency of 2-12MHz, the power of 1-15W and the time of 0.5-5min in a sound control mode. Verifying the delivery of the target gene and the expression in tumor infiltrating T cells and DC cells through tissue section and immunohistochemistry; the in situ immune response enhancement effect is verified by detecting the ratio of CD8+ T cells to CD4+ T cells in tumor infiltrating T cells; the effect of immune escape relief is verified by detecting the expression levels of CD80 and CD86 of tumor-infiltrating DC cells; the effect of enhancing the systemic immune response is verified by detecting the proportion of CD8+ T cells to CD4+ T cells in T cells of lymph nodes and blood circulation. On the basis, the kit is used for detecting the expression content of molecular indexes such as IL-6, IL-2, IFN-gamma and the like in blood, dynamically monitoring the change of the size of the in-situ tumor through ultrasonic imaging, drawing a survival curve and further verifying the effect of the acoustic control tumor immunotherapy.
A mouse tumor metastasis model is constructed, the proportion of CD8+ T cells to CD4+ T cells in T cells at a metastatic tumor part and the proportion of CD8+ T cells to CD4+ T cells in T cells of a whole lymph node and blood circulation are detected through flow cytometry, on the basis, the expression content of molecular indexes such as IL-6, IL-2 and IFN-gamma in blood is detected through a kit, and the growth condition of nodes of a metastatic tumor in the lung is observed to draw a survival curve and study the tumor metastasis inhibition effect.
Constructing a mouse tumor subcutaneous recurrence model, and observing the growth condition of in-situ tumor tissues through tissue sections and HE staining experiments; the ratio of CD8+ T cells to CD4+ T cells in T cells at the position of the recurrent tumor and the content of memory T cells in spleen are detected by flow cytometry, and the level of the generated immunological memory is evaluated; on the basis, the recurrence prevention effect is further researched by detecting the expression content of molecular indexes such as TNF-alpha, IFN-gamma and the like.
Example 1
(1) Preparation of polymer poly (dipiperidinopropane-quaternary ammonium dithioate): A0.1M dimethylaminopropionic acid solution was prepared from anhydrous dichloromethane, 2g EDC hydrochloride and 100mg dimethylaminopyridine were added under argon protection, 4g dithiodiethanol was added and the reaction was allowed to proceed overnight at room temperature. And purifying by silica gel chromatography to obtain the reduction sensitive monomer dithio-quaternary ammonium salt molecule. 2g of monomer dithio quaternary ammonium salt molecule, 4mL of dipiperidine propane (TDP) and 0.5g of 2-azido ethyl acrylate are weighed and added into a Schlenk tube, and the reaction is carried out under the protection of argon, and the product is dialyzed and purified by a dialysis bag to obtain cationic polymer poly (dipiperidine propane-dithio quaternary ammonium salt) (P (TDP-ANSAN)) with the molecular weight of about 5000.
(2) Preparing gene-loaded nanoparticles: the polymer P (TDP-ANSSAN) and the sound sensitive protein MscL gene are mixed and dissolved in deionized water, the nitrogen-phosphorus ratio of the polymer P and the sound sensitive protein MscL gene is 10:1, and the polymer P and the sound sensitive protein MscL gene are self-assembled to form the nano-particles. Then 50 mu g T cell or DC cell antibody is added respectively to obtain the gene-loaded nano-particle which can target tumor tissue T cells or DC cells respectively.
(3) Constructing a visual micro-nano carrier system for the sound-control efficient synergistic immunotherapy of tumors: preparing the liposome microbubble loaded with the ultrasonic imaging agent by a double emulsification-solvent volatilization method, and adding 5mL of the gene-loaded nanoparticle solution to obtain the ultrasonic imaging microbubble coated with the gene-loaded nanoparticles.
Example 2
(1) Preparation of polymer poly (dipiperidinopropane-quaternary ammonium dithioate): A0.1M dimethylaminopropionic acid solution was prepared from anhydrous dichloromethane, 2.5g of EDC hydrochloride and 110mg of dimethylaminopyridine were added under argon protection, 5g of dithiodiethanol was added, and the reaction was allowed to proceed overnight at room temperature. And purifying by silica gel chromatography to obtain the reduction sensitive monomer dithio-quaternary ammonium salt molecule. 4g of monomer dithio quaternary ammonium salt molecule, 5mL of dipiperidine propane (TDP) and 1g of 2-azido ethyl acrylate are weighed and added into a Schlenk tube, and then reacted under the protection of argon, and the product is dialyzed and purified by a dialysis bag to obtain cationic polymer poly (dipiperidine propane-dithio quaternary ammonium salt) (P (TDP-ANSAN)) with the molecular weight of about 10000.
(2) Preparing gene-loaded nanoparticles: the polymer P (TDP-ANSSAN) and the sound sensitive protein MscL gene are mixed and dissolved in deionized water, the nitrogen-phosphorus ratio of the polymer P and the sound sensitive protein MscL gene is 30:1, and the polymer P and the sound sensitive protein MscL gene are self-assembled to form the nano-particles. Then 75 mu g T cell or DC cell antibody is added respectively to obtain the gene-loaded nano-particle which can target tumor tissue T cells or DC cells respectively.
(3) Constructing a visual micro-nano carrier system for the sound-control efficient synergistic immunotherapy of tumors: preparing the liposome microbubble loaded with the ultrasonic imaging agent by a double emulsification-solvent volatilization method, and adding 10mL of the gene-loaded nanoparticle solution to obtain the ultrasonic imaging microbubble coated with the gene-loaded nanoparticles.
Example 3
(1) Preparation of polymer poly (dipiperidinopropane-quaternary ammonium dithioate): A0.1M dimethylaminopropionic acid solution was prepared from anhydrous dichloromethane, 3g of EDC hydrochloride and 120mg of dimethylaminopyridine were added under argon protection, 6g of dithiodiethanol was added, and the reaction was allowed to proceed overnight at room temperature. And purifying by silica gel chromatography to obtain the reduction sensitive monomer dithio-quaternary ammonium salt molecule. 6g of monomer dithio quaternary ammonium salt molecule, 6mL of dipiperidine propane (TDP) and 1.5g of 2-azido ethyl acrylate are weighed into a Schlenk tube, and reacted under protection of argon gas, and the product is dialyzed and purified by a dialysis bag to obtain cationic polymer poly (dipiperidine propane-dithio quaternary ammonium salt) (P (TDP-ANSAN)) with the molecular weight of about 15000.
(2) Preparing gene-loaded nanoparticles: the polymer P (TDP-ANSSAN) and the sound sensitive protein MscL gene are mixed and dissolved in deionized water, the nitrogen-phosphorus ratio of the polymer P and the sound sensitive protein MscL gene is 50:1, and the polymer P and the sound sensitive protein MscL gene are self-assembled to form the nano-particles. Then 100 mu g T cell or DC cell antibody is added respectively to obtain the gene-loaded nano-particle which can target tumor tissue T cells or DC cells respectively.
(3) Constructing a visual micro-nano carrier system for the sound-control efficient synergistic immunotherapy of tumors: preparing the liposome microbubble loaded with the ultrasonic imaging agent by a double emulsification-solvent volatilization method, and adding 15mL of the gene-loaded nanoparticle solution to obtain the ultrasonic imaging microbubble coated with the gene-loaded nanoparticles.
Example 4
And transfecting the constructed MscL plasmid into T cells and DC cells, and verifying the expression effect of the MscL plasmid on cell membranes by using a fluorescence microscope and Western blot. Ultrasonic treatment with frequency of 6MHz, power of 15W and time of 3min is carried out on the two cells, calcium ion inflow conditions are observed through a confocal microscope by using a calcium indicator, the expression content of IL-2 and IFN-gamma of the T cell is detected by using a kit, the expression levels of CD80 and CD86 proteins of the DC cell are detected by using flow cytometry, and the activation conditions of the T cell and the DC cell are verified.
Example 5
Co-culturing the optimally prepared nano gene carrier and a T cell or a DC cell, and performing qualitative and quantitative analysis on the endocytosis effect of the nano carrier in the T cell or the DC cell by using a fluorescence microscope and a flow cytometer. After culturing for a certain time, observing the distribution of the nano gene carrier in the cell by using a confocal microscope, observing and detecting the expression of the target protein by using methods such as a fluorescence microscope, a confocal microscope, flow cytometry and the like through a Green Fluorescent Protein (GFP) label contained in the target gene, and further analyzing the content of the intracellular precursor protein by using WesternBlot. Observing the calcium ion inflow condition after ultrasonic treatment with the frequency of 6MHz, the power of 15W and the time of 3min by using a confocal microscope, detecting the IL-2 and IFN-gamma expression content of T cells by using a kit, and detecting the expression levels of CD80 and CD86 proteins of DC cells by using a flow cytometer.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (1)
1. A preparation method of a visual micro-nano carrier system for sound-control efficient synergistic immunotherapy of tumors is characterized by comprising the following steps: the system comprises an intelligent module consisting of microenvironment response type nano particles, a voice control module consisting of voice sensitive protein MscL genes, a targeting module consisting of T cells and DC cell antibodies, and a visual module consisting of microbubble-loaded ultrasonic imaging agents;
finally forming a structure of microvesicle-wrapped gene-loaded nanoparticles;
the particle size of the microbubbles is 1-5 microns, and the particle size of the gene-loaded nanoparticles is 20-100 nanometers;
the preparation method of the visual micro-nano carrier system for the acoustic control tumor high-efficiency synergetic immunotherapy comprises the following steps:
1) preparation of polymer poly (dipiperidinopropane-quaternary ammonium dithioate):
(1) preparing 0.1M dimethylaminopropionic acid solution by using anhydrous dichloromethane, adding 2-3g of EDC hydrochloride and 100-120mg of dimethylaminopyridine, performing argon protection, adding 4-6g of dithiodiethanol, reacting at room temperature overnight, and performing silica gel chromatography purification on a product to obtain a reduction sensitive monomer dithio-quaternary ammonium salt molecule;
(2) weighing 2-6g of monomer dithio quaternary ammonium salt molecule and 0.5-1.5g of 2-azido ethyl acrylate, adding 4-6mL of dipiperidine propane (TDP), reacting under the protection of argon gas, and dialyzing and purifying the product by a dialysis bag to obtain a cationic polymer poly (dipiperidine propane-dithio quaternary ammonium salt) P (TDP-ANSAN) with the molecular weight of 5000-15000;
2) preparing gene-loaded nanoparticles:
(1) mixing and dissolving a polymer P (TDP-ANSSAN) and a voice sensitive protein MscL gene in deionized water, wherein the nitrogen-phosphorus ratio of the polymer P and the voice sensitive protein MscL gene is 5:1-50:1, and self-assembling to form nanoparticles;
(2) adding 50-100 mu g T cell antibody to obtain gene-loaded nanoparticles of the target T cells;
(3) adding 50-100 mug of DC cell antibody to obtain the gene-loaded nano-particles of the targeted DC cells;
3) constructing a visual micro-nano carrier system for the sound-control efficient synergistic immunotherapy of tumors: preparing liposome microvesicle loaded with ultrasonic imaging agent by a double emulsification-solvent volatilization method, and adding 5-15mL of the gene-loaded nanoparticle solution to obtain the ultrasonic imaging microvesicle coated with the gene-loaded nanoparticles.
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