CN110917346B - Method for biomimetic simulated synthesis of photothermal tumor combined treatment nano preparation - Google Patents
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Abstract
The invention discloses a method for biomimetic simulated synthesis of a photothermal tumor combined treatment nano preparation. The main steps comprise 1) preparing PLGA organic solution; 2) Synthesizing (SEV + aCD 47) @ PLGA nano-particles by a thin film hydration method. Sendai virus (SEV) can activate organism immunity, and kill and eliminate tumor in all directions. The aCD47 can block a signaling pathway between CD47 and SIRPa, and activate the ability of macrophages to phagocytose tumor cells. Aggregation-induced emission (AIE), which is highly efficient in its aggregated state, visualizes the tumor treatment process. PLGA has good biocompatibility, no toxicity, and good properties of encapsulation and film formation, and is widely applied to the fields of pharmacy, medical engineering materials and modern industry. The composite nano-particle for the collective virus immunotherapy can improve the effect of tumor therapy.
Description
Technical Field
The invention relates to a preparation method of a nano preparation, in particular to a method for biomimetically simulating and synthesizing a photothermal tumor combined treatment nano preparation by a strategy of coating Sendai virus (SEV) and CD47 antibodies with PLGA.
Background
Currently, tumors are one of the most threatening diseases to humans in the world. Immunotherapy achieves the effect of treating cancer by activating the immune system of a human body and killing cancer cells and tumor tissues by means of the autoimmune function. Therefore, immunotherapy is one of the most effective methods for treating tumors. In view of the fact that the inactivated sendai virus (SEV) can be used as a foreign substance to stimulate the whole body immune system to generate cytokine storm (such as interferon, tumor necrosis factor, various interleukins and the like) in vivo, and enables dendritic cells (DC cells, presenting tumor antigens), macrophages (cells, phagocytic tumor cells) and natural killer cells (NK cells, killing tumor cells) and the like which play an important role in the anti-tumor treatment process to be activated in a large quantity, the proliferation efficiency of T cells is greatly improved subsequently. The composite nanometer preparation based on Sendai virus can activate the immune function of the organism systemically, cause all-round killing and removal to the tumor, and compared with the traditional tumor treatment method, the strategy ensures that the tumor treatment is more efficient.
CD47 is a protein expressed on the cell surface of macrophages, erythrocytes and the like, and when the CD47 is expressed on the cell surface of macrophages, the CD47 and SIRPa form a protein channel. SIRPa is expressed on tumor cells, and the pathway can cause macrophages to lose the capability of killing cancer cells. The addition of aCD47 can block the channel formed by CD47 and SIRPa and activate the anti-cancer capability of macrophages.
PLGA is formed by random polymerization of two monomers, namely lactic acid and glycolic acid, is a degradable functional polymer organic compound, the degradation products are lactic acid and glycolic acid, and are byproducts of human metabolic pathways, so that the PLGA does not have toxic or side effect when being applied to medicines and biological materials, has good biocompatibility, no toxicity and good encapsulation and film forming performances, and is widely applied to the fields of pharmacy, medical engineering materials and modern industry.
Biomimetic synthesis, as an effective strategy for preparing nanoparticles emerging in recent years, has several advantages: 1) The reaction condition is mild, no organic solvent participates in the whole process, and the synthesis can be called green synthesis; 2) The product property can be accurately regulated and controlled; 3) The biocompatibility is good; 4) Wide raw material source, relatively low cost and good repeatability. Solves the problems of complicated process, strict environmental requirement and certain pollution of the traditional synthetic method.
Disclosure of Invention
The invention provides a method for biomimetically simulating and synthesizing photothermal tumor combined treatment nano preparation by using a strategy of encapsulating Sendai virus (SEV) and CD47 antibodies by PLGA (poly lactic-co-glycolic acid) to overcome the defects of the prior art
The technical scheme of the invention is a method for biomimetically simulating and synthesizing photothermal tumor combined treatment nano preparation, which adopts a strategy of coating Sendai virus (SEV) and CD47 antibody by PLGA, and comprises the following specific steps:
1) Weighing PLGA material, adding dichloromethane, and ultrasonically dissolving to obtain PLGA organic solution with the concentration of 0.25-1 mg/ml;
2) Weighing AIE material, adding dichloromethane, and ultrasonically dissolving to obtain AIE organic solution with concentration of 0.5-2 mg/ml;
3) Synthesizing (SEV + aCD 47) @ PLGA nano-particles by a thin film hydration method.
(1) Placing a single-mouth bottle containing 2ml of PLGA solution on an ultrasonic crusher, and starting ultrasonic treatment for 10min at 0 ℃ and at a frequency of 3s and 1s;
(2) Adding 80uL of AIE solution and 1ml of PVA aqueous solution dropwise while carrying out ultrasonic treatment, and continuing ultrasonic treatment until the mixture is completely mixed;
(3) The sonicated product was immediately added to a round bottom flask and rotary evaporated on a rotary evaporator until the dichloromethane and water were all evaporated to dryness and a thin film formed at the bottom of the flask.
(4) Adding 1mLSEV virus into a round-bottomed bottle filled with liposome, adding a proper amount of diluted CD47 antibody, blowing, suspending and stirring until uniform mixing to finally obtain (SEV + aCD 47) @ PLGA nanoparticles.
The invention has the advantages that:
1. sendai virus SEV can activate cellular immunity for anti-tumor therapy.
2. The aCD47 can block a signaling pathway between CD47 and SIRPa, and activate the capability of macrophages to phagocytose tumor cells.
3. Aggregation-induced emission (AIE), which is highly efficient in its aggregated state, visualizes the tumor treatment process.
4. PLGA has good biocompatibility, no toxicity, and good properties of encapsulation and film formation, and is widely applied to the fields of pharmacy, medical engineering materials and modern industry.
5. The tumor combined treatment nano preparation with the Sendai virus (SEV) and CD47 antibodies encapsulated by PLGA combines the antibodies with tumor immunotherapy, and has obvious effect.
Drawings
FIG. 1: (SEV + aCD 47) @ PLGA nanoparticles stimulate interferon production.
Detailed Description
The invention is further described below with reference to the following figures and specific examples.
Example 1
1) Accurately weighing 5mg of PLGA material, adding 20mL of dichloromethane, and performing ultrasonic dissolution to obtain a PLGA organic solution with the concentration of 0.25 mg/mL.
2) 5mg of AIE material was weighed out accurately, and dissolved by sonication after adding 10mL of dichloromethane to obtain an AIE organic solution with a concentration of 0.5 mg/mL.
3) The method for synthesizing (SEV + aCD 47) @ PLGA nano-particles by using a film hydration method comprises the following steps:
(1) Placing a single-mouth bottle containing 2ml of PLGA solution on an ultrasonic crusher, and starting ultrasonic treatment for 10min at 0 ℃ and at a frequency of 3s and 1s;
(2) Adding 80uL of AIE solution and 1ml of PVA aqueous solution dropwise while carrying out ultrasonic treatment, and continuing ultrasonic treatment until the mixture is completely mixed;
(3) The sonicated product was immediately added to a round bottom flask and rotary evaporated on a rotary evaporator until the dichloromethane and water were all evaporated to dryness and a thin film formed at the bottom of the flask.
(4) Adding 1ml of LSEV virus into a round-bottomed bottle filled with liposome, adding 1ml of 2mg/ml of CD47 antibody, and stirring by blowing and suspending until the mixture is uniformly mixed to finally obtain (SEV + aCD 47) @ PLGA nano particles.
Example 2
1) Accurately weighing 10mg of PLGA material, adding 20mL of dichloromethane, and performing ultrasonic dissolution to obtain a PLGA organic solution with the concentration of 0.5 mg/mL.
2) 10mg of AIE material was accurately weighed, and dissolved by sonication after adding 10mL of dichloromethane to obtain an AIE organic solution with a concentration of 1 mg/mL.
3) The method for synthesizing (SEV + aCD 47) @ PLGA nano-particles by using a thin film hydration method comprises the following steps:
(1) Placing a single-mouth bottle containing 2ml of PLGA solution on an ultrasonic crusher, and starting ultrasonic treatment for 10min at 0 ℃ and at a frequency of 3s and 1s;
(2) Adding 80uL of AIE solution and 1ml of PVA aqueous solution dropwise while carrying out ultrasonic treatment, and continuing ultrasonic treatment until the mixture is completely mixed;
(3) The sonicated product was immediately added to a round bottom flask and rotary evaporated on a rotary evaporator until the dichloromethane and water were all evaporated to dryness and a thin film formed at the bottom of the flask.
(4) Adding 1ml of LSEV virus into a round-bottomed bottle filled with liposome, adding 1ml of diluted CD47 antibody, and blowing, suspending and stirring until uniform mixing to finally obtain (SEV + aCD 47) @ PLGA nanoparticles.
Example 3
1) Accurately weighing 20mg of PLGA material, adding 20mL of dichloromethane, and performing ultrasonic dissolution to obtain a PLGA organic solution with the concentration of 1 mg/mL.
2) 20mg of AIE material was weighed out accurately, and dissolved by sonication after adding 10mL of dichloromethane to give an AIE organic solution with a concentration of 2 mg/mL.
3) The method for synthesizing (SEV + aCD 47) @ PLGA nano-particles by using a thin film hydration method comprises the following steps:
(1) Placing a single-mouth bottle containing 2ml of PLGA solution on an ultrasonic crusher, and starting ultrasonic treatment for 10min at 0 ℃ and at a frequency of 3s and 1s;
(2) Adding 80uL of AIE solution and 1ml of PVA aqueous solution dropwise while carrying out ultrasonic treatment, and continuing ultrasonic treatment until the mixture is completely mixed;
(3) And immediately adding the ultrasonically treated product into a round-bottomed bottle, and performing rotary evaporation on a rotary evaporator until dichloromethane and water are completely evaporated to dryness to form a film at the bottom of the bottle.
(4) Adding 1ml LSEV virus into a round-bottomed bottle filled with liposome, adding 1ml of diluted CD47 antibody, blowing, suspending and stirring until uniform mixing to finally obtain (SEV + aCD 47) @ PLGA nano-particles.
Example 4
ELASA detection for stimulated interferon production
Taking dendritic cell DC growing in logarithmic phase, adjusting the concentration of cell suspension, and plating on a 6-well plate. The cells were allowed to adhere to the walls by placing at 37 ℃ in a 5% CO2 incubator and culturing for 24 hours.
Fresh medium containing (SEV + aCD 47) @ PLGA was added separately and the culture was continued for 48h.
Collecting culture medium supernatant for detecting cytokine, and centrifuging for 10min to remove supernatant precipitate.
Cells were incubated with diluted antibody for 1.5h, washed and incubated with horseradish peroxidase for 30min.
And (3) adding a developing solution for developing after washing the plate, adding a stop solution after the developing is finished, and detecting the absorbance at 450nm of an enzyme-labeling instrument.
Example 5
Taking the mouse breast cancer cells 4T1 growing in the logarithmic phase, adjusting the concentration of the cell suspension, and plating on a 6-well plate. The cells were allowed to adhere to the wall by placing in a 37 ℃ C., 5% CO2 incubator, and cultured for 24 hours. Fresh culture media containing different materials were added separately, and the culture was continued for 48h.
Meanwhile, 264.7 of mouse macrophage growing in logarithmic phase is taken, the concentration of cell suspension is adjusted, and the cell suspension is plated on a 6-well plate. The cells were allowed to adhere to the wall by placing in a 37 ℃ C., 5% CO2 incubator, and cultured for 24 hours.
The medium was removed and macrophages were stained with cell tracker green 30min, cell tracker dark red stained with 4T1 cells for 30min.
The 4T1 cells and macrophages are co-cultured in a serum-free culture medium, incubated for 3 hours at 37 ℃, and then observed for phagocytosis effect by a confocal microscope.
Claims (1)
1. A method for biomimetically simulating and synthesizing a photothermal tumor combined treatment nano preparation is characterized in that a strategy of coating Sendai virus (SEV) and CD47 antibodies by PLGA (polylactic-co-glycolic acid) comprises the following specific steps:
1) Weighing PLGA material, adding dichloromethane, and ultrasonically dissolving to obtain PLGA organic solution with the concentration of 0.25-1 mg/ml;
2) Weighing AIE material, adding dichloromethane, and ultrasonically dissolving to obtain AIE organic solution with concentration of 0.5-2 mg/ml;
3) Synthesizing (SEV + aCD 47) @ PLGA nano-particles by a thin film hydration method, which comprises the following steps:
(1) Placing a single-mouth bottle containing 2ml of PLGA solution on an ultrasonic crusher, and starting ultrasonic treatment for 10min at 0 ℃ and at a frequency of 3s and 1s;
(2) Adding 80uL of AIE solution and 1ml of PVA aqueous solution dropwise while carrying out ultrasonic treatment, and continuing to carry out ultrasonic treatment until the mixture is completely mixed;
(3) Immediately adding the ultrasonic-treated product into a round-bottomed bottle, and performing rotary evaporation on a rotary evaporator until dichloromethane and water are completely evaporated to dryness to form a film at the bottom of the bottle;
(4) Adding 1ml of LSEV virus into a round-bottomed bottle filled with liposome, adding 1ml of diluted CD47 antibody, and blowing, suspending and stirring until uniform mixing to finally obtain (SEV + aCD 47) @ PLGA nanoparticles.
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CN110179836A (en) * | 2019-05-08 | 2019-08-30 | 天津大学 | The synthetic method of the tumor combined therapeutic nanometer formulation of CuS mineralising sendai virus |
CN110179837A (en) * | 2019-05-08 | 2019-08-30 | 天津大学 | The synthetic method of the visualization guidance tumour combined immunization treatment nanometer formulation of Gd:CuS mineralising influenza virus |
CN110201005A (en) * | 2019-05-08 | 2019-09-06 | 天津大学 | The synthetic method of the visualization guidance tumour combined immunization treatment nanometer formulation of Gd:CuS mineralising sendai virus |
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