CN110917346A

CN110917346A - Method for biomimetic simulated synthesis of photothermal tumor combined treatment nano preparation

Info

Publication number: CN110917346A
Application number: CN201911206087.7A
Authority: CN
Inventors: 郑斌; 彭文畅; 明东; 刘爽; 甘霖
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2019-11-30
Filing date: 2019-11-30
Publication date: 2020-03-27
Anticipated expiration: 2039-11-30
Also published as: CN110917346B

Abstract

The invention discloses a method for biomimetic simulated synthesis of a photothermal tumor combined treatment nano preparation. The main steps include 1) preparation of PLGA organic solution; 2) synthesizing (SEV + aCD47) @ 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 the signal path of 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

Method for biomimetic simulated synthesis of photothermal tumor combined treatment nano preparation

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 human immune system and killing cancer cells and tumor tissues by means of 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, and can kill and eliminate tumors in all directions, 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 macrophage, erythrocyte, etc. when expressed on the macrophage surface, 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 (polylactic-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, and the method comprises the following specific steps through a strategy of coating Sendai virus (SEV) and CD47 antibodies by PLGA:

1) weighing a PLGA material, adding dichloromethane, and then carrying out ultrasonic dissolution to obtain a 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 + aCD47) @ PLGA nano-particles by using 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 3s and 1 s;

(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 is achieved, and finally obtaining (SEV + aCD47) @ PLGA nano particles.

The invention has the advantages that:

1. sendai virus SEV can activate cellular immunity for anti-tumor therapy.

2. The aCD47 can block the signal path of CD47 and SIRPa, and activate the ability 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 + aCD47) @ 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 accurately weighed, added with 10mL of dichloromethane and dissolved by sonication to give an AIE organic solution with a concentration of 0.5 mg/mL.

3) The method for synthesizing (SEV + aCD47) @ PLGA nano-particles by using a thin film hydration method comprises the following steps:

(4) Adding 1ml of LSEV virus into a round-bottomed bottle filled with liposome, adding 1ml of 2mg/ml CD47 antibody, and blowing, suspending and stirring until uniform mixing to finally obtain (SEV + aCD47) @ PLGA nano particles.

Example 2

1) 10mg of PLGA material is accurately weighed, 20mL of dichloromethane is added, and ultrasonic dissolution is carried out to obtain 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.

(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 + aCD47) @ 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.

(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 + aCD47) @ PLGA nano-particles.

Example 4

ELASA detection for stimulating interferon production

Dendritic cell DCs grown in log phase were taken, cell suspension concentration was adjusted, and plated on 6-well plates. The cells were attached to the wall in a 37 ℃ 5% CO2 incubator for 24 hours.

Fresh medium containing (SEV + aCD47) @ PLGA was added separately and the culture was continued for 48 h.

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 30 min.

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

Mouse breast cancer cells grown in logarithmic phase 4T1 were taken, cell suspension concentration was adjusted, and plated on 6-well plates. The cells were attached to the wall in a 37 ℃ 5% CO2 incubator for 24 hours. Fresh culture media containing different materials were added separately and the culture was continued for 48 h.

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 attached to the wall in a 37 ℃ 5% CO2 incubator for 24 hours.

The medium was removed and macrophages were stained with cell tracker green for 30min and 4T1 cell tracker dark red for 30 min.

4T1 cells and macrophages were co-cultured in serum-free medium, incubated at 37 ℃ for 3h, and then observed for phagocytosis effect by confocal microscopy.

Claims

1. A method for biomimetically simulating and synthesizing photothermal tumor combined therapy nanometer preparation is characterized in that a strategy of coating Sendai virus (SEV) and CD47 antibodies by PLGA comprises the following specific steps:

2. The method for biomimetically simulating and synthesizing photothermal tumor combination therapy nano preparation according to claim 1, wherein the step 3) is specifically as follows:

(1) placing the single-mouth bottle containing the PLGA solution on an ultrasonic crusher, and starting ultrasonic treatment for 10min at 0 ℃ and 3s and 1 s;

(3) immediately adding the ultrasonic 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 SEV virus into a round-bottomed bottle filled with liposome, adding the diluted CD47 antibody, and blowing, suspending and stirring until uniform mixing to finally obtain (SEV + aCD47) @ PLGA nanoparticles.