CN112843016A - Artificial antigen presenting nanoparticles and preparation method and application thereof - Google Patents
Artificial antigen presenting nanoparticles and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biological medicines, in particular to an artificial antigen presenting nanoparticle, which mainly comprises PEG and CD47-Fc modified PLGA nanoparticles, a tumor target antigen and an anti-CD 28 antibody, wherein the tumor target antigen can change according to the change of tumor cells, so that activated and amplified T cells are ensured to have specific killing effect on corresponding tumor cells, and meanwhile, the PEG and CD47-Fc modification shown by the PLGA nanoparticles can ensure that the artificial antigen presenting nanoparticle has the advantages of long circulation in vivo and phagocytosis resistance. The invention also relates to a preparation method of the artificial antigen presenting nanoparticles, which has simple operation steps, high repeatability and short preparation period and is beneficial to mass production.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to an artificial antigen presenting nanoparticle and a preparation method and application thereof.
Background
Although Antigen Presenting Cells (APCs), such as Dendritic Cells (DCs), are effective tools for amplifying antigen-specific T cells in vitro and in vivo, they still have many drawbacks and need to overcome, such as difficulty in amplification technology, long amplification period, high capital cost, non-specific stimulation, and biosafety caused by living cells. With the rapid development of non-cellular artificial antigen presenting cell technology, the concept of nanoscale artificial antigen presenting cells (aapcs) has been proposed in recent years. Aapcs, while having the advantages of better biodistribution and lower embolization rates than cell-sized aapcs, are more readily taken up by macrophages, have less surface area in contact with T cells, and result in a relatively weak ability of aapcs to expand T cells. Therefore, how to optimize aapcs, improve the in vivo circulation ability and anti-phagocytosis ability of aapcs to improve the T cell expansion/activation ability of aapcs, and further induce more complete immune response is a problem to be solved.
Disclosure of Invention
One of the purposes of the invention is to provide an artificial antigen presenting nanoparticle with long circulation and anti-phagocytosis effect.
In order to achieve the purpose, the invention adopts the technical scheme that: an artificial antigen presenting nanoparticle with long circulation and anti-phagocytosis effects comprises a PEG PLGA nanoparticle, wherein the surface of the PEG PLGA nanoparticle is modified with CD47-Fc, a tumor target antigen and an anti-CD 28 antibody to obtain the artificial antigen presenting nanoparticle.
Preferably, the artificial antigen-presenting nanoparticles have a particle size ranging from 20nm to 500nm, and the deviation in particle size between different artificial antigen-presenting nanoparticles is in the range of 0nm to 100 nm.
Preferably, the length of the PEG molecule modified on the surface of the PLGA nanoparticle is 800-6000.
Preferably, the weight part ratio of the PLGA nanoparticles to the PEG is 1.5-2.0: 0.03-0.01; the weight part ratio of the PEG PLGA nanoparticles to the CD47-Fc, the tumor target antigen and the anti-CD 28 antibody is 5-10: 0.03-0.01: 0.03-0.01.
When intravenous administration is carried out, the PEG and CD47-Fc molecules on the surface of the artificial antigen presenting nanoparticle can ensure that the nanoparticle has longer circulation time in vivo, and the CD47-Fc molecules on the surface of the nanoparticle interact with signal regulatory protein-alpha on phagocytes, so that the phagocytosis of the artificial antigen presenting nanoparticle by the phagocytes is reduced, and T cells can be continuously and efficiently activated and amplified in vivo. The surface modification density of the nanoparticles and the contact area of the nanoparticles and T cells can be easily adjusted by adjusting the particle size and the particle size uniformity of the artificial antigen presenting nanoparticles, and the adjustment can ensure that the nanoparticles can play a more efficient role in activating and amplifying the T cells in vivo.
The second purpose of the invention is to provide a preparation method of the artificial antigen presenting nanoparticle with long circulation and anti-phagocytosis effect.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for preparing artificial antigen presenting nano-particles with long circulation and anti-phagocytosis effect comprises the following operation steps: obtaining PEG PLGA nano-particles, and then modifying the surface of the PEG PLGA nano-particles with tumor targeting antigens, anti-CD 28 antibodies and CD47-Fc under the activation of a surfactant to obtain the artificial antigen presenting nano-particles.
Preferably, the surfactant is (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride or N-hydroxysuccinimide.
Preferably, the method specifically comprises the following steps:
1) mixing 5-10 parts by weight of PEG PLGA nanoparticle solution with 0.2-0.4 part by weight of EDC & HCl solution and 0.2-0.4 part by weight of NHS solution, and stirring at room temperature for 1-4 hours; then centrifuging the mixture under the conditions of 8000-12000rpm for 5-10 minutes to obtain a precipitate B;
2) dissolving the precipitate B in 5-20 weight parts of 1% polyethyleneimine solution, stirring at room temperature for 4-12 hr, centrifuging for precipitation under 8000-12000rpm for 5-10 min to obtain precipitate C;
3) placing 5-10 parts by weight of the precipitate C in a 1mL EP tube, resuspending the precipitate C with 0.1-0.5 part by weight of PBS, then placing 0.03-0.01 part by weight of tumor target antigen, 0.03-0.01 part by weight of anti-CD 28 antibody and 0.03-0.01 part by weight of CD47-Fc, then placing the EP tube on a rotating disc for rotating reaction for 12-16 hours at the reaction temperature of 4 ℃, further sealing the reaction liquid with 2-5 parts by weight of bovine serum albumin for 12-16 hours at the reaction temperature of room temperature, finally centrifuging the sealed reaction liquid under the conditions of 8000-12000rpm for 5-10 minutes, and washing with deionized water for 3 times to obtain the final artificial antigen presenting nanoparticles.
Preferably, the preparation method of the PEGylated PLGA nanoparticle solution comprises the following steps:
1) dissolving 5-10 parts by weight of PLGA nanoparticles in 6-12 parts by weight of isotonic buffer salt solution which is 0.1M MES with the pH value of 4.5-6.0;
2) taking 0.2-0.4 weight part of EDC & HCl solution and 0.2-0.4 weight part of NHS solution, stirring for 1-2 hours at room temperature, and then carrying out centrifugal precipitation under the conditions of 8000-12000rpm for 5-10 minutes to obtain a precipitate A;
3) dissolving precipitate A in 4-8 weight parts of deionized water at room temperature, and adding 0.01-0.03 weight part of NH2PEG-COOH, standing for 8-12 hours, centrifuging the reactant under the centrifugation condition of 8000-12000rpm for 5-10 minutes, and resuspending the centrifuged product with 4-8 parts by weight of deionized water to obtain the PEG PLGA nanoparticle solution.
Preferably, the preparation method of the PLGA nanoparticles comprises the following steps:
1) dissolving 5-10 parts by weight of PLGA in 5-10 parts by weight of dichloromethane, and carrying out ultrasonic emulsification under the ultrasonic condition that the ultrasonic treatment is stopped for 10 seconds every 10-15 seconds and is continued for 5-10 cycles to form PLGA solution;
2) adding 30-60 parts by weight of 1% PVA into the PLGA solution after ultrasonic emulsification, and performing ultrasonic emulsification under the ultrasonic condition that the ultrasonic treatment is stopped for 10 seconds every 10-15 seconds and is continued for 5-10 cycles to obtain an emulsion A;
3) adding 60-120 parts by weight of 0.5% PVA into the emulsion A, and then carrying out ultrasonic emulsification under the ultrasonic condition that the mixture stops for 10 seconds every 10-15 seconds and lasts for 5-10 cycles to obtain emulsion B;
4) stirring the emulsion B for 4-12h at room temperature by using a magnetic stirrer to obtain emulsion C;
5) and (3) carrying out centrifugal precipitation by using excessive deionized water under the centrifugal condition of 4000-8000rpm for 5-10 minutes, repeating for 3-5 times, and carrying out freeze drying to obtain the PLGA nanoparticles.
The method has the advantages of simple operation steps, high repeatability and short preparation period, and is beneficial to mass production. The particle size and the particle size uniformity of the artificial antigen presenting nanoparticles can be controlled by adjusting ultrasonic emulsification parameters, so that the surface modification ratio of the artificial antigen presenting nanoparticles is ensured to be at a higher level, the nanoparticles have longer circulation time in vivo, phagocytosis of the artificial antigen presenting nanoparticles by phagocytes can be reduced, and T cells can be continuously and efficiently activated and amplified in vivo.
The invention also aims to provide the application of the artificial antigen presenting nanoparticle with long circulation and anti-phagocytosis effect.
In order to achieve the purpose, the invention adopts the technical scheme that: the application of the artificial antigen presenting nanoparticles with long circulation and anti-phagocytosis effect is characterized in that the artificial antigen presenting nanoparticles are used for preparing anti-tumor drugs.
The artificial antigen presenting nano particle can be independently prepared into an antitumor drug, can also be used as an active ingredient to be prepared into the antitumor drug together with other medicinal ingredients, and can continuously and efficiently activate and amplify T cells in vivo after intravenous administration so as to induce perfect immune reaction and clear the tumor cells in vivo.
In summary, compared with the prior art, the invention has the following beneficial effects:
1. when intravenous administration is carried out, the PEG and CD47-Fc molecules on the surface of the artificial antigen presenting nanoparticle can ensure that the nanoparticle has longer circulation time in vivo, and the CD47-Fc molecules on the surface of the nanoparticle interact with signal regulatory protein-alpha on phagocytes, so that the phagocytosis of the artificial antigen presenting nanoparticle by the phagocytes is reduced, and T cells can be continuously and efficiently activated and amplified in vivo. The surface modification density of the nanoparticles and the contact area of the nanoparticles and T cells can be easily adjusted by adjusting the particle size and the particle size uniformity of the artificial antigen presenting nanoparticles, and the adjustment can ensure that the nanoparticles can play a more efficient role in activating and amplifying the T cells in vivo;
2. the method has the advantages of simple operation steps, high repeatability and short preparation period, and is beneficial to mass production. The particle size and the particle size uniformity of the artificial antigen presenting nanoparticles can be controlled by adjusting ultrasonic emulsification parameters, so that the surface modification ratio of the artificial antigen presenting nanoparticles is ensured to be at a higher level, the nanoparticles have longer circulation time in vivo, phagocytosis of the artificial antigen presenting nanoparticles by phagocytes can be reduced, and T cells can be continuously and efficiently activated and amplified in vivo;
3. the artificial antigen presenting nano particle can be independently prepared into an antitumor drug, can also be used as an active ingredient to be prepared into the antitumor drug together with other medicinal ingredients, and can continuously and efficiently activate and amplify T cells in vivo after intravenous administration so as to induce perfect immune reaction and clear the tumor cells in vivo.
Drawings
FIG. 1 is a graph showing the results of an antitumor drug efficacy test in the artificial antigen-presenting nanoparticle of the present invention;
FIG. 2 is a graph showing the survival rate of mice in the in vivo antitumor drug efficacy test using the artificial antigen-presenting nanoparticles of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Example 1:
a preparation method of artificial antigen presenting nanoparticles with long circulation and anti-phagocytosis effect specifically comprises the following steps:
1) mixing 5 parts by weight of PEG PLGA nanoparticle solution with 0.2 part by weight of EDC & HCl solution and 0.2 part by weight of NHS solution, and stirring at room temperature for 1 hour; centrifuging the mixture at 8000rpm for 5 min to obtain precipitate B;
2) dissolving the precipitate B in 5 parts by weight of 1% polyethyleneimine solution, stirring at room temperature for 4 hours, and centrifuging at 8000rpm for 5 minutes to obtain precipitate C;
3) placing 5 parts by weight of the precipitate C in a 1mL EP tube, resuspending the precipitate C with 0.1 part by weight of PBS, then placing 0.03 part by weight of tumor target antigen, 0.03 part by weight of anti-CD 28 antibody and 0.03 part by weight of CD47-Fc on the EP tube on a rotating disc, rotating and reacting for 12 hours at the reaction temperature of 4 ℃, then further sealing the reaction solution with 2 parts by weight of bovine serum albumin for 12 hours at the reaction temperature of room temperature, finally centrifuging the sealed reaction solution at the centrifugation condition of 8000rpm for 5 minutes, and washing with deionized water for 3 times to obtain the final artificial antigen presenting nanoparticle.
Example 2:
a preparation method of artificial antigen presenting nanoparticles with long circulation and anti-phagocytosis effect specifically comprises the following steps:
1) mixing 10 parts by weight of PEG PLGA nanoparticle solution with 0.4 part by weight of EDC & HCl solution and 0.4 part by weight of NHS solution, and stirring at room temperature for 4 hours; centrifuging the mixture at 12000rpm for 10 min to obtain precipitate B;
2) dissolving 20 parts by weight of 1% polyethyleneimine solution into the precipitate B, stirring at room temperature for 12 hours, and centrifuging and precipitating at 12000rpm for 10 minutes to obtain a precipitate C;
3) placing 10 parts by weight of the precipitate C in a 1mL EP tube, resuspending the precipitate C with 0.5 part by weight of PBS, then placing 0.01 part by weight of tumor target antigen, 0.01 part by weight of anti-CD 28 antibody and 0.01 part by weight of CD47-Fc on a rotating disc, rotating the EP tube on the rotating disc for 16 hours at the reaction temperature of 4 ℃, then further sealing the reaction solution with 5 parts by weight of bovine serum albumin for 16 hours at the reaction temperature of room temperature, finally centrifuging the sealed reaction solution at the centrifugation condition of 12000rpm for 10 minutes, and washing with deionized water for 3 times to obtain the final artificial antigen presenting nanoparticles.
Example 3:
the difference from example 1 is that the preparation method of the pegylated PLGA nanoparticle solution comprises the following steps:
1) taking 5 parts by weight of PLGA nanoparticles, and dissolving the PLGA nanoparticles in 6 parts by weight of isotonic buffer salt solution, wherein the isotonic buffer salt solution is 0.1M MES with the pH value of 4.5;
2) taking 0.2 weight part of EDC & HCl solution and 0.2 weight part of NHS solution, stirring at room temperature for 1 hour, and then carrying out centrifugal precipitation under the centrifugal condition of 8000rpm for 5 minutes to obtain a precipitate A;
3) 4 parts by weight of deionized water were taken to dissolve the precipitate A completely at room temperature, followed by addition of 0.01 part by weight of NH2And (3) PEG-COOH, standing for 8 hours, centrifuging the reactant under the centrifugation condition of 8000rpm for 5 minutes, and resuspending the centrifuged product with 4 parts by weight of deionized water to obtain the PEG PLGA nanoparticle solution.
Example 4:
the difference from example 1 is that the preparation method of the pegylated PLGA nanoparticle solution comprises the following steps:
1) taking 10 parts by weight of PLGA nanoparticles, and dissolving the PLGA nanoparticles in 12 parts by weight of isotonic buffer salt solution which is 0.1M MES with the pH value of 6.0;
2) taking 0.4 weight part of EDC & HCl solution and 0.4 weight part of NHS solution, stirring at room temperature for 2 hours, and then carrying out centrifugal precipitation under the centrifugal condition of 12000rpm for 10 minutes to obtain a precipitate A;
3) the precipitate A was completely dissolved at room temperature with 8 parts by weight of deionized water, followed by addition of 0.03 parts by weight of NH2And (3) PEG-COOH, standing for 12 hours, centrifuging the reactant at 12000rpm for 10 minutes, and resuspending the centrifuged product with 8 parts by weight of deionized water to obtain the PEG PLGA nanoparticle solution.
Example 5:
the difference from example 3 is that the preparation method of the PLGA nanoparticle comprises the following steps:
1) dissolving 5 parts by weight of PLGA in 5 parts by weight of dichloromethane, and carrying out ultrasonic emulsification under the ultrasonic condition that the ultrasonic treatment is stopped for 10 seconds every 10 seconds, and continuing for 5 cycles to form a PLGA solution;
2) adding 30 parts by weight of 1% PVA into the PLGA solution after ultrasonic emulsification, and performing ultrasonic emulsification under the ultrasonic condition that the ultrasonic treatment is stopped for 10 seconds every 10 seconds and is continued for 5 cycles to obtain an emulsion A;
3) adding 60 parts by weight of 0.5% PVA into the emulsion A, and then carrying out ultrasonic emulsification under the ultrasonic condition that the mixture stops for 10 seconds every 10 seconds of ultrasonic treatment and lasts for 5 cycles to obtain emulsion B;
4) stirring the emulsion B for 4 hours at room temperature by using a magnetic stirrer to obtain emulsion C;
5) and (3) carrying out centrifugal precipitation by using excessive deionized water under the centrifugal condition of 4000rpm for 5 minutes, repeating for 3 times, and carrying out freeze drying to obtain the PLGA nano-particles.
Example 6:
the difference from example 3 is that the preparation method of the PLGA nanoparticle comprises the following steps:
1) dissolving 10 parts by weight of PLGA in 10 parts by weight of dichloromethane, and carrying out ultrasonic emulsification under the ultrasonic condition that the ultrasonic treatment is stopped for 10 seconds every 15 seconds, and continuing for 10 cycles to form a PLGA solution;
2) adding 60 parts by weight of 1% PVA into the PLGA solution after ultrasonic emulsification, and performing ultrasonic emulsification under the ultrasonic condition that the ultrasonic treatment is stopped for 10 seconds every 15 seconds and is continued for 10 cycles to obtain an emulsion A;
3) adding 120 parts by weight of 0.5% PVA into the emulsion A, and then carrying out ultrasonic emulsification under the ultrasonic condition that the ultrasonic treatment is stopped for 10 seconds every 15 seconds, and continuing for 10 cycles to obtain emulsion B;
4) stirring the emulsion B for 12 hours at room temperature by using a magnetic stirrer to obtain emulsion C;
5) and (3) carrying out centrifugal precipitation by using excessive deionized water under the centrifugal condition of 8000rpm for 10 minutes, repeating for 5 times, and carrying out freeze drying to obtain the PLGA nano-particles.
Example 7:
a method for preparing artificial antigen presenting nanoparticles with long circulation and anti-phagocytosis effect comprises the following steps:
the method comprises the following steps: dissolving 10mg PLGA in 6mL dichloromethane, and carrying out ultrasonic treatment on the solution by using an ultrasonic crusher for 30s with 40% amplitude;
step two: mixing the emulsified PLGA solution with 50mL of 1% PVA solution, and carrying out ultrasonic emulsification, wherein the emulsification parameters are 10 seconds for 10 seconds per ultrasonic treatment and 5 cycles; obtaining emulsion A;
step three: slowly pouring the emulsion A into 100mL of 0.5% PVA solution, stirring for 12 hours at room temperature by using a magnetic stirring instrument, and finally ensuring that the organic solution is completely evaporated to obtain a solution B;
step four: centrifuging the solution B (4000rpm for 5 minutes) and collecting the supernatant, repeating the centrifugation for 3 times to ensure that no precipitate is generated after the final supernatant is centrifuged (4000rpm for 5 minutes), thus obtaining a solution C;
step five: solution C was centrifuged (12000rpm, 10 min), the pellet was collected and resuspended in 4mL of deionized water, and repeated 3 times for removal of excess PVA. Freeze-drying the solution to obtain PLGA nanoparticles;
step six: taking 10.0mg of the freeze-dried PLGA nanoparticles obtained in the fifth step, and dissolving the freeze-dried PLGA nanoparticles in 10mL of isotonic buffer salt solution (0.1M MES, pH 5.0) to obtain a solution D;
step seven: to the above solution D, EDC (0.2mg) and NHS (0.05mg) were added and stirred at room temperature for 2 hours, followed by centrifugation (12000rpm, 10 minutes) of the mixture for removal of excess EDC and NHS to give precipitate a;
step eight: resuspending the precipitate A in 5mL of deionized water, adding 2mg of NH2-PEG2000-COOH at room temperature, standing for reaction for 12 hours, centrifuging the reactant (12000rpm for 10 minutes) to remove unbound NH2-PEG2000-COOH, and finally resuspending the reactant with deionized water to obtain PEG-modified PLGA nanoparticles with the concentration of 2 mg/mL;
step nine: taking 1mL of the PEG PLGA nanoparticles obtained in the step eight, sterilizing the PEG PLGA nanoparticles for 30 minutes by ultraviolet, and then placing the nanoparticles in a water bath for ultrasonic treatment for 5 minutes to obtain a solution E;
step ten: to the above solution E, EDC (0.2mg) and NHS (0.05mg) were added and stirred at room temperature for 2 hours, followed by centrifugation (12000rpm, 10 minutes) of the mixture for removal of excess EDC and NHS to give a precipitate B;
step eleven: adding 10mL of 1% Polyethyleneimine (PEI) solution into the precipitate B for resuspension, stirring at room temperature for 4 hours, and centrifuging (12000rpm for 10 minutes) after the reaction is finished for removing free PEI to obtain a precipitate C;
step twelve: 10mg of the pellet C was placed in a 1mL EP tube and resuspended with 100. mu.L of PBS, followed by addition of 0.03mg of tumor targeting antigen (GPC-3), 0.03mg of anti-CD 28 antibody and 0.03mg of CD 47-Fc; and then placing the EP tube on a rotating disc for rotating reaction (12 hours and 4 ℃), further sealing the reaction liquid by using 2mL of Bovine Serum Albumin (BSA) (12 hours and room temperature), finally centrifuging the sealed reaction liquid (12000rpm and 10 minutes), and washing the reaction liquid for 3 times by using deionized water to obtain the final artificial antigen presenting nanoparticles.
Example 8:
the difference from example 7 is that:
the method comprises the following steps: dissolving 8mg of PLGA in 6mL of dichloromethane, and carrying out ultrasonic treatment on the solution by using an ultrasonic crusher for 45s at 50% of amplitude;
step two: mixing the emulsified PLGA solution with 60mL of 1% PVA solution, and carrying out ultrasonic emulsification, wherein the emulsification parameters are 10 seconds for 15 seconds per ultrasonic treatment and 8 cycles; to obtain emulsion A
Step three: slowly pouring the emulsion A into 100mL of 0.5% PVA solution, stirring for 12 hours at room temperature by using a magnetic stirring instrument, and finally ensuring that the organic solution is completely evaporated to obtain a solution B;
step four: centrifuging the solution B (4000rpm for 5 minutes) and collecting the supernatant, repeating the centrifugation for 3 times to ensure that no precipitate is generated after the final supernatant is centrifuged (4000rpm for 5 minutes), thus obtaining a solution C;
step five: solution C was centrifuged (12000rpm, 10 min), the pellet was collected and resuspended in 2mL of deionized water and repeated 3 times for removal of excess PVA. And freeze-drying the solution to obtain the PLGA nanoparticles.
Example 9:
the difference from example 7 is that:
step six: 10mg of the lyophilized PLGA nanoparticles obtained in step five were dissolved in 10mL of isotonic buffered saline (0.1M MES, pH 6.0) to obtain solution D.
Step seven: to the above solution D, EDC (0.3mg) and NHS (0.1mg) were added and stirred at room temperature for 1 hour, and then the mixture was centrifuged (12000rpm, 10 minutes) for removing excess EDC and NHS, to give precipitate A.
Step eight: and (3) resuspending the precipitate A in 5mL of deionized water, adding 2mg of NH2-PEG1000-COOH at room temperature, standing for reaction for 12 hours, centrifuging the reaction (12000rpm for 10 minutes) to remove unbound NH2-PEG1000-COOH, and finally resuspending the precipitate A in deionized water to obtain the PEG PLGA nanoparticles with the concentration of 2 mg/mL.
Step nine: taking 1mL of the PEG PLGA nanoparticles obtained in the step eight, sterilizing the PEG PLGA nanoparticles for 30 minutes by ultraviolet, and then placing the nanoparticles in a water bath for ultrasonic treatment for 5 minutes to obtain a solution E;
step ten: to the above solution E, EDC (0.3mg) and NHS (0.1mg) were added and stirred at room temperature for 2 hours, and then the mixture was centrifuged (12000rpm, 10 minutes) for removing excess EDC and NHS, to give precipitate B.
Step eleven: 10mL of a 1% Polyethyleneimine (PEI) solution was added to the precipitate B, and the mixture was resuspended with stirring at room temperature for 8 hours, and after completion of the reaction, the mixture was centrifuged (12000rpm, 10 minutes) to remove free PEI, thereby obtaining a precipitate C.
Step twelve: 10mg of the pellet C was placed in a 1mL EP tube and resuspended with 100. mu.L of PBS, followed by addition of 0.01mg of tumor targeting antigen (GPC-3), 0.01mg of anti-CD 28 antibody and 0.01mg of CD 47-Fc; and then placing the EP tube on a rotating disc for rotating reaction (14 hours and 4 ℃), further sealing the reaction solution by using 2mL of Bovine Serum Albumin (BSA) (14 hours and room temperature), finally centrifuging the sealed reaction solution (12000rpm and 10 minutes), and washing for 3 times by using deionized water to obtain the final artificial antigen presenting nanoparticles.
Example 10:
the difference from example 7 is that:
step eight: and (3) resuspending the precipitate A in 5mL of deionized water, adding 0mg of NH2-PEG2000-COOH at room temperature, standing for reaction for 12 hours, centrifuging the reaction (12000rpm for 10 minutes) to remove unbound NH2-PEG2000-COOH, and finally resuspending the precipitate A in deionized water to obtain the PEG PLGA nanoparticles with the concentration of 2 mg/mL.
Step twelve: 10mg of the precipitate C was placed in a 1mL EP tube, and 100. mu.L of PBS was added for resuspension, followed by addition of 0.03mg of tumor targeting antigen (GPC-3), 0.03mg of anti-CD 28 antibody; and then placing the EP tube on a rotating disc for rotating reaction (12 hours and 4 ℃), further sealing the reaction liquid by using 2mL of Bovine Serum Albumin (BSA) (12 hours and room temperature), finally centrifuging the sealed reaction liquid (12000rpm and 10 minutes), and washing the reaction liquid for 3 times by using deionized water to obtain the final artificial antigen presenting nanoparticles.
Comparative example 1:
10mg of PLGA nanoparticles, 0.03mg of tumor target antigen (GPC-3), 0.03mg of anti-CD 28 antibody and 0.03mg of CD47-Fc were weighed and mixed, and dissolved in 1mL of sterile deionized water to obtain a mixed solution.
Comparative example 2:
in vivo antitumor drug effect test, 65 female C57BL/6 mice of 6 weeks old were housed in a pathogen-free facility, of which 5 were used as an environmental monitoring group, and 60 mice were inoculated with subcutaneous hepatoma cell (HepG2) tumor cells, and 100. mu.L of tumor fluid (containing 2X 10^6 tumor cells) was subcutaneously injected on the upper side of the left hind limb of each mouse, and 7 days after tumor implantation, the 60 mice were randomly divided into 6 groups, 4 of them were treated by injecting the artificial antigen-presenting nanoparticles of examples 7, 8, 9 and 10, respectively, as an experimental group A, B, C, D, 1 of the remaining 2 groups were treated by injecting the mixed solution of comparative example 1, as a control group A, and the remaining 1 group was treated by injecting PBS buffer, as a control group B, and the growth, systemic and neurotoxicity of the animal tumors were observed and the death status was recorded. The antitumor effect and the survival curve of the mice are shown in fig. 1 and fig. 2.
As can be seen from fig. 1 and 2, the compositions provided in experimental groups a-C have different inhibitory effects on tumor growth, and the tumor of control group B treated with PBS rapidly grows, while the tumor of tumor-bearing mice in experimental groups a-C all regressed or increased at a reduced rate and the survival time of the mice increased significantly, compared to control group B;
however, there was still a significant difference in drug efficacy between experimental groups a-C, with experimental group a being the best because the ratio of the components and the preparation process are most preferred in the preparation of the nanoparticles. Compared with the experimental group A, the ultrasonic emulsification parameters of the nanoparticles in the experimental group B are not optimal parameters, so that the obtained final particle size of the nanoparticles is smaller and nonuniform, and the subsequent surface modification ratio of the nanoparticles is lower, so that the anti-tumor effect is poor; the experimental group C is that the molecular weight of the selected PEG modifier is smaller, the circulation effect of the nanoparticles in vivo cannot be ensured, and simultaneously the addition amounts of CD47-Fc, the tumor target antigen and the anti-CD 28 antibody are also reduced, which are the reasons for directly causing the poor anti-tumor effect of the final nanoparticles.
Compared with experimental groups A-C, the preparation formula of the experimental group D does not contain PEG and CD47-Fc modification, so that the nanoparticles do not have the advantages of long circulation and phagocytosis resistance in vivo, and the nanoparticles are easily phagocytized by phagocytes in vivo or cleared by blood circulation; the control group A directly administers PLGA nanoparticles, PEG molecules, CD47-Fc and tumor target antigen in a free form, and obviously, the experimental results show that the mode can not completely exert the drug effect, and the experimental results show that the administration mode of the nanoparticles without long circulation and anti-phagocytosis function or the free drug is not suitable for in vivo administration.
While one embodiment of the present invention has been described in detail, the description is only a preferred embodiment of the present invention and should not be taken as limiting the scope of the invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
Claims (10)
1. An artificial antigen presenting nanoparticle with long circulation and anti-phagocytosis effect is characterized by comprising a PEG PLGA nanoparticle, wherein the surface of the PEG PLGA nanoparticle is modified with CD47-Fc, a tumor target antigen and an anti-CD 28 antibody to obtain the artificial antigen presenting nanoparticle.
2. The artificial antigen-presenting nanoparticle with long-circulating anti-phagocytosis according to claim 1, wherein the particle size of the artificial antigen-presenting nanoparticle is in the range of 20nm to 500nm, and the deviation of the particle size between different artificial antigen-presenting nanoparticles is in the range of 0nm to 100 nm.
3. The artificial antigen presenting nanoparticle with long circulation and anti-phagocytosis effect according to claim 1, wherein the molecular weight of PEG modified on the surface of the PLGA nanoparticle is 800-6000.
4. The artificial antigen-presenting nanoparticle with long circulation and anti-phagocytosis according to any one of claims 1 to 3, wherein the weight ratio of PLGA nanoparticles to PEG is 1.5-2.0: 0.03-0.01; the weight part ratio of the PEG PLGA nanoparticles to the CD47-Fc, the tumor target antigen and the anti-CD 28 antibody is 5-10: 0.03-0.01: 0.03-0.01.
5. A method for preparing long-circulating anti-phagocytosis artificial antigen-presenting nanoparticles, characterized by preparing long-circulating anti-phagocytosis artificial antigen-presenting nanoparticles according to any one of claims 1 to 3, comprising the steps of: obtaining PEG PLGA nano-particles, and then modifying the surface of the PEG PLGA nano-particles with tumor targeting antigens, anti-CD 28 antibodies and CD47-Fc under the activation of a surfactant to obtain the artificial antigen presenting nano-particles.
6. The method for preparing the artificial antigen-presenting nanoparticle with long circulation and anti-phagocytosis according to claim 5, wherein the surfactant is (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide.
7. The method for preparing the artificial antigen presenting nanoparticle with long circulation and anti-phagocytosis according to claim 6, comprising the following steps:
1) mixing 5-10 parts by weight of PEG PLGA nanoparticle solution with 0.2-0.4 part by weight of EDC & HCl solution and 0.2-0.4 part by weight of NHS solution, and stirring at room temperature for 1-4 hours; then centrifuging the mixture under the conditions of 8000-12000rpm for 5-10 minutes to obtain a precipitate B;
2) dissolving the precipitate B in 5-20 weight parts of 1% polyethyleneimine solution, stirring at room temperature for 4-12 hr, centrifuging for precipitation under 8000-12000rpm for 5-10 min to obtain precipitate C;
3) placing 5-10 parts by weight of the precipitate C in a 1mL EP tube, resuspending the precipitate C with 0.1-0.5 part by weight of PBS, then adding 0.03-0.01 part by weight of tumor target antigen, 0.03-0.01 part by weight of anti-CD 28 antibody and 0.03-0.01 part by weight of CD47-Fc, then placing the EP tube on a rotating disc for rotating reaction for 12-16 hours at the reaction temperature of 0-8 ℃, further sealing the reaction liquid with 2-5 parts by weight of bovine serum albumin for 12-16 hours at the reaction temperature of room temperature, finally centrifuging the sealed reaction liquid under the conditions of 8000-12000rpm for 5-10 minutes, and washing with deionized water for 3 times to obtain the final artificial antigen presenting nanoparticles.
8. The method for preparing artificial antigen presenting nanoparticles with long circulation and anti-phagocytosis effect according to claim 7, wherein the method for preparing the PEGylated PLGA nanoparticle solution comprises the following steps:
1) dissolving 5-10 parts by weight of PLGA nanoparticles in 6-12 parts by weight of isotonic buffer salt solution which is 0.1M MES with the pH value of 4.5-6.0;
2) taking 0.2-0.4 weight part of EDC & HCl solution and 0.2-0.4 weight part of NHS solution, stirring for 1-2 hours at room temperature, and then carrying out centrifugal precipitation under the conditions of 8000-12000rpm for 5-10 minutes to obtain a precipitate A;
3) dissolving precipitate A in 4-8 weight parts of deionized water at room temperature, and adding 0.01-0.03 weight part of NH2PEG-COOH, standing for 8-12 hours, centrifuging the reactant under the centrifugation condition of 8000-12000rpm for 5-10 minutes, and resuspending the centrifuged product with 4-8 parts by weight of deionized water to obtain the PEG PLGA nanoparticle solution.
9. The method for preparing artificial antigen presenting nanoparticles with long circulation and anti-phagocytosis according to claim 8, wherein the PLGA nanoparticles are prepared by the following steps:
1) dissolving 5-10 parts by weight of PLGA in 5-10 parts by weight of dichloromethane, and carrying out ultrasonic emulsification under the ultrasonic condition that the ultrasonic treatment is stopped for 10 seconds every 10-15 seconds and is continued for 5-10 cycles to form PLGA solution;
2) adding 30-60 parts by weight of 1% PVA into the PLGA solution after ultrasonic emulsification, and performing ultrasonic emulsification under the ultrasonic condition that the ultrasonic treatment is stopped for 10 seconds every 10-15 seconds and is continued for 5-10 cycles to obtain an emulsion A;
3) adding 60-120 parts by weight of 0.5% PVA into the emulsion A, and then carrying out ultrasonic emulsification under the ultrasonic condition that the mixture stops for 10 seconds every 10-15 seconds and lasts for 5-10 cycles to obtain emulsion B;
4) stirring the emulsion B for 4-12h at room temperature by using a magnetic stirrer to obtain emulsion C;
5) and (3) carrying out centrifugal precipitation by using excessive deionized water under the centrifugal condition of 4000-8000rpm for 5-10 minutes, repeating for 3-5 times, and carrying out freeze drying to obtain the PLGA nanoparticles.
10. Use of the artificial antigen-presenting nanoparticles having long circulation and anti-phagocytosis effect for the preparation of antitumor drugs, characterized in that the artificial antigen-presenting nanoparticles of any one of claims 1 to 3 are used.
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| SONG S, JIN X, ZHANG L, ET AL.: "PEGylated and CD47-conjugated nanoellipsoidal artificial antigen-presenting cells minimize phagocytosis and augment anti-tumor T-cell responses", 《INTERNATIONAL JOURNAL OF NANOMEDICINE》 * |
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