CN112336853A - Liposome nano vaccine, preparation method and application - Google Patents

Liposome nano vaccine, preparation method and application Download PDF

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CN112336853A
CN112336853A CN202011130845.4A CN202011130845A CN112336853A CN 112336853 A CN112336853 A CN 112336853A CN 202011130845 A CN202011130845 A CN 202011130845A CN 112336853 A CN112336853 A CN 112336853A
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dopc
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金萍
张婧欣
周玥
周建大
曹科
王少华
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Third Xiangya Hospital of Central South University
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Abstract

The invention relates to the technical field of nano-medicine, and discloses a preparation method of a liposome nano-vaccine; also disclosed is a liposome nano vaccine; also discloses the application of the liposome nano vaccine in the preparation of the medicine for stimulating the anti-tumor immunity of the organism. The vaccine provided by the invention induces and generates specific immune T cells aiming at tumors through phagocytosis, processing and presentation of antigen presenting cells to exert a tumor killing effect, has immunogenicity and immunostimulation, and is a high-efficiency and safe tumor therapeutic vaccine.

Description

Liposome nano vaccine, preparation method and application
Technical Field
The invention relates to the technical field of nano-medicine, in particular to a liposome nano-vaccine, a preparation method and application thereof.
Background
Tumor formation is the result of immune escape of tumor antigens, and the growth and metastasis of tumors are promoted by the immune suppression and immune tolerance of the body, so that how to start and enhance the inherent immune response of tumor patients to suppress tumors and reduce the possibility of escape immune surveillance is the main target of modern tumor treatment. Vaccinology has changed enormously since its early development, but the classical vaccine strategies based on attenuated or inactivated pathogens are still used today, the most common problems of this vaccine are the risk of infection in immunocompromised humans, the presence of impurities when isolating the pathogen in vitro, and instability.
Thus, scientists have sought to develop vaccines that use only a minimal pathogen component, which can carry a minimal fragment from the immunological information of the protein, the peptide epitope, and which can also include other adjuvants to enhance the effectiveness of the vaccine. Vaccines that encapsulate antigenic polypeptide chains have peptide components derived primarily from bacterial, viral or parasitic proteins, or from tumor cell lysates, which are necessary to stimulate an appropriate immune response. Since the composition of the peptide chain is extremely simple, there are many advantages compared to using the whole pathogenic microorganism or protein. However, removal of a significant amount of the "danger signal" component of the pathogen greatly reduces vaccine efficacy, and to compensate for this loss, the problem can be solved by the additional addition of adjuvants.
Lipid carriers have been widely used in the study of vaccine delivery, and liposomes are in fact a very common nano-vaccine. The nano-particles can transport hydrophobic and hydrophilic molecules, and can prolong the action time of the vaccine by prolonging the half life of the vaccine and slowly releasing the drug. The liposome has low toxicity or no toxicity, and has good biocompatibility and biodegradability. Scientists can evade detection of the immune system or improve the solubility of liposomal vaccines by chemically modifying the lipid nanoparticles. Lipid nano-vaccines can also be used in conjunction with other therapeutic strategies to improve patient response. The main component of the liposome nanoparticle is phospholipid, the phospholipid has amphiphilic property and can form a double-layer structure, the phospholipid can form vesicles in water, and once anticancer drugs or tumor vaccine related components are loaded into the structure of the liposome nanoparticle, the liposome vaccine can be prepared, and the delivery mode can improve the solubility and stability of the content of the liposome vaccine. Therefore, the inventor develops and constructs a liposome nanoparticle tumor vaccine, which has important significance in the field of tumor immunotherapy.
Disclosure of Invention
Based on the problems, the invention provides a liposome nano vaccine, a preparation method and an application, the vaccine induces and generates specific immune T cells aiming at tumors through phagocytosis, processing and presentation of antigen presenting cells to exert a tumor killing effect, has immunogenicity and immunostimulation, and is a high-efficiency and safe tumor therapeutic vaccine.
In order to solve the technical problems, the invention provides a preparation method of liposome nano vaccine, which comprises the following steps:
s1: mixing the tumor antigen polypeptide chain aqueous solution and the adjuvant aqueous solution capable of improving the immunity of the organism for 20s in a vortex manner to obtain an antigen/adjuvant mixed solution, adding a DOPC (dioctyl phthalate) tert-butyl alcohol solution into the antigen/adjuvant mixed solution, and mixing for 20s in a vortex manner to obtain an antigen/adjuvant/DOPC solution;
s2: adding a 1% tween 20 tert-butyl alcohol solution into the antigen/adjuvant/DOPC solution in the step S1, vortex-mixing for 20S to obtain an antigen/adjuvant/DOPC/tween 20 solution, placing the antigen/adjuvant/DOPC/tween 20 solution into a 1.5mL centrifuge tube, sealing the opening of the 1.5mL centrifuge tube with a sealing film, poking a small hole with a syringe needle, and placing the 1.5mL centrifuge tube at-80 ℃ overnight; and (3) freeze-drying the completely frozen antigen/adjuvant/DOPC/Tween 20 sample in a freeze-drying machine for more than 5 hours in the next day until the sample is completely dried into liposome, thus obtaining the liposome nano vaccine.
Further, in step S1, the adjuvants for enhancing immunity include mouse TLR9 agonist CpG and mouse STING agonist GAMP.
Further, the concentration of the tumor antigen polypeptide chain in the aqueous solution of the tumor antigen polypeptide chain in step S1 is 10mg/mL, the concentrations of the mouse TLR9 agonist CpG and the mouse STING agonist GAMP in the aqueous solution of the adjuvant in step S1 are 4mg/mL and 1mg/mL, respectively, and the adding volumes of the aqueous solution of the tumor antigen polypeptide chain, the mouse TLR9 agonist CpG and the mouse STING agonist GAMP in the antigen/adjuvant mixed solution in step S1 are 10. mu.l, 2.5. mu.l and 5. mu.l, respectively.
Further, the concentration of DOPC in the DOPC tert-butanol solution in step S1 was 20mg/mL, and the addition volume of the DOPC tert-butanol solution in the antigen/adjuvant/DOPC solution was 16 μ l.
Further, the aqueous solution of tumor antigen polypeptide chains in step S1 is an aqueous solution of P66 tumor antigen polypeptide chains.
In order to solve the technical problems, the invention also provides a liposome nano vaccine.
In order to solve the technical problems, the invention also provides the application of the liposome nano vaccine in preparing the medicine for stimulating the anti-tumor immunity of the organism.
Further, the tumor is breast cancer.
Compared with the prior art, the invention has the beneficial effects that: the liposome nano vaccine is loaded with proper P66 tumor antigen polypeptide chains and an adjuvant capable of improving the immunocompetence of an organism, wherein the adjuvant comprises a ligand CpG oligonucleotide adjuvant of a mouse Toll-like receptor 9(TLR9) and a ligand 2'3' -cGAMP adjuvant of a mouse interferon activated protein (STING) receptor, the two adjuvants are single-chain DNA products, and the two adjuvants can simultaneously activate two immune channels of TLR9 and STING, so that the reaction of the I-type interferon can be jointly stimulated, the immunity degree of the organism can be greatly increased, and the liposome nano vaccine is superior to a vaccine structure only containing one immune adjuvant; the vaccine provided by the invention induces and generates specific immune T cells aiming at tumors through phagocytosis, processing and presentation of antigen presenting cells to exert a tumor killing effect, has immunogenicity and immunostimulation, and is a high-efficiency and safe tumor therapeutic vaccine.
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FIG. 1 is a graph showing the therapeutic effect of the liposome nano vaccine of example 2 against breast cancer tumor.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
Example 1:
the preparation method of the relevant reagents used in this example is as follows.
The preparation method of the tumor antigen polypeptide chain aqueous solution comprises the following steps: dissolving 10mg of tumor antigen polypeptide chain in 1ml of deionized water, and performing vortex oscillation until the polypeptide chain is completely dissolved to obtain the tumor antigen polypeptide chain aqueous solution.
The preparation method of the mouse TLR9 agonist CpG adjuvant solution comprises the following steps: dissolving 1mg of adjuvant CpG capable of improving the immunity of the organism in 250 mul of deionized water, and whirling and shaking until the adjuvant CpG is completely dissolved to obtain the mouse TLR9 agonist CpG adjuvant aqueous solution.
The preparation method of the mouse STING agonist GAMP adjuvant solution comprises the following steps: dissolving 500 mu g adjuvant cGAMP capable of improving the immunity of the organism in 500 mu l of deionized water, and vortexing and shaking until the adjuvant is completely dissolved to obtain the mouse STING agonist GAMP adjuvant water solution.
The preparation method of DOPC tertiary butanol solution is as follows: dissolving 400mg of 1, 2-dioleoyl-sn-propanetriyl-3-choline phosphorus (DOPC) with 20ml of tert-butyl alcohol, and vortexing until the solid components completely disappear to obtain DOPC tert-butyl alcohol solution.
The preparation method of the 1% tween 20 tert-butanol solution is as follows: add 500. mu.l of Tween 20 solution to 50ml of tert-butanol and manually invert the tube until fully dissolved, yielding a 1% Tween 20-tert-butanol solution.
The embodiment provides a preparation method of a liposome nano vaccine, which comprises the following steps:
s1: mixing the tumor antigen polypeptide chain aqueous solution and the adjuvant aqueous solution capable of improving the immunity of the organism for 20s in a vortex manner to obtain an antigen/adjuvant mixed solution, adding a DOPC (dioctyl phthalate) tert-butyl alcohol solution into the antigen/adjuvant mixed solution, and mixing for 20s in a vortex manner to obtain an antigen/adjuvant/DOPC solution;
s2: adding a 1% tween 20 tert-butyl alcohol solution into the antigen/adjuvant/DOPC solution in the step S1, vortex-mixing for 20S to obtain an antigen/adjuvant/DOPC/tween 20 solution, placing the antigen/adjuvant/DOPC/tween 20 solution into a 1.5mL centrifuge tube, sealing the opening of the 1.5mL centrifuge tube with a sealing film, poking a small hole with a syringe needle, and placing the 1.5mL centrifuge tube at-80 ℃ overnight; and (3) freeze-drying the completely frozen antigen/adjuvant/DOPC/Tween 20 sample in a freeze-drying machine for more than 5 hours in the next day until the sample is completely dried into liposome, thus obtaining the liposome nano vaccine.
In this embodiment, the adjuvants capable of enhancing immunity in step S1 are the mouse TLR9 agonist CpG and the mouse STING agonist GAMP.
In this example, the concentration of the tumor antigen polypeptide chain in the aqueous solution of tumor antigen polypeptide chain in step S1 was 10mg/mL, the concentrations of the mouse TLR9 agonist CpG and the mouse STING agonist GAMP in the aqueous solution of adjuvant in step S1 were 4mg/mL and 1mg/mL, respectively, and the addition volumes of the aqueous solution of tumor antigen polypeptide chain, the mouse TLR9 agonist CpG, and the mouse STING agonist GAMP in the antigen/adjuvant mixed solution in step S1 were 10. mu.l, 2.5. mu.l, and 5. mu.l, respectively.
The concentration of DOPC in the DOPC t-butanol solution in step S1 of this example was 20mg/mL, and the volume of the DOPC t-butanol solution added in the antigen/adjuvant/DOPC solution was 16. mu.l.
The dose of the vaccine prepared by the method is the dose of one mouse.
The liposome nano vaccine prepared by the method can be applied to the preparation of medicaments for stimulating the anti-tumor immunity of organisms.
The liposome nano vaccine finally prepared in the embodiment can be stored at-20 ℃, and is dissolved in water before use, and the liposome nano vaccine in the embodiment is injected in an intradermal mode.
Example 2:
the present embodiment provides a method for preparing a liposome nano vaccine, wherein reagents and apparatuses required for preparing the liposome nano vaccine are as follows: mouse TLR9 agonist ODN 1826(CpG, invitrogen usa), mouse STING agonist 2'3' -cGAMP (cGAMP, invitrogen usa), P66 antigen polypeptide chain (TYVPANASL, peptide2.0 usa), dimethyl sulfoxide (DMSO), 1, 2-dioleoyl-sn-propanetriyl-3-choline phosphate (DOPC), tert-butanol (t-butanol), Tween 20(Tween-20), sonicator, freeze dryer.
The preparation method of the P66 tumor antigen polypeptide chain aqueous solution comprises the following steps: 10mg of P66 tumor antigen polypeptide chain is dissolved in 1ml of deionized water, and vortex shaking is carried out until the polypeptide chain is completely dissolved, so as to obtain the P66 tumor antigen polypeptide chain aqueous solution.
The preparation method of the mouse TLR9 agonist CpG adjuvant solution comprises the following steps: dissolving 1mg of adjuvant CpG capable of improving the immunity of the organism in 250 mul of deionized water, and whirling and shaking until the adjuvant CpG is completely dissolved to obtain the mouse TLR9 agonist CpG adjuvant aqueous solution.
The preparation method of the mouse STING agonist GAMP adjuvant solution comprises the following steps: dissolving 500 mu g adjuvant cGAMP capable of improving the immunity of the organism in 500 mu l of deionized water, and vortexing and shaking until the adjuvant is completely dissolved to obtain the mouse STING agonist GAMP adjuvant water solution.
The preparation method of DOPC tertiary butanol solution is as follows: dissolving 400mg of 1, 2-dioleoyl-sn-propanetriyl-3-choline phosphorus (DOPC) with 20ml of tert-butyl alcohol, and vortexing until the solid components completely disappear to obtain DOPC tert-butyl alcohol solution.
The preparation method of the 1% tween 20 tert-butanol solution is as follows: add 500. mu.l of Tween 20 solution to 50ml of tert-butanol and manually invert the tube until fully dissolved, yielding a 1% Tween 20-tert-butanol solution.
The preparation of the liposome nano vaccine of the embodiment comprises the following steps:
s1: vortex mixing a P66 tumor antigen polypeptide chain aqueous solution and an adjuvant aqueous solution capable of improving the immunity of the organism for 20s to obtain a P66/adjuvant mixed solution, adding a 1, 2-dioleoyl-sn-propanetriyl-3-choline phosphorus tert-butyl alcohol solution into the P66/adjuvant mixed solution, and vortex mixing for 20s to obtain a P66/adjuvant/DOPC solution, wherein the adjuvants capable of improving the immunity of the organism in the embodiment are a mouse TLR9 agonist CpG and a mouse STING agonist GAMP, but are not limited to the two adjuvants; the concentration of P66 tumor antigen polypeptide chain in the P66 tumor antigen polypeptide chain aqueous solution in the embodiment is 10mg/mL, the concentrations of the mouse TLR9 agonist CpG and the mouse STING agonist GAMP in the adjuvant aqueous solution are 4mg/mL and 1mg/mL respectively, the adding volumes of the P66 tumor antigen polypeptide chain aqueous solution, the mouse TLR9 agonist CpG and the mouse STING agonist GAMP in the P66/adjuvant mixed solution in the embodiment are 10 mul, 2.5 mul and 5 mul respectively, the concentration of DOPC in the DOPC tert-butanol solution in the embodiment is 20mg/mL, and the adding volume of the DOPC tert-butanol solution in the P66/adjuvant/DOPC solution is 16 mul.
S2: adding a 1% tween 20 tert-butyl alcohol solution into the P66/adjuvant/DOPC solution in the step S1, vortex-mixing for 20S to obtain a P66/adjuvant/DOPC/Tween 20 solution, placing the P66/adjuvant/DOPC/Tween 20 solution into a 1.5mL centrifuge tube, sealing the opening of the 1.5mL centrifuge tube with a sealing film, poking a small hole with a syringe needle, and placing the 1.5mL centrifuge tube at-80 ℃ for overnight; and (3) freeze-drying the completely frozen P66/adjuvant/DOPC/Tween 20 sample in a freeze dryer for more than 5 hours in the next day until the sample is completely dried into liposome, thus obtaining the liposome nano vaccine.
The dose of the vaccine prepared by the method is the dose of one mouse.
The liposome nano vaccine prepared by the method can be applied to the preparation of medicaments for stimulating the immunity of organisms against breast cancer.
The liposome nano vaccine finally prepared in the embodiment can be stored at-20 ℃, and is dissolved in water before use, and the liposome nano vaccine in the embodiment is injected in an intradermal mode.
The therapeutic dose of the liposome nano vaccine for 1 mouse used once in the embodiment is 100 mul, wherein the effective components are 10 mug of mouse TLR9 agonist CpG, 5 mug of mouse STING agonist GAMP and 100 mug g P66 tumor antigen polypeptide chain.
In this example, healthy female Balb/c mice of 6-8 weeks old were divided into 2 groups of 10 mice each, one group was injected with PBS as a blank control group, and the other group was injected with the liposome nano-vaccine prepared in this example as an experimental group. Mice were injected with TUBO cells per 2.5X 10 mammary fat pad on day 0 on the lower left side of the mammary gland5(ii) individual cells; on days 3 and 10, mice were injected bilaterally intraplantarly (i.d.) with PBS and the liposome nano-vaccine prepared in this example, and tumor formation was recorded. The length and width of the tumor were measured with a vernier caliper every 2 or 3 days from day 11, and the tumor volume was calculated as length × width 2/2. Mice were sacrificed when tumors reached 2cm in length (endpoint) and the date was recorded. The results are shown in figure 1, the tumor growth rate of the mice in the nano vaccine treatment group is far less than that of the blank control group, and P can be obtained by t test<0.05, which shows that the tumor growth curves of the two groups have statistical difference, the results show that the liposome nano vaccine used in the present example has good curative effect on breast cancer.
The TUBO cell of the embodiment is a HER2 positive breast cancer tumor cell, the tumor antigen polypeptide which is acknowledged to have specificity and effectiveness aiming at the cell is P66, the amino acid sequence of the P66 tumor antigen polypeptide chain is TYVPANASL, and the antigen polypeptide can stimulate T cells to differentiate specific T cells which specially kill HER2 positive tumor cells after being processed by antigen presenting cells (mainly dendritic cells) to cause effective tumor immunoreaction.
The above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the verification process of the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all equivalent structural changes made by using the contents of the specification and the drawings of the present invention should be covered by the scope of the present invention.
Sequence listing
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Claims (8)

1. A preparation method of liposome nano vaccine is characterized by comprising the following steps:
s1: mixing the tumor antigen polypeptide chain aqueous solution and the adjuvant aqueous solution capable of improving the immunity of the organism for 20s in a vortex manner to obtain an antigen/adjuvant mixed solution, adding a DOPC (dioctyl phthalate) tert-butyl alcohol solution into the antigen/adjuvant mixed solution, and mixing for 20s in a vortex manner to obtain an antigen/adjuvant/DOPC solution;
s2: adding a 1% tween 20 tert-butyl alcohol solution into the antigen/adjuvant/DOPC solution in the step S1, vortex-mixing for 20S to obtain an antigen/adjuvant/DOPC/tween 20 solution, placing the antigen/adjuvant/DOPC/tween 20 solution into a 1.5mL centrifuge tube, sealing the opening of the 1.5mL centrifuge tube with a sealing film, poking a small hole with a syringe needle, and placing the 1.5mL centrifuge tube at-80 ℃ overnight; and (3) freeze-drying the completely frozen antigen/adjuvant/DOPC/Tween 20 sample in a freeze-drying machine for more than 5 hours in the next day until the sample is completely dried into liposome, thus obtaining the liposome nano vaccine.
2. The method of claim 1, wherein the adjuvant capable of enhancing immunity in step S1 is the mouse TLR9 agonist CpG and the mouse STING agonist GAMP.
3. The method according to claim 2, wherein the concentration of the tumor antigen polypeptide chain in the aqueous solution of the tumor antigen polypeptide chain in step S1 is 10mg/mL, the concentrations of the mouse TLR9 agonist CpG and the mouse STING agonist GAMP in the aqueous solution of the adjuvant in step S1 are 4mg/mL and 1mg/mL, respectively, and the addition volumes of the aqueous solution of the tumor antigen polypeptide chain, the mouse TLR9 agonist CpG, and the mouse STING agonist GAMP in the antigen/adjuvant mixture in step S1 are 10. mu.l, 2.5. mu.l, and 5. mu.l, respectively.
4. The method for producing according to claim 1, wherein the concentration of DOPC in the DOPC tert-butanol solution in step S1 is 20mg/mL, and the addition volume of the DOPC tert-butanol solution in the antigen/adjuvant/DOPC solution is 16 μ l.
5. The method of claim 1, wherein the aqueous solution of tumor antigen polypeptide chains in step S1 is an aqueous solution of P66 tumor antigen polypeptide chains.
6. The liposomal nano-vaccine prepared by the method of any one of claims 1-5.
7. Use of the liposomal nanobvaccine of any of claims 1-4 for the preparation of a medicament for stimulating anti-tumor immunity in the body.
8. The use of claim 7, wherein the tumor is breast cancer.
CN202011130845.4A 2020-10-21 2020-10-21 Liposome nano vaccine, preparation method and application Pending CN112336853A (en)

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CN114366808A (en) * 2021-12-14 2022-04-19 南京农业大学 Polysaccharide and virus antigen co-delivery nano vaccine, preparation method and application thereof

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