CN115414494B - Polypeptide nanometer vaccine and preparation method and application thereof - Google Patents

Polypeptide nanometer vaccine and preparation method and application thereof Download PDF

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CN115414494B
CN115414494B CN202210987549.9A CN202210987549A CN115414494B CN 115414494 B CN115414494 B CN 115414494B CN 202210987549 A CN202210987549 A CN 202210987549A CN 115414494 B CN115414494 B CN 115414494B
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CN115414494A (en
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王晶晶
宋立铨
金喆彤
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Nanjing Xiaoyu Biotechnology Co ltd
Nanjing University of Chinese Medicine
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Nanjing University of Chinese Medicine
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Abstract

The invention provides a polypeptide nanometer vaccine and a preparation method thereof, wherein the polypeptide nanometer vaccine comprises a carrier and an adjuvant loaded on the carrier, the carrier is nanometer particles obtained by crosslinking beta-cyclodextrin modified polyethyleneimine and antigen peptide through a crosslinking agent, and the crosslinking agent is a reduction response type crosslinking agent. The polypeptide nanometer vaccine can realize the co-delivery of antigen peptide and adjuvant, can be efficiently enriched in lymph nodes, can crack and release polypeptide antigen under the condition of reducibility in cells, can fully stimulate antigen presenting cells, enhance the cross presentation of the antigen and improve immune response. The polypeptide nanometer vaccine has low raw material cost, easy acquisition, simple preparation method, and good application prospect, and is universal for all peptide antigens.

Description

Polypeptide nanometer vaccine and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological immunity, in particular to a polypeptide nanometer vaccine and a preparation method and application thereof.
Background
The vaccine has great contribution to guaranteeing human health, improving life quality and promoting social development.
Vaccines are generally divided into two categories: prophylactic vaccines and therapeutic vaccines. Well-known prophylactic vaccines are mainly used for the prevention of diseases, the recipients being healthy individuals or newborns, such as the pertussis vaccine, the novel coronavirus pneumonitis vaccine, etc.; the therapeutic vaccine is mainly used for individuals suffering from diseases, and the receiver is a patient, and is a product or product expressed by natural, artificial synthesis or gene recombination technology, such as therapeutic hepatitis B vaccine, therapeutic pancreatic cancer vaccine and the like, which can treat or prevent disease deterioration by inducing specific immune response in organisms infected with pathogenic microorganisms or suffering from certain diseases, and belongs to specific active immunotherapy. Currently, therapeutic vaccines mainly include subunit vaccines, immune complex vaccines, polypeptide vaccines, and the like.
In recent years, the incidence and mortality of malignant tumors worldwide are increased by about 3.9% and 2.5% each year, and malignant tumor death accounts for 23.91% of all resident death factors, so that the search for a treatment mode for effectively improving the survival rate of cancer patients and improving the survival quality of patients has become a great research difficulty. At this time, immunological-based tumor immunotherapy has demonstrated great advantages in the field of cancer treatment. Tumor immunotherapy is a treatment method for specifically killing tumor cells by using an immune system of an organism, and the method can inhibit the growth of the tumor and enable the organism to generate immunological memory, can effectively inhibit proliferation, metastasis and recurrence of malignant tumors, and mainly comprises monoclonal antibody treatment, adoptive cell therapy, tumor vaccine and the like at present.
Tumor vaccines can utilize tumor-associated antigens (TAAs) such as DNA, RNA, proteins, or peptides to modulate the immune system. After the vaccine enters the human body, the antigen is recognized by Pattern Recognition Receptors (PRRs) present on the surface of Antigen Presenting Cells (APCs) such as macrophages or Dendritic Cells (DCs) present on the periphery, and transported to immune organs such as draining lymph nodes or spleen. Because of the difference of antigens, some are directly recognized by B cells, and some are presented to T cells in immune organs to generate specific T cells aiming at different tumor antigens, and dormant and disabled T cells can be reactivated, so that new or existing anti-tumor immune responses are stimulated, and the induction and enhancement of tumor specific T cells to cancer responses are realized. And the immunity is different from the wide immunity induced by blocking of immune check points, and the tumor vaccine can realize accurate immunity to tumors. In recent years, related tumor vaccines such as cervical cancer, prostate cancer and the like are also marketed.
The polypeptide vaccine prepared by utilizing the tumor surface specific antigen polypeptide provides the most direct mode of targeting specific epitopes, and the specific polypeptide epitopes contained in the polypeptide vaccine are identified by T cell receptors, so that the tumor specific T cells can be stimulated and generated, the tumor can be killed accurately, and the adverse inflammatory reaction of the polypeptide vaccine is reduced.
Along with the identification of the cancer sharing antigen and the mutant antigen, the application range of the polypeptide cancer vaccine is expected to be greatly expanded, and the polypeptide cancer vaccine has great potential for benefiting patients. And cancer vaccines based on antigenic polypeptides are highly safe, easy to manufacture, and they can directly monitor T cell responses to cd4+ and cd8+ T cells of the antigenic peptides, which is critical in vaccine development.
However, most of the current polypeptide-based tumor vaccines have the following problems: the first antigenic peptide is susceptible to degradation during delivery; second, co-delivery of an immunoadjuvant is difficult, while in the absence of an immunoadjuvant, the immature antigen presenting cells have a lower chance of triggering an immune response upon contact with the antigen. Thirdly, the peptide sequences of the polypeptide vaccines are various, the physicochemical properties are different, and the existing carrier is difficult to be applied to all peptide antigens.
Disclosure of Invention
The invention provides a polypeptide nanometer vaccine, a preparation method and application thereof, which can be suitable for various antigen peptides with various types, structures and physicochemical properties, and provides an antigen-adjuvant integrated delivery scheme.
In order to solve the technical problems, the invention adopts the following scheme:
a polypeptide nanometer vaccine comprises a carrier and an adjuvant loaded on the carrier,
The carrier is nano particles obtained by crosslinking beta-cyclodextrin modified polyethyleneimine (beta-CD-PEI) and antigen peptide through a crosslinking agent, and the crosslinking agent is a reduction response type crosslinking agent.
Preferably, the adjuvant is encapsulated in the cavities of the beta-cyclodextrin or is supported on the surface of the carrier by electrostatic action.
Preferably, the adjuvant is a hydrophobic adjuvant or an ionic adjuvant, the hydrophobic adjuvant is wrapped in a cavity of the beta-cyclodextrin, and the ionic adjuvant is loaded on the surface of the carrier through electrostatic action.
Preferably, the hydrophobic adjuvant is remiquinimod (R848) or imiquimod (R837).
Preferably, the ionic adjuvant is an oligodeoxynucleotide (CpG) or a polyinosinic cytidylic acid (Poly I: C).
Preferably, the beta-cyclodextrin modified polyethyleneimine has the structural formula:
；
wherein n is a natural number.
Preferably, the reduction-responsive crosslinking agent (PNC-DTDE-PNC) has the structural formula:
。
Preferably, the antigenic peptide is a prophylactic or therapeutic antigen. Such as neoantigens derived from tumor genome mutations, polypeptides specifically expressed on the viral surface, and the like.
Preferably, the antigen peptide is an antigen ovalbumin OVA antigen peptide SIINFEKL, an antigen peptide QAIVRGCSMPGPWRSGRLLVSRRWSVE targeting CT26, an antigen peptide LCPGNKYEM targeting B16F10M27, an antigen peptide ASMTNMELM targeting MC38, an antigen peptide SLIVHLNEV targeting hepatoma cell HHCC, an HLA-A2 restricted antigen peptide YLEPGPVTA targeting melanoma, an antigen peptide ELAGIGILTV, an antigen peptide FVGEFFTDV, a tetanus toxoid peptide AQYIKANSKFIGITEL, and a model peptide FFQNK.
Preferably, the particle size of the carrier is 30-1000 nm.
The invention also provides a preparation method of the polypeptide nanometer vaccine, which comprises the following steps:
(1) Crosslinking beta-CD-PEI, a reduction response type crosslinking agent and antigen peptide in dimethyl sulfoxide to obtain the carrier;
(2) And incubating the adjuvant and the carrier in a solution to obtain the polypeptide nanometer vaccine.
Preferably, in the step (1), the molar ratio of the beta-CD-PEI, the reduction-responsive cross-linking agent and the antigenic peptide is 0.5-4:1-3.5.
Preferably, the beta-CD-PEI of step (1) is prepared by the following method:
(1-1-1) adding p-toluenesulfonyl chloride into a beta-cyclodextrin (beta-CD) NaOH solution, and stirring for reaction to obtain a product beta-CD-OTs;
(1-1-2) beta-CD-OTs and Polyethyleneimine (PEI) were reacted with stirring in Dimethylsulfoxide (DMSO) to give beta-cyclodextrin modified polyethyleneimine (beta-CD-PEI).
The structural formula of the beta-CD-OTs is as follows:。
preferably, the steps (1-1-1) and (1-1-2) further comprise a step of post-treatment of the obtained product.
Preferably, the reduction-responsive crosslinking agent in step (1) is prepared by the following method:
(1-2-1) 2-hydroxyethyl disulfide (DTDE), pyridine and phenyl p-nitrochloroformate (PNC) were reacted in anhydrous methylene chloride to give reduction-responsive crosslinking agent PNC-DTDE-PNC.
Preferably, the molar ratio of the beta-CD in the carrier to the hydrophobic adjuvant is 1-20:1.
Preferably, the time of the crosslinking reaction in the step (1) is 12-72 hours.
Preferably, the co-incubation time in the step (2) is 1-12 h.
The invention also provides application of the polypeptide nanometer vaccine in preparing an anti-tumor vaccine.
The invention has the beneficial effects that:
(1) The hollow cavity and electrostatic effect of the beta-CD-PEI in the carrier of the nano vaccine can realize the loading of different antigens and adjuvants.
(2) The invention uses two end groups of PNC-DTDE-PNC to crosslink the amino of beta-CD-PEI and the amino of polypeptide, and further, the disulfide bond on the crosslinking agent is reduced and cracked in the cell, thus realizing the release of antigen polypeptide in the cytoplasm of the cell. Each peptide chain at least contains 1 free amino group, and the cross-linking agent PNC-DTDE-PNC adopted by the invention is suitable for connecting any peptide chain, thereby providing possibility for constructing a universal nanometer vaccine carrier.
(3) The nano vaccine provided by the invention is in a nano particle form, can increase the uptake of Dendritic Cells (DCs) to antigens and adjuvants, and is beneficial to lymph node targeted delivery, so that the nano vaccine is beneficial to further biological application, and the medicine is more enriched in lymph node parts. It can up regulate the secretion of surface co-stimulatory molecules and cytokines, and is beneficial to stimulating immune response of organism and improving antitumor effect. Traditional tumor vaccines in free state are difficult to target lymph nodes and have limited drug enrichment.
(4) The raw materials adopted by the invention are low in price, easy to obtain, simple in preparation method and easy to produce, have universality in preparation of antigen polypeptide, and have good application prospects.
Drawings
FIG. 1 is an infrared absorption spectrum of the antigen peptide multi-adjuvant vaccine prepared in example 1.
FIG. 2 is a graph showing the particle size distribution of the antigen peptide multi-adjuvant nanovaccine prepared in example 1.
FIG. 3 is an electron micrograph of the antigenic peptide multi-adjuvant nanovaccine prepared in example 1.
FIG. 4 is a lymph node IVIS (infrared imaging system) image of the antigenic peptide multi-adjuvant nanovaccine prepared in example 1.
FIG. 5 is a tumor growth inhibition curve of the antigenic peptide multi-adjuvant nanovaccine prepared in example 1 and other comparative materials.
FIG. 6 is an infrared absorption spectrum of the model peptide multi-adjuvant nanovaccine prepared in example 3.
FIG. 7 is an electron micrograph of the antigenic peptide multi-adjuvant nanovaccine prepared in example 4.
FIG. 8 is a lymph node IVIS image of the antigenic peptide multi-adjuvant nanovaccine prepared in example 4.
Fig. 9 is a tumor growth inhibition curve of the antigenic peptide multi-adjuvant nanovaccine and other comparative materials prepared in example 4.
FIG. 10 is a graph of tumor weights of the antigenic peptide multi-adjuvant nanovaccine prepared in example 4 and other comparative materials.
FIG. 11 is a tumor map of the antigenic peptide multi-adjuvant nanovaccine and other comparative materials prepared in example 4.
FIG. 12 is an electron micrograph of the antigenic peptide multi-adjuvant nanovaccine prepared in example 5.
FIG. 13 is a tumor growth inhibition curve of the antigenic peptide multi-adjuvant nanovaccine and other comparative materials prepared in example 5.
FIG. 14 is a tumor map of the antigenic peptide multi-adjuvant nanovaccine prepared in example 5 and other comparative materials.
Figure 15 is a prophylactic tumor suppression curve for the antigenic peptide multi-adjuvant nanovaccine prepared in example 5 and other comparative materials.
Detailed Description
Example 1
1. Beta-CD-PEI synthesis:
(1) First step, synthesizing beta-CD-OTs: beta-CD (50 g, 44.0 mmol) was dissolved in 500mL of 0.4N NaOH solution, cooled to 5℃in an ice-water bath, and p-toluenesulfonyl chloride (35 g, 184 mmol) was slowly added to the continuously stirred solution over 5 minutes. The mixed solution was stirred at 5 ℃ for 30 minutes and then filtered. The filtrate was neutralized with saturated hydrochloric acid and stirred for 1h. And then filtering the sample, washing the filter cake with water for 3 times, and drying the washed sample in a vacuum drying oven at 60 ℃ for one night to obtain the product beta-CD-OTs.
(2) Second step, synthesizing beta-CD-PEI: PEI600 (1.1 g,1.8 mmol) and beta-CD-OTs (2.1 g,1.8 mmol) were dissolved in an appropriate amount of DMSO and the mixture was stirred under nitrogen for 3 days, followed by dialysis of the solution and lyophilization to give beta-CD-PEI. And adopting nuclear magnetic resonance and mass spectrum to carry out structural identification.
2. Synthesis of PNC-DTDE-PNC crosslinker:
DTDE (4.0 g,46.7 mmol) and pyridine (7.4 g,93.4 mmol) were dissolved in 10 mL anhydrous dichloromethane with 5A molecular sieve standing overnight to remove water before use, N 2 protected, and PNC in dichloromethane (10.3 g,5.1 mmol, dissolved in 10 mL anhydrous dichloromethane) was slowly added over 2h under ice water bath conditions followed by a light-protected reaction of 20 h. After spin-drying, the mixture was purified by column chromatography on silica gel (developing solvent: methylene chloride). The synthesis of PNC-DTDE-PNC was determined using nuclear magnetic resonance and mass spectrometry.
3.1. Construction of the nano vaccine vector:
Dissolving antigen PEPTIDE PEPTIDE (sequence QAIVRGCSMPGPWRSGRLLVSRRWSVE), beta-CD-PEI and PNC-DTDE-PNC in 1 mL DMSO, stirring at 25deg.C for 24-72 h, dialyzing with 1500 Da dialysis bag in pure water to remove DMSO, and controlling particle diameter, morphology and stability of nanoparticle by adjusting feeding ratio and reaction time.
The structural formula of the prepared polypeptide nanometer vaccine carrier is as follows:
。
3.2 Construction of adjuvant-loaded nanovaccine:
Incubating R848 and CpG with the prepared polypeptide nanometer vaccine carrier in aqueous solution for 1-12 h, wherein R848 is included by a beta-CD cavity, and CpG is adsorbed on the carrier by electrostatic action. The loading amount of the adjuvant can be adjusted by controlling the feeding proportion and the co-incubation time so as to control the immunological adjuvant with the optimal proportion.
Fig. 1 is an infrared absorption spectrum diagram of a double-adjuvant nano vaccine loaded with antigen peptide, fig. 2 is a particle size of the nano vaccine, and fig. 3 is an electron microscope diagram of the nano vaccine, and it is known from the figure that there is an N-H absorption peak at 3324.12 cm -1, which indicates that in this embodiment, a cross-linking agent is connected with an amino group on a polypeptide, and the particle size of the nano vaccine carrier obtained in this embodiment is 346±39.7 nm.
Example 2
Nano vaccine lymph node enrichment ability investigation:
According to the functional group ratio beta-CD-PEI 600:PEPTIDE:PNC-DTDE-PNC=1:3:4, dissolving antigen PEPTIDE PEPTIDE (sequence is QAIVRGCSMPGPWRSGRLLVSRRWSVE), beta-CD-PEI and PNC-DTDE-PNC in 1 mL DMSO, controlling pH to be about 7.4, adding 20 uLCy-7 fluorescent dye, and incubating for 24-72 h to obtain the fluorescent-labeled nano vaccine.
100 UL nm vaccine was subcutaneously injected into the root tail of BALB\C mice (50 uL on both left and right sides). Mice were sacrificed at 6, 12 and 24 h post-injection, respectively, and inguinal Lymph Nodes (LNs) were removed for imaging in vitro.
As shown in FIG. 4, the bilateral inguinal regions of the nano vaccine group mice have stronger fluorescence in 6h, 12 h and 24 h, and the free group (injected with free beta-CD-PEI, PEPTIDE, R848 and CpG) has weak fluorescence in 12 h, which indicates that the prepared nano vaccine has stronger lymph node enrichment capability.
The tumor inhibiting effect of the beta-CD-PEI-antigen peptide double-adjuvant nano vaccine is examined:
tumor cells were injected into the right upper arm of Day0 mice, and were randomly divided into PBS, free, and nanovaccine groups prior to administration, and were administered at the tail root of Day7 and Day 14.
As shown in fig. 5, the remaining two groups were reduced compared to the PBS group, and the nanovaccine group was also different from the free group, which was statistically significant.
Example 3
Model PEPTIDEs (sequence FFQNK, amino only), β -CD-PEI and PNC-DTDE-PNC were dissolved in 1mL DMSO according to the ratio of functional groups β -CD-PEI600:PEPTIDE: PNC-DTDE-PNC=1:3:4, stirred at 25℃for 24-72 h, and dialyzed in pure water using a 1500 Da dialysis bag to remove DMSO. And co-incubating the R848 and CpG with the prepared polypeptide nanometer vaccine carrier in an aqueous solution for 1-12 h to prepare the double-adjuvant nanometer vaccine loaded with the model peptide. FIG. 6 is an infrared absorption spectrum of a model peptide-loaded double-adjuvant nanovaccine, which shows that the cross-linking agent is linked to the amino group on the polypeptide and has an N-H absorption peak at 3312.65 cm -1 in this example.
Example 4
1. Construction of a double-adjuvant nanovaccine loaded with an antigenic peptide (SIINFEKL sequence):
beta-CD-PEI synthesized in the same manner as in example 1, PNC-DTDE-PNC crosslinking agent synthesized in the same manner as in example 1 and antigenic PEPTIDE PEPTIDE (sequence SIINFEKL) were dissolved in 1mL DMSO according to the functional ratio beta-CD-PEI 600:PEPTIDE: PNC-DTDE-PNC=1:3:4, stirred at 25℃for 24 to 48 hours, and DMSO was removed by dialysis in pure water using a 1500 Da dialysis bag. And co-incubating the R848 and CpG with the prepared polypeptide nanometer vaccine carrier for 12 hours in an aqueous solution to prepare the double-adjuvant loaded polypeptide nanometer vaccine.
Fig. 7 is an electron microscope image of a nano vaccine, and the particle size of the nano vaccine carrier obtained in this example is 197.8±11.1 nm.
2. Nano vaccine lymph node enrichment ability investigation:
According to the functional group ratio beta-CD-PEI 600:PEPTIDE:PNC-DTDE-PNC=1:3:4, dissolving antigen PEPTIDE (sequence is SIINFEKL), beta-CD-PEI and PNC-DTDE-PNC in 1mL DMSO, controlling pH to be about 7.4, adding 20 uLCy-7 fluorescent dye, and incubating for 24-72 h to obtain the fluorescent marked nano vaccine.
100 UL nm vaccine was subcutaneously injected into the root tail of BALB\C mice (50 uL on both left and right sides). Mice were sacrificed at 6, 12 and 24 h post-injection, respectively, and inguinal Lymph Nodes (LNs) were removed for imaging in vitro.
As shown in FIG. 8, the bilateral inguinal regions of the nano vaccine group mice have stronger fluorescence in 6h,12 h and 24h, the free group has fluorescence in 6h and 12h but the fluorescence intensity is obviously weaker than that of the nano vaccine group, and the 24h fluorescence is basically disappeared, which shows that the prepared nano vaccine has stronger lymph node enrichment capability.
3. Examining the tumor inhibiting effect of the nano vaccine:
tumor cells were injected into the right upper arm of Day0 mice, and were randomly divided into PBS, free, and nanovaccine groups prior to administration, and were administered at the tail root of Day7 and Day 14.
As shown in fig. 9, 10 and 11, the tumor volume and tumor weight of the nano vaccine group mice are obviously reduced compared with those of the PBS group and the free group, and the difference between the nano vaccine group and the PBS group and the free group has statistical significance, which indicates that the nano vaccine can inhibit tumor growth.
Example 5
1. Construction of antigen peptide (sequence ASMTNMELM) -loaded double-adjuvant nanovaccine:
beta-CD-PEI synthesized in the same manner as in example 1, PNC-DTDE-PNC crosslinking agent synthesized in the same manner as in example 1 and antigen PEPTIDE PEPTIDE (sequence ASMTNMELM) were dissolved in 1 mL DMSO according to the functional group ratio beta-CD-PEI 600:PEPTIDE: PNC-DTDE-PNC=1:3:4, stirred at 25℃for 24 to 48 hours, and DMSO was removed by dialysis in pure water using a 1500 Da dialysis bag. And co-incubating the R848 and the CpG and the prepared polypeptide nanometer vaccine carrier in an aqueous solution for 1-12 h to prepare the double-adjuvant loaded polypeptide nanometer vaccine.
Fig. 12 is an electron microscope image of the prepared nano vaccine, and the particle size of the nano vaccine carrier obtained in this example is 309.8±4.9 nm.
2. Examining the tumor inhibiting effect of the nano vaccine:
Tumor cells were injected into the right upper arm of Day0 mice, and were randomly divided into PBS, free and nanovaccine groups prior to administration, and were administered at the tail roots of Day7, day14 and Day 21.
As shown in fig. 13 and 14, the tumor volumes of the nano vaccine group mice are smaller than those of the PBS group and the free group, and the tumor of 2/5 mice of the nano vaccine group gradually becomes smaller until the tumor disappears, which indicates that the nano vaccine can inhibit the tumor growth.
3. And (3) evaluating tumor preventive capability of the nano vaccine:
The PBS group, the free group and the nano vaccine group are randomly divided before administration, and tumor cells are injected into the right upper arm of the mice at Day-21, day-14 and Day-7 at the tail root.
As shown in fig. 15, the tumor growth curves of the nanovaccine group were all gentler than those of the PBS group and the free group, indicating that the prophylactic administration of the nanovaccine can inhibit the growth rate of tumor to some extent.
Example 6
Beta-CD-PEI synthesized in the same manner as in example 1, PNC-DTDE-PNC crosslinking agent synthesized in the same manner as in example 1 and antigen PEPTIDE PEPTIDE (sequence YLEPGPVTA) were dissolved in 1 mL DMSO, stirred at 25 ℃ for 12-36 h, and dialyzed in pure water with a 1500 Da dialysis bag to remove DMSO. Functional group ratio β -CD-PEI: PEPTIDE: PNC-DTDE-pnc=0.5:3.5:4.
And co-incubating polyinosinic acid (Poly I: C) and the prepared polypeptide nanometer vaccine carrier in an aqueous solution for 12 hours, and adsorbing the Poly I: C on the carrier through electrostatic action to prepare the polypeptide nanometer vaccine loaded with the Poly I: C. The average particle size of the nanovaccine vector obtained in this example was about 140 nm.
Example 7
Beta-CD-PEI synthesized in the same manner as in example 1, PNC-DTDE-PNC crosslinking agent synthesized in the same manner as in example 1 and antigen PEPTIDE PEPTIDE (sequence LCPGNKYEM) were dissolved in 1 mL DMSO, stirred at 25℃for 24-48 h, and dialyzed in pure water using a 1500 Da dialysis bag to remove DMSO. Functional group ratio β -CD-PEI: PEPTIDE: PNC-DTDE-pnc=4:1:3.5.
And co-incubating the R837 and the prepared polypeptide nanometer vaccine carrier in an aqueous solution for 12h, wherein the R837 is included in the carrier through a beta-CD cavity, and the polypeptide nanometer vaccine carrying the R837 is prepared. The average particle size of the nanovaccine vector obtained in this example was about 430 nm.
Example 8
Beta-CD-PEI synthesized in the same manner as in example 1, PNC-DTDE-PNC crosslinking agent synthesized in the same manner as in example 1 and model peptide (sequence FFQNK, amino group only) were dissolved in 1 mL DMSO, stirred at 25℃for 24 to 48 hours, and DMSO was removed by dialysis in pure water using a 1500 Da dialysis bag. Functional group ratio β -CD-PEI: PEPTIDE: PNC-DTDE-pnc=4:1:1.
And co-incubating the R848 and the prepared polypeptide nanometer vaccine carrier in an aqueous solution for 12h, wherein the R848 is included in the carrier through a beta-CD cavity, and the polypeptide nanometer vaccine carrying the R848 is prepared. The average particle size of the nanovaccine vector obtained in this example was about 30 nm.

Claims (5)

1. A polypeptide nanometer vaccine is characterized by comprising a carrier and an adjuvant loaded on the carrier,
The carrier is nano particles obtained by crosslinking beta-cyclodextrin modified polyethyleneimine and antigenic peptide through a crosslinking agent, and the crosslinking agent is a reduction response type crosslinking agent;
The adjuvant is wrapped in a cavity of the beta-cyclodextrin or is loaded on the carrier through electrostatic action; the adjuvant is a hydrophobic adjuvant or an ionic adjuvant, the hydrophobic adjuvant is wrapped in a cavity of the beta-cyclodextrin, and the ionic adjuvant is loaded on the carrier through electrostatic action; the hydrophobic adjuvant is resiquimod, the ionic adjuvant is oligodeoxynucleotide,
The structural formula of the reduction-responsive cross-linking agent is as follows:
；
The antigen peptide is antigen ovalbumin OVA antigen peptide SIINFEKL, CT26 targeting antigen peptide QAIVRGCSMPGPWRSGRLLVSRRWSVE, MC38 targeting antigen peptide ASMTNMELM or model peptide FFQNK.
2. The polypeptide nanometer vaccine according to claim 1, wherein the particle size of the carrier is 30-1000 nm.
3. The method for preparing the polypeptide nanometer vaccine according to any one of claims 1-2, which is characterized by comprising the following steps:
(1) Crosslinking beta-cyclodextrin modified polyethyleneimine, a reduction response type crosslinking agent and antigenic peptide in dimethyl sulfoxide to obtain the carrier;
(2) And incubating the adjuvant and the carrier in a solution to obtain the polypeptide nanometer vaccine.
4. A process according to claim 3, wherein the β -cyclodextrin modified polyethyleneimine of step (1) is prepared by:
(1-1-1) adding p-toluenesulfonyl chloride into NaOH solution of beta-cyclodextrin, and stirring to react to obtain a product beta-CD-OTs;
(1-1-2) the beta-CD-OTs and the polyethyleneimine are stirred and reacted in dimethyl sulfoxide to obtain the polyethyleneimine modified by beta-cyclodextrin.
5. Use of the polypeptide nanometer vaccine according to any one of claims 1-2 for preparing an anti-tumor vaccine.
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