CN113855634A

CN113855634A - Polypeptide-modified liposome, mRNA (messenger ribonucleic acid) delivery system and dendritic cell vaccine

Info

Publication number: CN113855634A
Application number: CN202010615002.7A
Authority: CN
Inventors: 杨莉; 张瑞
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-06-30
Filing date: 2020-06-30
Publication date: 2021-12-31

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a liposome modified by a hydrophobic modified polypeptide, an mRNA (messenger ribonucleic acid) delivery system and a dendritic cell vaccine. The invention aims to solve the technical problem of improving the immune effect of the DCs vaccine based on the mRNA vaccine and mRNA sensitization. The technical scheme for solving the technical problem is to provide a liposome modified by hydrophobic modified cationic polypeptide. The modified liposome of the polypeptide can effectively deliver nucleic acid into cells; especially, mRNA of the coded antigen can be efficiently delivered to DCs, the efficiency of delivering the mRNA to the DCs by the cationic liposome is obviously improved, the immune effect of the mRNA vaccine and the mRNA sensitized DC vaccine is enhanced, and the method has good clinical application prospect.

Description

Polypeptide-modified liposome, mRNA (messenger ribonucleic acid) delivery system and dendritic cell vaccine

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a liposome modified by a hydrophobic modified polypeptide, an mRNA (messenger ribonucleic acid) delivery system and a dendritic cell vaccine.

Background

In recent years, the application of mRNA to the fields of gene therapy, immunotherapy, and stem cell biomedicine has attracted a great deal of attention. Compared to other types of gene therapy and tumor immunotherapy, the advantages of mRNA are mainly reflected in: 1) unlike viral vectors, mRNA does not need to be integrated into the genome of the host, so possible insertional mutations and aberrant transcription can be avoided; 2) compared to plasmid DNA, mRNA is simple in structure, requires fewer elements for translation into protein, and is biodegradable, acting only for a short period, thereby reducing the risk of side effects; 3) in the course of personalized immunotherapy, mRNA has distinct advantages over peptide vaccines, such as low synthesis cost, short synthesis cycle, simultaneous encoding of multiple antigen sequences and freedom from MHC haplotypes. In addition, with the development of mRNA structural engineering, such as modification of a 5 'end cap substructure and a 3' end polyA tail, the defects of poor mRNA stability, low translation efficiency and easy degradation are greatly improved, and the application of mRNA therapy is promoted. In recent years, mRNA has been widely used in cell programming and vaccine research, and shows great potential for application. In vaccine studies, mRNA vaccines have been shown to have the ability to activate an effective immune response, including cytotoxic T cells. In addition, mRNA vaccines can activate innate immunity through specific recognition by pattern recognition receptors. At present, in clinical trials of tumor immunotherapy applications, many studies have evaluated the anti-tumor effect of mRNA vaccines, indicating that mRNA vaccines are feasible for preventing and treating tumors. Meanwhile, mRNA vaccines are also applied to the research of various infectious disease vaccines, such as CMV vaccine and Zika vaccine of Moderna company in the United states, and the like are clinically researched.

Efficient mRNA delivery into cells and successful activation of the immune response are key to the clinical transformation of mRNA vaccines. Modified mrnas, mRNA conjugates, viral vectors, microparticles, and nanoparticles have been developed for mRNA delivery in recent years. Although the modified mRNA is somewhat resistant to degradation, it readily binds to serum proteins and aggregates, leading to vascular occlusion. Viral vectors have been highly efficient vectors for nucleic acid delivery through natural evolution and thus can be used as delivery vectors for mRNA. However, the high immunogenicity, potential carcinogenesis, broad tropism, reduced packaging capacity after modification and the limitation of the difficulty of production of viral vectors themselves limit their widespread use. In contrast to viral vectors, non-viral vectors are less efficient in transfection but less immunogenic than viral vectors. In addition, non-viral vectors are easy to synthesize, can insert large fragments, and have the potential to load larger mrnas. With the development of new materials and techniques for their preparation, and a better understanding of the mechanisms involved, non-viral vectors are becoming the vehicles of choice for the delivery of mRNA. Most common are liposomes, polymers, followed by peptides and inorganic nanoparticles. Wherein liposomes can be synthesized relatively easily in a scalable manner, protect mRNA from degradation, facilitate endosomal escape, and can be targeted to specific cell types by surface modification. The liposome can also deliver mRNA and adjuvant into cells together according to requirements, and has potential application prospect.

Although mRNA vaccines have achieved certain effects in clinical studies, the efficiency of liposome delivery of mRNA and the magnitude of immune response induced by the vaccine remain key factors affecting the effectiveness of mRNA vaccines. Therefore, there is a need to develop a vector that can deliver mRNA into cells with high efficiency and can stimulate a strong immune response to assist mRNA vaccines in their action.

Meanwhile, in order to obtain better immune effect, a method of culturing DC cells in vitro and loading antigens to the DC cells in different modes for direct immunization can be adopted. The way of loading antigen to DC cells mainly includes: polypeptide, antigen or whole cell lysate is directly incubated, viral vector (such as adenovirus) carries antigen gene transduction, mRNA transfection of coded antigen, etc. The DC vaccine sensitized by the mRNA is considered to be an ideal mode for preparing the DC vaccine due to the advantages that the mRNA can carry various antigens, multiple antigen epitopes are expressed in cells, the antigen cross presentation is induced, the safety is high compared with a virus vector, and the like. To enhance the effect of mRNA into DC cells, electroporation, liposomes, sonication, and the like are commonly used. In comparison, liposomes mediated mRNA entry into DCs is less harmful to cells, but transfection efficiency is to be improved. Therefore, there is also a need to develop novel mRNA vectors that transfect mRNA into DC cells more efficiently and that are more capable of activating DC cells.

Disclosure of Invention

The technical problem to be solved by the invention is to improve the immune effect of the DCs vaccine based on the mRNA vaccine and mRNA sensitization. The technical scheme for solving the technical problems is to provide a liposome modified by hydrophobic modified cationic polypeptide.

In the liposome, the sequence of the cationic polypeptide is VQWRIRVAVIRK, and the hydrophobic modification is that a hydrophobic fragment is coupled at the nitrogen tail end of the polypeptide; the modified liposome consists of cationic lipid and auxiliary lipid.

Wherein the cationic lipid in the liposome is trimethyl-2, 3-dioleyloxypropylammonium chloride (DOTMA), trimethyl-2, 3-dioleyloxypropylammonium bromide (DOTAP), dimethyl-2, 3-dioleyloxypropyl-2- (2-spermicharacterised carboxamido) ethylammonium trifluoroacetate (DOSPA), trimethyl dodecylammonium bromide (DTAB), trimethyl tetradecylammonium bromide (TTAB), trimethyl hexadecylammonium bromide (CTAB), dimethyl dioctadecylammonium bromide (DDAB), dimethyl-2-hydroxyethyl-2, 3-dioleyloxypropylammonium bromide (DORI), dimethyl-2-hydroxyethyl-2, 3-dioleyloxypropylammonium bromide (DORIE), dimethyl-3-hydroxypropyl-2, 3-dioleyloxypropylammonium (DORIE-HP), dimethyl-4-hydroxybutyl-2, 3-dioleyloxypropylammonium bromide (DORIE-HB), dimethyl-5-hydroxypentyl-2, 3-dioleyloxypropylammonium bromide (DORIE-HPc), dimethyl-2-hydroxyethyl-2, 3-dihexadecyloxypropylammonium bromide (DPRIE), dimethyl-2-hydroxyethyl-2, 3-dioctadecylpropylammonium bromide (DSRIE), dimethyl-2-hydroxyethyl-2, 3-ditetradecyloxypropylammonium bromide (DMRIE), N- (2-sperminoyl) -N ', N' -Dioctadecylglycinamide (DOGS), 1, 2-dioleoyl-3-succinyl-sn-glycerocholine ester (DC) -Chol), 3 β - [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol (DOSC) or lipid poly-L-lysine (LPLL).

Wherein the auxiliary lipid in the liposome is at least one of cholesterol (Chol), Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), Dioleoylphosphatidylethanolamine (DOPE) and Diphosphatidylcholine (DOPC).

Further, the mass ratio between the cationic lipid and the helper lipid in the liposome is as follows: cationic lipid: the auxiliary lipid is 0.5-20: 1. Preferably, the cationic lipid: auxiliary lipid is 0.8-1.25: 1

Further, the mass ratio of the hydrophobic modified cationic polypeptide to the modified liposome moiety in the liposome is as follows: hydrophobically modified polypeptides: the ratio of the liposome is 0.025-0.5: 1. Preferably, the hydrophobically modified polypeptide: the liposome ratio is 0.025-0.2: 1.

Wherein the liposome is prepared by at least one of ethanol injection method, thin film dispersion method, ultrasonic dispersion method, reverse evaporation method, high pressure homogenization method, carbon dioxide supercritical method or freeze drying method.

Wherein, the liposome is characterized by being prepared by the following method:

a. weighing cationic lipid and auxiliary lipid, dissolving in solvent, and removing organic solvent by rotary evaporation under reduced pressure; the solvent can be at least one of chloroform, ethanol, methanol, dichloromethane or ethyl acetate;

b. b, adding an aqueous solution of the hydrophobic modified cationic polypeptide or a solution formed by dissolving the hydrophobic modified cationic polypeptide in the solvent in the step a into the system for hydration and demoulding to obtain liposome suspension;

c. carrying out ultrasonic treatment on the obtained liposome suspension to obtain liposome;

or, the preparation method comprises the following steps:

a. weighing cationic lipid and auxiliary lipid, dissolving in solvent to prepare organic phase, and dissolving in reaction container to prepare organic phase; the solvent is ethanol or diethyl ether;

b. injecting DP7-C solution into the organic phase and stirring; evaporating under vacuum and reduced pressure to 1/5-1/2 of the original volume; homogenizing and extruding to obtain blank liposome solution; the DP7-C solution is an aqueous solution, an ethanol solution or an ether solution thereof;

c. carrying out ultrasonic treatment on the blank liposome solution to obtain DP7-C modified liposome.

Wherein the liposome is prepared from the following raw materials in parts by weight: the mass ratio of the cationic lipid to the helper lipid to the hydrophobic modified cationic polypeptide is 10-40: 0.5-10.

Furthermore, the liposome is prepared from the following raw materials in proportion: cationic lipid to helper lipid in mass ratio: the hydrophobic modified cationic polypeptide is 10-40: 1-6.

Preferably, the liposome is prepared from the following raw materials in proportion: according to the mass ratio of DOTAP to cholesterol: hydrophobization modified cationic polypeptide 20:20: 3.

Wherein, the liposome is modified to VQWRIRVAVIRK-NH2 through amidation of the carbon terminal of the cationic polypeptide VQWRIRVAVIRK.

Wherein the hydrophobic segment of the modified cationic polypeptide in the liposome is sterol compound, saturated straight chain fatty acid, and PEG derivative.

Wherein the sterol compound in the liposome is a cholesterol compound or a cholic acid compound. The sterol compound is at least one of succinylated cholesterol, cholic acid or deoxycholic acid.

Wherein the saturated straight chain fatty acid in the liposome is at least one of C6-C20. Preferably, the saturated straight-chain fatty acid is at least one of C8 to C18. The straight chain fatty acid is at least one of stearic acid (C18), palmitic acid (C16), lauric acid (C12) or n-caprylic acid (C8).

Wherein the PEG derivative in the liposome is 1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine-polyethylene glycol (DSPE-PEG), distearoylphosphatidylethanolamine-polyethylene glycol (DOPE-PEG) or dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG).

Furthermore, the nitrogen end of the cationic polypeptide in the liposome is coupled with the hydrophobic segment in a mode of passing-CO-OH on the hydrophobic segment and-NH on the polypeptide₂Amidation reaction.

Wherein, the hydrophobic modified cationic polypeptide structure in the liposome is as follows:

and R is a sterol compound, saturated straight-chain fatty acid or a PEG derivative.

Further, R in the structure of the hydrophobic modified cationic polypeptide is:

at least one of (1).

The invention also provides application of the liposome modified by the hydrophobic modified cationic polypeptide in preparation of a nucleic acid delivery system.

The invention also provides a nucleic acid delivery system. The nucleic acid delivery system is prepared by loading nucleic acid into liposome modified by the hydrophobic modified polypeptide.

Wherein the nucleic acid in the nucleic acid delivery system is at least one of DNA or RNA.

Further, the RNA is at least one of messenger RNA, siRNA, sgRNA, or mRNA.

Wherein the nucleic acid delivery system is prepared by using the liposome modified by the hydrophobic modified cationic polypeptide and nucleic acid as raw materials according to the mass ratio of 1-10: 1. Preferably, the mass ratio of the liposome modified by the hydrophobic modified cationic polypeptide to the nucleic acid is 1-3: 1. More preferably, the liposome and the nucleic acid modified by the hydrophobic modified cationic polypeptide are in a mass ratio of 2: 1.

Furthermore, the nucleic acid delivery system is prepared by incubating liposome modified by hydrophobic modified cationic polypeptide and nucleic acid. Wherein the co-incubation is performed in an aqueous solution or in a culture medium. Wherein the culture time is 4-15 minutes. Wherein the culture medium is at least one of RPMI 1640, DMEM double non-culture medium and Optim culture medium.

Wherein the length of the nucleic acid loaded in the nucleic acid delivery system is 20-7000 bp.

Wherein the nucleic acid in the nucleic acid delivery system is mRNA. Further, the mRNA is mRNA having a 5 'cap structure and a 3' polyA tail structure.

Further, the mRNA in the above-mentioned nucleic acid delivery system is an antigen-encoding mRNA.

The invention further provides the application of the mRNA delivery system in preparing a dendritic cell vaccine.

The invention also provides a dendritic cell vaccine. The dendritic cell vaccine comprises dendritic cells loaded with the mRNA delivery system as a main active ingredient.

Wherein, the dendritic cell is a myeloid DC or a lymphoid DC.

Further, the dendritic cells are ex vivo dendritic cells obtained from patients.

Further, the dendritic cell vaccine is prepared by the following method:

a. taking immature dendritic cells (imDCs) after induction culture;

b. adding the nucleic acid delivery system to a culture medium for incubation

c. And after incubation, digesting the treated cells by using pancreatin, washing the cells, centrifugally suspending, and detecting to obtain mature dendritic cells.

Furthermore, the dendritic cell vaccine also comprises pharmaceutically acceptable auxiliary components. The pharmaceutically acceptable auxiliary component is at least one of protective agent, excipient, immunologic adjuvant, dispersant or cell culture medium.

The invention has the beneficial effects that: according to the invention, the hydrophobic modified VQWRIRVAVIRK polypeptide is used for modifying the cationic liposome, and the obtained modified liposome can efficiently deliver nucleic acid into cells; especially, mRNA for coding antigen can be efficiently delivered to DCs, the efficiency of delivering the mRNA to the DCs by the cationic liposome is obviously improved, and the immune effect of liposome mRNA vaccine and mRNA-loaded DCs vaccine is enhanced. Meanwhile, the modified liposome can also show stronger immune function, such as more effective stimulation of maturation of DCs and secretion of proinflammatory cytokines, and obviously enhance the immune function of the DCs as APC. In the embodiment of the invention, the modified cationic liposome subcutaneous immune mouse loaded with antigen mRNA and the DCs subcutaneous immune mouse loaded with liposome/mRNA compound show obviously enhanced anti-tumor effect and spleen lymphocyte reaction, which shows that the technical scheme of the invention can comprehensively promote the final in vivo effect of the DCs cell vaccine through various effects such as high-efficiency delivery of mRNA, promotion of maturation of DCs, enhancement of the immunity effect of DCs and the like. Meanwhile, the technical scheme of the invention has the advantages of convenient preparation, low cost, low toxicity and safety, short preparation period, nearly half of time reduction compared with the existing preparation of the antigen peptide-loaded DCs vaccine, good clinical application prospect, and especially greater effect in the individualized DCs cellular immunotherapy scheme.

Drawings

FIG. 1 preparation and characterization of DP7-C modified liposomes. Particle size and potential distribution of DP7-C modified DOTAP liposomes prepared by thin film dispersion. a. Particle size distribution; b. and (4) electric potential. C-d. characterization of DP7-C modified DOTAP liposomes prepared by ethanol injection. c. Particle size distribution; d. and (4) electric potential. Characterization of DP7-C modified DC-chol/DOPE liposomes. e. Particle size distribution; f. and (4) electric potential.

FIG. 2 characterization and proportion screening for binding to mRNA of DP7-C modified DOTAP liposomes. a-b.particle size distribution and zeta potential before and after the DOTAP liposome modified by DP7-C is compounded with mRNA; performing transmission electron microscope characterization on DP7-C modified DOTAP liposome before and after compounding mRNA; d. gel block electrophoresis was used to determine the ratio of DP7-C modified liposomes complexed with mRNA.

FIG. 3 detection of the efficiency of transfection of 293T, dendritic cells with liposome/mRNA complexes. a. Efficiency of transfection of 293T cells; mean fluorescence intensity in 293t cells; JAWSII transfection efficiency; d.DC2.4 transfection efficiency; e, transfection efficiency of BMDCs; liposome/mRNA complexes were detected by the DC uptake pathway. (xp <0.01, x p < 0.001).

FIG. 4 serum stability assay of liposome/mRNA complexes. a. Serum stability of liposome/mRNA complexes; b. serum stability of DP7-C modified liposome/mRNA complexes.

Figure 5 liposome cytotoxicity assay.

Figure 6 lymph node targeting of liposome/eGFP mRNA complexes following subcutaneous injection perilymph nodes in mice (./p < 0.05).

FIG. 7 Liposome in vitro induction of DCs maturation, CD103⁺DC ratio and cytokine secretion assay. a. Detecting liposome-induced DC maturation; b. liposome-induced CD103⁺A DC ratio; ELISA detecting IL-1 beta content in culture liquid supernatant; ELISA detects the content of TNF-alpha in the culture solution supernatant; ELISA detecting the IL-6 content of the culture solution supernatant; ELISA detecting the content of IL-12p70 in the culture solution supernatant; ELISA detects the IL-10 content of the culture solution supernatant; IL-12p70 to IL-10 ratio. (. about.p)<0.05， **p<0.01，***p<0.001)。

FIG. 8 Liposome-induced maturation of DCs in lymph nodes, CD103⁺DC ratio and cytokine secretion in serum. a. Subcutaneous injection of liposomes beside the lymph nodes in mice recruits DCs into the lymph nodes. b. The mouse lymph node is injected with liposome subcutaneously to increase the proportion of mature DCs in lymph node. c. Liposome-induced CD103⁺A DC ratio; d. IL-6 content in serum; e. the content of TNF-alpha in serum; f. IL-12p70 content in serum; g. IL-10 content in serum; h. IL-12p70 in serum was compared to IL-10. (. about.p)<0.05，**p<0.01，***p<0.001)。

FIG. 9. anti-tumor effect of liposome/OVA mRNA complexes in the treatment of EG7-OVA subcutaneous tumor model. a. A tumor growth curve; b. detecting spleen antigen specific lymphocyte. (. p <0.05,. p < 0.001).

Figure 10 anti-tumor effect of liposome/neoantigen mRNA complexes on LL2 orthotopic tumor model. a. The number of lung nodules; b. mean lung weight; c. wild-type antigenic peptide, mutant antigenic peptide and spleen CD8 after mRNA treatment⁺Activation of T lymphocytes; d. wild-type antigenic peptide, mutant antigenic peptide and spleen CD8 after mRNA treatment⁺The case where T lymphocytes secrete IFN-. gamma.. (. about.p)<0.05，**p<0.05，***p<0.001)。

FIG. 11. antitumor Effect of DCs vaccine loaded with liposome/OVA mRNA complexes on the treatment of EG7-OVA subcutaneous tumor model. a. A tumor growth curve; b. detecting spleen antigen specific lymphocyte. (. p <0.05,. p < 0.001).

Figure 12 anti-tumor effect of liposome/neoantigen mRNA complex loaded DCs vaccine treatment on LL2 orthotopic tumor model. a. A tumor growth curve; b. tumor weight; c. wild-type antigenic peptide, mutant antigenic peptide and spleen CD8 after mRNA treatment⁺The secretion of IFN-gamma by T lymphocytes. (. about.p)<0.05，**p<0.05，***p<0.001)。

Detailed Description

The present invention will be described in more detail with reference to the following description of specific embodiments.

In the previous studies of the present invention, a polypeptide having an excellent antibacterial effect was obtained, its sequence was VQWRIRVAVIRK (SEQ ID No.1), which was named DP 7. In further research, the DP7 polypeptide is subjected to hydrophobic modification to become an amphiphilic compound with the capability of self-assembling into micelles, so that the cytotoxicity of the DP7 polypeptide can be reduced, and the antibacterial activity is maintained; on the other hand, the nano-particle can be used as a delivery carrier of some medicines after being assembled into nano-particles.

The inventors innovatively considered modification of cationic liposomes with the hydrophobically modified DP7 polypeptide and then examined whether the resulting complex obtained by its modification functions as a carrier for mRNA delivery. In experiments, surprisingly, the use of the DP7 polypeptide modified by the hydrophobic modification not only obviously improves the efficiency of the cationic liposome in transfecting mRNA into DCs; meanwhile, the DC cell maturation and the proinflammatory cytokine secretion can be better promoted in vitro and in vivo, and the huge potential in the preparation of mRNA (messenger ribonucleic acid) transmission systems, mRNA vaccines and mRNA-loaded DC vaccines is embodied. On the basis, the technical schemes of the invention are obtained.

In some embodiments, various conventional cationic liposomes can be modified using a hydrophobically modified DP7 polypeptide. It is well known to those skilled in the art that cationic lipids are difficult to form liposomes with good stability and desired delivery ability alone, and therefore, it is necessary to add auxiliary lipids to form cationic liposomes. Therefore, the cationic liposome involved in the invention is also mainly a liposome with positive charges, which is composed of cationic lipid and auxiliary lipid together.

The cationic lipids that can be used in the present invention can be selected from cationic lipid molecules commonly used in the art for the preparation of cationic liposomes. For example, trimethyl-2, 3-dioleyloxypropylammonium chloride (DOTMA), trimethyl-2, 3-dioleyloxypropylammonium bromide (DOTAP), dimethyl-2, 3-dioleyloxypropyl-2- (2-spermicarbonamido) ethylammonium trifluoroacetate (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), dimethyldioctadecylammonium bromide (DDAB), dimethyl-2-hydroxyethyl-2, 3-dioleyloxypropylammonium bromide (DORI), dimethyl-2-hydroxyethyl-2, 3-dioleyloxypropylammonium bromide (DORIE), dimethyl-3-hydroxypropyl-2-bromide, 3-dioleyloxypropylammonium (DORIE-HP), dimethyl-4-hydroxybutyl-2, 3-dioleyloxypropylammonium bromide (DORIE-HB), dimethyl-5-hydroxypentyl-2, 3-dioleyloxypropylammonium bromide (DORIE-HPc), dimethyl-2-hydroxyethyl-2, 3-dihexadecyloxypropylammonium bromide (DPRIE), dimethyl-2-hydroxyethyl-2, 3-dioctadecyloxypropylammonium bromide (DSRIE), dimethyl-2-hydroxyethyl-2, 3-ditetradecyloxypropylammonium bromide (DMRIE), N- (2-arginoyl) -N ', N' -Dioctadecylglycinamide (DOGS), 1, 2-dioleoyl-3-succinyl-sn-glycerocholine ester (DC- Chol), 3 beta- [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol (DOSC), lipid poly-L-lysine (LPLL).

While helper lipids cationic lipids that may be used in the present invention may be selected from helper lipids commonly used in the art for preparing cationic liposomes. For example, at least one of cholesterol (Chol), Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), Dioleoylphosphatidylethanolamine (DOPE), and Diphosphatidylcholine (DOPC) may be selected.

The cationic liposome in the product of the present invention can be prepared by methods known in the art, including but not limited to at least one of ethanol injection, thin film dispersion, ultrasonic dispersion, reverse evaporation, high pressure homogenization, carbon dioxide supercritical method, or freeze drying. Also included are combinations and improved methods based on the above methods, such as prior art methods for preparing cationic liposomes, e.g., film-freeze-thaw methods, film-extrusion methods, vacuum drying-sonication methods, and the like.

When preparing the cationic liposome, the dosage ratio between the cationic lipid and the auxiliary lipid can be adjusted according to the specific used raw materials and requirements. However, considering the addition of the hydrophobic modified cationic polypeptide, the mass ratio of the cationic lipid to the auxiliary lipid can be controlled to be 0.5-1.5: 1. Preferably, the mass ratio of the cationic lipid to the auxiliary lipid is 0.8-1.25: 1.

The hydrophobic modified cationic polypeptide used for modifying the cationic liposome is modified by connecting a hydrophobic fragment to the nitrogen end of the VQWRIRVAVIRK polypeptide. And the carbon end of VQWRIRVAVIRK polypeptide can be amidated and modified to VQWRIRVAVIRK-NH₂。

The hydrophobic segment used for hydrophobic modification of VQWRIRVAVIRK polypeptide can be sterol compound, saturated straight chain fatty acid or PEG derivative. The sterol compound is a cholesterol compound or a cholic acid compound. For example, it may be selected from succinylated cholesterol, cholic acid or deoxycholic acid. The saturated straight chain fatty acid is at least one of C6-C20. Preferably, the saturated straight-chain fatty acid is at least one of C8 to C18. For example, it may be selected from stearic acid (C18), palmitic acid (C16), lauric acid (C12) or n-octanoic acid (C8). The PEG derivative may be selected from 1, 2-dioleoyl-SN-glycerol-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG), distearoylphosphatidylethanolamine-polyethylene glycol (DOPE-PEG) or dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG).

Generally, the nitrogen terminus of the cationic polypeptide is coupled to the hydrophobic moiety (hydrophobic compound) by coupling via the hydrophobic moiety (hydrophobic compound)-CO-OH and-NH on antimicrobial peptides₂Amidation reaction and coupling.

The structure of the hydrophobically modified cationic polypeptide can be represented as:

wherein, R is a sterol compound or a saturated straight chain fatty acid or a PEG derivative.

In some examples, R in the above formula is:

at least one of (1).

However, the mass ratio between the hydrophobic modified cationic polypeptide and the liposome can be generally selected from the following ranges: the hydrophobic modified polypeptide: liposome is 0.025-0.5: 1. Preferably, the hydrophobic modified polypeptide: liposome ═ 0.025-0.2: 1.

In one embodiment of the present invention, the cationic polypeptide is modified with cholesterol as the hydrophobated moiety, i.e., R in the above formula is:

the polypeptide obtained by modifying VQWRIRVAVIRK-NH2 with cholesterol is named DP7-C in the invention.

The invention further provides a reference method for preparing the liposome modified by the hydrophobic modified cationic polypeptide.

One of the methods is based on a thin film dispersion method, comprising the steps of:

a. weighing cationic lipid and auxiliary lipid, dissolving in solvent, and removing organic solvent by rotary evaporation under reduced pressure;

b. b, adding an aqueous solution of the hydrophobic modified cationic polypeptide or a solution formed by dissolving the hydrophobic modified cationic polypeptide in the solvent available in the step a into the system for hydration and demoulding to obtain liposome suspension;

the solvent used in step a is generally an organic solvent, and commonly used solvents include chloroform, ethanol, methanol, dichloromethane, ethyl acetate, and the like.

Another method is based on an ethanol injection method, comprising the steps of:

a. weighing cationic lipid and auxiliary lipid, dissolving in solvent to prepare organic phase, and dissolving in reaction container to prepare organic phase;

b. injecting DP7-C solution into the organic phase and stirring; evaporating under vacuum and reduced pressure to 1/5-1/2 of the original volume; homogenizing and extruding to obtain blank liposome solution;

The method comprises the following main raw materials in proportion: the mass ratio of the cationic lipid to the helper lipid to the hydrophobic modified cationic polypeptide is 10-40: 0.5-10.

The method further comprises the following main raw materials in proportion: cationic lipid to helper lipid in mass ratio: the hydrophobic modified cationic polypeptide is 10-40: 1-6.

The solvent used in step a is typically ethanol. However, if ether injection is used, the solvent is ether here. The DP7-C solution of step b may be in aqueous, ethanolic or ethereal solution, as appropriate.

For example, in one embodiment of the invention liposomes are further formed using DP7-C with DOTAP and cholesterol as starting materials. And provides a typical preparation method based on a thin film dispersion method, which comprises the following steps:

a. weighing DOTAP and cholesterol, dissolving in a solvent, and performing reduced pressure rotary evaporation to remove the solvent;

b. adding the water solution of the hydrophobic modified cationic polypeptide into the system for hydration and demoulding to obtain liposome suspension;

c. and (c) carrying out ultrasonic treatment on the liposome suspension obtained in the step b to obtain the liposome.

The solvent used in step a is generally an organic solvent, and commonly used solvents include chloroform, ethanol, methanol, dichloromethane, ethyl acetate and the like.

The temperature of the system is generally required to be controlled not to be too high during ultrasonic treatment, and is preferably below 25 ℃, for example, the DP7-C polypeptide modified DPTAP liposome with a proper morphology can be prepared by adopting ice bath, intermittent ultrasonic treatment and the like.

In the preparation process, the mass ratio of the components is DOTAP: cholesterol: DP 7-C: 10-40: 0.5-10. Preferably, the mass ratio is DOTAP: cholesterol: DP 7-C: 10-40: 1-6. The optimal mass ratio is as follows: DOTAP: cholesterol: DP 7-C: 20: 3.

Similarly, in another embodiment of the present invention, DP7-C modified DC-chol/DOPE liposomes were prepared based on the membrane dispersion method.

In addition, another embodiment of the invention also adopts a method based on ethanol injection to prepare the DP7-C modified DOTAP liposome.

These examples also show that the cationic liposomes of the products of the invention can be prepared by methods known in the art and then modified with DP7-C, and that the resulting modified products have more stable properties.

The liposome modified by the hydrophobic modified cationic polypeptide has the function of loading nucleic acid and transferring the nucleic acid into cells, so that a nucleic acid transfer system can be formed by loading the nucleic acid. These target cells may be cells of various animals and plants including human. The cells may be in vivo or in an ex vivo culture.

The nucleic acid delivery system can be used to deliver at least one nucleic acid, either DNA or RNA. And the nucleic acid with the length of 20-7000 bp can obtain good transmission efficiency.

The nucleic acid delivery system can be widely applied to deliver at least one of various specific RNA molecules, such as messenger RNA, siRNA, sgRNA or mRNA. In particular, mRNA for obtaining a target protein or polypeptide by translation in a cell, which usually has a 5 'cap structure and a 3' polyA tail structure, can be efficiently transferred.

More importantly, this delivery system can be made very simply. A typical convenient protocol is to co-incubate liposomes modified with the above hydrophobically modified cationic polypeptides with nucleic acids in a liquid. The co-incubation can be performed in an aqueous solution or in a commonly used liquid medium, such as a commonly used cell culture medium, e.g., RPMI 1640 medium, DMEM medium, Optim medium, and the like, and a mixed medium prepared by combining the two media. The period of co-incubation in the system for 4 to 15min is generally sufficient to load a sufficient amount of nucleic acid onto the liposomes to obtain the nucleic acid delivery system. The nucleic acid delivery system can be prepared from liposome modified by hydrophobic modified cationic polypeptide and nucleic acid according to the mass ratio of 1-10: 1. Preferably, the liposome is prepared from the liposome and nucleic acid according to the mass ratio of 1-3: 1.

Since it has been found in previous studies that the DP7-C polypeptide alone is capable of self-assembling in water to form micelles, it can be loaded with a range of small molecule drugs, polypeptides, or even short nucleic acid molecules, such as siRNA. However, the properties and functions of the liposomes obtained by modifying cationic liposomes with DP7-C are not known.

First, the present inventors have found that cationic liposomes modified with DP7-C are capable of delivering long nucleic acid molecules, such as mRNA, in addition to the substances that a DP7-C polypeptide alone is capable of delivering.

Unexpected results were obtained when DP7-C modified cationic liposomes loaded with mRNA were used to transfect DC cells, and DP7-C modified cationic liposomes were able to improve the transfection efficiency of mRNA into DC cells. Further, it was confirmed that DP7-C modified cationic liposomes significantly improved the mRNA transfer efficiency in three different DC cells, JAWSII, DC2.4 and BMDC, and also significantly better than the commercial transfection reagents Lipo2000 and PEI 25K.

In further experiments, the invention finds that the cationic liposome modified with DP7-C not only improves the delivery efficiency of mRNA, but also has better serum stability and lower cytotoxicity. Intensive research also finds that the unmodified DOTAP liposome/mRNA complex is mainly internalized by DC cells through a megalocytosis pathway, while the cationic liposome/mRNA complex modified with DP7-C is mainly internalized by the DC cells through two pathways of caveolin and clathrin, namely that the DP7 polypeptide after hydrophobic modification can change the uptake pathway of cationic liposome by the DC cells, and the reason is probably that the transfection efficiency of mRNA is greatly improved.

Thus, it was determined that hydrophobically modified DP7 polypeptide modified cationic liposomes are particularly suitable as delivery vehicles for delivering nucleic acid molecules, particularly mRNA, to a variety of DC cells. This can just solve the problem of limiting the effectiveness of vaccines by the intracellular delivery efficiency that is widely present in the art when preparing mRNA loaded DC vaccines.

In the subsequent research, the method also unexpectedly discovers that the cationic liposome is endowed with stronger lymph node targeting by using the DP7 polypeptide modified by hydrophobic modification, has obviously better capability of promoting DC cell maturation and promoting secretion of various inflammatory cytokines in vitro and in vivo, and shows a wide prospect in the aspect of preparing DC cell mRNA vaccines.

Based on this, the present invention provides the use of the nucleic acid delivery system described above in the preparation of a dendritic cell vaccine. The dendritic cell vaccine comprises dendritic cells loaded with the nucleic acid delivery system of the invention as a main active ingredient, wherein the nucleic acid loaded by the nucleic acid delivery system is mRNA.

Naturally, to prepare a dendritic cell vaccine, the nucleic acid delivery system described above requires loading and delivering the antigen mRNA into the DC cells. The reed antigen can be any antigen that can be encoded by mRNA and is desirably expressed and presented after entry into a DC cell. The antigen may be a full-length antigen with a proteinaceous component, such as a protein or peptide, or may be an antigen that is further modified after translation, for example, certain glycoproteins or lipoproteins.

The antigen may be a tumor antigen, including a specific protein or polypeptide expressed by a tumor cell. Such as WT1, MUC1, EGFRvIII, HER-2, MAGE-A3, NY-ESO-1, PSMA, GD2 or MART1, or individualized mutant neo-antigen combinations based on patient tumor sequence determination.

The antigen may also be a viral antigen, such as a protein or polypeptide that makes up a viral portion, or a specific protein or polypeptide that is expressed in cells infected with a virus under the control of a viral expression machinery. Such as EBV, LMP2, HPV E6E7, adenovirus 5Hexon or HCMV pp 65.

The antigen may also be a bacterial antigen, including a protein or polypeptide expressed by a bacterium. Such as Pseudomonas aeruginosa antigen, tetanus bacillus antigen, Streptococcus pneumoniae, Salmonella, etc.

It will also be understood by those skilled in the art that the antigen may also be an antigen associated with a disease, including autoimmune-related antigens, such as antigens involved in or over-expressed during an autoimmune disease or disorder. Examples of such antigens include autoimmune-related antigens such as ppipp, IGRP, GAD65, and myelin basic protein antigen.

In addition, the loaded mRNA molecule may encode one or more immunogenic epitopes. These epitopes can generally be peptide fragments of between 3 and 50 amino acid residues of any length, generally in linear arrangement, and can be further post-translationally modified after expression in DC. It is to be understood that each immunogenic epitope of more than one immunogenic epitope may be the same or different.

For example, in one embodiment of the present invention, based on 5 LL2 mutant neoantigens with immunogenicity, a new mRNA is constructed, which can be translated in vivo and processed to form a peptide chain containing the 5 antigenic peptides. The polypeptide is loaded on a cationic liposome modified by the DP7 polypeptide after hydrophobic modification, and the polypeptide shows obvious anti-tumor effect in both in-situ and subcutaneous tumor models.

For preparing a dendritic cell vaccine, the dendritic cells can be selected from Myeloid DC (MDC) cells or Lymphoid DC (LDC) cells.

According to the development of related fields based on immunology, bioinformatics and the like at present, the preparation of personalized vaccines of patients can be realized. For example, the DC cell vaccine for a specific tumor patient can be generated by searching for a tumor specific antigen of the tumor patient, designing a personalized mRNA according to the specific antigen, loading the mRNA on a hydrophobic modified DP7 polypeptide modified cationic liposome, and delivering the cationic liposome into a DC cell. In this case, ex vivo dendritic cells obtained from patients themselves are often used.

The method for preparing the dendritic cell vaccine comprises the following main steps:

a. taking immature dendritic cells (imDCs) after induction culture;

b. adding the nucleic acid delivery system to a culture medium for incubation

Detection of mature dendritic cells various means are available in the art. For example, after the antibody CD11c, CD80 and CD86 is added to the DC cells after the centrifugal resuspension, the DC cells can be stained, and then whether the dendritic cells are mature or not can be judged according to the staining result.

It will be appreciated by those skilled in the art that either the cationic liposomes modified with the hydrophobically modified DP7 polypeptide loaded with antigen mRNA are used directly or DC cell vaccines prepared on the basis thereof may also comprise pharmaceutically acceptable auxiliary components.

The pharmaceutically acceptable auxiliary components are at least one of protective agent, excipient, immunological adjuvant, dispersant or cell culture medium.

For example, a DC live cell vaccine will be handled, formulated and used in a solution that may therefore contain pharmaceutically acceptable concentrations of salts, buffers, preservatives and various media to assist in maintaining the viability of the live cells in the vaccine preparation and to reduce or prevent cell lysis. If the vaccine is to be stored frozen, the vaccine may include a cryoprotectant, such as DMSO.

If desired, the vaccine may further comprise an adjuvant to assist in the induction or re-stimulation of the immune response, including prolonging or boosting the immune response. The adjuvant may be selected among various adjuvants known in the art according to the actual use of the DC vaccine.

The present invention will be described in further detail by way of examples.

The experimental materials and equipment mainly used in the examples are as follows:

1. cell line for experiment and experimental animal

The 293T, JAWSII, DC2.4 cell line and the mouse lung cancer cell line LL2 were purchased from American Type Culture Collection (ATCC). Cell culture was performed using DMEM (Gibico) medium containing 10% Fetal bovine serum (Fetal bone serum, FBS, Gibico). 6-8 week old C57BL/6J female mice used in the experiment were purchased from Peking Wintonlifa laboratory animals Co., Ltd and were bred in SPF-grade environment.

2. Main reagent material and kit

Cell culture media for experiments: 1640 medium (RPMI-1640), DMEM medium and Fetal Bovine Serum (FBS) were purchased from Gibco, USA.

A FLAG polypeptide: DYKDDDDK (SEQ ID No.2) (available from sigma corporation)

OVA mRNA was purchased from Tebu-bio.

Mouse IFN-gamma pre-coated ELISpot kit (# DKW22-2000-096), lymphocyte separation medium and ELISPOT special culture medium (DKW 34-EU0100 in David) were purchased from David as a biotechnology limited liability company.

Cytokines and agonists: rmGM-CSF (122-03) was purchased from Shanghai Puxin Biotech, Inc.; PolyIC is available from Thermo corporation; agonist CPG1826 was synthesized by invitrogen.

Flow-through antibody: the following flow-through antibodies were purchased from BD: rat Anti-Mouse CD86, Hamster Anti-Mouse CD11c, Rat Anti-Mouse CD8 alpha, Rat Anti-Mouse CD4, Rat Anti-Mouse I-A/I-E, Hamster Anti-Mouse CD103, Hamster Anti-Mouse CD80, Hamster Anti-Mouse CD3E, Rat Anti-Mouse IFN-gamma.

Western Blot antibody: anti-GAPDH antibody and anti-MyD88antibody were purchased from Abcam.

The TLR2 receptor inhibitor C29, the macropinocytic pathway inhibitor, the caveolin pathway inhibitor and the clathrin pathway inhibitor were all purchased from MCE corporation.

DOTAP, DOPE, DC-Chol cholesterol were purchased from Shanghai Avena corporation; dialysis cards were purchased from Sigma-aldrich; e-GFP mRNA was purchased from Thermo; PEI25K reagent was purchased from Sigma-aldrich; lipofectamine 2000 transfection reagent was purchased from Invitrogen; RiboRuler High Range RNA Ladder from Thermo.

3. Main instrument equipment

Rotary evaporator, V rotary evaporator RV 10, german moxa card; a small animal in vivo imaging system, Caliper Life Sciences, IVIS spectra; malvern particle size potentiometer: zetasizer Nano-ZS Zen 3600, Malvern; transmission electron microscopy: h-600 transmission electron microscope, Hitachi; flow cytometry: FACSCalibur, BD; an enzyme-labeling instrument: multiskan Mk3, Thermo Scientific; DNA concentration measurement instrument: nanodrop 2000, Thermo Scientific; laser confocal microscopy: FV1000, Olympus; a constant-temperature incubator: MCO-18, Thermo Scientific.

EXAMPLE I preparation and characterization of DP7-C modified liposomes

In this example, different methods (membrane dispersion method and ethanol injection method) were used to prepare DP7-C modified DOTAP/cholesterol cationic liposome, and DP7-C modified DC-chol/DOPE cationic liposome was prepared by membrane dispersion method.

Preparation and characterization of DP7-C modified DOTAP liposome

1) DP7-C modified DOTAP liposome prepared by adopting film dispersion method

20mg of DOTAP and 20mg of cholesterol were weighed out and dissolved in 4ml of chloroform, and the solution was put into a 100ml round bottom bottle. The organic solvent was removed by rotary evaporation under reduced pressure in a 37 ℃ water bath at a set rotation speed of 50 rpm/min. 10ml of 300 mu g/ml DP7-C aqueous solution is added into an eggplant-shaped bottle for hydration and demoulding, the temperature is set at 60 ℃, the rotation speed is 75rpm/min, and the hydration is continued for 30 min. Finally, the resulting liposome suspension was subjected to ice bath sonication for 3min (set conditions of 110W, sonication for 3s, and cessation for 3s) to prepare DP7-C modified DOTAP liposomes (in the following examples, DOTAP/cholesterol liposomes are simply referred to as DOTAP liposomes, and DOTAP/cholesterol liposomes are labeled as DOTAP in the drawings).

The DP7-C modified DOTAP liposomes prepared above were dialyzed in a dialysis card to examine the efficiency of the DP7-C modified liposomes. The cut-off molecular weight of the dialysis card is 2000Da, the prepared liposome is added into the dialysis card, and the dialysis card is placed in a beaker with 1L of water for dialysis for 24 h. Taking out a sample in the dialysis card, detecting DP7-C in the sample before and after dialysis by using HPLC, and reflecting the change condition of the concentration of DP7-C by peak area comparison.

The experimental results are as follows: and comparing peak areas obtained by HPLC detection of the sample in the dialysis card after 24h and the sample before dialysis. The time of the peak appearance of DP7-C was about 15.5min, the peak area of the sample before dialysis was 1747.38, and the peak area after dialysis was 900.2, and the proportion of DP7-C stably modified to the liposomes was calculated from the standard curve. The results show that: after dialysis 57.63% of DP7-C was stably inserted into DOTAP liposomes. The particle size distribution and the zeta potential of the liposome are characterized by using a particle size potentiometer (Malvern Zetasizer, UK), the particle size distribution and the zeta potential change condition after the liposome is compounded with mRNA are measured, and all experiments are repeated for three times to obtain a middle value. The particle size of the DOTAP liposome modified by DP7-C was 100.23 + -7.50 nm, and the zeta potential was 53.02 + -5.51 mV (FIGS. 1a-1 b).

2) DP7-C modified DOTAP liposome prepared by ethanol injection method

The method comprises the following steps: weighing 20mg of DOTAP and 20mg of cholesterol, adding 10ml of ethanol into a reaction container to dissolve and prepare an organic phase, injecting DP7-C aqueous solution into the organic phase to enable the final concentration to be 300 mu g/ml, and stirring for 10 min; evaporating under vacuum and reduced pressure to 1/5-1/2 of the original volume; homogenizing and extruding to obtain blank liposome solution. Ultrasonic treatment at 200W for 10min in ice bath. The final efficiency of DP7-C modification to DOTAP liposomes was detected to be 53.24%.

DP7-C modified liposomes prepared by ethanol injection method had particle size of about 113.35 + -13.34 nm and zeta potential of 52.33 + -4.32 mV as measured by a particle size potentiometer (FIGS. 1C-1 d).

Preparation and characterization of DP7-C modified DC-chol/DOPE cationic liposome

20mg of the cationic lipid DC-cholesterol (3b- [ N- (N ', N' -dimethylaminoethane) -carbamoyl)]Cholesterol hydrochloride) and 40mg of the neutral lipid DOPE (1, 2-diol-sn-glycerol-3-phosphoethanolamine) as 1: 2 is dissolved in chloroform and placed in a glass bottle. In the absence of O₂The lipids were dried under argon flow. After evaporation, the lipid layer was hydrated by adding 10ml of 300. mu.g/ml aqueous DP7-C at 60 ℃ and 75rpm/min for 30 min. After vortexing for 3min, the glass flask with the multilamellar liposomes formed was placed in an ultrasonic bath for 30min to form unilamellar liposomes. Finally, DP7-C modified DC-chol/DOPE liposomes were obtained by extrusion with a micro-extruder using a membrane with 200nm pores. The efficiency of DP7-C modification was 52.31% after dialysis.

The liposome particle size was approximately 185.18 + -25.47 nm and the zeta potential was 23.21 + -3.45 mV as measured using a particle size potentiometer (FIGS. 1e-1 f).

This example shows that DP7-C can be successfully modified to cationic liposomes made by different methods and from different starting materials, resulting in products with similar particle size and zeta potential properties. Modification of DOTAP liposomes with DP7-C resulted in stable liposomes of about 100nm in size and a zeta potential of about 50 mV. Modification of DC-chol/DOPE cationic liposomes with DP7-C resulted in stable liposomes having a particle size of about 180nm and a zeta potential of about 20 mV. In the following examples, experiments relating to DP7-C modified DOTAP liposome-loaded mRNA and DCs vaccine preparation are provided.

Example II preparation and characterization of DP7-C modified DOTAP Liposome Complex mRNA

DP7-C modified DOTAP liposome and mRNA compounding method and optimal proportion

DP7-C modified DOTAP liposomes prepared in example one were diluted in aqueous solution to the use concentration. According to liposome: and adding mRNA in a ratio of 2:1, gently mixing uniformly, and incubating at room temperature for 10min to obtain the DP7-C modified DOTAP liposome. Gel block electrophoresis detected the binding of DP7-C modified liposomes to different mass ratios of mRNA (mRNA encoding a novel antigen shown in SEQ ID No.4, the same below), with DP7-C modified liposomes to mRNA mass ratios of 0:1, 0.25:1, 0.5:1, 1:1, 2:1, respectively.

The experimental results show that: DP7-C modified DOTAP Liposome complex mRNA had a particle size of 130.45. + -. 9.32nm and a zeta potential of 34.67. + -. 7.45mV (FIGS. 2 a-b). Transmission electron microscope observation shows that the prepared DOTAP liposome modified by DP7-C has uniform particle size and good dispersibility, and the diameter is equivalent to the size measured by a particle size potentiometer and is about 100 nm. DP7-C modified DOTPA liposome after mRNA complexation has good dispersibility, uniform particle size, diameter of 120-130nm, and consistent with the result of particle size analysis (FIG. 2C). Gel blocking electrophoresis results show that DP7-C modified DOTAP liposome and mRNA have the mass ratio of 2:1, free mRNA bands were not present, and both mRNA bands were bound to DP7-C modified DOTAP liposomes, indicating that DP7-C modified DOTAP liposomes incubated with mRNA at an optimal ratio of 2:1 (fig. 2 d).

EXAMPLE III study of DP7-C modified DOTAP liposomes as a delivery vehicle for mRNA

Efficiency of DP7-C modified DOTAP Liposomal transfection of mRNA into 293T cells

Mu.g of EGFP mRNA and 1. mu.g of liposomes, 1. mu.g of Lipo2000 and 1. mu.g of PEI25K were co-incubated for 10min in RPMI 1640 medium and transfected into 2X 10 plates⁵In 24-well plates of 293T cells, the RPMI 1640 medium was replaced with RPMI 1640+ 10% FBS + 1% PS medium 4h later and transfected for 24 h. The green fluorescent protein expression was observed using a fluorescence microscope and photographed. Cells were digested with pancreatin and the proportion of GFP positive cells was examined using flow cytometry to determine transfection efficiency, and the mean fluorescence intensity was analyzed.

The experimental results showed that the efficiency of transfecting mRNA into 293T cells by unmodified DOTAP liposome was 84.89 + -2.13%, and the efficiency of transfecting mRNA into 293T cells by DP7-C modified DOTAP liposome was 84.87 + -3.21%. Whereas PEI transfected mRNA into 293T cells with 80.89 + -5.56% efficiency and Lipo2000 transfected mRNA into 293T cells with 80.89 + -10.23% efficiency, indicating that DP7-C modified DOTAP liposomes were comparable to unmodified DOTAP liposomes as well as Lipo2000 and PEI25K in transfecting eGFP mRNA into 293T cells (FIGS. 3a-3 b).

Efficiency of DP7-C modified DOTAP Liposomal transfection of mRNA into dendritic cells

In this experiment, GFP mRNA was targeted, and mRNA was transferred to DCs, expressed as protein and presented by DCs, and the transfection efficiency of DCs was examined. The specific method comprises co-incubating EGFP mRNA 0.5 μ g and liposome 1 μ g, Lipo2000 1 μ g and PEI25K 1 μ g in RPMI 1640 medium, adding into the medium coated with 2 × 10⁵In 24-well plates of JAWSII, DC2.4 and BMDC, RPMI 1640 medium was changed to RPMI 1640+ 10% FBS + 1% PS medium after 4h, and transfection was continued for 24 h. The green fluorescent protein expression was observed using a fluorescence microscope and photographed. Cells were trypsinized, centrifuged at 12000 rpm for 3min, resuspended in 100. mu.l PBS, incubated for 40min with 1. mu.l CD11c antibody, excess antibody was washed off after the incubation was complete, and the proportion of GFP-positive cells in CD11 c-positive cells was examined using a flow cytometer to determine transfection efficiency.

The results show that: the efficiency of transferring mRNA into JAWSII cells by the unmodified DOTAP liposome is 4.51 +/-1.03 percent, the efficiency of transferring mRNA into DC2.4 cells is 5.05 +/-2.12 percent, and the efficiency of transferring mRNA into BMDCs is 8.40 +/-1.31 percent; the efficiency of transferring mRNA into JAWSII cells by the DOTAP liposome modified with DP7-C is 12.23 +/-1.35%, the efficiency of transferring mRNA into DC2.4 cells is 28.49 +/-2.46%, and the efficiency of transferring mRNA into BMDCs is 14.51 +/-2.35%; the transfer efficiency of Lipo2000 to JAWSII cells is 3.65 +/-1.12%, the transfer efficiency of mRNA to DC2.4 cells is 4.56 +/-1.37%, and the transfer efficiency of mRNA to BMDCs is 6.40 +/-1.32%; the efficiency of transferring mRNA into JAWSII cells by PEI is 4.63 +/-1.11%, the efficiency of transferring mRNA into DC2.4 cells is 5.07 +/-1.32%, and the efficiency of transferring mRNA into BMDCs is 6.73 +/-1.27%. This result demonstrates that DOTAP liposomes modified with DP7-C unexpectedly increased the efficiency of mRNA transfer into DCs, and that the transfection efficiency in all three DC cells was significantly different from the other controls (p <0.001) (fig. 3C-3 e).

3. Study of the mechanism associated with uptake of mRNA-loaded DP7-C modified DOTAP liposomes by DCs

Endocytosis is the process of transporting extracellular substances into cells by the movement of plasma membrane deformation. Mainly divided into macroendocytosis, clathrin and caveolin pathways. In order to determine which uptake pathway the liposome/mRNA complex enters the DCs is related to, the DCs are treated by inhibitors of each uptake pathway, and the uptake efficiency of the DCs on the liposome/mRNA complex after the pathway is inhibited is tested, so that the entry of the mRNA carried by the liposome into the DCs is related to which endocytic pathway. Study was carried out using the clathrin-mediated endocytosis pathway inhibitor Chloroprazine, the macropinocytic pathway inhibitor Amiloride, and the caveolin-mediated endocytosis pathway inhibitor Genistein.

The specific experimental method is to lay DC2.4 on a 24-hole plate with each hole being 2 multiplied by 10⁵Cells were treated for 2h with 5. mu.M of chloromazine, 20. mu.M of amiloride and 30. mu.M of genistein, respectively. Subsequently, a complex of DOTAP liposome (1. mu.g) incubated with eGFP mRNA (0.5. mu.g) and a complex of DP7-C modified DOTAP liposome (1. mu.g) incubated with eGFP mRNA (0.5. mu.g) were added and incubated at 37 ℃ for 24 hours. Cells were harvested and the proportion of GFP positive cells was examined using flow cytometry.

From the results, it can be seen that: the transfection efficiency of DC2.4 with unmodified DOTAP liposome/mRNA complex was 5.05 + -2.12%, the transfection efficiency after using macroendocytosis pathway inhibitor was 0.78 + -0.43%, which was significantly lower than that of the group not treated with inhibitor (p <0.001), and the transfection efficiency after using clathrin and caveolin pathways was not significantly different from that of the group not treated with inhibitor (p >0.05), indicating that the unmodified DOTAP liposome/mRNA complex was mainly taken up by DCs via the macroendocytosis pathway. Whereas the efficiency of transfection of DC2.4 with DP7-C modified DOTAP liposome/mRNA complex was 28.49 + -2.46%, the uptake efficiency did not change significantly after treatment with the megalocytosis pathway inhibitor (p >0.05), whereas the uptake efficiency decreased to 10.47 + -1.35% after treatment with clathrin inhibitor and was significantly lower than that of the group without inhibitor (p < 0.001). Whereas the intake efficiency after treatment with the pit protein inhibitor decreased to 15.23 ± 2.67% and was significantly lower than the group without inhibitor treatment (p < 0.01). Indicating that DP7-C modified DOTAP liposome/mRNA complex was internalized by DCs primarily in both the caveolin and clathrin pathways (fig. 3 f).

4. Serum stability assay for liposome/mRNA complexes

The liposome/mRNA complex must be serum stable to function in vivo. We therefore set up different concentrations of serum in 293T cells for transfection to determine the serum stability of the liposome/mRNA complexes. Transfection efficiency was checked 24h after transfection by fluorescence microscopy and flow cytometry. The results show that the transfection efficiency of the DOTAP liposome/mRNA complex before and after modification in serum with different concentrations is equivalent to that in serum-free medium, and no significant difference exists among groups (p >0.05), indicating that the liposome/mRNA complex has better serum stability (fig. 4a-4 b).

Cytotoxicity assays for DOTAP liposomes before and after DP7-C modification

Cells were treated with different concentrations of DP7-C, liposomes, DP7-C modified DOTAP liposomes, PEI25K or Lipo2000 for 24h, comparing cytotoxicity at different concentrations. The specific method comprises spreading 293T cells on 96-well plate with each well at 1 × 10⁴And (4) cells. After 24h of incubation, cells were treated with varying concentrations of DOTAP liposomes, DP7-C modified DOTAP liposomes, PEI25K or Lipo2000 for 24h, then 10 μ L of MTT was added to each well, incubated at 37 ℃ for 4h, the medium was aspirated off, and 200 μ L DMSO was added to each well. Finally, the absorbance value at 570nm was read using a SpectramaxM5 microplate luminometer (molecular devices, Sunnyvale, Calif., USA).

As a result, the cell viability was significantly reduced with the increase of the concentrations of PEI25K and Lipo2000, and the PEI25K treatment group at 100. mu.g/ml had only 13.47. + -. 1.23% of the cells survived, and the Lipo2000 treatment group at 100. mu.g/ml had only 15.26. + -. 1.37% of the cells survived, indicating that the two transfection reagents were more cytotoxic. The cell viability of the DOTAP liposome before and after modification is slowly reduced along with the increase of the concentration, 65.27 +/-6.14% of cells still exist in a DOTAP liposome treatment group with 100 mu g/ml, 71.27 +/-3.14% of cells still exist in a DP7-C modified DOTAP liposome treatment group with 100 mu g/ml, and the cytotoxicity of the DOTAP liposome before and after DP7-C modification is lower than that of Lipo2000 and PEI 25K. While the cell viability did not change significantly in the DP7-C group with increasing DP7-C concentration, about 92.31. + -. 1.34% of the cells survived with 100. mu.g/ml treatment group, with lower cytotoxicity (FIG. 5).

Efficiency test of DP7-C modified DOTAP Liposome transfection of mRNA into cells in lymph nodes

Previous experiments have demonstrated in vitro that DP7-C modified DOTAP liposomes indeed improve the efficiency of transferring mRNA into DCs, and this experiment was to investigate whether they are targeted.

After 10 mu geGFP mRNA and 20 mu g of liposome are co-incubated, the mixture is injected to C57BL/6J female mice with the age of 6-8 weeks by the subcutaneous lymph node, and after 24 hours, the proximal lymph node is taken out and the expression of green fluorescent protein in the lymph node is photographed by using a living body imager to reflect the expression efficiency of the eGFP mRNA in the lymph node.

As can be seen from the results of in vivo imaging technique, the efficiency of transferring eGFP mRNA into lymph nodes by the DP7-C modified DOTAP liposome is obviously higher than that of the unmodified liposome (p is less than 0.05), and the fluorescence intensity of eGFP in the lymph nodes of the DP7-C modified DOTAP liposome/eGFP mRNA complex group is obviously higher than that of eGFP in the lymph nodes of the DOTAP liposome/eGFP mRNA complex group (FIG. 6).

EXAMPLE IV DP7-C modified DOTAP liposomes as a study to promote DC maturation and cytokine secretion

In the experiments in this section, it was mainly verified whether DP7-C modified DOTAP liposomes are more advantageous in promoting DCs maturation, and the effect of DP7-C modified DOTAP liposomes in inducing DCs maturation was evaluated in comparison with the commonly used adjuvants polyIC and CPG. In addition, differences between DOTAP liposomes and DP7-C modified DOTAP liposomes were compared in terms of cytokine secretion from DCs to elucidate whether the two had differences in activating DCs function.

1. Liposome in vitro induction of DCs maturation

BMDCs cultured up to day 8 were treated with 5. mu.g/ml unmodified liposomes and DP7-C modified liposomes for 24h, respectively, with 5. mu.g/ml DP7-C, CPG and polyIC as positive controls. After 24h, cells were harvested and stained for CD11c, CD80, CD86 to examine BMDCs for maturation. Cells were collected and stained for CD11c, CD103, and BMDCs typing was examined.

From the results of flow cytometry, DP7-C alone induced maturation of 23.51. + -. 7.23% of DCs, CPG alone induced maturation of 44.58. + -. 4.32% of DCs, and polyIC alone induced maturation of 11.40. + -. 2.32% of DCs; the unmodified DOTAP liposome can induce the maturation of 19.61 +/-1.34 percent of DCs, and the DOTAP liposome modified by DP7-C can induce the maturation of 26.31 +/-1.42 percent of DCs; whereas the PBS group induced maturation of only 4.04. + -. 0.58% of DCs. From the rate of induction of DCs maturation, DP7-C modified DOTAP liposomes were more advantageous in inducing DCs maturation (p <0.05) than unmodified liposomes and were significantly higher than the ability of the commonly used adjuvant poly ic to induce DCs maturation (p <0.01), but lower than the ability of CPG to stimulate DC maturation at equivalent doses (p <0.01) (fig. 7a-7 b).

2. Liposome external induction DCs secretion cell factor

BMDCs cultured up to day 8 were treated with 5. mu.g/ml DP7-C, 5. mu.g/ml unmodified liposomes, and 5. mu.g/ml DP7-C modified liposomes, respectively, for 24h, and the supernatants were collected for ELISA testing.

From the results, it can be seen that the secretion of IL-1 β after treatment with DP7-C modified DOTAP liposome increased from 437.24 ± 35.23pg/ml of unmodified DOTAP liposome control group to 1478.87 ± 75.23pg/ml with a significant difference (p <0.001) between the two (fig. 7C); after DP7-C modified DOTAP liposome treatment, the secreted amount of TNF- α increased from 3452.23 ± 123.21pg/ml of unmodified DOTAP liposome control to 4359.30 ± 77.25pg/ml with a significant difference (p <0.001) (fig. 7 d); after stimulation with DP7-C modified liposomes, the amount of IL-6 secreted increased from 3617.23 ± 62.14pg/ml to 4225.24 ± 73.23pg/ml for the unmodified liposome control group with a significant difference between the two (p <0.05) (fig. 7 e); after stimulation with DP7-C modified DOTAP liposomes, the secretion of IL-12p70 increased from 72.14 ± 11.34pg/ml of the unmodified DOTAP liposome control group to 367.24 ± 23.23pg/ml with a significant difference between the two (p <0.001) (fig. 7 f); for IL-10 secretion, the secretion of unmodified DOTAP liposome control group was 182.14 + -23.34 pg/ml, and IL-10 secretion after stimulation with DP7-C modified liposomes was 182.24 + -13.13 pg/ml, with no significant difference (p >0.05) between the two groups (FIG. 7 g). However, from the ratio of IL-12p70/IL-10, treatment with DP7-C modified DOTAP liposomes significantly increased the ratio of IL-12p70/IL-10 (p <0.001) compared to unmodified DOTAP liposomes (FIG. 7 h).

The experimental results show that the DP7-C modified DOTAP liposome has the capability of inducing DCs to secrete cytokines and has a certain synergistic addition effect. And DCs can be in an activated state rather than a suppressed state, which is more beneficial to the efficacy of the vaccine.

3. Liposome-induced lymph node DCs maturation

Injecting 20 mu g of liposome beside subcutaneous lymph nodes of C57BL/6J female mice with the age of 6-8 weeks, taking out the proximal and distal lymph nodes after 24h, collecting cells, staining CD11C and MHC II, and detecting the maturation of DCs in the lymph nodes. Cells were collected for CD11c and CD103 staining and for detection of CD103 in lymph nodes⁺The ratio of DCs.

The results show that the ratio of DCs in the proximal and distal lymph nodes was significantly increased compared to the unmodified DOTAP liposome group following subcutaneous injection of DP7-C modified DOTAP liposomes perilymph nodes in mice, where the ratio of DCs in the proximal lymph nodes increased from 18.31 ± 2.12% for the unmodified DOTAP liposome group to 22.03 ± 1.45% for the DP7-C modified DOTAP liposome group (p < 0.001). The DP7-C group, the unmodified DOTAP liposome group and the modified DOTAP liposome group were all effective in recruiting DCs to lymph nodes increasing the proportion of DCs in lymph nodes (p <0.001) compared to the proportion of DCs in the control group (7.79 ± 1.12%) (fig. 8 a). From the ratio of matured DCs in lymph nodes, only 0.76. + -. 0.23% of DCs matured in the PBS group, 1.13. + -. 0.21% of DCs matured in the DP7-C group, 1.27. + -. 0.24% of DCs matured in the unmodified DOTAP liposome group, and 1.40. + -. 0.36% of DCs matured in the DP7-C modified DOTAP liposome group. From the results, DP7-C modified DOTAP liposomes were superior to unmodified DOTAP liposomes in inducing DCs maturation in vivo (proximal lymph node p <0.001, distal lymph node p <0.01) (FIG. 8 b).

4. Effect of subcutaneous paralymph node injection liposome on mouse serum cytokine content

The cytokine content in serum after injecting liposome to mouse lymph node side subcutaneously is detected to evaluate the condition of in vivo activated immunity of liposome before and after modification. Injecting 20 μ g liposome to C57BL/6J female mice of 6-8 weeks old by subcutaneous lymph node, removing blood from eyeball 24h later, placing the blood at 37 deg.C for 1h, centrifuging at 13000rpm for 15min, and separating serum for ELISA detection.

The results show that DP7-C modified DOTAP liposomes are more advantageous than unmodified DOTAP liposomes in inducing DCs to secrete proinflammatory cytokines. From the results, it can be seen that the secretion of IL-6 increased from 3.14 + -1.23 pg/ml of the unmodified DOTAP liposome control group to 66.87 + -5.14 pg/ml with a significant difference (p <0.001) between the two after treatment with DP7-C modified DOTAP liposome (FIG. 8 d); after DP7-C modified DOTAP liposome treatment, the secreted amount of TNF- α increased from 15.21 ± 2.32pg/ml to 22.32 ± 4.25pg/ml of the unmodified DOTAP liposome control group with a significant difference between the two (p <0.01) (fig. 8 e); after stimulation with DP7-C modified DOTAP liposomes, the secretion of IL-12p70 increased from 0.53 ± 0.14pg/ml of the unmodified DOTAP liposome control group to 52.24 ± 27.53 pg/ml with a significant difference between the two (p <0.05) (fig. 8 f); for IL-10 secretion, the unmodified DOTAP liposome control group secreted 27.12. + -. 4.34pg/ml, and the DP7-C modified DOTAP liposome stimulated IL-10 secretion of 12.34. + -. 1.43pg/ml, with a significant difference (p <0.01) (FIG. 8 g). From the proportion of IL-12p70/IL-10, compared with unmodified DOTAP liposome, the DP7-C modified DOTAP liposome can obviously improve the proportion of IL-12p70/IL-10 (p <0.01) (figure 8h), which indicates that DP7-C modified liposome has the capability of inducing DCs to secrete cytokines, and can enable the DCs to be in an activated state rather than an inhibited state, thereby being more beneficial to the efficacy of vaccines. This result is essentially consistent with the in vitro results.

5. In vivo and in vitro processing of liposomes affects the typing of DCs

There are reports in the literature of CD103⁺DCs contribute to antigen cross-presentation. CD103⁺The higher the proportion of DCs, the stronger the ability to cross-present antigens, and the higher the efficiency. CD103 was tested 24h after treatment of DCs with 5. mu.g/ml of different stimuli (DP7-C, CPG, polyIC, DOTAP liposomes and DP7-C modified DOTAP liposomes)⁺The ratio of DCs.

In vitro results show CD103 in PBS group⁺The proportion of DCs was 0.70. + -. 0.43%, DP7-C treated group CD103⁺The proportion of DCs was 6.18. + -. 1.23%, CPG-treated group CD103⁺The proportion of DCs was 1.74. + -. 0.83%, polyIC-treated group CD103⁺The proportion of DCs was 1.75. + -. 0.71% and the DOTAP liposome treatment group CD103⁺The proportion of DCs was 2.33. + -. 0.93%, DP7-C modified DOTAP Liposome treated group CD103⁺The proportion of DCs was 6.72. + -. 1.42%. Wherein the DP7-C group and DP7-C modified DOTAP liposome treated group have significant differences (p) from the unmodified DOTAP liposome treated group<0.01) (fig. 7 c).

In vivo results showed CD103 in PBS group⁺The proportion of DCs was 0.32. + -. 0.13%, DP7-C treated group CD103⁺The proportion of DCs was 0.70. + -. 0.23%, DOTAP Liposome treatment group CD103⁺Proportion of DCs 0.39. + -. 0.16%, DP7-C modified DOTAP Liposome treatment group CD103⁺The proportion of DCs was 0.72. + -. 0.18%. CD103 in proximal and distal lymph nodes of DOTAP Liposome treated group with DP7-C modification⁺The ratio of DCs was significantly higher than that of the unmodified DOTAP liposome group (p)<0.001) (fig. 8 c).

Example five functional verification of DP7-C modified Liposome/mRNA Complex vaccine

1. Functional validation of DP7-C modified liposome/mRNA vaccine based on model antigen OVA

(1) Preparation of Liposome/OVA mRNA Complex

20 μ g each of DOTAP liposome before and after DP7-C modification and 10 μ g of OVA mRNA were gently mixed in a total volume of 100 μ L of aqueous solution, and incubated at room temperature for 5-15 min.

(2) Antitumor effect of the invention in EG7-OVA subcutaneous tumor model

Will be 1 × 10⁶EG7-OVA cells (purchased from cell banks and stored in the laboratory) were resuspended in 100. mu.l DMEM double medium-free medium and then injected subcutaneously into mice. The liposome/mRNA complexes prepared above were injected at subcutaneous lymph nodes on

days

4, 11, and 18 at a dose of 20. mu.g liposomes and 10. mu.g mRNA per mouse. Changes in mouse tumor volume were recorded every 2 days. Mice were sacrificed on day 21 post inoculation.

The experimental results showed that the mean tumor volume of the PBS group was 2324.17 + -421.22 mm from the mean tumor volume³The mean tumor volume of the unmodified DOTAP liposome/OVA mRNA complex immune group is 1247.34 +/-178.21 mm³DP7-C modified DOTAP liposome/OVA mRNA Complex immunization group mean tumor volume of 157.34 +/-123.21 mm³. Wherein the mean tumor volume of the DP7-C modified DOTAP liposome/OVA mRNA complex immunized group was significantly different compared to the PBS group (p<0.001) was also significantly different (p) compared to the unmodified DOTAP liposome/OVA mRNA complex immunization group<0.01) (fig. 9 a). Indicating that the DP7-C modified DOTAP liposome/OVA mRNA complex group exerts more effective antitumor effects than the unmodified DOTAP liposome/OVA mRNA complex group.

(3) Spleen CD8 detection by ELISPOT⁺Activation of T cells

To further elucidate the immune effect of the liposome/OVA mRNA complex vaccine and the ability to trigger antigen-specific lymphocyte responses, the activation of antigen-specific spleen lymphocytes against OVA after vaccine immunization was examined using the ELISPOT assay.

At day 21 post-inoculation, when mice were sacrificed, mouse spleens were removed and mouse splenic lymphocytes were isolated using lymphocyte isolates. Will be 5X 10⁵Spleen lymphocytes were plated in 24-well plates with 100. mu.l RPMI 1640+ 10% FBS + 1% PS medium, treated with 10. mu.g/ml OVA for 72h, and assayed for IFN-. gamma.secretion according to the standard protocol of the ELISPOT kit instructions.

The results show that antigen-specific lymphocyte responses were stronger in both the group of unmodified liposomes after OVA treatment and the group of modified liposomes than in the PBS group. And the DP7-C modified liposome group induced a significantly stronger antigen-specific lymphocyte response than the unmodified liposome group (fig. 9 b).

2. Functional validation of DP7-C modified liposome/mRNA vaccines based on neoantigen (neoantigen)

(1) Design of mutant neoantigen and acquisition of mRNA

The five new antigen sequences used were those previously screened from the LL2 cell line and verified for immunogenicity and anti-tumor efficacy. The mutation site of each mutant neoantigen is taken as the center, 13 amino acids are respectively extended from front to back, namely, the short peptide sequence of each neoantigen comprises 27 amino acid sequences, and is connected with an linker and an MITD sequence (MHC class I tracing signal), so that the neoantigen with five neoantigen short peptides is designed.

The amino acid sequence of the finally designed wild-type antigen is as follows (SEQ ID No. 3):

MRVTAPRTLLLLLSGALALTETWAGSGGSGGGGSGGHSGAEQYFKEWFSRPANLHGIILPR LSGGSGGGGSGGLSPRHYYSGYSSSPEYSSESTHKIWERGGSGGGGSGGPPTVDSVCLKWA PPKHKQVKLSKKGGSGGGGSGGTKTELELALSPIHDSSAIPAAGSNQVTGGSGGGGSGGFS HSSNLTLHYRTHLVDRPYDCKCGKAGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCR RKSSGGKGGSYSQAASSDSAQGSDVSLTA。

the amino acid sequence of the finally designed mutant antigen is as follows (SEQ ID No. 4):

MRVTAPRTLLLLLSGALALTETWAGSGGSGGGGSGGHSGAEQYFKEWFSIPANLHGIILPRL SGGSGGGGSGGLSPRHYYSGYSSSLEYSSESTHKIWERGGSGGGGSGGPPTVDSVCLKWAP LKHKQVKLSKKGGSGGGGSGGTKTELELALSPIHYSSAIPAAGSNQVTGGSGGGGSGGFSH SSNLTLHYRTLLVDRPYDCKCGKAGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRR KSSGGKGGSYSQAASSDSAQGSDVSLTA。

the structure of the designed mRNA sequence is sequentially an SP signal peptide coding sequence (MHC class I signal peptide fragment which mainly acts for guiding polypeptide to enter a cavity through endoplasmic reticulum membrane), a start linker coding sequence, a new antigen 1 coding sequence, a middle linker coding sequence, a new antigen 2 coding sequence, a middle linker coding sequence, a new antigen 3 coding sequence, a middle linker coding sequence, a new antigen 4 coding sequence, a middle linker coding sequence, a new antigen 5 coding sequence, an end linker coding sequence, an MITD coding sequence (MHC class I transfection signal which improves mRNA stability and translation efficiency) and a polyA tail.

The DNA sequence encoding the mutant antigen was designed as (SEQ ID No. 5):

TAATACGACTCACTATAGGGCTAGCATGCGAGTCACCGCACCTCGCACACTCCTTCTGCT CTTGTCTGGAGCATTGGCTCTCACCGAAACCTGGGCTGGCTCTGGCGGCAGTGGAGGC GGTGGATCAGGCGGCCACTCAGGGGCAGAGCAATACTTCAAAGAATGGTTTAGTATCCC TGCTAATTTGCATGGGATCATACTTCCCCGACTCAGTGGCGGTTCCGGCGGGGGCGGAA GTGGGGGGTTGTCACCTCGACATTATTACAGTGGGTACTCTAGCTCTCTTGAATACTCTA GTGAGAGCACCCATAAAATCTGGGAGCGCGGTGGATCTGGAGGGGGGGGTTCTGGGGG GCCACCAACCGTTGATTCCGTATGCCTGAAGTGGGCCCCTTTGAAACACAAACAGGTTA AGCTTAGTAAGAAAGGAGGAAGCGGCGGTGGGGGATCTGGCGGAACTAAGACCGAGCT CGAACTTGCACTTAGTCCCATACACTATTCAAGCGCAATTCCAGCTGCAGGTTCCAACCA GGTCACCGGTGGAAGTGGTGGTGGCGGTTCAGGAGGCTTCTCACATTCCAGCAATTTGA CATTGCACTACAGAACTTTGCTTGTTGATCGCCCATATGACTGCAAATGCGGTAAGGCTG GTGGGAGTGGCGGTGGAGGCAGTGGGGGTATCGTTGGCATAGTTGCCGGCCTTGCAGTT CTTGCAGTGGTCGTGATAGGGGCCGTTGTGGCTACAGTAATGTGTCGCAGAAAGTCTAG TGGTGGCAAGGGGGGGTCATATTCACAGGCCGCATCTTCCGATTCTGCACAAGGATCAG ATGTTTCACTTACAGCTTAGACCGGTTTACTCGAGAGCTCGCTTTCTTGCTGTCCAATTTC TATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGC CTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCTGCGTCGACTAATT ACTCGAGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTC CAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAA AAACATTTATTTTCATTGCTGCGTCGACTAATCTAGAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。

the PCR template was transcribed using the transcription kit according to the protocol and the size and purity of the mRNA was detected by agarose gel electrophoresis, finding that the transcribed mRNA was 1287bp in size, consistent with expectations. Indicating successful in vitro transcription of the designed mRNA encoding the novel antigen sequence. The purified neoantigen mRNA was stored at-80 ℃ for further use.

(2) Preparation of Liposome/mRNA Complex

20 mu g of DOTAP liposome before and after modification and 10 mu g of mRNA are mixed in 100 mu L water solution and incubated for 5-15min at room temperature.

(3) Antitumor Effect of the invention in situ tumor model

Will be 3X 10⁵The mouse LL2 cells were resuspended in 100. mu.l DMEM double-medium without medium and injected into the mouse via tail vein to establish an in situ lung cancer model. The liposome/mRNA complexes prepared above were injected at subcutaneous lymph nodes on

days

4, 11, and 18 at a dose of 20. mu.g liposomes and 10. mu.g mRNA per mouse. Mice body weight changes were recorded every 2 days. Mice were sacrificed on day 21 post inoculation, major tissue organs were fixed in 4% paraformaldehyde, and lung tissues were photographed.

An in situ tumor model of LL2 was established to assess whether the liposome/neoantigen mRNA complexes before and after modification could exert anti-tumor effects. The results show that the DP7-C modified DOTAP liposome/neoantigen mRNA complex immunization group has better effect of inhibiting tumor growth than the DOTAP liposome/neoantigen mRNA complex immunization group. From the growth of tumors on the lung, the mean tumor node number of the DP7-C modified DOTAP liposome/neoantigen mRNA complex immunized group was 3 ± 1, while the mean tumor node number of the unmodified DOTAP liposome/mRNA complex immunized group was 10 ± 3 with statistical differences (p <0.05) (fig. 10 a). From the lung weight, the mean lung weight of the DP7-C modified DOTAP liposome/neoantigen mRNA complex immunization group was 0.22 + -0.03 g, which is significantly lower than the mean lung weight of the unmodified DOTAP liposome/mRNA complex subcutaneous immunization group (0.37 + -0.08 g), with statistical differences (p < 0.05). These results indicate that DP7-C modified DOTAP liposome/neoantigen mRNA complexes are more superior in anti-tumor effect than unmodified DOTAP liposome/neoantigen mRNA (FIG. 10 b).

(4) Flow cytometry detection of spleen CD8⁺Activation of T cells

To further elucidate the immunological effects of the liposome/neoantigen mRNA complexes and their ability to elicit antigen-specific lymphocyte responses, flow cytometry was used to examine spleen lymphocyte activation following mixed peptide treatment and mRNA treatment.

At day 21 post-inoculation, when mice were sacrificed, mouse spleens were removed and mouse splenic lymphocytes were isolated using lymphocyte isolates. Will be 3X 10⁶Spleen lymphocytes were plated in 6-well plates with 1ml of RPMI 1640+ 10% FBS + 1% PS medium, treated with 10. mu.g/ml of a flag polypeptide, wild-type peptides (2. mu.g/ml of each of the wild-type peptides corresponding to five antigens), mutant peptides (2. mu.g/ml of each of the mutant peptides corresponding to five antigens), and mRNA for 3 hours, respectively, and then 2. mu.l of Golgi blocker was added. After an additional 11h of culture, cells were harvested for CD3, CD8, and IFN-. gamma.flow staining. Flow cytometry for CD8 detection⁺Activation of T cells.

The results show that DP7-C modified DOTAP liposome/neoantigen mRNA complex immunization group CD8 after mutant peptide treatment⁺The T cell proportion was 17.79. + -. 0.63%, statistically different (p) from 12.26. + -. 0.74% for the unmodified DOTAP liposome group<0.01). CD8 using DOTAP liposome set before and after mRNA treatment modification⁺T cell ratio is obviously up-regulated compared with a control group, but no obvious difference is found between the DOTAP liposome groups before and after modification. After the mixed peptide treatment, the proportion of IFN-gamma-secreting cells of the immune group of the DP7-C modified DOTAP liposome/neoantigen mRNA complex is 2.66 +/-0.32 percent and is higher than the proportion (0.81 +/-0.14 percent) of IFN-gamma-secreting cells of the immune group of the unmodified DOTAP liposome/neoantigen mRNA complex after the mixed peptide treatment, and the statistical difference (p) exists<0.01); after mRNA treatment, the cell proportion of IFN-gamma secretion of the DOTAP liposome/new antigen mRNA compound immune group before and after modification is obviously improved, and the cell proportion of IFN-gamma secretion of the DOTAP liposome/new antigen mRNA compound immune group after DP7-C modification is 4.13 +/-0.72 percent, which is obviously higher than that of the unmodified liposome/new antigen mRNA compound immune group (0.92 +/-0.14 percent), and has statistical difference (p is<0.01) (fig. 10c-10 d). Indicating that DP7-C modified DOTAP liposome/neoantigen mRNA complexes are more effective in activating antigen-specific lymphocyte responses after subcutaneous injection than the unmodified liposome/neoantigen mRNA complexes of the immunization group.

EXAMPLE sixthly functional validation of DCs vaccine loaded with DP7-C modified Liposome/mRNA Complex

1. Functional validation of mRNA-loaded DCs vaccines based on model antigen OVA

(1) Preparation of DCs vaccine carrying liposome/mRNA complexes

Firstly, taking shin bones and fibulas of adult C57BL/6J female mice of about 6 weeks old, soaking in 75% ethanol for 5min to kill bacteria, then removing muscle tissues, and soaking leg bones in a RPMI 1640+ 1% PS culture medium; shearing two ends of the leg bone by using a sterilized scissors, sucking a fresh RPMI 1640+ 1% PS culture medium by using a syringe, and blowing out bone marrow cells until all the bone marrow cells are blown out;

② filtering the collected culture medium containing bone marrow cells by using 70 μm screen, centrifuging at 1200rpm for 3min, discarding supernatant, and then using erythrocyte lysate (weighing 1.3g Tris-base and 3.74g NH)₄Dissolving Cl in 490ml of ultrapure water, adjusting the pH value of the solution to 7.2-7.4 by using concentrated hydrochloric acid, adding ultrapure water to a constant volume of 500ml, removing bacteria by using a 0.22-micron filter, preserving at 4 ℃, preparing for use) and suspending cells, standing at room temperature for 3min, centrifuging at 1200rpm for 3min, finally washing out a erythrocyte lysate by using RPMI 1640+ 10% FBS + 1% PS medium, and suspending the cells again;

③ divide the resuspended cells into culture dishes, 2X 10 cells per dish⁶-3×10⁶Cells were cultured in a cell culture chamber at 37 ℃ with 10ml of RPMI 1640+ 10% FBS + 1% PS medium and 20ng/ml of GM-CSF cytokine added to each dish, and fresh RPMI 1640+ 10% FBS + 1% PS medium containing 20ng/ml GM-CSF was added on the third day of culture until immature DCs (imDCs) were obtained by culturing until day 8. DCs cultured until day 8 are taken, 1ml of PBS is used for washing out the culture medium, 100 mu l of PBS is used for resuspending cells, 1 mu l of APC Hamster Anti-Mouse CD11c flow antibody is added, the mixture is gently mixed and placed at 4 ℃ for incubation for 40min in the dark; after incubation, excess antibody was washed with PBS, cells were resuspended in 200. mu.l PBS, and CD11c was detected using flow cytometry⁺The proportion of DCs. When the ratio is higher than 80%, the subsequent processing may be performed.

(iv) adding liposomes (2. mu.g/ml)/mRNA (1. mu.g/ml) to the imDCs induced to day 8The complex was treated for 24 h. Collection 10⁶Cells were incubated with 1. mu.l APC Hamster Anti-Mouse CD11c, 1. mu.l Percp-Cy5.5Hamster Anti-Mouse CD80, and 1. mu.l FITC Rat Anti-Mouse CD86 antibody at 4 ℃ for 40min protected from light. After the incubation, excess antibody was washed away with PBS, cells were resuspended in 200 μ l PBS, and the proportion of each antibody-positive cell was determined by flow cytometry. When the proportion of the mature DCs is more than 90%, the DCs vaccine is successfully prepared. Can be used for subsequent tests.

(2) Antitumor effect of the invention in EG7-OVA subcutaneous tumor model

Will be 1 × 10⁶EG7-OVA cells were resuspended in 100. mu.l DMEM medium and injected subcutaneously into mice. Injection of the liposome/mRNA complex-loaded DCs vaccine at subcutaneous lymph nodes on

days

4, 11, and 18 at a dose of 2X 10 per mouse⁶And (4) DCs. Changes in mouse tumor volume were recorded every 2 days. Mice were sacrificed on day 23 post inoculation.

The experimental results showed that the mean tumor volume of the PBS group was 3343.17 + -221.22 mm from the mean tumor volume³The mean tumor volume of DCs immune group loaded with unmodified DOTAP liposome/OVA mRNA complex is 2642.34 +/-173.11 mm³The mean tumor volume of DCs immune group loaded with DP7-C modified DOTAP liposome/OVA mRNA complex was 1356.34 + -113.12 mm³. The mean tumor volume of the DCs immunized group loaded with DP7-C modified DOTAP liposome/OVA mRNA complex was significantly different (p) compared to the PBS group<0.001) also had significant differences (p) compared to the DCs immune group loaded with unmodified DOTAP liposome/OVA mRNA complex<0.01) (fig. 11 a). Indicating that the DCs group loaded with DP7-C modified DOTAP liposome/OVA mRNA complex exerted more potent antitumor effects than the DCs group loaded with unmodified DOTAP liposome/OVAmRNA complex.

(3) Spleen CD8 detection by ELISPOT⁺Activation of T cells

To further elucidate the immune effect and the ability to stimulate antigen-specific lymphocyte reaction of the liposome/OVAmRNA complex-loaded DCs vaccine, the activation of antigen-specific spleen lymphocytes against OVA after vaccine immunization was examined using an ELISPOT assay.

At day 23 post-inoculation, when mice were sacrificed, mouse spleens were removed and mouse splenic lymphocytes were isolated using lymphocyte isolates. Will be 5X 10⁵Spleen lymphocytes were plated in 24-well plates with 100. mu.l RPMI 1640+ 10% FBS + 1% PS medium, treated with 10. mu.g/ml OVA for 72h, and assayed for IFN-. gamma.secretion according to the standard protocol of the ELISPOT kit instructions.

The results show that antigen-specific lymphocyte responses of both the OVA-treated DCs group loaded with unmodified liposomes/OVAmRNA and the modified DCs group loaded with liposomes/OVAmRNA were stronger than those of the PBS group. And the DP7-C modified liposome/OVAmRNA loaded DCs group induced a significantly stronger antigen-specific lymphocyte response than the unmodified liposome/OVAmRNA loaded vaccine group (fig. 11 b).

2. Functional validation of mRNA-loaded DCs vaccines based on neoantigen (neoantigen)

(1) Antitumor effect of the invention in LL2 subcutaneous tumor model

The LL2 subcutaneous tumor model was established to evaluate whether the lipid/neoantigen mRNA complex-loaded DCs vaccine could exert anti-tumor effects.

Will be 5X 10⁵Individual mouse LL2 cells were resuspended in 100 μ l DMEM double no medium and then injected subcutaneously into mice to establish a subcutaneous tumor model. Injection of 2X 10 at subcutaneous lymph nodes on

days

4, 11, and 18⁶And (3) DCs cells loaded with liposome/mRNA complexes. Mice were sacrificed on day 21 post-inoculation and the anti-tumor effect of the vaccines was evaluated by plotting the subcutaneous tumor growth curve of the mice. The experimental groups were PBS control group, DCs immunization group loaded with unmodified DOTAP liposome/neoantigen mRNA complex, DCs vaccine immunization group loaded with DP7-C modified DOTAP liposome/neoantigen mRNA complex, 6 mice per group.

The experimental results showed that the mean tumor volume of the PBS group was 1324.17 + -221.22 mm from the mean tumor volume³The mean tumor volume of DCs immune group loaded with unmodified DOTAP liposome/neoantigen mRNA complex is 677.34 +/-178.21 mm³Load DP7-CThe mean tumor volume of DCs immune group of the modified DOTAP liposome/neoantigen mRNA complex was 356.34 + -47.21 mm³. The mean tumor volume of the DCs immunization group loaded with DP7-C modified DOTAP liposome/neoantigen mRNA complexes was significantly different compared to the PBS group (p)<0.001) compared to the DCs immune group loaded with unmodified DOTAP liposome/neoantigen mRNA complex (p)<0.01) (fig. 12 a). And from the in vitro tumor weight, the average tumor weight of the PBS group is 1.22 +/-0.42 g, the average tumor weight of the DCs immune group loaded with the unmodified DOTAP liposome/new antigen mRNA complex is 0.93 +/-0.16 g, and the average tumor weight of the DCs immune group loaded with the DP7-C modified DOTAP liposome/new antigen mRNA complex is 0.58 +/-0.07 g. The mean tumor weight of the DCs immunized group loaded with DP7-C modified DOTAP liposome/neoantigen mRNA complexes was significantly lower than that of the PBS group (p)<0.001) and DCs vaccine group (p) loaded with unmodified DOTAP liposomes/mRNA<0.01) (fig. 12 b).

(2) Flow cytometry detection of spleen CD8⁺Activation of T cells

To further elucidate the immune efficacy and ability to elicit antigen-specific lymphocyte responses of the liposome/neoantigen mRNA complex-loaded DCs vaccines, spleen lymphocyte activation following mixed peptide treatment and mRNA treatment was examined using flow cytometry.

At day 21 post-inoculation, when mice were sacrificed, mouse spleens were removed and mouse splenic lymphocytes were isolated using lymphocyte isolates. Will be 3X 10⁶Spleen lymphocytes were plated in 6-well plates with 1ml of RPMI 1640+ 10% FBS + 1% PS medium, treated with 10. mu.g/ml of a flag polypeptide, wild-type peptides (2. mu.g/ml of each of the wild-type peptides corresponding to five antigens), mixed mutant peptides (2. mu.g/ml of each of the mutant peptides corresponding to five antigens), and mRNA for 3 hours, respectively, and then 2. mu.l of Golgi blocker was added. After an additional 11h of culture, cells were harvested for CD3, CD8, and IFN-. gamma.flow staining. Flow cytometry for CD8 detection⁺Proportion of T cells.

The results showed that the fraction of cells secreting IFN- γ in DCs immune groups loaded with DP7-C modified DOTAP liposome/neoantigen mRNA complexes after mixed mutant peptide treatment was 3.79 ± 0.62%, with statistical differences (p <0.05) compared to 1.46 ± 0.34% in the unmodified DOTAP liposome group. The IFN-gamma secreting cell ratios of DCs immunized groups loaded with both DOTAP liposomes/mRNA before and after modification after mRNA treatment were all significantly up-regulated compared to the control group. After mRNA treatment, the cell proportion of IFN-gamma secretion of the DCs immune group loaded with the DOTAP liposome/new antigen mRNA compound before and after modification is obviously improved, and the cell proportion of IFN-gamma secretion of the DCs immune group loaded with the DOTAP liposome/new antigen mRNA compound after DP7-C modification is 5.15 +/-0.74 percent which is obviously higher than that of the DCs immune group loaded with the unmodified liposome/new antigen mRNA compound (1.45 +/-0.25 percent) and has statistical difference (p <0.01) (figure 12C). Indicating that DCs loaded with DP7-C modified DOTAP liposome/neoantigen mRNA complex can more effectively activate antigen-specific lymphocyte reaction after subcutaneous injection compared with DCs immune group loaded with unmodified liposome/neoantigen mRNA complex.

In the previous research, the cholesterol modified cationic polypeptide DP7-C has the dual effects of a carrier and an adjuvant, and can be used for improving the anti-tumor effect of a DCs vaccine loaded with a neoantigen peptide. In the embodiment of the invention, DP7-C with double effects of a carrier and an immune adjuvant is modified on a DOTAP liposome capable of delivering mRNA into cells, so that the efficiency of delivering the mRNA into the cells by the liposome and the immune response induced by the liposome are successfully improved.

In the above examples of the invention, we prepared DP7-C modified DOTAP liposome and DP7-C modified DC-chol liposome respectively, and demonstrated that DP7-C can be stably modified on the liposome. It was demonstrated that DP7-C modified DOTAP liposomes increased the efficiency of liposomes in transfecting mRNA into DCs. And DP7-C modified DOTAP liposome has the characteristic of promoting the secretion of DCs, maturation and proinflammatory cytokines in vitro and in vivo better than the unmodified liposome. Finally, the anti-tumor effect and the spleen lymphocyte reaction exciting capability of the DOTAP liposome/OVAmRNA composite vaccine before and after DP7-C modification, the DP7-C liposome/mRNA composite vaccine encoding individualized new antigen and the DCs vaccine loading the liposome/mRNA composite are respectively verified by establishing a tumor model, and the modified liposome/mRNA vaccine and the DCs vaccine loading the modified liposome/mRNA composite are more advantageous in the anti-tumor effect and the activation of spleen antigen specific lymphocyte reaction. In summary, DP7-C is modified on the DOTAP liposome, so that the uptake efficiency of DCs to mRNA can be enhanced, the maturation and proinflammatory cytokines secretion of the DCs can be promoted, and antigen-specific cytotoxic T lymphocyte reaction is triggered, so that the anti-tumor effect of the mRNA vaccine is enhanced, and the antigen-specific lymphocyte reaction has more advantages in the aspects of anti-tumor effect and induction of antigen-specific lymphocyte reaction.

Therefore, the present invention provides the art with a new and effective option to further improve the intracellular delivery efficiency of mRNA, enhance the effect of liposome/mRNA complex vaccines and liposome/mRNA complex-loaded DCs vaccines.

Sequence listing

<110> Sichuan university

<120> polypeptide-modified liposome, mRNA delivery system and dendritic cell vaccine

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Val Gln Trp Arg Ile Arg Val Ala Val Ile Arg Lys

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<210> 2

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Asp Tyr Lys Asp Asp Asp Asp Lys

1 5

<210> 3

<211> 273

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Met Arg Val Thr Ala Pro Arg Thr Leu Leu Leu Leu Leu Ser Gly Ala

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Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser Gly Gly Ser Gly Gly Gly

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Gly Ser Gly Gly His Ser Gly Ala Glu Gln Tyr Phe Lys Glu Trp Phe

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Ser Arg Pro Ala Asn Leu His Gly Ile Ile Leu Pro Arg Leu Ser Gly

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Gly Ser Gly Gly Gly Gly Ser Gly Gly Leu Ser Pro Arg His Tyr Tyr

65 70 75 80

Ser Gly Tyr Ser Ser Ser Pro Glu Tyr Ser Ser Glu Ser Thr His Lys

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Ile Trp Glu Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Pro Pro

100 105 110

Thr Val Asp Ser Val Cys Leu Lys Trp Ala Pro Pro Lys His Lys Gln

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Val Lys Leu Ser Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Thr Lys Thr Glu Leu Glu Leu Ala Leu Ser Pro Ile His Asp Ser Ser

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Ala Ile Pro Ala Ala Gly Ser Asn Gln Val Thr Gly Gly Ser Gly Gly

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Arg Thr His Leu Val Asp Arg Pro Tyr Asp Cys Lys Cys Gly Lys Ala

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Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val Val Ala Thr

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Ala

<210> 4

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Met Arg Val Thr Ala Pro Arg Thr Leu Leu Leu Leu Leu Ser Gly Ala

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Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser Gly Gly Ser Gly Gly Gly

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Gly Ser Gly Gly His Ser Gly Ala Glu Gln Tyr Phe Lys Glu Trp Phe

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Ser Ile Pro Ala Asn Leu His Gly Ile Ile Leu Pro Arg Leu Ser Gly

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Gly Ser Gly Gly Gly Gly Ser Gly Gly Leu Ser Pro Arg His Tyr Tyr

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Ser Gly Tyr Ser Ser Ser Leu Glu Tyr Ser Ser Glu Ser Thr His Lys

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Ile Trp Glu Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Pro Pro

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Thr Val Asp Ser Val Cys Leu Lys Trp Ala Pro Leu Lys His Lys Gln

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Val Lys Leu Ser Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

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Thr Lys Thr Glu Leu Glu Leu Ala Leu Ser Pro Ile His Tyr Ser Ser

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Ala Ile Pro Ala Ala Gly Ser Asn Gln Val Thr Gly Gly Ser Gly Gly

165 170 175

Gly Gly Ser Gly Gly Phe Ser His Ser Ser Asn Leu Thr Leu His Tyr

180 185 190

Arg Thr Leu Leu Val Asp Arg Pro Tyr Asp Cys Lys Cys Gly Lys Ala

195 200 205

Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile Val Gly Ile Val Ala

210 215 220

Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val Val Ala Thr

225 230 235 240

Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser

245 250 255

Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr

260 265 270

Ala

<210> 5

<211> 1287

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

taatacgact cactataggg ctagcatgcg agtcaccgca cctcgcacac tccttctgct 60

cttgtctgga gcattggctc tcaccgaaac ctgggctggc tctggcggca gtggaggcgg 120

tggatcaggc ggccactcag gggcagagca atacttcaaa gaatggttta gtatccctgc 180

taatttgcat gggatcatac ttccccgact cagtggcggt tccggcgggg gcggaagtgg 240

ggggttgtca cctcgacatt attacagtgg gtactctagc tctcttgaat actctagtga 300

gagcacccat aaaatctggg agcgcggtgg atctggaggg gggggttctg gggggccacc 360

aaccgttgat tccgtatgcc tgaagtgggc ccctttgaaa cacaaacagg ttaagcttag 420

taagaaagga ggaagcggcg gtgggggatc tggcggaact aagaccgagc tcgaacttgc 480

acttagtccc atacactatt caagcgcaat tccagctgca ggttccaacc aggtcaccgg 540

tggaagtggt ggtggcggtt caggaggctt ctcacattcc agcaatttga cattgcacta 600

cagaactttg cttgttgatc gcccatatga ctgcaaatgc ggtaaggctg gtgggagtgg 660

cggtggaggc agtgggggta tcgttggcat agttgccggc cttgcagttc ttgcagtggt 720

cgtgataggg gccgttgtgg ctacagtaat gtgtcgcaga aagtctagtg gtggcaaggg 780

ggggtcatat tcacaggccg catcttccga ttctgcacaa ggatcagatg tttcacttac 840

agcttagacc ggtttactcg agagctcgct ttcttgctgt ccaatttcta ttaaaggttc 900

ctttgttccc taagtccaac tactaaactg ggggatatta tgaagggcct tgagcatctg 960

gattctgcct aataaaaaac atttattttc attgctgcgt cgactaatta ctcgagagct 1020

cgctttcttg ctgtccaatt tctattaaag gttcctttgt tccctaagtc caactactaa 1080

actgggggat attatgaagg gccttgagca tctggattct gcctaataaa aaacatttat 1140

tttcattgct gcgtcgacta atctagaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260

aaaaaaaaaa aaaaaaaaaa aaaaaaa 1287

Claims

1. A liposome modified by hydrophobic modified cationic polypeptide, wherein the sequence of the cationic polypeptide is VQWRIRVAVIRK, and the hydrophobic modification is that a hydrophobic fragment is coupled at the nitrogen terminal of the cationic polypeptide; the liposome consists of cationic lipid and auxiliary lipid.

2. Liposome modified with a hydrophobically modified cationic polypeptide according to claim 1, characterized in that the cationic lipids constituting the liposome are trimethyl-2, 3-dioleyloxypropylammonium chloride, trimethyl-2, 3-dioleyloxypropylammonium bromide, dimethyl-2, 3-dioleyloxypropyl-2- (2-sperminylamido) ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide, dimethyl-2-hydroxyethyl-2, 3-dioleyloxypropylammonium bromide, dimethyl-3-hydroxypropyl-2, 3-dioleyloxypropylammonium bromide dimethyl-4-hydroxybutyl-2, 3-dioleyloxypropylammonium bromide dimethyl-5-hydroxypentyl-2, 3-dioleyloxypropylammonium bromide dimethyl-2-hydroxyethyl-2, 3-dihexadecyloxypropylammonium bromide dimethyl-2-hydroxyethyl-2, 3-dioctadecylpropylammonium bromide dimethyl-2-hydroxyethyl-2, 3-ditetradecyloxypropylammonium bromide, N- (2-sperminoyl) -N ', N ' -dioctadecyl glycinamide, 1, 2-dioleoyl-3-succinyl-sn-glycerocholine ester, 3 β - [ N- (N ', n' -dimethylaminoethyl) carbamoyl ] cholesterol, lipid poly-L-lysine.

3. The liposome modified by the hydrophobic modified cationic polypeptide of claim 1, wherein the helper lipid constituting the liposome is at least one of cholesterol, phosphatidylethanolamine, phosphatidylcholine, dioleoylphosphatidylethanolamine and diphosphatidylcholine.

4. The liposome modified by the hydrophobic modified cationic polypeptide of any one of claims 1 to 3, wherein the liposome is prepared from cationic lipid and helper lipid, and the mass ratio of the cationic lipid to the helper lipid is as follows: cationic lipid: the auxiliary lipid is 0.5-20: 1.

5. The liposome modified by the hydrophobic modified cationic polypeptide of any one of claims 1 to 4, wherein the mass ratio of the hydrophobic modified cationic polypeptide to the liposome composed of the cationic lipid and the helper lipid is: hydrophobically modified cationic polypeptides: the ratio of the liposome is 0.025-0.5: 1.

6. The liposome modified by the hydrophobic modified cationic polypeptide of claim 5, wherein the mass ratio of the hydrophobic modified cationic polypeptide to the liposome consisting of the cationic lipid and the helper lipid is: hydrophobically modified cationic polypeptides: the ratio of the liposome is 0.025-0.2: 1.

7. The liposome modified by the hydrophobic modified cationic polypeptide of any one of claims 1 to 6, wherein: is prepared by at least one of an ethanol injection method, a film dispersion method, an ultrasonic dispersion method, a reverse evaporation method, a high-pressure homogenization method, a carbon dioxide supercritical method or a freeze drying method.

8. The liposome modified by the hydrophobic modified cationic polypeptide of any one of claims 1 to 7, which is prepared by the following method:

or, the preparation method comprises the following steps:

b. injecting DP7-C solution into the organic phase and stirring; evaporating under vacuum and reduced pressure to 1/5-1/2 of the original volume; homogenizing and extruding to obtain blank liposome solution; the DP7-C solution is an aqueous solution, an ethanol solution or an ether solution;

9. The liposome modified by the hydrophobic modified cationic polypeptide of claim 8, which is prepared by using the following raw materials in proportion: the mass ratio of the cationic lipid to the helper lipid to the hydrophobic modified cationic polypeptide is 10-40: 0.5-10.

10. The liposome modified by the hydrophobic modified cationic polypeptide of claim 9, which is prepared by using the following raw materials in proportion: cationic lipid to helper lipid in mass ratio: hydrophobization modified cationic polypeptide is 10-40: 1-6; preferably, the liposome is prepared from the following raw materials in proportion: according to the mass ratio of DOTAP to cholesterol: hydrophobization modified cationic polypeptide 20:20: 3.

11. The liposome modified by the hydrophobic modified cationic polypeptide of any one of claims 1-7, wherein the cationic lipid constituting the liposome is trimethyl-2, 3-dioleoyloxypropylammonium bromide (DOTAP), and the helper lipid is cholesterol; alternatively, the cationic lipid constituting the liposome is 1, 2-dioleoyl-3-succinyl-sn-glycerocholine ester (DC-Chol), and the helper lipid is dioleoyl phosphatidylethanolamine (DOPE).

12. The liposome modified by the hydrophobic modified polypeptide of any one of claims 1-11, wherein the carbon terminal amidation modification of the cationic polypeptide VQWRIRVAVIRK is VQWRIRVAVIRK-NH 2.

13. The liposome modified by the hydrophobic modified polypeptide of any one of claims 1-12, wherein the hydrophobic segment is a sterol compound or a saturated straight chain fatty acid or a PEG derivative.

14. The liposome modified by hydrophobic modified polypeptide of claim 13, wherein the sterol compound is cholesterol compound or cholic acid compound.

15. The liposome modified by the hydrophobic modified polypeptide of claim 13, wherein the saturated straight chain fatty acid is C₆-C₂₀At least one of (1).

16. Liposome modified by a hydrophobically modified polypeptide according to claim 13, wherein the PEG derivative is at least one of 1, 2-dioleoyl-SN-glycero-3-phosphoethanolamine-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol or dipalmitoylphosphatidylethanolamine-polyethylene glycol.

17. The liposome modified by the hydrophobic modified polypeptide of any one of claims 1-16, wherein the nitrogen terminal of the polypeptide is coupled to the hydrophobic segment via-CO-OH on the hydrophobic segment and-NH on the polypeptide₂Amidation reaction.

18. A liposome modified by a hydrophobic modified polypeptide according to claim 17, wherein the hydrophobic modified polypeptide has the structure:

19. A liposome modified by a hydrophobic modified polypeptide as claimed in claim 18, wherein R in the polypeptide structure is:

at least one of (1).

20. Use of a liposome modified with a hydrophobically modified cationic polypeptide as described in any one of claims 1 to 19 in the preparation of a nucleic acid delivery system.

21. A nucleic acid delivery system comprising a liposome-loaded nucleic acid modified with the hydrophobically modified polypeptide of any of claims 1-19.

22. The nucleic acid delivery system of claim 21, wherein said nucleic acid is at least one of DNA or RNA.

23. The nucleic acid delivery system of claim 22, wherein the RNA is at least one of messenger RNA, siRNA, sgRNA, or mRNA.

24. The nucleic acid delivery system of any one of claims 20 to 23, wherein the liposome modified by the hydrophobic modified cationic polypeptide and the nucleic acid are prepared from raw materials at a mass ratio of 1-10: 1.

25. The nucleic acid delivery system of claim 24, wherein: the preparation method comprises the step of preparing a liposome modified by hydrophobic modified cationic polypeptide and nucleic acid by using the liposome and the nucleic acid as raw materials according to the mass ratio of 1-3: 1.

26. A nucleic acid delivery system according to any one of claims 20 to 25, wherein: is prepared by incubating liposome modified by hydrophobic modified cationic polypeptide with nucleic acid.

27. The nucleic acid delivery system of claim 26, wherein: the liposome is obtained by co-incubating liposome modified by hydrophobic modified cationic polypeptide and nucleic acid in a culture medium for 4-15 min, wherein the culture medium is at least one of RPMI 1640, DMEM double-non culture medium or Optim culture medium.

28. The mRNA delivery system according to any one of claims 20 to 27, wherein: the length of the nucleic acid is 20-7000 bp.

29. The mRNA delivery system according to any one of claims 20 to 28, wherein: the mRNA is mRNA with a 5 'cap structure and a 3' polyA tail structure.

30. Use of an mRNA delivery system according to any one of claims 20 to 29 for the preparation of a dendritic cell vaccine.

31. A dendritic cell vaccine characterized by: a dendritic cell loaded with the mRNA delivery system according to any one of claims 20 to 29 as a main active ingredient.

32. The dendritic cell vaccine of claim 31, wherein: the dendritic cells are myeloid DC cells or lymphoid DC cells.

33. The dendritic cell vaccine of claim 31, wherein: the dendritic cells are in vitro dendritic cells obtained by a patient.

34. The dendritic cell vaccine of any one of claims 31 to 33, prepared using the following method:

a. taking immature dendritic cells (imDCs) after induction culture;

b. adding the nucleic acid delivery system to a culture medium for incubation

35. A dendritic cell vaccine according to any one of claims 31 to 34. The method is characterized in that: also comprises pharmaceutically acceptable auxiliary components.

36. The dendritic cell vaccine of claim 35. The method is characterized in that: the pharmaceutically acceptable auxiliary component is at least one of a protective agent, an excipient, an immunologic adjuvant, a dispersing agent or a cell culture medium.