Peptide-modified liposomes, mRNA delivery systems and dendritic cell vaccines

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to liposomes modified by hydrophobic modified polypeptides, mRNA delivery systems and dendritic cell vaccines. The technical problem to be solved by the present invention is to improve the immune effect of mRNA vaccines and DCs vaccines sensitized by mRNA. The technical solution to solve the technical problem is to provide a liposome modified by a hydrophobic modified cationic polypeptide. The modified liposomes of the polypeptides of the present invention can efficiently deliver nucleic acids into cells; in particular, they can efficiently deliver mRNA encoding antigens into DCs, significantly improve the efficiency of cationic liposomes in delivering mRNA to DCs, enhance the immune effect of mRNA vaccines and DC vaccines sensitized by mRNA, and have good clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a liposome modified by a hydrophobic modified polypeptide, an mRNA delivery system and a dendritic cell vaccine. Background Art

[0002] In recent years, the application of mRNA in gene therapy, immunotherapy and stem cell biomedicine has attracted widespread attention. Compared with 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 host genome, so possible insertion mutations and abnormal transcription can be avoided; 2) Compared with plasmid DNA, mRNA has a simple structure, fewer elements are required for translation into protein, and it is biodegradable and only works in the short term, thus reducing the risk of side effects; 3) In the process of personalized immunotherapy, mRNA has obvious advantages over peptide vaccines, such as low synthesis cost, short synthesis cycle, simultaneous encoding of multiple antigen sequences and no restriction on MHC haplotype. In addition, with the development of mRNA structural engineering, such as the modification of the 5' end cap structure and the 3' end polyA tail, the shortcomings of poor stability, low translation efficiency and easy degradation of mRNA have been greatly improved, which has promoted the application of mRNA therapy. In recent years, mRNA has been widely used in the research of cell programming and vaccines, and has shown great application potential. In vaccine research, mRNA vaccines have shown the ability to activate effective immune responses including cytotoxic T cells. In addition, mRNA vaccines can also activate innate immunity through specific recognition of pattern recognition receptors. Currently, in clinical trials of tumor immunotherapy, many studies have evaluated the anti-tumor effects of mRNA vaccines, indicating that it is feasible to use mRNA vaccines for the prevention and treatment of tumors. At the same time, mRNA vaccines are also used in the research of vaccines for various infectious diseases, such as the CMV vaccine and Zika vaccine of Moderna in the United States, which have entered clinical research.

[0003] Effective delivery of mRNA into cells and successful activation of immune responses are key to the clinical transformation of mRNA vaccines. In recent years, modified mRNA, mRNA conjugates, viral vectors, microparticles, and nanoparticles have been developed for the delivery of mRNA. Although modified mRNA is resistant to degradation to some extent, it is prone to bind to serum proteins and aggregate, leading to vascular occlusion. Viral vectors have evolved to be efficient carriers for nucleic acid delivery and can therefore be used as delivery vectors for mRNA. However, the high immunogenicity, potential carcinogenicity, broad tropism, decreased packaging capacity after modification, and difficulty in production of viral vectors themselves limit their widespread application. In contrast to viral vectors, although non-viral vectors have lower transfection efficiency, they have lower immunogenicity than viral vectors. In addition, non-viral vectors are easy to synthesize, can be inserted into large fragments, and have the potential to carry larger mRNA loads. With the development of new materials and their preparation technologies, as well as a better understanding of the mechanisms involved, non-viral vectors are becoming the preferred carriers for delivering mRNA. The most common are liposomes and polymers, followed by peptides and inorganic nanoparticles. Among them, liposomes can be synthesized relatively easily in a scalable manner, protect mRNA from degradation, promote endosomal escape, and can be targeted to specific cell types through surface modification. Liposomes can also deliver mRNA and adjuvants into cells as needed, which has potential application prospects.

[0004] Although mRNA vaccines have achieved certain results in clinical studies, the efficiency of liposome delivery of mRNA and the strength of vaccine-induced immune response are still key factors affecting the effectiveness of mRNA vaccines. Therefore, it is necessary to develop carriers that can efficiently deliver mRNA into cells and stimulate strong immune responses to assist mRNA vaccines in their effectiveness.

[0005] At the same time, in order to obtain a better immune effect, in vitro cultured DC cells can also be used to load antigens into DC cells in different ways for direct immunization. The main ways to load antigens into DC cells include: direct incubation of peptides, antigens or whole cell lysates, viral vectors (such as adenovirus) carrying antigen gene transduction, mRNA transfection encoding antigens, etc. Due to the advantages of mRNA carrying multiple antigens, expressing multiple antigen epitopes intracellularly, inducing antigen cross-presentation, and high safety compared to viral vectors, DC vaccines sensitized with mRNA are considered to be an ideal way to prepare DC vaccines. In order to improve the effect of mRNA entering DC cells, electroporation, liposomes, and ultrasonic treatment are generally used. In comparison, liposome-mediated mRNA entry into DC is less harmful to cells, but the transfection efficiency needs to be improved. Therefore, it is also necessary to develop new mRNA vectors that are more efficient in transfecting mRNA into DC cells and can better activate DC cells. Summary of the invention

[0006] The technical problem to be solved by the present invention is to improve the immune effect of mRNA vaccines and DCs vaccines sensitized by mRNA. The technical solution of the present invention to solve the above technical problem is to provide a liposome modified by a hydrophobic modified cationic polypeptide.

[0007] In the liposome, the sequence of the cationic polypeptide is VQWRIRVAVIRK, the hydrophobic modification is coupling a hydrophobic fragment to the nitrogen end of the polypeptide; and the modified liposome is composed of a cationic lipid and an auxiliary lipid.

[0008] The cationic lipids in the liposomes are trimethyl-2,3-dioleyloxypropylammonium chloride (DOTMA), trimethyl-2,3-dioleyloxypropylammonium bromide (DOTAP), dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)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-2,3-dioleyloxypropylammonium bromide (DORIE-HP), dimethyl-4-hydroxyethyl ...OTMA), trimethyl-2,3-dioleyloxypropylammonium bromide (DOTMA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), dimethyldioctadecylammonium bromide (DDAB), di -hydroxybutyl-2,3-dioleyloxypropylammonium (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-sperminecarbonyl)-N',N'-dioctadecylglycinamide (DOGS), 1,2-dioleoyl-3-succinyl-sn-glycerocholine ester (DC-Chol), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DOSC) or lipid poly-L-lysine (LPLL).

[0009] Wherein, the auxiliary lipid in the above liposome is at least one of cholesterol (Chol), phosphatidylethanolamine (PE), phosphatidylcholine (PC), dioleoylphosphatidylethanolamine (DOPE), and diphosphatidylcholine (DOPC).

[0010] Furthermore, the mass ratio between the cationic lipid and the auxiliary lipid in the above liposome is: cationic lipid: auxiliary lipid = 0.5-20:1. Preferably, cationic lipid: auxiliary lipid = 0.8-1.25:1

[0011] Furthermore, the mass ratio between the hydrophobic modified cationic polypeptide and the modified liposome part in the above liposome is: hydrophobic modified polypeptide: liposome = 0.025-0.5: 1. Preferably, the hydrophobic modified polypeptide: liposome = 0.025-0.2: 1.

[0012] The liposomes are prepared by at least one of ethanol injection, thin film dispersion, ultrasonic dispersion, reverse evaporation, high pressure homogenization, supercritical carbon dioxide or freeze drying.

[0013] The liposome is prepared by the following method:

[0014] a. Weigh the cationic lipid and the auxiliary lipid, dissolve them in a solvent, and remove the organic solvent by rotary evaporation under reduced pressure; the solvent may be at least one of chloroform, ethanol, methanol, dichloromethane or ethyl acetate;

[0015] b. adding an aqueous solution of the hydrophobically modified cationic polypeptide or a solution formed by dissolving the hydrophobically modified cationic polypeptide in the solvent of step a into the system for hydration and demembranation to obtain a liposome suspension;

[0016] c. ultrasonically treating the obtained liposome suspension to obtain liposomes;

[0017] Alternatively, made by:

[0018] a. Weigh the cationic lipid and the auxiliary lipid, dissolve them in a solvent to prepare an organic phase, and dissolve the organic phase in a reaction container; the solvent is ethanol or ether;

[0019] b. Inject the DP7-C solution into the organic phase and stir; evaporate to 1 / 5 to 1 / 2 of the original volume under vacuum; and homogenize and extrude to obtain a blank liposome solution; the DP7-C solution is an aqueous solution, an ethanol solution or an ether solution;

[0020] c. The blank liposome solution was treated with ultrasound to obtain DP7-C modified liposomes.

[0021] The liposomes are prepared using raw materials in the following ratio: cationic lipid: auxiliary lipid: hydrophobic modified cationic polypeptide = 10-40: 10-40: 0.5-10 by mass ratio.

[0022] Furthermore, the above liposomes are prepared using raw materials in the following ratio: mass ratio of cationic lipid: auxiliary lipid: hydrophobic modified cationic polypeptide = 10-40:10-40:1-6.

[0023] Preferably, the liposomes are prepared using raw materials in the following ratio: mass ratio of DOTAP: cholesterol: hydrophobic modified cationic polypeptide = 20:20:3.

[0024] Wherein, the cationic polypeptide VQWRIRVAVIRK described in the above liposome is amidated and modified at the carbon terminus to VQWRIRVAVIRK-NH2.

[0025] Wherein, the hydrophobic fragment of the modified cationic polypeptide in the above liposome is a sterol compound, a saturated straight-chain fatty acid, or a PEG derivative.

[0026] Wherein, the sterol compound in the liposome is a cholesterol compound or a bile acid compound. The sterol compound is further at least one of succinylated cholesterol, bile acid or deoxycholic acid.

[0027] Wherein, the saturated straight-chain fatty acid in the liposome is at least one of C6 to 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-octanoic acid (C8).

[0028] Wherein, the PEG derivative described in the above liposome is 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG), distearoyl phosphatidylethanolamine-polyethylene glycol (DOPE-PEG) or dipalmitoyl phosphatidylethanolamine-polyethylene glycol (DPPE-PEG).

[0029] Furthermore, the nitrogen end of the cationic polypeptide in the above liposome is coupled to the hydrophobic segment by an amidation reaction between -CO-OH on the hydrophobic segment and -NH2 on the polypeptide.

[0030] Wherein, the hydrophobic modified cationic polypeptide structure described in the above liposome is:

[0031]

[0032] The R is a sterol compound, a saturated straight-chain fatty acid or a PEG derivative.

[0033] Furthermore, R in the hydrophobically modified cationic polypeptide structure is:

[0034] At least one of .

[0035] The present invention also provides the use of the liposome modified by the hydrophobic modified cationic polypeptide in the preparation of a nucleic acid delivery system.

[0036] The present invention also provides a nucleic acid delivery system, which is made of liposomes modified by the hydrophobic modified polypeptide and loaded with nucleic acid.

[0037] Wherein, the nucleic acid described in the above nucleic acid delivery system is at least one of DNA or RNA.

[0038] Furthermore, the above-mentioned RNA is at least one of messenger RNA, siRNA, sgRNA or mRNA.

[0039] Wherein, the nucleic acid delivery system is prepared from the liposomes modified by the hydrophobic modified cationic polypeptide and the nucleic acid in a mass ratio of 1 to 10:1 as raw materials. Preferably, the liposomes modified by the hydrophobic modified cationic polypeptide and the nucleic acid in a mass ratio of 1 to 3:1. More preferably, the liposomes modified by the hydrophobic modified cationic polypeptide and the nucleic acid in a mass ratio of 2:1.

[0040] Furthermore, the nucleic acid delivery system is prepared by co-incubating a liposome modified with a hydrophobic modified cationic polypeptide with a nucleic acid. The co-incubation is carried out in an aqueous solution or a culture medium. The culture time is 4 to 15 minutes. The culture medium is at least one of RPMI 1640, DMEM double-free culture medium, and Optim culture medium.

[0041] Wherein, the length of the nucleic acid loaded in the above-mentioned nucleic acid delivery system is 20 to 7000 bp.

[0042] Wherein, the nucleic acid in the nucleic acid delivery system is mRNA. Further, the mRNA is mRNA with a 5' cap structure and a 3' polyA tail structure.

[0043] Furthermore, the mRNA described in the above nucleic acid delivery system is mRNA encoding an antigen.

[0044] The present invention further provides the use of the above mRNA delivery system in preparing dendritic cell vaccines.

[0045] The present invention also provides a dendritic cell vaccine, which comprises dendritic cells loaded with the mRNA delivery system as the main active component.

[0046] Wherein, the dendritic cells are myeloid DCs or lymphoid DCs.

[0047] Furthermore, the dendritic cells are in vitro dendritic cells obtained from the patient himself.

[0048] Furthermore, the above dendritic cell vaccine is prepared by the following method:

[0049] a. Take immature dendritic cells (imDCs) after induction culture;

[0050] b. Add the nucleic acid delivery system to the culture medium for incubation

[0051] c. After the incubation is completed, the cells are digested with trypsin, washed, resuspended by centrifugation, and tested to obtain mature dendritic cells.

[0052] Furthermore, the dendritic cell vaccine mentioned above also includes a pharmaceutically acceptable auxiliary component, and the pharmaceutically acceptable auxiliary component is at least one of a protective agent, an excipient, an immune adjuvant, a dispersant or a cell culture medium.

[0053] The beneficial effects of the present invention are: the present invention uses a hydrophobic modified VQWRIRVAVIRK polypeptide to modify cationic liposomes, and the modified liposomes obtained can efficiently deliver nucleic acids to cells; in particular, the mRNA encoding antigen can be efficiently delivered to DCs, significantly improving the efficiency of cationic liposomes in delivering mRNA to DCs, and enhancing the immune effects of liposome mRNA vaccines and DCs vaccines loaded with mRNA. At the same time, the modified liposomes can also show stronger immune functions, such as more effectively stimulating the maturation of DCs and the secretion of proinflammatory cytokines, and significantly enhancing the immune function of DCs as APCs. In the embodiments of the present invention, mice were subcutaneously immunized with modified cationic liposomes loaded with antigen mRNA, and mice were subcutaneously immunized with DCs loaded with liposome / mRNA complexes, all of which showed significantly enhanced anti-tumor effects and spleen lymphocyte responses, indicating that the technical solution of the present invention can comprehensively promote the final in vivo effect of DCs cell vaccines through multiple effects such as efficient delivery of mRNA, promotion of maturation of DCs, and enhancement of the immune efficacy of DCs. At the same time, the technical solution of the present invention is easy to prepare, low in cost, low in toxicity and safety, and has a short preparation cycle. It can shorten the time for preparing DCs vaccines loaded with antigen peptides by nearly half, and has good clinical application prospects, especially in personalized DCs cell immunotherapy programs. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1.Preparation and characterization of DP7-C modified liposomes. ab. Particle size and potential distribution of DP7-C modified DOTAP liposomes prepared by thin film dispersion method. a. Particle size distribution; b. Potential. cd. Characterization of DP7-C modified DOTAP liposomes prepared by ethanol injection method. c. Particle size distribution; d. Potential. ef. Characterization of DP7-C modified DC-chol / DOPE liposomes. e. Particle size distribution; f. Potential.

[0055] Figure 2 .Characterization of DP7-C modified DOTAP liposomes and screening of their ratio of binding to mRNA. ab.Particle size distribution and zeta potential of DP7-C modified DOTAP liposomes before and after mRNA complexing; c.Transmission electron microscopy characterization of DP7-C modified DOTAP liposomes before and after mRNA complexing; d.Gel retardation electrophoresis to detect the ratio of DP7-C modified liposomes to mRNA complexing.

[0056] Figure 3 . Detection of the efficiency of liposome / mRNA complex transfection of 293T and dendritic cells. a. Efficiency of transfection of 293T cells; b. Average fluorescence intensity in 293T cells; c. JAWSII transfection efficiency; d. DC2.4 transfection efficiency; e. BMDCs transfection efficiency; f. Detection of the uptake pathway of liposome / mRNA complex by DC. (**p<0.01, ***p<0.001).

[0057] Figure 4 . Serum stability test of liposome / mRNA complex. a. Serum stability of liposome / mRNA complex; b. Serum stability of DP7-C modified liposome / mRNA complex.

[0058] Figure 5 .Liposome cytotoxicity assay.

[0059] Figure 6 .Lymph node targeting of liposome / eGFP mRNA complexes after subcutaneous injection into the lymph nodes of mice (*p<0.05).

[0060] Figure 7 .Liposomes induce DCs maturation in vitro, CD103 + DC ratio and cytokine secretion detection. a. Liposome-induced DC maturation detection; b. Liposome-induced CD103 +DC ratio; c. ELISA detection of IL-1β content in culture supernatant; d. ELISA detection of TNF-α content in culture supernatant; e. ELISA detection of IL-6 content in culture supernatant; f. ELISA detection of IL-12p70 content in culture supernatant; g. ELISA detection of IL-10 content in culture supernatant; h. IL-12p70 to IL-10 ratio. (*p<0.05, **p<0.01, ***p<0.001).

[0061] Figure 8 .Liposomes induce DCs maturation in lymph nodes, CD103 + DC ratio and cytokine secretion in serum were detected. a. Subcutaneous injection of liposomes near the lymph nodes of mice can recruit DCs into the lymph nodes. b. Subcutaneous injection of liposomes near the lymph nodes of mice can increase the ratio of mature DCs in the lymph nodes. c. Liposomes induce CD103 + DC ratio; d. serum IL-6 content; e. serum TNF-α content; f. serum IL-12p70 content; g. serum IL-10 content; h. serum IL-12p70 to IL-10 ratio (*p<0.05, **p<0.01, ***p<0.001).

[0062] Fig. 9 . Anti-tumor effect of liposome / OVA mRNA complex in treating EG7-OVA subcutaneous tumor model. a. Tumor growth curve; b. Detection of spleen antigen-specific lymphocytes. (*p<0.05, **p<0.05, ***p<0.001).

[0063] Fig.10 . Anti-tumor effect of liposome / neoantigen mRNA complex in treating LL2 orthotopic tumor model. a. Number of lung nodules; b. Average lung weight; c. Splenic CD8 after treatment with wild-type antigen peptide, mutant antigen peptide and mRNA + Activation of T lymphocytes; d. Splenic CD8 + IFN-γ secretion by T lymphocytes (*p<0.05, **p<0.05, ***p<0.001).

[0064] Fig.11 . Anti-tumor effect of DCs vaccine loaded with liposome / OVAmRNA complex in treating EG7-OVA subcutaneous tumor model. a. Tumor growth curve; b. Splenic antigen-specific lymphocyte detection. (*p<0.05, ***p<0.001).

[0065] Fig.12. Anti-tumor effect of DCs vaccine loaded with liposome / neoantigen mRNA complex in treating LL2 orthotopic tumor model. a. Tumor growth curve; b. Tumor weight; c. Splenic CD8 after wild-type antigen peptide, mutant antigen peptide and mRNA treatment + IFN-γ secretion by T lymphocytes (*p<0.05, **p<0.05, ***p<0.001). DETAILED DESCRIPTION

[0066] The present invention is described in detail below through the introduction of specific implementation methods.

[0067] In the preliminary study of the present invention, a polypeptide with excellent antibacterial effect was obtained, whose sequence is VQWRIRVAVIRK (SEQ ID No. 1), and it was named DP7. In further research, it was found that the DP7 polypeptide became an amphiphilic compound with the ability to self-assemble into micelles after hydrophobic modification, which can reduce the cytotoxicity of the DP7 polypeptide and maintain the antibacterial activity on the one hand; on the other hand, it can be used as a delivery carrier for some drugs after being assembled into nanoparticles.

[0068] The inventor creatively considered using the hydrophobic modified DP7 polypeptide to modify the cationic liposome, and then investigated whether the complex obtained by the modification has the function of an mRNA delivery carrier. In the experiment, it was unexpectedly found that the modification method using the hydrophobic modified DP7 polypeptide not only significantly improved the efficiency of cationic liposomes in transfecting mRNA into DCs; it also better promoted DC cell maturation and secretion of pro-inflammatory cytokines in vivo and in vitro, reflecting the great potential in the preparation of mRNA delivery systems, mRNA vaccines, and DC vaccines loaded with mRNA. On this basis, various technical solutions of the present invention were obtained.

[0069] In some embodiments, various conventional cationic liposomes can be modified using hydrophobic modified DP7 polypeptides. It is well known to those skilled in the art that cationic lipids are difficult to form liposomes with good stability and expected delivery capacity alone, so auxiliary lipids need to be added to form cationic liposomes. Therefore, the cationic liposomes involved in the present invention are also mainly liposomes with positive charges composed of cationic lipids and auxiliary lipids.

[0070] The cationic lipids that can be used in the present invention can be selected from the cationic lipid molecules commonly used in the preparation of cationic liposomes in the art. For example, trimethyl-2,3-dioleyloxypropylammonium chloride (DOTMA), trimethyl-2,3-dioleyloxypropylammonium bromide (DOTAP), dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)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-2,3-dioleyloxypropylammonium bromide (DORIE-HP), dimethyl-4-hydroxybutyl bromide -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-sperminecarbonyl)-N',N'-dioctadecylglycinamide (DOGS), 1,2-dioleoyl-3-succinyl-sn-glycerocholine ester (DC-Chol), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DOSC), lipid poly-L-lysine (LPLL).

[0071] The cationic lipids that can be used in the present invention can be selected from the commonly used helper lipids for preparing cationic liposomes in the art. For example, at least one of cholesterol (Chol), phosphatidylethanolamine (PE), phosphatidylcholine (PC), dioleoylphosphatidylethanolamine (DOPE), and diphosphatidylcholine (DOPC) can be selected but not limited to.

[0072] The cationic liposomes in the product of the present invention can be prepared by existing methods in the art, including but not limited to 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. It also includes a combination and improved method based on the above methods, such as thin film-freeze-thaw method, thin film-extrusion method, vacuum drying-ultrasound method and other methods used in the prior art for preparing cationic liposomes.

[0073] When preparing cationic liposomes, the ratio of cationic lipid to auxiliary lipid can be adjusted according to the specific raw materials and requirements. However, considering the addition of hydrophobic modified cationic polypeptide, the mass ratio of cationic lipid to auxiliary lipid can be controlled to be 0.5-1.5:1. Preferably, the mass ratio is cationic lipid: auxiliary lipid = 0.8-1.25:1.

[0074] The hydrophobic modified cationic polypeptide used to modify cationic liposomes in the present invention is modified by connecting a hydrophobic fragment to the nitrogen end of the VQWRIRVAVIRK polypeptide. The carbon end of the VQWRIRVAVIRK polypeptide can be amidated to form VQWRIRVAVIRK-NH2.

[0075] The hydrophobic fragment used for the hydrophobic modification of the VQWRIRVAVIRK polypeptide can be a sterol compound, a saturated straight-chain fatty acid or a PEG derivative. The sterol compound is a cholesterol compound or a bile acid compound. For example, it can be selected from succinylated cholesterol, bile acid or deoxycholic acid. The saturated straight-chain fatty acid is at least one of C6 to C20. Preferably, the saturated straight-chain fatty acid is at least one of C8 to C18. For example, it can be selected from stearic acid (C18), palmitic acid (C16), lauric acid (C12) or n-octanoic acid (C8). The PEG derivative can be selected from 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG), distearoyl phosphatidylethanolamine-polyethylene glycol (DOPE-PEG) or dipalmitoyl phosphatidylethanolamine-polyethylene glycol (DPPE-PEG).

[0076] Generally speaking, the nitrogen end of the cationic polypeptide is coupled to the hydrophobic fragment (hydrophobic compound) by an amidation reaction between -CO-OH on the hydrophobic fragment (hydrophobic compound) and -NH2 on the antimicrobial peptide.

[0077] The structure of the hydrophobic modified cationic peptide can be expressed as:

[0078]

[0079] Wherein, the R is a sterol compound, a saturated straight-chain fatty acid or a PEG derivative.

[0080] In some embodiments, R in the above structural formula is:

[0081] At least one of .

[0082] After investigation, the mass ratio between the hydrophobic modified cationic polypeptide and the liposome can generally be selected within the following range: hydrophobic modified polypeptide: liposome = 0.025-0.5: 1. Preferably, the hydrophobic modified polypeptide: liposome = 0.025-0.2:1.

[0083] In one embodiment of the present invention, cholesterol is used as a hydrophobic fragment to modify the cationic polypeptide, that is, R in the above formula is: The polypeptide obtained by modifying VQWRIRVAVIRK-NH2 with cholesterol is named DP7-C in the present invention.

[0084] The present invention further provides a reference method for preparing liposomes modified by the hydrophobic modified cationic polypeptide.

[0085] One method is based on a thin film dispersion method and includes the following steps:

[0086] a. Weigh the cationic lipid and the auxiliary lipid, dissolve them in a solvent, and remove the organic solvent by vacuum rotary evaporation;

[0087] b. adding an aqueous solution of the hydrophobically modified cationic polypeptide or a solution formed by dissolving the hydrophobically modified cationic polypeptide in a solvent applicable to step a into the system for hydration and demembrane removal to obtain a liposome suspension;

[0088] c. ultrasonically treating the obtained liposome suspension to obtain liposomes;

[0089] The solvent used in step a is generally an organic solvent, and commonly used ones include chloroform, ethanol, methanol, dichloromethane, ethyl acetate, etc.

[0090] Another method is based on ethanol injection and includes the following steps:

[0091] a. Weigh the cationic lipid and the auxiliary lipid, dissolve them in a solvent to prepare an organic phase, and dissolve the organic phase in a reaction vessel;

[0092] b. Inject the DP7-C solution into the organic phase and stir; evaporate to 1 / 5 to 1 / 2 of the original volume under vacuum; homogenize and extrude to obtain a blank liposome solution;

[0093] c. The blank liposome solution was treated with ultrasound to obtain DP7-C modified liposomes.

[0094] The ratio of the main raw materials in the above method is: cationic lipid: auxiliary lipid: hydrophobic modified cationic polypeptide = 10-40:10-40:0.5-10 by mass ratio.

[0095] Furthermore, the ratio of the main raw materials in the above method is: cationic lipid: auxiliary lipid: hydrophobic modified cationic polypeptide = 10-40:10-40:1-6 in mass ratio.

[0096] The solvent used in step a is generally ethanol. However, if the ether injection method is used, the solvent here is ether. The DP7-C solution in step b can be an aqueous solution, an ethanol solution or an ether solution according to the circumstances.

[0097] For example, in one embodiment of the present invention, liposomes are prepared using DP7-C, DOTAP and cholesterol as raw materials. A typical preparation method based on thin film dispersion method is provided, and the steps of the method are as follows:

[0098] a. Weigh DOTAP and cholesterol and dissolve them in a solvent, and remove the solvent by vacuum rotary evaporation;

[0099] b. adding an aqueous solution of a hydrophobically modified cationic polypeptide into the system for hydration and de-membrane removal to obtain a liposome suspension;

[0100] c. Ultrasonic treatment of the liposome suspension obtained in b is performed to obtain liposomes.

[0101] The solvent used in step a is generally an organic solvent, and commonly used ones include chloroform, ethanol, methanol, dichloromethane, ethyl acetate, etc.

[0102] During ultrasonic treatment, it is generally necessary to control the temperature of the system not to be too high, preferably below 25°C. For example, ice bath, intermittent ultrasonic treatment, etc. can be used to ensure that DP7-C polypeptide-modified DPTAP liposomes with suitable morphology are prepared.

[0103] During the preparation process, the mass ratio of each component is: DOTAP: cholesterol: DP7-C = 10-40: 10-40: 0.5-10. Preferably, the mass ratio is DOTAP: cholesterol: DP7-C = 10-40: 10-40: 1-6. The optimal mass ratio is: DOTAP: cholesterol: DP7-C = 20: 20: 3.

[0104] Similarly, based on the thin film dispersion method, DP7-C modified DC-chol / DOPE liposomes were prepared in another embodiment of the present invention.

[0105] In addition, another example of the present invention also uses an ethanol injection method to prepare DP7-C modified DOTAP liposomes.

[0106] These examples also show that the cationic liposomes in the product of the present invention can be prepared by existing methods in the art and then modified with DP7-C. The resulting modified products have relatively stable properties.

[0107] The liposome modified by the hydrophobic modified cationic polypeptide provided by the present invention has the function of loading nucleic acid and delivering it to cells, so nucleic acid can be loaded to form a nucleic acid delivery system. These target cells can be various animals and plants including human cells. They can be cells in vivo or in vitro culture.

[0108] The nucleic acid delivery system can be used to deliver at least one nucleic acid of DNA or RNA, and nucleic acids with a length between 20 and 7000 bp can achieve good delivery efficiency.

[0109] This nucleic acid delivery system can be widely used to deliver various specific RNA molecules, such as at least one of messenger RNA, siRNA, sgRNA or mRNA. In particular, it can efficiently deliver mRNAs used to translate target proteins and polypeptides in cells, which generally have 5' cap structures and 3' polyA tail structures.

[0110] More importantly, this delivery system can be prepared very simply. A typical simple preparation scheme is to co-incubate the liposomes modified by the above-mentioned hydrophobic modified cationic polypeptide with nucleic acids in a liquid. Co-incubation can be carried out in an aqueous solution or in a commonly used liquid culture medium, such as RPMI 1640 culture medium, DMEM culture medium, Optim culture medium and other commonly used culture media for cell culture, as well as mixed culture media obtained by combining them. Co-incubation time of 4 to 15 minutes in the system is generally sufficient to load a sufficient amount of nucleic acid onto the liposome to obtain the nucleic acid delivery system. This nucleic acid delivery system can be prepared by liposomes modified by hydrophobic modified cationic polypeptides and nucleic acids in a mass ratio of 1 to 10:1. Preferably, it is prepared by the above-mentioned liposomes and nucleic acids in a mass ratio of 1 to 3:1.

[0111] Previous studies have shown that DP7-C peptides can self-assemble in water to form micelles, which can be loaded with small molecule drugs, peptides, and even short nucleic acid molecules, such as siRNA. However, the properties and functions of liposomes obtained by modifying cationic liposomes with DP7-C are still unknown.

[0112] First, the present invention discovered that the cationic liposomes modified with DP7-C can deliver not only substances that can be delivered by the DP7-C polypeptide alone, but also long nucleic acid molecules such as mRNA.

[0113] When using cationic liposomes modified with DP7-C to load mRNA to transfect DC cells, unexpected results were obtained. Cationic liposomes modified with DP7-C can improve the transfection efficiency of mRNA into DC cells. Further, in three different DC cells, JAWSII, DC2.4 and BMDC, it was confirmed that cationic liposomes modified with DP7-C can significantly improve the transfer efficiency of mRNA, and are also significantly better than commercial transfection reagents Lipo2000 and PEI25K.

[0114] In further experiments, the present invention found that the cationic liposome modified with DP7-C not only improves the delivery efficiency of mRNA, but also has good serum stability and low cytotoxicity. In-depth research also found that the unmodified DOTAP liposome / mRNA complex is mainly internalized by DC cells through the macropinocytosis pathway, while the cationic liposome / mRNA complex modified with DP7-C is mainly internalized by DC cells through the two pathways of caveolin and clathrin, that is, the hydrophobically modified DP7 polypeptide can change the pathway of cationic liposome uptake by DC cells, which is also likely to be the reason for its significant improvement in mRNA transfection efficiency.

[0115] Therefore, it can be basically determined that the cationic liposomes modified with the hydrophobically modified DP7 polypeptide are particularly suitable as delivery carriers for delivering nucleic acid molecules, especially mRNA, to various DC cells. This can solve the problem of intracellular delivery efficiency limiting the effectiveness of the vaccine when preparing DC vaccines loaded with mRNA.

[0116] In subsequent studies, it was unexpectedly discovered that the use of hydrophobic-modified DP7 polypeptide modification also gave cationic liposomes stronger lymph node targeting, and had significantly better ability to promote DC cell maturation and the secretion of various inflammatory cytokines in vivo and in vitro, reflecting broad prospects in the preparation of DC cell mRNA vaccines.

[0117] Based on this, the present invention provides the use of the above nucleic acid delivery system in the preparation of dendritic cell vaccines. The dendritic cell vaccine comprises dendritic cells loaded with the nucleic acid delivery system of the present invention as the main active ingredient, and the nucleic acid loaded in the nucleic acid delivery system is mRNA.

[0118] Naturally, to prepare a dendritic cell vaccine, the above nucleic acid delivery system needs to be loaded with antigen mRNA and delivered into DC cells. The antigen here can be any antigen that can be encoded by mRNA and is expected to be expressed and presented after entering the DC cells. The antigen can be a full-length antigen with protein components, such as proteins or peptides, or it can be an antigen that is further modified after translation, for example, certain glycoproteins or lipoproteins.

[0119] The antigen may be a tumor antigen, including specific proteins or peptides expressed by tumor cells, such as WT1, MUC1, EGFRvIII, HER-2, MAGE-A3, NY-ESO-1, PSMA, GD2 or MART1, etc., or a personalized combination of mutated new antigens determined based on the patient's tumor sequence.

[0120] The antigen may also be a viral antigen, such as a protein or polypeptide that constitutes part of a virus, or a specific protein or polypeptide expressed in a virus-infected cell controlled by a viral expression mechanism, such as EBV, LMP2, HPV E6 E7, adenovirus 5 Hexon or HCMV pp65 and other virus-related antigens.

[0121] The antigen may also be a bacterial antigen, including proteins or polypeptides expressed by bacteria, such as Pseudomonas aeruginosa antigens, Clostridium tetani antigens, Streptococcus pneumoniae, Salmonella and other bacterial antigens.

[0122] Those skilled in the art will also understand that the antigens may also be disease-related antigens, including autoimmune-related antigens, such as antigens involved in autoimmune diseases or disorders or overexpressed in the process of autoimmune diseases, such as autoimmune-related antigens such as ppIAPP, IGRP, GAD65 or myelin basic protein antigens.

[0123] In addition, the loaded mRNA molecules may encode one or more immunogenic epitopes. These epitopes may generally be peptides of any length between 3 and 50 amino acid residues, generally arranged linearly, and may be further post-translationally modified after expression in DCs. It is understood that each of more than one immunogenic epitope may be the same or different.

[0124] For example, in one embodiment of the present invention, based on the five LL2 mutant neoantigens screened out with immunogenicity, a new mRNA was constructed, which can be translated and processed into peptide chains containing these five antigenic peptides in vivo. It was loaded onto cationic liposomes modified with hydrophobic-modified DP7 polypeptides, and showed significant anti-tumor effects in both in situ and subcutaneous tumor models.

[0125] To prepare a dendritic cell vaccine, the dendritic cells can be selected from myeloid dendritic cells (MDC) or lymphoid dendritic cells (LDC).

[0126] According to the current development of related fields such as immunology and bioinformatics, it is possible to prepare personalized vaccines for patients. For example, by finding tumor-specific antigens of tumor patients, designing personalized mRNA based on specific antigens, loading it onto cationic liposomes modified with hydrophobic DP7 peptides, and delivering it to DC cells, DC cell vaccines for specific tumor patients can be generated. In this case, ex vivo dendritic cells obtained from the patient are often used.

[0127] The method for preparing the above-mentioned dendritic cell vaccine comprises the following main steps:

[0128] a. Take immature dendritic cells (imDCs) after induction culture;

[0129] b. Add the nucleic acid delivery system to the culture medium for incubation

[0130] c. After the incubation is completed, the cells are digested with trypsin, washed, resuspended by centrifugation, and tested to obtain mature dendritic cells.

[0131] There are many methods available in the art for detecting and obtaining mature dendritic cells. For example, CD11c, CD80 and CD86 antibodies can be added to DC cells after centrifugation and resuspending, and then the DC cells can be stained to determine whether they are mature based on the staining results.

[0132] It will be understood by those skilled in the art that, whether the cationic liposomes modified with the hydrophobically modified DP7 polypeptide loaded with antigen mRNA are directly used, or the DC cell vaccine prepared based on the cationic liposomes is used, pharmaceutically acceptable auxiliary components may also be included.

[0133] The pharmaceutically acceptable auxiliary components are at least one of a protective agent, an excipient, an immune adjuvant, a dispersant or a cell culture medium.

[0134] For example, DC live cell vaccines are handled, prepared and used in solutions, which may therefore contain pharmaceutically acceptable concentrations of salts, buffers, preservatives and various culture media to help maintain the viability of the live cells in the vaccine formulation and reduce or prevent cell lysis. If the vaccine is to be stored frozen, the vaccine may include a cryoprotectant, such as DMSO.

[0135] If necessary, the vaccine may further include an adjuvant to assist in the induction or re-stimulation of an immune response, including prolonging or strengthening an immune response. The adjuvant may be selected from various adjuvants known in the art according to the actual use of the DC vaccine.

[0136] The present invention is described in further detail below through examples.

[0137] The main experimental materials and equipment used in the examples are as follows:

[0138] 1. Experimental cell lines and experimental animals

[0139] 293T, JAWSII, DC2.4 cell lines and mouse lung cancer cell line LL2 were purchased from the American Type Culture Collection (ATCC). Cell culture was performed using DMEM (Gibico) medium containing 10% fetal bovine serum (FBS, Gibico). 6-8 week old C57BL / 6J female mice were purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd. and raised in an SPF environment.

[0140] 2. Main reagent materials and kits

[0141] Experimental cell culture medium: RPMI-1640 culture medium, DMEM culture medium and fetal bovine serum (FBS) were purchased from Gibco, USA.

[0142] FLAG polypeptide: DYKDDDDK (SEQ ID No. 2) (purchased from Sigma)

[0143] OVA mRNA was purchased from Tebu-bio.

[0144] Mouse IFN-gamma precoated ELISpot kit (#DKW22-2000-096), lymphocyte separation fluid and ELISPOT special culture medium (Dakowie DKW34-EU0100) were purchased from Dakowie Biotechnology Co., Ltd.

[0145] Cytokines and agonists: rmGM-CSF (122-03) was purchased from Shanghai Puxin Biotechnology Co., Ltd.; polyIC was purchased from Thermo Company; agonist CPG1826 was synthesized by Invitrogen.

[0146] Flow cytometry antibodies: The following flow cytometry antibodies were purchased from BD: Rat Anti-Mouse CD86, Hamster Anti-Mouse CD11c, Rat Anti-Mouse CD8α, Rat Anti-Mouse CD4, Rat Anti-Mouse IA / IE, Hamster Anti-Mouse CD103, Hamster Anti-Mouse CD80, Hamster Anti-Mouse CD3e, RatAnti-Mouse IFN-γ.

[0147] Western Blot antibodies: anti-GAPDH antibody and anti-MyD88 antibody were purchased from Abcam.

[0148] TLR2 receptor inhibitor C29, macropinocytosis pathway inhibitor, caveolin pathway inhibitor and clathrin pathway inhibitor were all purchased from MCE.

[0149] DOTAP, DOPE, and DC-Chol cholesterol were purchased from Shanghai Avituo Company; dialysis cards were purchased from Sigma-aldrich Company; e-GFP mRNA was purchased from Thermo Company; PEI25K reagent was purchased from Sigma-aldrich Company; Lipofectamine2000 transfection reagent was purchased from Invitrogen Company; and RiboRuler High Range RNA Ladder was purchased from Thermo Company.

[0150] 3. Main instruments and equipment

[0151] Rotary evaporator, V rotary evaporator RV 10, Aika, Germany; small animal in vivo imaging system, Caliper LifeSciences, IVIS Spectrum; Malvern particle size potential instrument: ZetaSizer Nano-ZS Zen 3600, Malvern; transmission electron microscope: H-600 transmission electron microscope, Hitachi; flow cytometer: FACSCalibur, BD; microplate reader: MultiskanMk3, Thermo Scientific; DNA concentration measurement instrument: Nanodrop 2000, Thermo Scientific; laser confocal microscope: FV1000, Olympus; constant temperature incubator: MCO-18, Thermo Scientific.

[0152] Example 1. Preparation and characterization of DP7-C modified liposomes

[0153] In this example, we used different methods (thin film dispersion method and ethanol injection method) to prepare DP7-C modified DOTAP / cholesterol cationic liposomes, and used thin film dispersion method to prepare DP7-C modified DC-chol / DOPE cationic liposomes.

[0154] 1. Preparation and characterization of DP7-C modified DOTAP liposomes

[0155] 1) Preparation of DP7-C modified DOTAP liposomes by thin film dispersion method

[0156] Weigh 20 mg DOTAP and 20 mg cholesterol, dissolve in 4 ml chloroform, and add to a 100 ml eggplant-shaped bottle. Remove the organic solvent by vacuum rotary evaporation at 37 ° C in a water bath, and set the speed to 50 rpm / min. Add 10 ml of 300 μg / ml DP7-C aqueous solution to the eggplant-shaped bottle for hydration and demolding, set the temperature to 60 ° C, the speed to 75 rpm / min, and continue hydration for 30 min. Finally, the obtained liposome suspension is ultrasonicated in an ice bath for 3 min (the setting conditions are 110W, ultrasonication for 3s, and stop for 3s) to prepare DP7-C modified DOTAP liposomes (DOTAP / cholesterol liposomes are referred to as DOTAP liposomes in the following examples, and DOTAP / cholesterol liposomes are marked as DOTAP in each figure).

[0157] The prepared DP7-C modified DOTAP liposomes were placed in a dialysis card for dialysis to detect the efficiency of the DP7-C modified liposomes. The molecular weight cutoff of the dialysis card is 2000Da. The prepared liposomes were added to the dialysis card, which was placed in a beaker with 1L of water for 24 hours. The samples in the dialysis card were taken out, and HPLC was used to detect the DP7-C in the samples before and after dialysis. The peak area comparison can reflect the change in DP7-C concentration.

[0158] Experimental results: The peak areas obtained by HPLC detection of samples in the dialysis card after 24 hours and samples before dialysis were compared. The elution time of DP7-C was about 15.5 minutes, the peak area of ​​the sample before dialysis was 1747.38, and the peak area after dialysis was 900.2. The proportion of DP7-C stably modified on the liposomes was calculated based on the standard curve. The results showed that 57.63% of DP7-C was stably inserted into the DOTAP liposomes after dialysis. The particle size distribution and zeta potential of the liposomes were characterized using a particle size potentiometer (Malvern ZetaSizer, UK). At the same time, the particle size distribution and zeta potential changes after the liposomes were complexed with mRNA were determined. All experiments were repeated three times and the middle value was taken. The particle size of the DOTAP liposomes modified with DP7-C is 100.23±7.50nm, and the zeta potential is 53.02±5.51mV( Figure 1 a-1b).

[0159] 2) Preparation of DP7-C modified DOTAP liposomes by ethanol injection method

[0160] The method is as follows: weigh 20 mg DOTAP and 20 mg cholesterol, add 10 ml ethanol to dissolve in a reaction container to prepare an organic phase, inject DP7-C aqueous solution into the organic phase to make the final concentration 300 μg / ml, stir for 10 minutes; evaporate to 1 / 5 to 1 / 2 of the original volume under vacuum; homogenize and extrude to obtain a blank liposome solution. Ultrasound at 200W for 10 minutes under ice bath. Finally, the efficiency of DP7-C modification to DOTAP liposomes was detected to be 53.24%.

[0161] The particle size potential of the DP7-C modified liposome prepared by the ethanol injection method was detected by a particle size potential analyzer, and the particle size was about 113.35±13.34nm, and the zeta potential was 52.33±4.32mV ( Figure 1 c-1d).

[0162] 2. Preparation and characterization of DP7-C modified DC-chol / DOPE cationic liposomes

[0163] 20 mg of cationic lipid DC-cholesterol (3b-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride) and 40 mg of neutral lipid DOPE (1,2-diol-sn-glycero-3-phosphoethanolamine) were dissolved in chloroform in a ratio of 1:2 and placed in a glass bottle. The lipids were dried under an argon flow without O2. After evaporation, the lipid layer was hydrated by adding 10 ml of 300 μg / ml DP7-C aqueous solution at 60°C and 75 rpm / min for 30 min. After vortexing for 3 min, the glass flask with the formed multilayer liposomes was placed in an ultrasonic bath for 30 min to form unilamellar liposomes. Finally, DP7-C modified DC-chol / DOPE liposomes were obtained by extrusion using a membrane with 200 nm pores using a micro extruder. The modification efficiency of DP7-C was measured to be 52.31% after dialysis.

[0164] The particle size potential of the liposomes was detected by a particle size potential analyzer, and the particle size was approximately 185.18±25.47nm, and the zeta potential was 23.21±3.45mV ( Figure 1 e-1f).

[0165] This example shows that DP7-C can successfully modify cationic liposomes prepared by different methods and different raw materials, and the resulting products have similar particle size and zeta potential properties. Modification of DOTAP liposomes with DP7-C can obtain stable liposomes with a size of about 100nm and a zeta potential of about 50mV. Modification of DC-chol / DOPE cationic liposomes with DP7-C can obtain stable liposomes with a particle size of about 180nm and a zeta potential of about 20mV. In the following examples, the relevant experiments of DP7-C-modified DOTAP liposomes loading mRNA and making DCs vaccines are mainly provided.

[0166] Example 2. Preparation and characterization of DP7-C modified DOTAP liposomes complexed with mRNA

[0167] 1. Method and optimal ratio of DP7-C modified DOTAP liposomes and mRNA complex

[0168] Take the DP7-C modified DOTAP liposomes prepared in Example 1 and dilute them in aqueous solution to the use concentration. Add mRNA at a ratio of liposome: mRNA = 2:1, mix gently, and incubate at room temperature for 10 minutes to obtain DP7-C modified DOTAP liposomes. Gel retardation electrophoresis detected the binding of DP7-C modified liposomes with mRNA of different mass ratios (mRNA encoding new antigens shown in SEQ ID No.4, the same below), and the mass ratios of DP7-C modified liposomes to mRNA were 0:1, 0.25:1, 0.5:1, 1:1, and 2:1, respectively.

[0169] The experimental results showed that the particle size of DP7-C modified DOTAP liposomes complexed with mRNA was 130.45±9.32nm and the zeta potential was 34.67±7.45mV( Figure 2 ab). Transmission electron microscopy observations showed that the prepared DP7-C modified DOTAP liposomes had uniform particle size and good dispersibility, and the diameter was comparable to the size measured by the particle size potentiometer, which was about 100 nm. The DP7-C modified DOTPA liposomes after mRNA complexing had good dispersibility and uniform particle size, with a diameter of 120-130 nm, which was consistent with the particle size analysis results ( Figure 2 c). The gel retardation electrophoresis results showed that there was no free mRNA band when the mass ratio of DP7-C modified DOTAP liposomes to mRNA was 2:1, and the mRNA bands were all bound to the DP7-C modified DOTAP liposomes, indicating that the optimal ratio of DP7-C modified DOTAP liposomes to mRNA incubation was 2:1 ( Figure 2 d).

[0170] Example 3: Study on DP7-C modified DOTAP liposomes as mRNA delivery vectors

[0171] 1. Efficiency of transfection of mRNA into 293T cells by DP7-C modified DOTAP liposomes

[0172] 0.5 μg EGFP mRNA, 1 μg liposome, 1 μg Lipo2000 and 1 μg PEI25K were co-incubated in RPMI 1640 medium for 10 min and then transfected into 2×10 5 293T cells were transfected in 24-well plates. After 4 hours, the RPMI 1640 medium was replaced with RPMI 1640+10% FBS+1% PS medium and transfected for 24 hours. The expression of green fluorescent protein was observed using a fluorescence microscope and photographed. The cells were digested with trypsin, and the proportion of GFP-positive cells was detected using a flow cytometer to determine the transfection efficiency and analyze the mean fluorescence intensity.

[0173] The experimental results showed that the efficiency of mRNA transfection into 293T cells by unmodified DOTAP liposomes was 84.89±2.13%, and the efficiency of mRNA transfection into 293T by DP7-C-modified DOTAP liposomes was 84.87±3.21%. The efficiency of mRNA transfection into 293T cells by PEI was 80.89±5.56%, and the efficiency of mRNA transfection into 293T cells by Lipo2000 was 80.89±10.23%, indicating that the efficiency of eGFP mRNA transfection into 293T cells by DOTAP liposomes modified with DP7-C was comparable to that of unmodified DOTAP liposomes, Lipo2000, and PEI25K ( Figure 3 a-3b).

[0174] 2. Efficiency of mRNA transfection into dendritic cells by DP7-C modified DOTAP liposomes

[0175] In this part of the experiment, GFP mRNA was used as the target, and mRNA was transferred into DCs to express it into protein and presented by DCs to investigate the transfection efficiency of DCs. The specific method was to co-incubate 0.5 μg EGFP mRNA with 1 μg liposome, 1 μg Lipo2000 and 1 μg PEI25K in RPMI 1640 medium and add them to 2×10 5 JAWSII, DC2.4 and BMDC were placed in 24-well plates. After 4 hours, the RPMI 1640 medium was replaced with RPMI 1640+10% FBS+1% PS medium, and the transfection was continued for 24 hours. The expression of green fluorescent protein was observed using a fluorescence microscope and photographed. The cells were digested with trypsin, centrifuged at 12000rpm for 3 minutes, resuspended with 100μl PBS, and incubated with 1μl CD11c antibody for 40 minutes. After the incubation, the excess antibody was washed away, and the proportion of GFP-positive cells in CD11c-positive cells was detected by flow cytometry to determine the transfection efficiency.

[0176] The results showed that the efficiency of mRNA transfer into JAWSII cells by unmodified DOTAP liposomes was 4.51±1.03%, the efficiency of mRNA transfer into DC2.4 cells was 5.05±2.12%, and the efficiency of mRNA transfer into BMDCs was 8.40±1.31%; while the efficiency of mRNA transfer into JAWSII cells by DP7-C-modified DOTAP liposomes was 12.23±1.35%, the efficiency of mRNA transfer into DC2.4 cells was 28.49±2.46%, and the efficiency of mRNA transfer into BMDCs was The efficiency of mRNA transfer into JAWSII cells by Lipo2000 was 3.65±1.12%, the efficiency of mRNA transfer into DC2.4 cells was 4.56±1.37%, and the efficiency of mRNA transfer into BMDCs was 6.40±1.32%; the efficiency of mRNA transfer into JAWSII cells by PEI was 4.63±1.11%, the efficiency of mRNA transfer into DC2.4 cells was 5.07±1.32%, and the efficiency of mRNA transfer into BMDCs was 6.73±1.27%. These results indicate that DOTAP liposomes modified with DP7-C can unexpectedly improve the efficiency of mRNA transfer into DCs, and the transfection efficiency in the three DC cells is significantly different from that of other control groups (p<0.001)( Figure 3 c-3e).

[0177] 3. Study on the mechanism of DCs uptake of DP7-C-modified DOTAP liposomes loaded with mRNA

[0178] Endocytosis is the process of transporting extracellular substances into cells through the deformation of the plasma membrane. It is mainly divided into macropinocytosis, clathrin pathway and caveolin pathway. In order to determine which uptake pathway is related to the entry of liposome / mRNA complexes into DCs, DCs were treated with inhibitors of each uptake pathway, and then the uptake efficiency of liposome / mRNA complexes by DCs after inhibiting the pathway was tested, so as to determine which endocytic pathway is related to the entry of liposomes into DCs carrying mRNA. The study used Chlorpromazine, an inhibitor of the clathrin-mediated endocytic pathway, Amiloride, an inhibitor of the macropinocytic pathway, and Genistein, an inhibitor of the caveolin-mediated endocytic pathway.

[0179] The specific experimental method is to plate DC2.4 in a 24-well plate, with 2×10 5Cells were treated with 5μM chlorpromazine, 20μM amiloride and 30μM genisterin for 2h. Then, the complex of DOTAP liposome (1μg) and eGFP mRNA (0.5μg) and the complex of DP7-C modified DOTAP liposome (1μg) and eGFP mRNA (0.5μg) were added and cultured at 37℃ for 24h. The cells were harvested and the proportion of GFP positive cells was detected by flow cytometry.

[0180] The results show that the efficiency of DC2.4 transfection by unmodified DOTAP liposome / mRNA complex is 5.05±2.12%, and the efficiency of transfection by macropinocytosis inhibitor is 0.78±0.43%, which is significantly lower than that of the group without inhibitor treatment (p<0.001). The efficiency of transfection by clathrin and caveolin inhibitor is not significantly different from that of the group without inhibitor treatment (p>0.05), indicating that the unmodified DOTAP liposome / mRNA complex is mainly taken up by DCs through macropinocytosis. The efficiency of DC2.4 transfection by DP7-C modified DOTAP liposome / mRNA complex is 28.49±2.46%, and the efficiency of uptake is not significantly changed after treatment with macropinocytosis inhibitor (p>0.05), while the efficiency of uptake is reduced to 10.47±1.35% after treatment with clathrin inhibitor, which is significantly lower than that of the group without inhibitor treatment (p<0.001). After treatment with caveolin inhibitor, the uptake efficiency was reduced to 15.23±2.67%, which was significantly lower than that of the group without inhibitor treatment (p<0.01). This indicates that the DP7-C modified DOTAP liposome / mRNA complex was internalized by DCs mainly through the two pathways of caveolin and clathrin. Figure 3 f).

[0181] 4. Serum stability test of liposome / mRNA complex

[0182] To be effective in vivo, liposome / mRNA complexes must be serum stable. Therefore, we set different concentrations of serum for transfection in 293T cells to determine the serum stability of liposome / mRNA complexes. Transfection efficiency was detected by fluorescence microscopy and flow cytometry 24 hours after transfection. The results showed that the transfection efficiency of DOTAP liposome / mRNA complexes before and after modification in different concentrations of serum was comparable to that in serum-free medium, and there was no significant difference between the groups (p>0.05), indicating that the liposome / mRNA complex has good serum stability ( Figure 4 a-4b).

[0183] 5. Cytotoxicity detection of DOTAP liposomes before and after DP7-C modification

[0184] The cells were treated with different concentrations of DP7-C, liposomes, DP7-C modified DOTAP liposomes, PEI25K or Lipo2000 for 24 h, and the cytotoxicity of different concentrations was compared. The specific method was to plate 293T cells on a 96-well plate, with 1×10 4 cells. After 24 h of culture, the cells were treated with different concentrations of DOTAP liposomes, DP7-C-modified DOTAP liposomes, PEI25K or Lipo2000 for 24 h, and then 10 μL of MTT was added to each well, cultured at 37 °C for 4 h, the culture medium was aspirated, and 200 μL of DMSO was added to each well. Finally, the absorbance value at 570 nm was read using a Spectramax M5 microplate photometer (Molecular Devices, Sunnyvale, CA, USA).

[0185] The results showed that with the increase of PEI25K and Lipo2000 concentrations, cell viability decreased significantly. Only 13.47±1.23% of cells survived in the 100μg / ml PEI25K treatment group and only 15.26±1.37% of cells survived in the 100μg / ml Lipo2000 treatment group, indicating that the two transfection reagents had high cytotoxicity. The cell viability of DOTAP liposomes before and after modification decreased slowly with the increase of concentration. There were still 65.27±6.14% of cells survived in the 100μg / ml DOTAP liposome treatment group and 71.27±3.14% of cells survived in the 100μg / ml DP7-C modified DOTAP liposome treatment group, indicating that the cytotoxicity of DOTAP liposomes before and after DP7-C modification was lower than that of Lipo2000 and PEI25K. In the DP7-C group, the cell viability did not change significantly with the increase of DP7-C concentration. About 92.31±1.34% of cells survived in the 100μg / ml treatment group, and the cytotoxicity was low ( Figure 5 ).

[0186] 6. Detection of the efficiency of transfection of mRNA into lymph node cells by DP7-C modified DOTAP liposomes

[0187] The previous experiments have verified in vitro that DP7-C-modified DOTAP liposomes can indeed improve the efficiency of mRNA transfer into DCs. This experiment studies whether it has targeting properties.

[0188] After co-incubation with 10 μg eGFP mRNA and 20 μg liposomes, the mixture was injected subcutaneously into the lymph nodes of 6-8 week old C57BL / 6J female mice. After 24 hours, the proximal lymph nodes were removed and the expression of green fluorescent protein in the lymph nodes was imaged using a live imaging device to reflect the expression efficiency of eGFP mRNA in the lymph nodes.

[0189] The results of in vivo imaging showed that the efficiency of DOTAP liposomes modified with DP7-C in transferring eGFP mRNA into lymph nodes was significantly higher than that of unmodified liposomes (p<0.05), as shown by the significantly higher fluorescence intensity of eGFP in lymph nodes of the DOTAP liposome / eGFP mRNA complex group modified with DP7-C than that of the DOTAP liposome / eGFP mRNA complex group ( Figure 6 ).

[0190] Example 4: Study on DP7-C-modified DOTAP liposomes as a catalyst for promoting DC maturation and cytokine secretion

[0191] In this part of the experiment, we mainly verified whether DP7-C modified DOTAP liposomes have more advantages in promoting DCs maturation, and compared them with the commonly used adjuvants polyIC and CPG to evaluate the effect of DP7-C modified DOTAP liposomes in inducing DCs maturation. In addition, we compared the differences between DOTAP liposomes and DP7-C modified DOTAP liposomes in terms of DCs secretion of cytokines to clarify whether there are differences between the two in activating DCs function.

[0192] 1. Liposomes induce DCs maturation in vitro

[0193] BMDCs cultured to day 8 were treated with 5 μg / ml unmodified liposomes and DP7-C modified liposomes for 24 hours, and 5 μg / ml DP7-C, CPG and polyIC were used as positive controls. After 24 hours, cells were collected for CD11c, CD80, and CD86 staining to detect the maturation of BMDCs. Cells were collected for CD11c and CD103 staining to detect the typing of BMDCs.

[0194] From the results of flow cytometry, DP7-C alone can induce 23.51±7.23% of DCs maturation, CPG alone can induce 44.58±4.32% of DCs maturation, and polyIC alone can induce 11.40±2.32% of DCs maturation; unmodified DOTAP liposomes can induce 19.61±1.34% of DCs maturation, and DOTAP liposomes modified with DP7-C can induce 26.31±1.42% of DCs maturation; while the PBS group can only induce 4.04±0.58% of DCs maturation. From the perspective of the proportion of inducing DCs maturation, DP7-C-modified DOTAP liposomes have more advantages than unmodified liposomes in inducing DCs maturation (p<0.05), and are significantly higher than the commonly used adjuvant polyIC in inducing DCs maturation (p<0.01), but lower than the ability of the same dose of CPG to stimulate DC maturation (p<0.01) ( Figure 7 a-7b).

[0195] 2. Liposomes induce DCs to secrete cytokines in vitro

[0196] BMDCs cultured on day 8 were treated with 5 μg / ml DP7-C, 5 μg / ml unmodified liposomes and 5 μg / ml DP7-C modified liposomes for 24 h, and the supernatants were collected for ELISA detection.

[0197] The results show that after treatment with DP7-C modified DOTAP liposomes, the secretion of IL-1β increased from 437.24±35.23pg / ml in the unmodified DOTAP liposome control group to 1478.87±75.23pg / ml, and there was a significant difference between the two (p<0.001)( Figure 7 c); After treatment with DP7-C modified DOTAP liposomes, the secretion of TNF-α increased from 3452.23±123.21pg / ml in the unmodified DOTAP liposome control group to 4359.30±77.25pg / ml, and there was a significant difference between the two (p<0.001) ( Figure 7 d); After stimulation by DP7-C modified liposomes, the secretion of IL-6 increased from 3617.23±62.14pg / ml in the unmodified liposome control group to 4225.24±73.23pg / ml, and there was a significant difference between the two (p<0.05) ( Figure 7e); After stimulation by DP7-C modified DOTAP liposomes, the secretion of IL-12p70 increased from 72.14±11.34pg / ml in the unmodified DOTAP liposome control group to 367.24±23.23pg / ml, and there was a significant difference between the two (p<0.001) ( Figure 7 f); For the secretion of IL-10, the secretion amount of the unmodified DOTAP liposome control group was 182.14±23.34pg / ml, and the secretion amount of IL-10 after stimulation with DP7-C modified liposomes was 182.24±13.13pg / ml. There was no significant difference between the two groups (p>0.05) ( Figure 7 g). However, from the perspective of the ratio of IL-12p70 / IL-10, the DP7-C-modified DOTAP liposomes significantly increased the ratio of IL-12p70 / IL-10 compared with the unmodified DOTAP liposomes (p<0.001)( Figure 7 h).

[0198] The above experimental results show that DP7-C modified DOTAP liposomes have a stronger ability to induce DCs to secrete cytokines and have a certain synergistic effect. It can also make DCs in an activated state rather than an inhibited state, which is more conducive to the efficacy of the vaccine.

[0199] 3. Liposome-induced maturation of lymph node DCs

[0200] 20 μg of liposomes were injected subcutaneously into the lymph nodes of 6-8 week old C57BL / 6J female mice. After 24 hours, the proximal and distal lymph nodes were removed and cells were collected for CD11c and MHC II staining to detect the maturation of DCs in the lymph nodes. + The proportion of DCs.

[0201] The results showed that after subcutaneous injection of DP7-C modified DOTAP liposomes near the lymph nodes of mice, the proportion of DCs in the proximal and distal lymph nodes was significantly increased compared with the unmodified DOTAP liposome group, among which the proportion of DCs in the proximal lymph nodes increased from 18.31±2.12% in the unmodified DOTAP liposome group to 22.03±1.45% in the DP7-C modified DOTAP liposome group (p<0.001). Compared with the proportion of DCs in the control group (7.79±1.12%), the DP7-C group, the unmodified DOTAP liposome group and the modified DOTAP liposome group were able to effectively recruit DCs to the lymph nodes and increase the proportion of DCs in the lymph nodes (p<0.001) ( Figure 8a). In terms of the proportion of mature DCs in lymph nodes, only 0.76±0.23% of DCs were mature in the PBS group, 1.13±0.21% of DCs were mature in the DP7-C group, 1.27±0.24% of DCs were mature in the unmodified DOTAP liposome group, and 1.40±0.36% of DCs were mature in the DP7-C modified DOTAP liposome group. The results show that DP7-C modified DOTAP liposomes are more advantageous than unmodified DOTAP liposomes in inducing DCs maturation in vivo (p<0.001 for proximal lymph nodes, p<0.01 for distal lymph nodes) ( Figure 8 b).

[0202] 4. Effect of subcutaneous lymph node injection of liposomes on serum cytokine levels in mice

[0203] The cytokine content in the serum after subcutaneous injection of liposomes near the lymph nodes of mice was detected to evaluate the in vivo immune activation of liposomes before and after modification. 20 μg of liposomes were injected subcutaneously near the lymph nodes of 6-8 week-old C57BL / 6J female mice. After 24 hours, the eyeballs were removed to collect blood. The blood was placed at 37°C for 1 hour and centrifuged at 13000 rpm for 15 minutes. The serum was separated for ELISA detection.

[0204] The results showed that DP7-C modified DOTAP liposomes were more advantageous than unmodified DOTAP liposomes in inducing DCs to secrete proinflammatory cytokines. The results showed that after treatment with DP7-C modified DOTAP liposomes, the secretion of IL-6 increased from 3.14±1.23pg / ml in the unmodified DOTAP liposome control group to 66.87±5.14pg / ml, and there was a significant difference between the two (p<0.001)( Figure 8 d); After treatment with DP7-C modified DOTAP liposomes, the secretion of TNF-α increased from 15.21±2.32pg / ml in the unmodified DOTAP liposome control group to 22.32±4.25pg / ml, and there was a significant difference between the two (p<0.01) ( Figure 8 e); After stimulation by DP7-C modified DOTAP liposomes, the secretion of IL-12p70 increased from 0.53±0.14pg / ml in the unmodified DOTAP liposome control group to 52.24±27.53pg / ml, and there was a significant difference between the two (p<0.05) ( Figure 8f); For the secretion of IL-10, the secretion amount of the unmodified DOTAP liposome control group was 27.12±4.34pg / ml, and the secretion amount of IL-10 after stimulation by DP7-C modified DOTAP liposomes was 12.34±1.43pg / ml, and there was a significant difference between the two (p<0.01) ( Figure 8 g). From the perspective of the ratio of IL-12p70 / IL-10, the DOTAP liposomes modified with DP7-C can significantly increase the ratio of IL-12p70 / IL-10 compared with the unmodified DOTAP liposomes (p<0.01) ( Figure 8 h), indicating that DP7-C modified liposomes have a stronger ability to induce DCs to secrete cytokines, and can make DCs in an activated state rather than an inhibited state, which is more conducive to the efficacy of the vaccine. This result is basically consistent with the in vitro results.

[0205] 5. Effects of liposome treatment in vivo and in vitro on DCs typing

[0206] There are reports that CD103 + DCs facilitate antigen cross-presentation. CD103 + The higher the proportion of DCs, the stronger the ability of antigen cross-presentation and the higher the efficiency. Here, the CD103 expression of DCs was detected after 24 hours of treatment with 5 μg / ml different stimulants (DP7-C, CPG, polyIC, DOTAP liposomes and DP7-C modified DOTAP liposomes). + The proportion of DCs.

[0207] In vitro results showed that CD103 + The proportion of DCs was 0.70±0.43%, and CD103 in the DP7-C treated group + The proportion of DCs was 6.18±1.23%, and CD103 in the CPG-treated group + The proportion of DCs was 1.74±0.83%, and CD103 in the polyIC-treated group + The proportion of DCs was 1.75±0.71%, and the proportion of CD103 in the DOTAP liposome-treated group was 1.75±0.71%. + The proportion of DCs was 2.33±0.93%, and the proportion of DCs in the DP7-C modified DOTAP liposome-treated group was 2.33±0.93%. + The proportion of DCs was 6.72±1.42%. Among them, the DP7-C group and the DP7-C modified DOTAP liposome treatment group were significantly different from the unmodified DOTAP liposome treatment group (p<0.01) ( Figure 7 c).

[0208] In vivo results showed that CD103 +The proportion of DCs was 0.32±0.13%, and CD103 in the DP7-C treated group + The proportion of DCs was 0.70±0.23%, and the proportion of CD103 in the DOTAP liposome-treated group was 0.70±0.23%. + The proportion of DCs was 0.39±0.16%, and the proportion of CD103 in the DP7-C modified DOTAP liposome-treated group was 0.39±0.16%. + The proportion of DCs was 0.72±0.18%. + The proportion of DCs was significantly higher than that of the unmodified DOTAP liposome group (p<0.001) ( Figure 8 c).

[0209] Example 5. Functional verification of DP7-C modified liposome / mRNA complex vaccine

[0210] 1. Functional validation of DP7-C modified liposome / mRNA vaccine based on model antigen OVA

[0211] (1) Preparation of liposome / OVA mRNA complex

[0212] 20 μg of DOTAP liposomes before and after DP7-C modification and 10 μg of OVA mRNA were gently mixed in a total volume of 100 μL of aqueous solution and incubated at room temperature for 5-15 min.

[0213] (2) Antitumor effect of the present invention in EG7-OVA subcutaneous tumor model

[0214] 1×10 6 EG7-OVA cells (purchased from a cell bank and stored in our laboratory) were resuspended in 100 μl DMEM double-free medium and injected subcutaneously into mice. The liposome / mRNA complex prepared above was injected into the subcutaneous lymph nodes on days 4, 11, and 18, with a dose of 20 μg liposome and 10 μg mRNA per mouse. The changes in mouse tumor volume were recorded every 2 days. Mice were killed on day 21 after inoculation.

[0215] The experimental results showed that the average tumor volume of the PBS group was 2324.17±421.22mm 3 The average tumor volume of the unmodified DOTAP liposome / OVA mRNA complex immunization group was 1247.34±178.21mm 3 The average tumor volume of the DP7-C modified DOTAP liposome / OVA mRNA complex immunization group was 157.34±123.21mm 3The average tumor volume of the DP7-C modified DOTAP liposome / OVA mRNA complex immunization group was significantly different from that of the PBS group (p<0.001), and was also significantly different from that of the unmodified DOTAP liposome / OVA mRNA complex immunization group (p<0.01) ( Fig. 9 a). This indicates that the DP7-C modified DOTAP liposome / OVA mRNA complex group exerted a more effective anti-tumor effect than the unmodified DOTAP liposome / OVA mRNA complex group.

[0216] (3) ELISPOT detection of spleen CD8 + T cell activation

[0217] To further clarify the immune effect of the liposome / OVA mRNA complex vaccine and its ability to stimulate antigen-specific lymphocyte responses, the ELISPOT assay was used to detect the activation of antigen-specific splenic lymphocytes against OVA after vaccination.

[0218] When mice were killed on the 21st day after inoculation, spleens were taken and lymphocytes were separated using lymphocyte separation fluid. 5 Each spleen lymphocyte was plated in a 24-well plate with 100 μl RPMI 1640+10% FBS+1% PS medium, treated with 10 μg / ml OVA for 72 h, and the IFN-γ secretion was detected according to the standard method in the ELISPOT kit instructions.

[0219] The results showed that the antigen-specific lymphocyte response of the unmodified liposome group and the modified liposome group after OVA treatment was stronger than that of the PBS group. In addition, the antigen-specific lymphocyte response induced by the DP7-C modified liposome group was significantly stronger than that of the unmodified liposome group ( Fig. 9 b).

[0220] 2. Functional verification of DP7-C modified liposome / mRNA vaccine based on neoantigen

[0221] (1) Design of mutant neoantigens and acquisition of mRNA

[0222] The five new antigen sequences used were previously screened from the LL2 cell line and have been proven to have immunogenicity and anti-tumor effects. With the mutation site of each mutant new antigen as the center, 13 amino acids are extended before and after, that is, each new antigen short peptide sequence includes 27 amino acid sequences, and connected with lingker and MITD sequences (MHC class I trafficking signal), and a new antigen with five new antigen short peptides is designed.

[0223] The amino acid sequence of the wild-type antigen finally designed is as follows (SEQ ID No.3):

[0224] MRVTAPRTLLLLLSGALALTETWAGSGGSGGGGSGGHSGAEQYFKEWFSRPANLHGIILPRLSGGSGGGGSGGLSPRHYYSGYSSSPEYSSESTHKIWERGGSGGGGSGGPPTVDSVCLKWAPPKHKQVKLSKKGGS GGGGSGGTKTELELALSPIHDSSAIPAAGSNQVTGGSGGGGSGGFSHSSNLTLHYRTHLVDRPYDCKCGKAGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA.

[0225] The amino acid sequence of the mutant antigen finally designed is as follows (SEQ ID No.4):

[0226] MRVTAPRTLLLLLSGALALTETWAGSGGSGGGGSGGHSGAEQYFKEWFSIPANLHGIILPRLSGGSGGGGSGGLSPRHYYSGYSSSLEYSSESTHKIWERGGSGGGGSGGPPTVDSVCLKWAPLKHKQVKLSKKGGS GGGGSGGTKTELELALSPIHYSSAIPAAGSNQVTGGSGGGGSGGFSHSSNLTLHYRTLLVDRPYDCKCGKAGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA.

[0227] The structure of the designed mRNA sequence is SP signal peptide coding sequence (MHC class I signal peptide fragment, the main function of which is to guide the polypeptide to pass through the endoplasmic reticulum membrane into the lumen), start linker coding sequence, neoantigen 1 coding sequence, middle linker coding sequence, neoantigen 2 coding sequence, middle linker coding sequence, neoantigen 3 coding sequence, middle linker coding sequence, neoantigen 4 coding sequence, middle linker coding sequence, neoantigen 5 coding sequence, end linker coding sequence, MITD coding sequence (MHC class I trafficking signal, which improves the stability and translation efficiency of mRNA) and polyA tail.

[0228] The designed DNA sequence encoding the mutant antigen is (SEQ ID No.5):

[0229]

[0230] The PCR template was transcribed using a transcription kit according to the instructions, and the size and purity of the mRNA were detected by agarose gel electrophoresis. It was found that the size of the transcribed mRNA was 1287 bp, which was in line with expectations. This indicates that the designed mRNA encoding the neoantigen sequence was successfully transcribed in vitro. The purified neoantigen mRNA was stored at -80°C for later use.

[0231] (2) Preparation of liposome / mRNA complex

[0232] 20 μg of DOTAP liposomes before and after modification and 10 μg of mRNA were gently mixed in a total volume of 100 μL of aqueous solution and incubated at room temperature for 5-15 min.

[0233] (3) Antitumor effect of the present invention in an orthotopic tumor model

[0234] 3×10 5 After resuspending each mouse LL2 cell in 100 μl DMEM double-free medium, the cells were injected into the mouse through the tail vein to establish an orthotopic lung cancer model. The liposome / mRNA complex prepared above was injected into the subcutaneous lymph nodes on days 4, 11, and 18, with a dose of 20 μg liposome and 10 μg mRNA per mouse. The weight changes of mice were recorded every 2 days. The mice were killed on the 21st day after inoculation, the main tissues and organs were fixed in 4% paraformaldehyde, and the lung tissues were photographed.

[0235] The LL2 orthotopic tumor model was established to evaluate whether the liposome / neoantigen mRNA complexes before and after modification can exert anti-tumor effects. The results showed that the DP7-C modified DOTAP liposome / neoantigen mRNA complex immunization group had a better effect on inhibiting tumor growth than the DOTAP liposome / neoantigen mRNA complex immunization group. From the perspective of lung tumor growth, the average number of tumor nodules in the DP7-C modified DOTAP liposome / neoantigen mRNA complex immunization group was 3±1, while the average number of tumor nodules in the unmodified DOTAP liposome / mRNA complex immunization group was 10±3, and there was a statistical difference between the two (p<0.05)( Fig.10 a). In terms of lung weight, the average lung weight of the DP7-C modified DOTAP liposome / neoantigen mRNA complex immunization group was 0.22±0.03g, which was significantly lower than the average lung weight of the unmodified DOTAP liposome / mRNA complex subcutaneous immunization group (0.37±0.08g), and there was a statistical difference between the two (p<0.05). These results indicate that the anti-tumor effect of the DP7-C modified DOTAP liposome / neoantigen mRNA complex is better than that of the unmodified DOTAP liposome / neoantigen mRNA ( Fig.10 b).

[0236] (4) Flow cytometry detection of spleen CD8 + T cell activation

[0237] To further elucidate the immune effect of liposome / neoantigen mRNA complexes and their ability to stimulate antigen-specific lymphocyte responses, flow cytometry was used to detect the activation of splenic lymphocytes after mixed peptide treatment and mRNA treatment.

[0238] When mice were killed on the 21st day after inoculation, spleens were taken and lymphocytes were separated using lymphocyte separation fluid. 6 Each spleen lymphocyte was plated in a 6-well plate with 1 ml RPMI 1640 + 10% FBS + 1% PS medium, and treated with 10 μg / ml flag polypeptide, wild-type peptide (2 μg / ml of each wild-type peptide corresponding to the five antigens), mutant peptide (2 μg / ml of each mutant peptide corresponding to the five antigens) and mRNA for 3 h, and then 2 μl of Golgi blocker was added. After culturing for 11 h, the cells were collected for CD3, CD8 and IFN-γ flow staining. Flow cytometry detection of CD8 + T cell activation.

[0239] The results showed that after mutant peptide treatment, CD8 + The proportion of T cells was 17.79±0.63%, which was statistically different from 12.26±0.74% in the unmodified DOTAP liposome group (p<0.01). + The proportion of T cells was significantly increased compared with the control group, but no significant difference was found between the DOTAP liposome groups before and after modification. After mixed peptide treatment, the proportion of cells secreting IFN-γ in the DP7-C modified DOTAP liposome / neoantigen mRNA complex immunization group was 2.66±0.32%, which was higher than the proportion of cells secreting IFN-γ in the unmodified DOTAP liposome / neoantigen mRNA complex immunization group after mixed peptide treatment (0.81±0.14%), with statistical difference (p<0.01); after mRNA treatment, the proportion of cells secreting IFN-γ in the DOTAP liposome / neoantigen mRNA complex immunization group before and after modification was significantly increased, and the proportion of cells secreting IFN-γ in the DP7-C modified DOTAP liposome / neoantigen mRNA complex immunization group was 4.13±0.72%, which was significantly higher than the unmodified liposome / neoantigen mRNA complex immunization group (0.92±0.14%), with statistical difference (p<0.01)( Fig.10c-10d). This indicates that the DP7-C modified DOTAP liposome / neoantigen mRNA complex can more effectively activate antigen-specific lymphocyte response after subcutaneous injection compared with the unmodified liposome / neoantigen mRNA complex immunization group.

[0240] Example 6. Functional verification of DCs vaccine loaded with DP7-C modified liposome / mRNA complex

[0241] 1. Functional validation of the mRNA-loaded DC vaccine based on the model antigen OVA

[0242] (1) Preparation of DCs vaccine loaded with liposome / mRNA complex

[0243] ①Take the tibia and fibula of adult C57BL / 6J female mice about 6 weeks old, soak them in 75% ethanol for 5 minutes to kill bacteria, then remove the muscle tissue and soak the leg bones in RPMI 1640+1% PS culture medium; cut off both ends of the leg bones with sterilized scissors, and then use a syringe to absorb fresh RPMI 1640+1% PS culture medium to blow out the bone marrow cells until all the bone marrow cells are blown out;

[0244] ② Filter the collected medium containing bone marrow cells with a 70μm mesh, centrifuge at 1200rpm for 3min, discard the supernatant, and resuspend the cells with red blood cell lysis buffer (weigh 1.3g Tris-base and 3.74g NH4Cl, dissolve with 490ml ultrapure water, adjust the pH value of the solution to 7.2-7.4 with concentrated hydrochloric acid, add ultrapure water to 500ml, remove bacteria with a 0.22μm filter, store at 4℃, prepare and use immediately), let stand at room temperature for 3min, centrifuge at 1200rpm for 3min, and finally use RPMI 1640+10% FBS+1% PS medium to wash away the red blood cell lysis buffer and resuspend the cells;

[0245] ③ Divide the resuspended cells into culture dishes, 2×10 6 -3×10 610 ml of RPMI1640 + 10% FBS + 1% PS culture medium was added to each dish, and 20 ng / ml GM-CSF cytokine was added. The culture dish was placed in a 37°C cell culture incubator for culture. On the third day of culture, fresh RPMI 1640 + 10% FBS + 1% PS culture medium containing 20 ng / ml GM-CSF was added until the 8th day of culture to obtain immature DCs (imDCs). Take the DCs cultured on the 8th day, wash off the culture medium with 1 ml PBS, resuspend the cells with 100 μl PBS, add 1 μl APC Hamster Anti-Mouse CD11c flow cytometry antibody, mix gently, and incubate at 4°C in the dark for 40 minutes; after the incubation is completed, wash off the excess antibody with PBS, resuspend the cells with 200 μl PBS, and use a flow cytometer to detect CD11c + When the proportion of DCs is higher than 80%, subsequent treatment can be performed.

[0246] ④ Add liposome (2 μg / ml) / mRNA (1 μg / ml) complex to imDCs induced on day 8 and continue treatment for 24 h. 6 For each cell, use 1μl APC Hamster Anti-Mouse CD11c, 1μl Percp-Cy5.5Hamster Anti-Mouse CD80 and 1μl FITC Rat Anti-Mouse CD86 antibodies, incubate at 4℃ in the dark for 40min. After incubation, wash off the excess antibodies with PBS, resuspend the cells with 200μl PBS, and use flow cytometry to detect the proportion of cells positive for each antibody. When the proportion of mature DCs is greater than 90%, the DCs vaccine is successfully prepared. It can be used for subsequent experiments.

[0247] (2) Antitumor effect of the present invention in EG7-OVA subcutaneous tumor model

[0248] 1×10 6 EG7-OVA cells were resuspended in 100 μl DMEM medium and injected subcutaneously into mice. DCs vaccine loaded with liposome / mRNA complex was injected into subcutaneous lymph nodes on days 4, 11, and 18. The dose per mouse was 2×10 6 DCs were added. The changes in tumor volume of mice were recorded every 2 days. Mice were killed on the 23rd day after inoculation.

[0249] The experimental results showed that the average tumor volume of the PBS group was 3343.17±221.22mm 3The average tumor volume of the DCs immunization group loaded with unmodified DOTAP liposomes / OVA mRNA complexes was 2642.34±173.11mm 3 The average tumor volume of the DCs immunization group loaded with DP7-C modified DOTAP liposomes / OVA mRNA complex was 1356.34±113.12mm 3 The average tumor volume of the DCs immunized group loaded with DP7-C modified DOTAP liposomes / OVA mRNA complexes was significantly different from that of the PBS group (p<0.001), and was also significantly different from that of the DCs immunized group loaded with unmodified DOTAP liposomes / OVA mRNA complexes (p<0.01) ( Fig.11 a). This indicates that the DCs group loaded with DP7-C modified DOTAP liposome / OVA mRNA complex has a more effective anti-tumor effect than the DCs group loaded with unmodified DOTAP liposome / OVA mRNA complex.

[0250] (3) ELISPOT detection of spleen CD8 + T cell activation

[0251] To further clarify the immune effect of DCs vaccine loaded with liposome / OVA mRNA complex and its ability to stimulate antigen-specific lymphocyte response, the ELISPOT assay was used to detect the activation of antigen-specific splenic lymphocytes against OVA after vaccine immunization.

[0252] When mice were killed on the 23rd day after inoculation, spleens were taken and lymphocytes were separated using lymphocyte separation fluid. 5 Each spleen lymphocyte was plated in a 24-well plate with 100 μl RPMI 1640+10% FBS+1% PS medium, treated with 10 μg / ml OVA for 72 h, and the IFN-γ secretion was detected according to the standard method in the ELISPOT kit instructions.

[0253] The results showed that the antigen-specific lymphocyte responses of the DCs loaded with unmodified liposomes / OVA mRNA and the DCs loaded with modified liposomes / OVA mRNA after OVA treatment were stronger than those of the PBS group. In addition, the antigen-specific lymphocyte responses induced by the DCs loaded with DP7-C modified liposomes / OVA mRNA were significantly stronger than those of the DCs loaded with unmodified liposomes / OVA mRNA vaccine ( Fig.11 b).

[0254] 2. Functional validation of mRNA-loaded DC vaccines based on neoantigens

[0255] (1) Antitumor effect of the present invention in LL2 subcutaneous tumor model

[0256] The LL2 subcutaneous tumor model was established to evaluate whether DCs vaccine loaded with liposome / neoantigen mRNA complexes can exert anti-tumor effects.

[0257] 5×10 5 After resuspending 100 μl of DMEM double-free medium, LL2 cells were injected subcutaneously into mice to establish a subcutaneous tumor model. On days 4, 11, and 18, 2×10 6 DCs cells loaded with liposome / mRNA complexes. The mice were killed on the 21st day after vaccination, and the anti-tumor effect of the vaccine was evaluated by drawing 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 complexes, and DCs vaccine immunization group loaded with DP7-C modified DOTAP liposome / neoantigen mRNA complexes, with 6 mice in each group.

[0258] The experimental results showed that the average tumor volume of the PBS group was 1324.17±221.22mm 3 The average tumor volume of the DCs immunization group loaded with unmodified DOTAP liposomes / neoantigen mRNA complexes was 677.34±178.21mm 3 The average tumor volume of the DCs immunization group loaded with DP7-C modified DOTAP liposomes / neoantigen mRNA complexes was 356.34±47.21mm 3 The average tumor volume of the DCs immunization group loaded with DP7-C modified DOTAP liposomes / neoantigen mRNA complexes was significantly different from that of the PBS group (p<0.001), and was also significantly different from that of the DCs immunization group loaded with unmodified DOTAP liposomes / neoantigen mRNA complexes (p<0.01) ( Fig.12 a). In terms of in vitro tumor weight, the average tumor weight of the PBS group was 1.22±0.42g, the average tumor weight of the DCs immunization group loaded with unmodified DOTAP liposomes / neoantigen mRNA complexes was 0.93±0.16g, and the average tumor weight of the DCs immunization group loaded with DP7-C-modified DOTAP liposomes / neoantigen mRNA complexes was 0.58±0.07g. Among them, the average tumor weight of the DCs immunization group loaded with DP7-C-modified DOTAP liposomes / neoantigen mRNA complexes was significantly lower than that of the PBS group (p<0.001) and the DCs vaccine group loaded with unmodified DOTAP liposomes / mRNA (p<0.01) ( Fig.12b).

[0259] (2) Flow cytometry detection of spleen CD8 + T cell activation

[0260] To further clarify the immune effect of DCs vaccine loaded with liposome / neoantigen mRNA complex and its ability to stimulate antigen-specific lymphocyte response, flow cytometry was used to detect the activation of splenic lymphocytes after mixed peptide treatment and mRNA treatment.

[0261] When mice were killed on the 21st day after inoculation, spleens were taken and lymphocytes were separated using lymphocyte separation fluid. 6 Each spleen lymphocyte was plated in a 6-well plate with 1 ml RPMI 1640 + 10% FBS + 1% PS medium, and treated with 10 μg / ml flag polypeptide, wild-type peptide (2 μg / ml of each wild-type peptide corresponding to the five antigens), mixed mutant peptide (2 μg / ml of each mutant peptide corresponding to the five antigens) and mRNA for 3 h, and then 2 μl of Golgi blocker was added. After culturing for 11 h, the cells were collected for CD3, CD8 and IFN-γ flow staining. Flow cytometry detection of CD8 + The proportion of T cells.

[0262] The results showed that the proportion of cells secreting IFN-γ in the DCs immunization group loaded with DP7-C modified DOTAP liposome / neoantigen mRNA complex after mixed mutant peptide treatment was 3.79±0.62%, which was statistically different from 1.46±0.34% in the unmodified DOTAP liposome group (p<0.05). The proportion of cells secreting IFN-γ in the DCs immunization group loaded with DOTAP liposome / mRNA before and after modification after mRNA treatment was significantly increased compared with the control group. After mRNA treatment, the proportion of cells secreting IFN-γ in the DCs immunization group loaded with DOTAP liposome / neoantigen mRNA complex before and after modification was significantly increased, and the proportion of cells secreting IFN-γ in the DCs immunization group loaded with DP7-C modified DOTAP liposome / neoantigen mRNA complex was 5.15±0.74%, which was significantly higher than the unmodified liposome / neoantigen mRNA complex immunization group (1.45±0.25%), with statistical differences (p<0.01)( Fig.12 c). This indicates that DCs loaded with DP7-C modified DOTAP liposome / neoantigen mRNA complexes can more effectively activate antigen-specific lymphocyte responses after subcutaneous injection compared with DCs immunization group loaded with unmodified liposome / neoantigen mRNA complexes.

[0263] In previous studies, we found that the cholesterol-modified cationic polypeptide DP7-C has the dual functions of a carrier and an adjuvant, and can be used to improve the anti-tumor effect of DCs vaccines loaded with new antigen peptides. In the embodiment of the present invention, DP7-C, which has the dual functions of a carrier and an immune adjuvant, is modified onto DOTAP liposomes that can deliver mRNA into cells, successfully improving the efficiency of liposomes in delivering mRNA into cells and the immune response induced by liposomes.

[0264] In the above examples of the present invention, we prepared DP7-C modified DOTAP liposomes and DP7-C modified DC-chol liposomes, respectively, and proved that DP7-C can be stably modified to the above liposomes. It was verified that the DP7-C modified DOTAP liposomes improved the efficiency of liposomes transfecting mRNA into DCs. And the DP7-C modified DOTAP liposomes have the characteristics of promoting DCs, maturation and pro-inflammatory cytokine secretion in vivo and in vitro, which are better than unmodified liposomes. Finally, by establishing tumor models, the anti-tumor effects and the ability to stimulate spleen lymphocyte responses of DOTAP liposome / OVA mRNA composite vaccines before and after modification of DP7-C, liposomes / mRNA composite vaccines encoding personalized new antigens before and after modification of DP7-C, and DCs vaccines loaded with liposomes / mRNA composites were verified respectively. It was found that the modified liposome / mRNA vaccine and the DCs vaccine loaded with modified liposomes / mRNA composites have more advantages in anti-tumor effects and activation of spleen antigen-specific lymphocyte responses. In summary, the present invention modifies DP7-C onto DOTAP liposomes, which can enhance the mRNA uptake efficiency of DCs, promote DCs maturation and pro-inflammatory cytokine secretion, induce antigen-specific cytotoxic T lymphocyte response, thereby enhancing the anti-tumor effect of mRNA vaccines, and has more advantages in anti-tumor effect and inducing antigen-specific lymphocyte response.

[0265] Therefore, the present invention provides a new and effective option for the art to further improve the delivery efficiency of mRNA in cells, enhance the effects of liposome / mRNA complex vaccines and DCs vaccines loaded with liposome / mRNA complexes. Sequence Listing <110> Sichuan University <120> Peptide-modified liposomes, mRNA delivery systems and dendritic cell vaccines <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <400> 1 Val Gln Trp Arg Ile Arg Val Ala Val Ile Arg Lys 1 5 10 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <400> 2 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 3 <211> 273 <212> PRT <213> Artificial Sequence <400> 3 Met Arg Val Thr Ala Pro Arg Thr Leu Leu Leu Leu Leu Ser Gly Ala 1 5 10 15 Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser Gly Gly Ser Gly Gly Gly 20 25 30 Gly Ser Gly Gly His Ser Gly Ala Glu Gln Tyr Phe Lys Glu Trp Phe 35 40 45 Ser Arg Pro Ala Asn Leu His Gly Ile Ile Leu Pro Arg Leu Ser Gly 50 55 60 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 85 90 95 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 115 120 125 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 145 150 155 160 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 His 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> 4 <211> 273 <212> PRT <213> Artificial Sequence <400> 4 Met Arg Val Thr Ala Pro Arg Thr Leu Leu Leu Leu Leu Ser Gly Ala 1 5 10 15 Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser Gly Gly Ser Gly Gly Gly 20 25 30 Gly Ser Gly Gly His Ser Gly Ala Glu Gln Tyr Phe Lys Glu Trp Phe 35 40 45 Ser Ile Pro Ala Asn Leu His Gly Ile Ile Leu Pro Arg Leu Ser Gly 50 55 60 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 Leu Glu Tyr Ser Ser Glu Ser Thr His Lys 85 90 95 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 Leu Lys His Lys Gln 115 120 125 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 Tyr Ser Ser 145 150 155 160 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 <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 aaaaaaaaaaaaa aaaaaaaaaaaaa aaaaaaaaa 1287

Claims

1. A liposome modified with a hydrophobic modified cationic polypeptide, wherein the sequence of the cationic polypeptide is VQWRIRVAVIRK, and the structure of the hydrophobic modified polypeptide is: ； R in the polypeptide structure is: The liposome is composed of cationic lipid and auxiliary lipid; the cationic lipid is trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), and the auxiliary lipid is cholesterol.

2. The liposome modified by the hydrophobic modified cationic polypeptide according to claim 1, characterized in that The mass ratio between the cationic lipid and the auxiliary lipid is: cationic lipid: auxiliary lipid = 0.5-20:

1.

3. The liposome modified with the hydrophobic modified cationic polypeptide according to any one of claims 1 to 2, characterized in that The mass ratio of the hydrophobic modified cationic polypeptide to the liposome composed of the cationic lipid and the auxiliary lipid is: hydrophobic modified cationic polypeptide: liposome = 0.025-0.5:

1.

4. The liposome modified by the hydrophobic modified cationic polypeptide according to claim 3, characterized in that The mass ratio of the hydrophobic modified cationic polypeptide to the liposome composed of the cationic lipid and the auxiliary lipid is: hydrophobic modified cationic polypeptide: liposome = 0.025-0.2:

1.

5. The liposome modified with the hydrophobic modified cationic polypeptide according to claim 1, characterized in that: The preparation method is 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.

6. The liposome modified with the hydrophobic modified cationic polypeptide according to any one of claims 1 to 5, characterized in that Made by: a. Weigh the cationic lipid and the auxiliary lipid, dissolve them in a solvent, and remove the organic solvent by rotary evaporation under reduced pressure; the solvent may be at least one of chloroform, ethanol, methanol, dichloromethane or ethyl acetate; b. adding an aqueous solution of the hydrophobically modified cationic polypeptide or a solution formed by dissolving the hydrophobically modified cationic polypeptide in the solvent of step a into the system for hydration and demembranation to obtain a liposome suspension; c. ultrasonically treating the obtained liposome suspension to obtain liposomes; Alternatively, made by: a. Weigh the cationic lipid and the auxiliary lipid, dissolve them in a solvent to prepare an organic phase, and dissolve the organic phase in a reaction container; the solvent is ethanol or ether; b. Inject the DP7-C solution into the organic phase and stir; evaporate under vacuum to 1 / 5 to 1 / 2 of the original volume; and homogenize and extrude to obtain a blank liposome solution; the DP7-C solution is an aqueous solution, an ethanol solution or an ether solution; c. The blank liposome solution was treated with ultrasound to obtain DP7-C modified liposomes.

7. The liposome modified with the hydrophobic modified cationic polypeptide according to claim 6, characterized in that The following raw materials are used to prepare the product: the mass ratio of cationic lipid: auxiliary lipid: hydrophobic modified cationic polypeptide = 10-40:10-40:0.5-10.

8. The liposome modified with the hydrophobic modified cationic polypeptide according to claim 7, characterized in that The following raw materials are used to prepare the product: the mass ratio of cationic lipid: auxiliary lipid: hydrophobic modified cationic polypeptide = 10-40:10-40:1-6.

9. The liposome modified with the hydrophobic modified cationic polypeptide according to claim 8, characterized in that The liposomes are prepared using raw materials in the following ratio: DOTAP: cholesterol: hydrophobic modified cationic polypeptide = 20:20:3 by mass ratio. The hydrophobic modified polypeptide structure is:

10. Use of the liposome modified with the hydrophobic modified cationic polypeptide according to any one of claims 1 to 9 in preparing a nucleic acid delivery system.

11. A nucleic acid delivery system, characterized in that The liposomes are modified with the hydrophobic modified polypeptide according to any one of claims 1 to 9 and loaded with nucleic acid.

12. The nucleic acid delivery system according to claim 11, characterized in that The nucleic acid is at least one of DNA or RNA.

13. The nucleic acid delivery system according to claim 12, characterized in that The RNA is at least one of messenger RNA, siRNA, sgRNA or mRNA.

14. The nucleic acid delivery system according to any one of claims 10 to 13, characterized in that The liposomes modified by the hydrophobic modified cationic polypeptide and the nucleic acid are prepared as raw materials in a mass ratio of 1 to 10:

1.

15. The nucleic acid delivery system according to claim 14, characterized in that: The liposomes modified by the hydrophobic modified cationic polypeptide and the nucleic acid are prepared as raw materials in a mass ratio of 1 to 3:

1.

16. The nucleic acid delivery system according to any one of claims 10 to 15, characterized in that: The liposome modified with a hydrophobic modified cationic polypeptide is prepared by co-incubating with nucleic acid.

17. The nucleic acid delivery system according to claim 16, characterized in that: The method is obtained by co-incubating liposomes modified by hydrophobic modified cationic polypeptides and nucleic acids in a culture medium for 4 to 15 minutes. The culture medium is at least one of RPMI 1640, DMEM double-free culture medium or Optim culture medium.

18. The mRNA delivery system according to any one of claims 10 to 17, characterized in that: The length of the nucleic acid is 20 to 7000 bp.

19. The mRNA delivery system according to any one of claims 10 to 18, characterized in that: The mRNA is an mRNA with a 5' cap structure and a 3' polyA tail structure.

20. Use of the mRNA delivery system according to any one of claims 10 to 19 in the preparation of dendritic cell vaccines.

21. A dendritic cell vaccine, characterized in that: The main active ingredient is dendritic cells loaded with the mRNA delivery system according to any one of claims 10 to 19.

22. The dendritic cell vaccine according to claim 21, characterized in that: The dendritic cells are myeloid DC cells or lymphoid DC cells.

23. The dendritic cell vaccine according to claim 21, characterized in that: The dendritic cells are in vitro dendritic cells obtained from the patient himself.

24. The dendritic cell vaccine according to any one of claims 21 to 23, characterized in that Prepared using the following method: a. Take immature dendritic cells (imDCs) after induction culture; b. Add the nucleic acid delivery system to the culture medium for incubation c. After the incubation is completed, the cells are digested with trypsin, washed, resuspended by centrifugation, and tested to obtain mature dendritic cells.

25. The dendritic cell vaccine according to any one of claims 21 to 24, characterized in that: Pharmaceutically acceptable auxiliary ingredients are also included.

26. The dendritic cell vaccine according to claim 25, characterized in that: The pharmaceutically acceptable auxiliary component is at least one of a protective agent, an excipient, an immune adjuvant, a dispersant or a cell culture medium.

Images