Lipidated melittin and application thereof as vaccine vector

Abstract

The invention relates to the technical field of novel biomedical materials, in particular to lipidated melittin and application of the lipidated melittin as a vaccine vector, and the lipidated melittin is obtained by sequentially modifying the N end of melittin with lysine, phenylalanine and C8-C18 alkyl carboxylic acid. The lipidated melittin has good mRNA wrapping, delivery and immune response enhancing capabilities, is simple to prepare and can be used as a carrier of a nano vaccine for preparing the nano vaccine, the prepared nano vaccine has a good immune cell activation effect after being injected in vivo, and compared with a control group, the lipidated melittin can effectively prevent and inhibit tumor growth and has a good application prospect. And a valuable vector tool is provided for the development of mRNA tumor vaccines.

Description

Technical Field

[0001] This invention relates to the field of new biomedical materials technology, and in particular to a lipid-modified bee venom peptide and its application as a vaccine carrier. Background Technology

[0002] mRNA vaccine delivery vectors are technologies used to safely and efficiently deliver messenger ribonucleic acid (mRNA) into target cells. These vectors play a crucial role in vaccine development. Currently, the main mRNA delivery methods can be categorized into lipid nanoparticles, polymer nanoparticles, viral vectors, and electroporation. Lipid nanoparticles (LNPs) offer numerous advantages as mRNA delivery vectors, such as preventing mRNA degradation, promoting cellular uptake, and ensuring safety, making them one of the most commonly used mRNA vaccine delivery systems.

[0003] For mRNA tumor vaccines, it is necessary to appropriately improve the immunogenicity of the delivery vector. While traditional lipid nanoparticle mRNA delivery vectors possess highly efficient mRNA delivery capabilities, the effectiveness of the immune response still needs improvement. Optimizing the lipid composition ratio in the lipid nanoparticle formulation and introducing immune agonists can enhance the immunogenicity of mRNA tumor vaccine vectors, but this also compromises the encapsulation and delivery efficiency of mRNA to varying degrees. Therefore, developing a vector with highly efficient mRNA delivery and the ability to appropriately improve immunogenicity to enhance the overall efficacy of mRNA tumor vaccines is of great significance. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a lipidated bee venom peptide and its application as a vaccine carrier, which can enhance the carrier's ability to activate immune responses.

[0005] To achieve the above objectives, the present invention provides a lipid-modified bee venom peptide, which is obtained by sequentially modifying the N-terminus of the bee venom peptide with lysine, phenylalanine, and C8-C18 alkyl carboxylic acids.

[0006] The modification involves lysine, phenylalanine, or C8-C18 alkylcarboxylic acids forming amide bonds through the reaction of carboxyl and amino groups to achieve linkage with bee venom peptides.

[0007] The structural formula of the lipid-modified bee venom peptide is shown below:

[0008] R-FKGIGAVLKVLTGLPALISWIKRKRQQ-NH2;

[0009] Wherein, R is preferably an alkyl carboxylic acid group of C8 to C18. In some specific embodiments, R is an alkyl carboxylic acid group of C8, C9, C10, C11, C12, C13, C14, C15, C16, C17 or C18, more preferably an alkyl carboxylic acid group of C8, C10, C12, C14, C16 or C18, and most preferably an alkyl carboxylic acid group of C14, i.e., myristic acid.

[0010] In this invention, R is preferably a C8-C18 alkyl carboxylic acid group, specifically referring to a C8-C18 alkyl carboxylic acid residue, that is, the remaining group after the carboxyl group of the alkyl carboxylic acid loses an OH group.

[0011] This invention modifies bee venom peptides with lysine, phenylalanine, and C8-C18 alkyl carboxylic acids, which can neutralize the positive charge they carry. The C8-C18 alkyl chains help regulate the hydrophilic and hydrophobic properties of bee venom peptides, making the lipidated bee venom peptides easier to insert into the surface of lipid nanoparticles and reducing the toxicity of bee venom peptides.

[0012] The present invention does not impose any special limitations on the preparation process of the above-mentioned lipidated bee venom peptides. They can be synthesized by solid-phase peptide synthesis method according to the amino acid sequence.

[0013] In some specific embodiments, the preparation process of the lipidated bee venom peptide is as follows:

[0014] R-FK-Melittin was obtained on 2-chloro resin by solid phase peptide synthesis (SPPS).

[0015] For the deprotection of Fmoc and the deprotection of peptide side chains, deprotection methods well known to those skilled in the art can be used.

[0016] In some specific implementations, Fmoc deprotection is carried out in a 20% piperidine DMF solution. Specifically, a 20% piperidine DMF solution can be bubbled for 30 minutes.

[0017] In some specific embodiments, the deprotection of the peptide side chains is carried out in an aqueous solution of 95% TFA. Specifically, an aqueous solution of 95% TFA can be stirred for 0.5 hours.

[0018] After the reaction is complete, R-FK-Melittin can be obtained by precipitation with diethyl ether.

[0019] This invention provides the application of the above-mentioned lipidated bee venom peptide in the preparation of vaccine delivery vectors.

[0020] The present invention also provides a vaccine carrier composition comprising the above-mentioned lipidated bee venom peptide and auxiliary lipids.

[0021] The present invention does not impose any particular limitation on the auxiliary lipids; any auxiliary lipids known to those skilled in the art for the preparation of liposome nanoparticles may be used. In some specific embodiments, the auxiliary lipids include:

[0022] Phospholipids, cholesterol, polyethylene glycol-modified lipids, and ionizable lipids.

[0023] In some specific embodiments, the phospholipid is distearate phosphatidylcholine.

[0024] In some specific embodiments, the PEGylated lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000).

[0025] In some specific embodiments, the ionizable lipid is heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecapoxy)hexyl)amino)octanoate (SM-102).

[0026] In some specific embodiments, the cholesterol is high-purity cholesterol (CHO-HP).

[0027] Based on this, the present invention also provides a vaccine carrier, the raw materials for which include the above-mentioned lipidated bee venom peptide and auxiliary lipids.

[0028] The selection of the assisting lipids is the same as described above and will not be repeated here.

[0029] The vaccine carrier is prepared by self-assembly of the above-mentioned lipidated bee venom peptide and auxiliary lipid in an organic solvent in water or a buffer solution.

[0030] The present invention does not impose any special limitation on the organic solvent, and can be any suitable organic solvent known to those skilled in the art. In some specific embodiments, the organic solvent is ethanol.

[0031] In some specific embodiments, the buffer solution is a sodium citrate buffer solution.

[0032] The pH value of the self-assembled material is preferably 1 to 5. In some specific embodiments, the pH value is 1, 2, 3, 4 or 5, or any of the above values ​​may be the upper or lower limit.

[0033] The present invention does not limit the above-mentioned self-assembly method, and can be a method known to those skilled in the art. In some specific embodiments, the self-assembly adopts a vortex mixing method to bring the organic phase and the aqueous phase into contact and perform self-assembly.

[0034] After self-assembly, nanoparticles are preferably obtained by dialysis.

[0035] The dialysis solvent can be a generally applicable buffer solution, including but not limited to phosphate buffer solution. The pH value of the buffer solution is preferably 7 to 8. In some specific embodiments, the pH value is 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, or any of the above values ​​as the upper or lower limit.

[0036] The molecular weight cutoff for dialysis is preferably 1000~3000 Da, more preferably 2000 Da.

[0037] The present invention also provides a nano-vaccine pharmaceutical composition comprising an antigen component and the above-described lipidated bee venom peptide or the above-described vaccine carrier composition.

[0038] The preferred mass ratio of the antigen to the lipidized bee venom peptide or the vaccine carrier composition is 1:(5~20). In some specific embodiments, the mass ratio is 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20, or any of the above values ​​as the upper or lower limit.

[0039] The present invention does not impose any special limitation on the above-mentioned antigen. In some specific embodiments, the antigen is mRNA.

[0040] The present invention also provides a vaccine formulation comprising the above-described nanovaccine drug composition.

[0041] The vaccine formulation is lipid nanoparticles formed by the self-assembly of the above-mentioned nanovaccine drug composition in water or a buffer solution.

[0042] In some specific embodiments of the present invention, the vaccine formulation is prepared according to the following method:

[0043] An organic phase comprising the above-mentioned lipotropic meliotide, phospholipids, cholesterol, polyethylene glycol-modified lipids, and ionizable lipids is contacted with an aqueous solution or buffer solution containing the antigen to undergo self-assembly, thereby obtaining antigen-loaded liposome nanoparticles.

[0044] The present invention does not impose any special limitation on the organic solvent of the organic phase, and can be any suitable organic solvent known to those skilled in the art. In some specific embodiments, the organic solvent is ethanol.

[0045] The present invention does not impose any special limitation on the buffer solution, and it can be any suitable buffer solution known to those skilled in the art. In some specific embodiments, the buffer solution is a sodium citrate buffer solution.

[0046] In some specific implementations, the antigen is mRNA.

[0047] The concentration of the mRNA in the buffer solution is preferably 1~100mM, more preferably 5~50mM, and even more preferably 5~20mM. In some specific embodiments, the concentration of the mRNA in the buffer solution is 5, 10, 15 or 20mM, or any of the above values ​​are the upper or lower limits.

[0048] The pH value of the self-assembled material is preferably 1 to 5. In some specific embodiments, the pH value is 1, 2, 3, 4 or 5, or any of the above values ​​may be the upper or lower limit.

[0049] The volume ratio of the organic phase to the aqueous solution or buffer solution containing the antigen is preferably 1:(1~10), more preferably 1:(1~5), and even more preferably 1:(2~4). In some specific embodiments, the volume ratio is 1:2, 1:3, or 1:4, or any of the above values ​​may be the upper or lower limit.

[0050] The present invention does not limit the above-mentioned self-assembly method, and can be a method known to those skilled in the art. In some specific embodiments, the self-assembly adopts a vortex mixing method to bring the organic phase and the aqueous phase into contact and perform self-assembly.

[0051] The vortex mixing time can be 1s to 100min, preferably 30s to 10min, more preferably 30s to 5min. In some specific embodiments, the vortex mixing time is 30s, 1min, 2min, 3min, 4min or 5min, or any of the above values ​​can be the upper or lower limit.

[0052] After self-assembly, lipid nanoparticles are preferably obtained by dialysis. The lipid nanoparticles have a particle size of 50-300 nm, more preferably 150-250 nm, and a morphology of spherical or near-spherical.

[0053] The dialysis solvent can be a generally applicable buffer solution, including but not limited to phosphate buffer solution. The pH value of the buffer solution is preferably 7 to 8. In some specific embodiments, the pH value is 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, or any of the above values ​​as the upper or lower limit.

[0054] The molecular weight cutoff for dialysis is preferably 1000~3000 Da, more preferably 2000 Da.

[0055] The dialysis time can be 1 to 48 hours, preferably 5 to 24 hours, and more preferably 8 to 15 hours. In some specific embodiments, the dialysis time is 8, 9, 10, 11, 12, 13, 14 or 15 hours, or any of the above values ​​as the upper or lower limit.

[0056] The vaccine formulation provided by this invention can be a powder injection or a liquid formulation, etc. Depending on the dosage form requirements, the vaccine formulation may also include pharmaceutically acceptable excipients. This invention does not impose special limitations on the selection of excipients; those skilled in the art can select them according to dosage form requirements.

[0057] The present invention also provides the use of the above-mentioned vaccine formulation in the preparation of medicaments for the prevention, treatment and / or reduction of tumors.

[0058] The mRNA tumor vaccine prepared in this invention, when co-incubated with bone marrow-derived dendritic cells, significantly promoted dendritic cell maturation compared to the control group. Furthermore, the vector-encapsulated EGFP mRNA exhibited excellent transfection capability into 293T cells. Intramuscular injection of the mOVA-encapsulated tumor vaccine into a B16F10-OVA tumor-bearing mouse model demonstrated superior tumor prevention and treatment effects compared to the control group. The peptide lipid nanoparticles (PLNPs) possess excellent mRNA delivery capabilities and effectively activate in vivo immune responses, showing significant tumor prevention and treatment effects compared to the control group.

[0059] Compared with existing technologies, this invention provides a lipid-modified bee venom peptide, obtained by sequentially modifying the C-terminus of the bee venom peptide with lysine, phenylalanine, and C8-C18 alkylcarboxylic acids. The aforementioned lipid-modified bee venom peptide exhibits excellent mRNA encapsulation, delivery, and immune response enhancement capabilities, and is simple to prepare. It can be used as a carrier for nanovaccines. The prepared nanovaccines, after in vivo injection, demonstrate excellent immune cell activation effects and, compared with the control group, can effectively prevent and inhibit tumor growth, providing a valuable carrier tool for the development of mRNA tumor vaccines. Attached Figure Description

[0060] Figure 1 This is a schematic diagram illustrating the preparation of polypeptide lipid nanoparticles according to the present invention;

[0061] Figure 2 The mass spectrum of C14-FK-Melittin;

[0062] Figure 3 SM-102 (left) and PLNP 10% (Right) Transmission electron microscope image;

[0063] Figure 4 These are fluorescence images of 293T cell lines transfected with EGFP mRNA encapsulated in different vectors obtained in Example 4;

[0064] Figure 5 The results of fluorescence quantification of 293T cell lines after transfection with EGFP mRNA encapsulated in different vectors obtained in Example 4;

[0065] Figure 6 The image shows the cell viability of the DC 2.4 cell line after treatment with different concentrations of the material obtained in Example 5.

[0066] Figure 7 This is a graph showing the immune response levels of BMDCs after treatment with OVA mRNA encapsulated in different vectors obtained in Example 6.

[0067] Figure 8 The preventive effect of OVA mRNA encapsulated in different vectors on B16F10-OVA tumors obtained in Example 8 is shown in the figure.

[0068] Figure 9 The image shows the therapeutic effects of OVA mRNA encapsulated in different vectors on B16F10-OVA tumor-bearing mice obtained in Example 9. Detailed Implementation

[0069] To further illustrate the present invention, a detailed description is provided below with reference to embodiments. However, it should be understood that these descriptions are merely for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims.

[0070] There are no particular restrictions on the source of any raw materials used in this invention; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0071] In the following examples, the phospholipid is distearate phosphatidylcholine (DSPC), the ionizable lipid is 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecapoxy)hexyl]amino]octanoate (SM-102), and the PEGylated lipid is 1,2-dimyristoyl-rac-glycerol-3-polyethylene glycol 2000 (DMG-PEG2000).

[0072] Example 1

[0073] Preparation of lipotropic bee venom peptides:

[0074] C14-FK-Melittin was obtained on 2-chloro resin via solid-phase peptide synthesis (SPPS): synthesis began from the C-terminus, first ligating the peptide to the resin, then synthesizing according to the meliostein sequence, followed by ligation of the FK sequence, and finally ligation of myristic acid. After sequence synthesis, the peptide was cleaved from the resin, and the peptide side chains were deprotected using a 95% TFA aqueous solution with stirring for 0.5 hours. C14-FK-Melittin was obtained by precipitation with diethyl ether, denoted as Myristate-FKGIGAVLKVLTGLPALISWIKRKRQQ-NH2.

[0075] The structure of C14-FK-Melittin was characterized by mass spectrometry, and the mass spectrum is shown below. Figure 2 .

[0076] Example 2

[0077] Preparation of mRNA vaccine: In this invention, polypeptide lipid nanoparticles with different mass ratios were prepared. The lipid-containing ethanol solution was prepared according to the ratio shown in Table 1 and added dropwise to a citrate buffer solution (10 mM, pH 3.0) containing mRNA. After vortex mixing, the mixture was dialyzed in a phosphate buffer solution (10 mM, pH 7.2) to obtain an mRNA tumor vaccine with polypeptide lipid nanoparticles as carriers.

[0078] Table 1. Mass content of each component in lipid-containing ethanol solutions

[0079]

[0080] Note: The mass content of lipotropic meliotide refers to its mass content in auxiliary lipids.

[0081] Table 2. Particle size, potential, PDI, and encapsulation efficiency of the polypeptide-lipid nanoparticles prepared in Example 1.

[0082]

[0083] Figure 3 SM-102 (left) and PLNP 10% The transmission electron microscope image (right) shows that the polypeptide lipid nanoparticles are still spherical, and their morphology is not significantly different from that of lipid nanoparticles. Example 3

[0084] Cell culture: 293T and B16F10-OVA cell lines were selected and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. BMDC cell lines were selected and cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The culture conditions were 5% carbon dioxide concentration and 37℃ in a cell culture incubator.

[0085] Example 4

[0086] Cell transfection: The 293T cell line was used for cell transfection evaluation. 1 × 10⁶ cells were seeded per well in a 96-well plate. 4 Cells were cultured for 12 hours. Different formulations containing EGFP mRNA were co-incubated with cells for 24 hours, followed by fluorescence imaging. The delivery capabilities of different materials were compared using quantitative fluorescence analysis. Compared to the control group, peptide-lipid nanoparticles (PLNPs) showed superior delivery. 10% The encapsulated EGFP mRNA showed a brighter green fluorescent signal, and this formulation will be used in subsequent in-depth studies.

[0087] The test results are as follows Figure 4 , Figure 5 As shown, Figure 4 These are fluorescence images of 293T cell lines after transfection with EGFP mRNA encapsulated in different vectors. Figure 5 This is the result of quantitative fluorescence analysis, by Figure 4 and Figure 5 It can be seen that PLNP 10% It has the best transfection effect.

[0088] Example 5

[0089] Cytotoxicity: The DC2.4 cell line was used for cytotoxicity evaluation. 1 × 10⁶ cells were seeded per well in a 96-well plate. 4 The number of cells was determined, and the cells were cultured for 12 hours. Different concentrations of material were then co-incubated with the cells for 24 hours. The culture medium was then changed, and 10 μL of CCK 8 detection solution was added to each well. The cells were cultured for another 0.5 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula.

[0090] Cell viability (%) = (A 样品 / A 空白 )×100%

[0091] The test results are as follows Figure 6 As shown, Figure 6 The results show that PLNP 10% It has superior 293T cell transfection capability.

[0092] Example 6

[0093] In vitro activation: BMDCs cell lines were seeded in 12-well plates. After co-incubating the cells with different materials encapsulating OVA mRNA for 24 hours, the culture medium was removed, and the cells were washed three times with phosphate-buffered saline (PBFS). The cells were then incubated with CD11c-FITC, CD80-APC, and CD86-PE-Cy7 antibodies by flow cytometry (4°C, 1 h). After incubation, the cells were washed three times with PBFS and fixed with 4% paraformaldehyde PBFS for 15 minutes. Cell analysis was performed by flow cytometry. Compared with the control, the OVA mRNA vaccine group encapsulated with peptide-lipid nanoparticles showed greater BMDC activation, which will contribute to the immune response of mRNA tumor vaccines.

[0094] The test results are as follows Figure 7 As shown, Figure 7 The results showed that the introduction of lipid-modified melitoxin did not cause significant cytotoxicity, demonstrating its good biocompatibility.

[0095] Example 7

[0096] In vivo transfection: C57BL / 6J mice weighing approximately 20 g were used. Luc mRNA encapsulated in peptide-lipid nanoparticles was injected into the inner side of the right leg of each mouse at a dosage of 10 μg of mRNA. Six hours after injection, luciferase substrate luciferin potassium salt was injected intraperitoneally at a dosage of 3 mg per mouse. Fifteen minutes after substrate injection, bioluminescence detection was performed at the injection site using a small animal in vivo imaging system. Compared with the control group, there was no significant difference in bioluminescence results between the peptide-lipid nanoparticle-encapsulated Luc mRNA group and the traditional lipid nanoparticle-encapsulated Luc mRNA group.

[0097] Example 8

[0098] Tumor prevention: The tumor prevention experiment used the B16F10-OVA tumor model and C57BL / 6J black mice weighing approximately 20 g. First, peptide-lipid nanoparticles encapsulating OVA mRNA were injected intramuscularly into the inner side of the right leg of each mouse, at a dose of 10 μg mRNA. Five days later, the vaccine was injected again. Seven days later, the dosage was 1.0 × 10⁻⁶ mRNA per mouse. 6 Cells were inoculated into the right back of mice, and tumor volume was measured every two days. The tumor was considered complete when it exceeded 1000 mm². 3 Mice were euthanized. Compared with the control group, the OVA mRNA vaccine encapsulated in peptide lipid nanoparticles showed better tumor prevention efficacy, with a tumor inhibition rate of up to 50%.

[0099] The test results are as follows Figure 8 As shown.

[0100] Example 9

[0101] Tumor treatment: The B16F10-OVA tumor model was used, with C57BL / 6J black mice weighing approximately 20 g each, administered 1.0 × 10⁻⁶ ppm per mouse. 6 Cells were inoculated into the right back of mice, and the tumor was allowed to grow to 100 mm in size. 3 In this study, peptide-lipid nanoparticles encapsulating OVA mRNA were injected intramuscularly into the medial side of the right leg of mice, with each mouse receiving 10 μg of mRNA. The vaccine was administered again five days later. Compared to the control group, the OVA mRNA vaccine encapsulated in peptide-lipid nanoparticles showed a significant inhibitory effect on tumor growth.

[0102] The test results are as follows Figure 9 As shown.

[0103] The above experimental results show that the mRNA tumor vaccine prepared in this invention, when injected intramuscularly, can induce a better immune response compared with the control group, and can effectively prevent and inhibit tumor growth.

[0104] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A lipid-modified bee venom peptide, characterized in that, It is obtained by sequentially modifying the N-terminus of bee venom peptide with lysine, phenylalanine, and C8-C18 alkylcarboxylic acids.

2. The lipotropic bee venom peptide according to claim 1, characterized in that, The structural formula of the lipid-modified bee venom peptide is shown below: R-FKGIGAVLKVLTGLPALISWIKRKRQQ-NH2; R is a C8~C18 alkyl carboxylic acid group.

3. The lipotropic bee venom peptide according to claim 2, characterized in that, R stands for myristic acid.

4. The use of the lipidated bee venom peptide according to any one of claims 1 to 3 in the preparation of a vaccine delivery vector.

5. A vaccine carrier composition, characterized in that, It includes the lipidated bee venom peptide and auxiliary lipids as described in any one of claims 1 to 3.

6. The vaccine carrier composition according to claim 5, characterized in that, The assisting lipids include: Phospholipids, cholesterol, polyethylene glycol-modified lipids, and ionizable lipids.

7. The vaccine carrier composition according to claim 6, characterized in that, The phospholipid is distearate phosphatidylcholine; The PEGylated lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000; The ionizable lipid is heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecapoxy)hexyl)amino)octanoate.

8. A nano-vaccine drug composition, characterized in that, It includes an antigen component, as well as the lipidized bee venom peptide according to any one of claims 1 to 3 or the vaccine carrier composition according to any one of claims 5 to 7.

9. A vaccine formulation, characterized in that, Includes the nano-vaccine drug composition of claim 8; The vaccine formulation is lipid nanoparticles formed by the self-assembly of the nanovaccine drug composition of claim 8 in water or a buffer solution.

10. The use of the nanovaccine composition of claim 8 or the vaccine formulation of claim 9 in the preparation of a medicament for the prevention, treatment and / or reduction of tumors.

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