CN118126131A - Self-assembled short peptides and their use as delivery vehicles - Google Patents
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- CN118126131A CN118126131A CN202410245379.6A CN202410245379A CN118126131A CN 118126131 A CN118126131 A CN 118126131A CN 202410245379 A CN202410245379 A CN 202410245379A CN 118126131 A CN118126131 A CN 118126131A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a self-assembled short peptide and application thereof as a delivery carrier, belonging to the medical application of nano biological delivery, wherein the amino acid sequences of the self-assembled short peptide are respectively as follows: d (Gln) d (Pro) d (Arg) d (Arg) d (Arg) Lys Lys Arg Arg Lys Lys Arg Gly and d (Asp) d (Gly) d (Arg) Arg Arg Lys Lys Ala Ala Ala Ala Ala Ala. The invention widens the new application field of the existing self-assembled short peptide, expands the application range type of the self-assembled short peptide, improves the research and development value of the self-assembled short peptide, provides a feasible research direction for the application of mRNA vaccine and nucleic acid medicine, and widens the research fields of medicine carrier, gene delivery and the like.
Description
Technical Field
The invention belongs to the medical field of nanometer biological drug delivery treatment, relates to self-assembled short peptide and application thereof as a delivery carrier, and in particular relates to application of the self-assembled short peptide as the delivery carrier in aspects of mRNA vaccine and nucleic acid drug delivery.
Background
The mRNA is widely researched in the fields of cell therapy, protein therapy and anti-tumor, and has important application prospect in the medical challenges in the fields of common infectious diseases and virulent infectious diseases prevention and treatment, immunotherapy, vaccine and the like. FAD approves mRNA therapeutic drug Onpattro ™ in 2018 for the treatment of hereditary amyloidosis, introduces mRNA therapy into the pharmaceutical market, and then two mRNA vaccines are promptly approved in 2019 and 2020, which technology is of great interest. mRNA research and development and application are two key technologies, namely mRNA primary structure sequence and mRNA high-efficiency delivery vector, especially in mRNA vaccine direction. Research further shows that the mRNA vaccine research platform shows strong immunogenicity and protectiveness in animal models, along with the deep penetration of bioinformatics and AI, the optimization and chemical modification of the structural sequence of the mRNA vaccine have made important progress, and the emphasis and the challenges of the current research are focused on the design and development of safe and effective delivery vectors.
Lipid Nanoparticle (LNP) technology derived from liposomes and their lipid compounds is one of the potential tools for drug delivery, has tremendous market space and social effect value, and has been applied to mRNA vaccines such as the new crown vaccine. But their effectiveness, biosafety and tolerability in vivo, especially in targeted therapies for genetic and neoplastic diseases, remain to be improved. Currently, the bottleneck of development of LNP-related new drugs is: (a) the difficulty of breaking through monopoly of the patent is high; (b) the components have high toxicity and are difficult to degrade in vivo; (c) The technical requirements of the production process are extremely high, the quality control difficulty is high, and the cost is high; (d) The mRNA release efficiency in vivo is extremely low, and effective drug concentrations are difficult to achieve. It is a challenge to design delivery materials that are degradable, non-toxic, safe, etc. in character and advantage, and that can encapsulate mRNA.
If classified from a self-assembly perspective, short peptides can be classified into two major classes, self-assembly and non-self-assembly: the non-self-assembled peptide small molecules can be used as mRNA vaccine vectors for use as immunoadjuvant functions in subunit vaccines; self-assembled short peptides (Self-Assembled Peptides) are used for the transport and controlled release of drug delivery. The inventor team of the application continuously researches for about twenty years in the field, further expands self-assembled short peptides into chiral self-assembled short peptides and research systems, and can self-assemble to form nano-structure aggregates through intermolecular interaction under physiological environment conditions, wherein the nano-structures comprise various forms such as nanospheres, nanotubes, nanocapsules, nanobranches, nanosheets, nanonets and the like, and have different application scenes or functions in different fields of science and different application directions, such as cell and stem cell engineering, tissue and organoid culture, protein delivery and stabilization, tissue repair and regenerative medicine, rapid hemostasis and scar repair, immunotherapy and vaccine, membrane protein structure and stabilization, energy instrument and electrochemical field, cosmetic anti-aging and medical engineering and the like.
The system has less research on mRNA vaccine vectors, but has special structure and directional function, and the degradation product is amino acid residue. If the modified polysaccharide is used as a delivery carrier material of an RNA vaccine, the serious problems of delivery efficiency, mechanism, degradability, cytotoxicity, safety and the like are not clear, and the modified polysaccharide is worthy of intensive study.
Disclosure of Invention
The invention provides a self-assembled short peptide and application thereof as a delivery carrier, which are used for solving the technical problems that the existing mRNA vaccine and nucleic acid medicine are difficult to effectively deliver and have larger toxic and side effects.
The technical scheme adopted by the invention is as follows:
A self-assembled short peptide comprising two amino acid sequences, one of which has the amino acid sequence: d (Gln) D (Pro) D (Arg) D (Arg) D (Arg) Lys Lys Arg Arg Lys Lys Arg Gly, wherein D () represents an amino acid of the D-form, i.e. the amino acid sequence can also be expressed as: gln-Pro-Arg-Arg-Arg-Lys-Lys-Arg-Arg-Lys-Lys-Arg-Gly and the first five amino acids Gln, pro, arg, arg, arg all represent D-type amino acids, the self-assembled short peptide is named CSP-1 in the application. And the amino acid sequence of the other is: d (Asp) D (Gly) D (Arg) Arg Arg Lys Lys Ala Ala Ala Ala Ala Ala, wherein D () represents an amino acid of the D type, i.e. the amino acid sequence can also be expressed as: asp-Gly-Arg-Arg-Arg-Lys-Lys-Ala-Ala-Ala-Ala, the first three amino acids Asp, gly, arg all represent D-amino acids, and the self-assembled short peptide is named CSP-2 in the application.
Amidation of the carbon end of the self-assembled short peptide, and the sequence after amidation is as follows:
The amino acid sequences of the amidated CSP-1 are respectively as follows: d (Gln) d (Pro) d (Arg) d (Arg) d (Arg) Lys Lys Arg Arg Lys Lys Arg Gly-NH 2;
The amino acid sequences of the amidated CSP-2 are respectively: d (Asp) d (Gly) d (Arg) Arg Arg Lys Lys Ala Ala Ala Ala Ala Ala-NH 2.
The self-assembled short peptide is used as a delivery carrier.
In particular, the self-assembled short peptide is used as a delivery carrier in mRNA vaccine.
When the self-assembled short peptide is used as a delivery carrier in an mRNA vaccine, the carrier is mRNA.
Further, mRNA includes mRNA vaccines, RNA drugs.
Experiments show that the self-assembled short peptides CSP-1 and CSP-2 have stable secondary structures, self-assembly is generated under the response of salt ions, when mRNA is not loaded, the CSP-1 self-assembles into loose nano vesicles, the CSP-2 self-assembles into loose nano tubular fibers, when mRNA is loaded, the self-assembly mechanism is triggered simultaneously due to the electrostatic adsorption effect, and the mRNA/CSP-1 and the mRNA/CSP-2 are both formed into compact nano vesicle structures with the particle size of about 200-350 nm.
Experiments show that the self-assembled short peptide CSP-1 and CSP-2 of the invention form a nano vesicle structure, and in the environment with neutral pH, the short peptide has positive charges, and can attract nucleic acid molecules with negative charges to wrap the nucleic acid molecules through electrostatic adsorption, thus having a certain protection effect on mRNA.
Experiments show that the self-assembled short peptides CSP-1, CSP-2 and mRNA can be mixed in different proportions to effectively load mRNA, and the formed mRNA/CSP compound has low toxicity to cells in the effective load of mRNA, and the survival rate of cells in the effective delivery mass ratio range is as shown in the figure to be more than 80%. Experiments show that the nano vesicles formed by the self-assembled short peptides CSP-1 and CSP-2 can effectively load mRNA, effectively deliver the mRNA to 293T cells and BALB/C mice to express corresponding antigen genes in vivo while protecting the mRNA, effectively deliver EGFP-mRNA to 293T cells in a list of preferred cells and Express Green Fluorescent Protein (EGFP) in the cells.
Experiments show that the nano-composite formed by the self-assembled short peptides CSP-1 and FIPV N MRNA can induce mice to generate specific antibodies of FIPV N after entering BALB/C mice, and activate the immune response of the mice after multiple immunization of the FIPV N-mRNA/CSP-1 composite, thereby effectively generating specific cellular immunity and humoral immunity of the mice. In the subsequent animal experiments, the preparation method has no obvious toxic or side effect on important organs of cells, such as liver, spleen, kidney and the like.
In conclusion, the self-assembled short peptides CSP-1 and CSP-2 can effectively load mRNA, avoid the degradation of mRNA by nuclease, and deliver mRNA to cells to express target gene proteins in vivo to play a role. The self-assembled short peptide CSP-1 can safely and effectively deliver nucleocapsid protein mRNA for expressing cat infectious peritonitis virus into BALB/C mice to induce humoral immunity and specific cellular immunity in the mice, and can be applied to the related fields of gene delivery, nucleic acid drug delivery as a drug delivery carrier and the like.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention provides a feasible research direction for the effective delivery of nucleic acid medicine and mRNA vaccine, and widens the research field of nucleic acid medicine delivery.
(2) The invention widens the new application field of the existing self-assembled short peptide, expands the application range type of the self-assembled short peptide and improves the research and development value of the self-assembled short peptide.
(3) The self-assembled short peptide can effectively send mRNA vaccine into cells and mice, can effectively express gene antigens in the cells, and can prevent nuclease from degrading mRNA.
(4) The novel self-assembled nano biomedical material can be widely applied to the fields of nano biomedical engineering, cell engineering, bioengineering and the like, and has obvious economic and social benefits.
(5) Provides a safe and effective nucleic acid delivery nano-carrier, which can be widely applied to the biomedical field.
Drawings
FIG. 1 is a block diagram of an assembled short peptide CSP-1 of the invention;
FIG. 2 is a block diagram of the assembled short peptide CSP-2 of the invention;
FIG. 3 is a schematic diagram showing particle size distribution of CSP/mRNA complexes of assembled short peptides CSP-1 and CSP-2 of the invention with different mass ratios (CSP-1/mRNA mass ratio of 2-6 and CSP-2/mRNA mass ratio of 5-9) as determined by Dynamic Light Scattering (DLS);
FIG. 4 is a schematic zeta potential of CSP/mRNA complexes of assembled short peptides CSP-1 and CSP-2 of the invention with different mass ratios (CSP-1/mRNA mass ratio of 2-6 and CSP-2/mRNA mass ratio of 5-9) as determined by Dynamic Light Scattering (DLS);
From FIGS. 3 to 4, it is understood that when the mass ratio of CSP-1/mRNA is 3 and the mass ratio of CSP-2/mRNA is 7, the potential and particle size of the different nanocomposites are shown, and the particle size of the formed nanocomposite is 250nm and 300nm, respectively;
FIG. 5 is a schematic representation of the morphology of the self-assembled short peptide and mRNA (mRNA/CSP) complexes of the invention under transmission electron microscopy of CSP-1 (upper left), CSP-2 (lower left), CSP-1/mRNA complex (upper right) and CSP-2/mRNA complex (lower right);
Wherein, when mRNA is not loaded, the CSP-1 self-assembly is in a vesicle structure, the CSP-2 self-assembly is in an irregular fiber shape, and after mRNA is loaded, the CSP-1/mRNA can be observed to form a compact spherical structure, and the CSP-2/mRNA forms a fiber-coated spherical structure, the sizes of which are respectively 250nm and 300nm and accord with the DLS result;
FIG. 6 is a graphical representation of the results of circular dichroism of CSP-1 and CSP-2 in the present invention;
Wherein, the chiral self-assembled short peptides CSP-1 and CSP-2 can both show stable secondary structures, CSP-1 shows a negative peak at 200nm and a positive peak near 220nm, which indicates that the secondary structure of CSP-1 is mainly random coil; in contrast, CSP-2 has a spectrum with positive peaks at 190nm and 220nm, with a negative peak near 215nm, indicating that CSP-2 forms a secondary structure with random coil as the main and partial beta sheets;
FIG. 7 is a schematic diagram showing the capacity of agarose gel electrophoresis to detect mRNA of different masses of CSP (CSP-1/mRNA mass ratio 0-4, CSP-1/mRNA mass ratio 0-8) in compression encapsulation;
Wherein both of the short peptides bind to the mRNA molecule, resulting in mRNA being compressed, the intensity of the mRNA band gradually decreasing and disappearing as the mass ratio of short peptide to mRNA increases;
FIG. 8 is a schematic representation of an in vitro toxicity assessment of mRNA/CSP complexes of the invention;
Wherein the effect of different CSP/mRNA mass ratios (CSP-1/mRNA mass ratio of 2-5 and CSP-1/mRNA mass ratio of 5-8) on 293T cell activity (left and middle panels) was examined by CCK8 experiments for viability of 293T cells in the presence of different concentrations of CSP-1 and CSP-2 (concentration range of 0-80. Mu.g/ml) at 24hours of co-transfection: when the mass ratio of CSP-1 to EGFP mRA is 2:1, the activity of the cells is close to 100%, and the mass ratio of CSP-1 to FIPV N MRNA is 3:1, the activity of the cells is close to 100%; at a mass ratio of 6:1 for CSP-2 and EGFP mRA, the viability of the cells was nearly 100% and for CSP-2 and FIPV N MRNA the mass ratio was 7:1, the activity of the cells was close to 100%.
FIG. 9 is a schematic representation of transfection and expression of in vitro CSP/EGFP mRNA complexes in 293T cells according to the present invention;
wherein, a and c are prepared with different mass ratios (a graph CSP-1/EGFP mRNA mass ratio is 2-4, c graph CSP-2/EGFP mRNA mass ratio is 5-7) and transfection efficiency of 293T cells by different mass vectors/EGFP mRNA self-assemblies. b and d RNase A enzyme treatment, the vector/mRNA with different mass ratios (b-graph CSP-1/EGFP mRNA mass ratio is 2-4, d-graph CSP-2/EGFP mRNA mass ratio is 5-7) is self-assembled to the transfection efficiency of 293T cells, and the result shows that when the CSP-1 and EGFP mRNA mass ratio is 3:1, CSP-1 and EGFP mRNA mass ratio of 7:1, the EGFP expression level in 293T cells was highest;
FIG. 10 is a graph showing the expression level of the N protein and the internal reference protein GAPDH in 293T cells from FIPV N-mRNA using chiral self-assembled short peptides CSP-1 and CSP-2 as delivery systems in the present invention;
Wherein, the mass ratio of CSP-1 to FIPV N-mRNA is 3:1-5:1, and the mass ratio of CSP-1 to FIPV N-mRNA is 6:1-8:1;
FIG. 11 is a schematic representation of the expression of the in vitro FIPV N MRNA/CSP complex of the invention in 293T cells;
wherein, the mass ratio of CSP-1 to FIPV N MRNA is 3:1, CSP-2 and FIPV N MRNA in a mass ratio of 7: FIPV N protein was expressed at the highest level in the cells at 1. In addition, similar to the cell fluorescence assay, CSP-1 delivers mRNA in a much higher capacity than CSP-2;
FIG. 12 is a schematic of the determination of FIPV N protein-specific IgG antibody titers by ELISA in the present invention;
Wherein the dashed line indicates the limit of detection, and the results indicate that CSP-1/FIPV N MRNA induced significant antibody titers in mice after primary immunization, significantly increased antibody titers after the third immunization, and reached specific antibody titers of 12800 upon the last booster immune response, as compared to the control groups (naked mRNA group, CSP-1 group, CSP-2 group). Although the antibody titer generated after CSP-2/mRNA complex inoculation was higher than that of the control group, it was not statistically significant, and no change in antibody titer was observed after booster needle injection;
FIG. 13 is a schematic of the determination of specific IL-4 antibody concentration in serum by ELISA in the present invention;
Wherein, CSP-1/FIPV N MRNA group, CSP-2/FIPV N MRNA group and bare FIPV N MRNA group are all significantly higher than CSP-1 and CSP-2 groups;
FIG. 14 is a schematic representation of the determination of specific TNF- α antibody concentration in serum by ELISA in the present invention;
Of these, CSP-1/FIPV N MRNA group was significantly higher than CSP-2/FIPV N MRNA group and bare FIPV N MRNA, CSP-1 and CSP-2 groups. CSP-2/FIPV N MRNA group was not significantly different from the naked FIPV N MRNA group, CSP-1 and CSP-2 groups;
FIG. 15 is a schematic representation of an ELISPot assay of the invention for detecting IFN-gamma secretion in spleen cells of immunized mice;
Wherein, when splenocytes were stimulated with FIPV N protein, IFN-gamma levels were significantly elevated in CSP-1/FIPV N MRNA groups compared to control groups, whereas IFN-gamma levels were not significantly different in CSP-2/FIPV N MRNA groups compared to control groups. Indicating that CSP-1/FIPV N MRNA group can effectively trigger IFN-gamma T cell immune response;
FIG. 16 is a schematic representation of the proportion of CD4+ and CD8+ cells in a mice immune response to T cells following delivery of FIPV N-mRNA into the mice using chiral self-assembled short peptide CSP-1 as a delivery system in accordance with the present invention;
Wherein the CD8+ positive proportion is 32% and the CD4+ positive proportion is 61.9%;
FIG. 17 is a schematic representation of the proportion of CD4+ and CD8+ cells in a mice after FIPV N-mRNA delivery to mice using chiral self-assembled short peptide CSP-2 as a delivery system in accordance with the present invention;
Wherein the CD8+ positive proportion is 13.9%, and the CD4+ positive proportion is 20.5%;
FIG. 18 is a schematic representation of the ratio of CD4+ to CD8+ cells in a mice after injection of naked FIPV N-mRNA to the mice in accordance with the present invention to elicit T cell immune response;
wherein the CD8+ positive proportion is 13.2% and the CD4+ positive proportion is 20.8%;
FIG. 19 is a schematic representation of the ratio of CD4+ to CD8+ cells in a mice upon injection of naked CSP-2 into the mice in accordance with the present invention to elicit T cell immune response in the mice;
Wherein, the positive proportion of CD8+ is 11.0%, and the positive proportion of CD4+ is 17.6%;
FIG. 20 is a schematic representation of the ratio of CD4+ to CD8+ cells in a mice upon injection of naked CSP-1 into the mice in accordance with the present invention to elicit T cell immune response in the mice;
Wherein the CD8+ positive proportion is 12.8% and the CD4+ positive proportion is 19.3%;
FIG. 21 is a graph showing the proportion of CD4+ and CD8+ cells in the present invention when FIPV N-mRNA/CSP-1 complex (20 ug), FIPV N-mRNA/CSP-2 complex (20 ug), naked mRNA (20 ug), CSP-1 (60 ug), CSP-2 (140 ug) were injected into mice to elicit T cell immune responses in mice;
Wherein, the CSP-1 is used as a delivery system to deliver FIPV N-mRNA, which can effectively induce cellular immune response and induce cellular immune response;
FIG. 22 is a graph showing the change in body weight of mice during immunization in the present invention;
The result shows that no obvious inflammatory reaction is caused at the injection part of the mice, and the weights of all groups of mice do not grow normally differently;
FIG. 23 is a graph showing HE staining of pathological tissues of liver, kidney and spleen 36 days after primary immunization in the present invention;
Wherein, CSP-1, CSP-2, CSP-1/FIPV N MRNA vaccine, CSP-2/FIPV N MRNA vaccine is compared with the control group naked FIPV N MRNA group) has no obvious pathological changes.
Detailed Description
The present invention is further described below in conjunction with embodiments, which are merely some, but not all embodiments of the present invention. Based on the embodiments of the present invention, other embodiments that may be used by those of ordinary skill in the art without making any inventive effort are within the scope of the present invention.
Example 1: design and synthesis of self-assembled short peptides CSP-1 and CSP-2
According to the characteristic structure of cell penetrating peptide and the physical and chemical characterization of chiral self-assembled short peptide, two chiral self-assembled short peptides CSP-1 and CSP-2 are designed, wherein the amino acid sequences of CSP-1 are respectively as follows: d (Gln) d (Pro) d (Arg) d (Arg) d (Arg) Lys Lys Arg Arg Lys Lys Arg Gly, the amidated sequence is: d (Gln) d (Pro) d (Arg) d (Arg) d (Arg) Lys Lys Arg Arg Lys Lys Arg Gly-NH 2, the structure of which is shown in FIG. 1. The amino acid sequences of CSP-2 are respectively: d (Asp) d (Gly) d (Arg) Arg Arg Lys Lys Ala Ala Ala Ala Ala Ala, the amidated sequence is: d (Asp) d (Gly) d (Arg) Arg Arg Lys Lys Ala Ala Ala Ala Ala Ala-NH 2, the structure of which is shown in FIG. 2. Using a CEM microwave polypeptide synthesizer, adopting an Fmoc strategy solid-phase synthesis method to synthesize target short peptide CSP-1 resin, taking a trifluoroacetic acid/m-cresol/water (volume ratio of 90:5:5) mixed solution as a lysate, stirring for 0.5h under ice bath, stirring for 1h at normal temperature for cracking, freeze-drying to obtain crude peptide, purifying the freeze-dried crude peptide by using a medium-pressure purification system to obtain freeze-dried powder with purity of pure peptide more than 95% (HPLC), and measuring molecular weight by mass spectrum to be 1750.11. Split charging into 10 mg/tube clean sterile EP tube, and placing in-20deg.C refrigerator for use.
Example 2: short peptide solution configuration and mRNA/CSP complex formation
Preparation of short peptide mother liquor: the purified lyophilized powder was prepared as a 10mg/ml polypeptide solution in ionized water and stored in a refrigerator at 4℃for use. The prepared CSP-1 mother liquor was diluted in PBS at a concentration of 1ug/ml, 2ug/ml, 6ug/ml, 8ug/ml,10 ug/ml. The dilution concentration of the CSP-2 mother solution is 10ug/ml, 12ug/ml, 14ug/ml and 16ug/ml. CSP/mRNA complexes were prepared according to the mass ratios shown for the two chiral self-assembled short peptides and mRNA in table 1, and incubated at room temperature for 30min to promote complex formation.
TABLE 1 chiral self-assembled short peptide delivery mRNA at different mass ratios
Example 3: physicochemical characterization of CSP and mRNA/CSP complexes
Dynamic Light Scattering (DLS): the mRNA/CSP complex prepared in Table 1 in example 2 was used to detect the particle size and the potential of the CSP-1/mRNA (mass ratio: 2-5) and CSP-2/mRNA (mass ratio: 5-8) complexes of different mass ratios by means of a nanoparticle size and potential analyzer.
The experimental results of fig. 3-4 show that: with the increase of CSP quality, the positive potential of the solution is continuously increased, the particle size of the CSP/mRNA compound is reduced, when the mass ratio of CSP-1 to mRNA is more than or equal to 3, the mass ratio of CSP-2 to mRNA is more than or equal to 6, mRNA can be compressed by short peptide to the maximum extent, at the moment, the particle size of the formed CSP-1/mRNA compound is about 250nm, and the particle size of the CSP-2/mRNA compound is about 350 nm.
Example 4: CSP and mRNA/CSP scanning electron microscope (TEM)
And (3) a Transmission Electron Microscope (TEM), detecting the self-assembled form of the CSP-1 (10 mg/ml), CSP-1/mRNA (mass ratio is 3) and CSP-2/mRNA (mass ratio is 7) compound by adopting a transmission electron microscope, adsorbing the CSP and CSP/mRNA compound in a copper mesh, staying for 30Sec, wiping the filter paper, staining the filter paper for 5min by saturated uranium acetate, and carrying out TEM detection after the filter paper volatilizes in the air.
The results in fig. 5 show that: when mRNA is not loaded, the CSP-1 self-assembly forms a loose vesicle structure, the CSP-2 self-assembly forms loose nano tubular (irregular) fibers, and after mRNA is loaded, the CSP-1/mRNA can be observed to form a compact spherical nano vesicle structure, and the CSP-2/mRNA forms a fiber-wrapped spherical nano vesicle structure, and the particle sizes of the fiber-wrapped spherical nano vesicle structure are respectively 250nm and 300nm, which are consistent with the DLS result.
Example 5: round dichroism (CD) spectrum analysis CSP secondary structure
And (3) performing Circular Dichroism (CD), detecting the secondary structure of CSP-1 and CSP-2 by using the circular dichroism, and placing 200 mu l of CSP-1 (1 mg/ml) and CSP-2 (1 mg/ml) solution into a 1mm absorption tank for CD scanning, wherein the scanning wavelength range is 190-270nm.
The results in fig. 6 show that: both chiral self-assembled short peptides CSP-1 and CSP-2 can show stable secondary structures, and the secondary structure of CSP-1 is based on random coil. CSP-2 forms a secondary structure with random coils as the main and partial beta sheets.
Example 6: gel blocking test of mRNA/CSP complexes
1. Mu.g of mRNA was taken and complexes of short peptides and mRNA were prepared in the mass ratio shown in Table (1) so that the final volume was 10. Mu.l. After adding 2. Mu.l of 6 XRNA loading buffer and mixing, electrophoresis was performed on a 120V 1% agarose gel for about 50 minutes, and the result was photographed by exposure on a nucleic acid agarose gel light-exposure meter and analyzed.
The results in FIG. 7 show that both peptides bind to mRNA molecules, resulting in mRNA compression. The intensity of the mRNA bands gradually decreased and disappeared as the mass ratio of short peptide to mRNA increased.
Example 7: cytotoxicity test
Cytotoxicity experiments were performed using CCK8 kit. 293T cells with good growth state are evenly spread into a 96-well plate, about 100 mu l of 293T cell suspension is added into each well, the cell density is adjusted to 3X 10 4 cells/well, and a culture plate is placed at 37 ℃ and is cultured in a 5% CO2 incubator overnight. A series of concentration gradients CSP-1 and CSP-2 and their respective mRNA complexes were configured at the mass ratios shown in Table 1 and were repeated 3 times in duplicate under each experimental condition. Subsequently, 10. Mu.l each of CSP and mRNA/CSP mixture was added to the cultured cells. At two time points of 12, 24hours after treatment, 10. Mu.l of CCK8 solution was added to each well and incubated for 1 hour in a 5% CO2 incubator at 37 ℃. After the incubation, the absorbance at OD450nm was measured using a protein concentration detector.
The experimental results showed (fig. 8): CSP/mRNA complex nanoparticles exceeded the cell activity of co-transfected 12hours at 24hours cell viability. At co-transfection 24hours: when the mass ratio of CSP-1 to EGFP mRA is 2:1, the activity of the cells is close to 100%, and the mass ratio of CSP-1 to FIPV N MRNA is 3:1, the activity of the cells is close to 100%; at a mass ratio of 6:1 for CSP-2 and EGFP mRA, the viability of the cells was nearly 100% and for CSP-2 and FIPV N MRNA the mass ratio was 7:1, the activity of the cells was close to 100%. Even at a maximum dose concentration of 80 μg/ml of CSP-treated cells, the viability of CSP-1-treated cells was higher than that of CSP-2-treated cells, with CSP-1-treated cells having an activity of 95% or more and CSP-2-treated cells having an activity of 78% or more.
Example 8:293T cell fluorescent body external transfection process
2Ml of the cell suspension was added dropwise to the six-well plate, the cell density was adjusted to 5X 10 6 cells/well, and the mixture was incubated in a 5% CO 2 incubator at 37℃for 12 hours. After incubation, the supernatant was aspirated, the cells were washed twice with PBS, 1ml of Opti-MEM medium was added to each well, 1. Mu.g each of EGFP-mRNA and FIPV N-mRNA mixtures of the two short peptides were prepared according to the gel blocking assay described above, incubated at room temperature for 30min, 500. Mu.l of the short peptide-EGFP mRNA mixture was added to each well, and a positive control (Lipofectamine 3000 as transfection reagent) was placed in a 5% CO 2 incubator overnight. After overnight incubation of 12-24hours, the sample was exposed to blue light excitation using an inverted fluorescence microscope, the cell fluorescence was observed and the transfected fluorescence was analyzed by photograph recording.
As can be seen from this example, the results in FIG. 9 show that both CSP-1 and CSP-2 promote the delivery of EGFP mRNA to the cytoplasm and the expression of the corresponding EGFP fluorescent protein. However, the expression efficiency of EGFP in cells with CSP-1 as a delivery vehicle was significantly higher than in cells with CSP-2 as a delivery vehicle. These findings demonstrate the excellent performance of CSP-1 as a vector for enhancing mRNA delivery and expression in 293T cells.
Example 9: RNase tolerance test
2Ml of the 293T cell suspension was evenly dropped into a six-well plate, the cell density was adjusted to 5X 106 cells/well, then after incubating the 6-well plate in a 5% CO2 incubator at 37℃for 12 hours, the supernatant was aspirated and the cells were thoroughly washed with PBS twice, and 1ml of Opti-MEM medium was added to each well. According to the results of the gel blocking test and the cell fluorescence test, a mixture of 2 short peptides and 1. Mu.g EGFP-mRNA and a mixture of 1. Mu. G FIPV MRNA complex were prepared, and incubated at room temperature for 30 min. 1. Mu.l of RNase A was added to the CSP/mRNA complex mixture, which was incubated in a 37℃water bath for 20min, and then the mixture was uniformly added to a six-well plate, and the cells were cultured overnight in a 5% CO2 incubator at 37 ℃. Cells were placed under blue excitation light using an inverted fluorescence microscope within 12-24hours of transfected EGFP-mRAN, and the fluorescence results of the cells were observed and recorded by photographing.
FIG. 9 shows that RNase-resistance protection experiments and fluorescent transfection experiments were performed simultaneously, and that the CSP/EGFP mRNA complex was treated with RNase A enzyme before the fluorescent transfection experiments and then co-transfected for 24 hours. The expression of EGFP was reduced after RNase A enzyme treatment, but the CSP/EGFP mRNA complex maintained efficient delivery of EGFP mRNA after RNase A enzyme treatment to produce the corresponding protein. The CSP/EGFP mRNA complex is shown to successfully alleviate mRNA degradation caused by RNase A enzyme and provide a certain degree of mRNA degradation protection.
Example 10: western blot detection
And collecting 293T cells transfected with FIPV N-mRNA in the six-hole plate, extracting total cell proteins after cell lysis, measuring the cell concentration by a BCA method, and detecting the protein expression condition of the FIPV in the cells by using a specific antibody of the FIPV N protein by using a Western blot experiment.
The results in FIG. 11 show that CSP-1 and FIPV N MRNA have a mass ratio of 3:1, CSP-2 and FIPV N MRNA in a mass ratio of 7: FIPV N protein was expressed at the highest level in the cells at 1. In addition, similar to the cell fluorescence assay, CSP-1 delivers mRNA in a much higher capacity than CSP-2.
Example 10: animal immunization experiment
In this example, the experimental animals were BALB/C female mice of 6-8 weeks of age, weighing 10-13g randomly distributed, 25 animals, supplied by the Chongqing medical university animal experiment center. The mice were randomly divided into 5 groups, each group of 5 mice, and each group was inoculated with CSP-1/FIPV N MRNA (20 ug), CSP-2/FIPV N MRNA (20 ug), naked FIPV N-mRN group (20 ug), CSP-1 (negative control group), CSP-2 (negative control group) for intramuscular injection three times at 10-day intervals. On day 12, 24, 36 days of the primary immunization, mandibular venous blood was collected, left standing at 4℃for 2h, centrifuged at 4000rpm for 30min, and the serum was isolated and stored in a-20℃refrigerator for use in detecting FIPV N-specific antibodies in serum, after 36 days, the mice were sacrificed to take tissue sites such as liver, kidney, spleen, etc. of the mice for tissue HE staining, and spleen cells in the spleen of the mice were isolated for ELISPOT experiments to assess cellular immune levels.
FIPV N-specific antibody detection in serum:
And (3) detecting FIPV N specific antibodies in mouse serum by using an ELISA kit, diluting a serum sample to be detected according to a volume ratio of 1:100 by a double ratio method, adding the diluted standard substance and the sample to be detected into a FIPV N protein coated 96-well plate, incubating for 30min at 37 ℃, washing for five times, incubating for 30min at 37 ℃ after enzyme addition, repeating washing for five times, adding a color reagent into each hole, developing for 15min at 37 ℃ in a dark place, adding a stop solution, and measuring the absorbance (OD 450 nm) of each hole at a wavelength of 450nm by using an enzyme-labeled instrument within 15min after the color development reaction is stopped. The titer of the antibody is the antibody titer at the maximum dilution of the positive reaction of the sample to be tested. The titer of the antibody is the antibody titer at the maximum dilution of the positive reaction of the sample to be tested. A similar method uses ELISA kits to detect the concentration of cytokine IL-4 and TNF-alpha antibodies in serum
Analysis of the results of fig. 12 shows that: compared with the control group (naked mRNA group, CSP-1 group and CSP-2 group), CSP-1/FIPV N MRNA induces significant antibody titer in mice after primary immunization, the antibody titer is significantly increased after the third immunization, and the final immune response is enhanced to achieve the specific antibody titer of 12800. Although the antibody titer generated after CSP-2/mRNA complex inoculation was higher than that of the control group, no statistical significance was achieved and no change in antibody titer was observed after booster needle injection. IL-4 detection by ELISA showed that CSP-1/FIPV N MRNA, CSP-2/FIPV N MRNA and nude FIPV N MRNA groups were significantly higher than CSP-1 and CSP-2 groups (FIG. 13). ELISA detection of TNF- α showed that the CSP-1/FIPV N MRNA group was significantly higher than the CSP-2/FIPV N MRNA group and the bare FIPV N MRNA, CSP-1 and CSP-2 groups (FIG. 14). The CSP-2/FIPV N MRNA group was not significantly different from the bare FIPV N MRNA group, CSP-1 and CSP-2 groups.
Detection of IFN-gamma in spleen cells:
The IFN-gamma in spleen cells is separated and detected by adopting ELISOPT kit, 1 x 106 cells/ml of separated spleen cells are prepared, added into an activated PVDF plate for 100ul, the positive control group (RIMP 1640 is used as a stimulator for 10 ul) and the negative control group (FIPN N protein is used as a stimulator for 2ug/ml,10 ul) are arranged, cells are lysed after overnight incubation at 37 ℃, the plate is washed three times and then incubated for about 1h (37 ℃, incubator), enzyme-labeled avidin is added after plate washing is carried out again, color development is carried out after plate washing, and the plate is counted by adopting a CTL counter.
The results in FIG. 15 show that when splenocytes were stimulated with FIPV N protein, IFN-gamma levels were significantly elevated in CSP-1/FIPV N MRNA versus control, whereas IFN-gamma levels were not significantly different in CSP-2/FIPV N MRNA versus control. The CSP-1/FIPV N MRNA group was shown to be effective in eliciting an immune response against IFN-gamma T cells.
Differentiation assay of T cell subpopulations in splenocytes:
The isolated spleen cells were prepared into cell suspensions at a concentration of 1X106 cells/ml, centrifuged at 1000rpm for 3min, the supernatant was discarded, 100ul PBS was added to the cell pellet to prepare cell suspensions, APC anti-mouse CD3 Antibody(2.5μl)、FITC anti-mouse CD4 Antibody(0.5μl)、PE- anti-mouse CD8a Antibody(1μl), were added to each group of mouse spleen cells, and the mixture was incubated for 20min in the absence of light after being blown and mixed uniformly, and after washing twice with PBS, the T cell subsets CD4+ and CD8+ were detected using a flow cytometer (U.S. A., cytoFLEX).
As shown in fig. 16-21, a significant increase in the cd4+ T cell percentage and cd8+ T cell percentage was observed for the CSP-1/FIPV N MRNA vaccine compared to the control group (CSP-1 and CSP-2), which indicated that the CSP-1/FIPV N MRNA vaccine effectively stimulated proliferation of the mouse cd4+ T cells and cd8+ T cells, enhancing overall immune function in the mice, consistent with the results of the previous experiment, without significant differences in the cd4+ T cell percentage and cd8+ T cell percentage for the CSP-2/FIPV N MRNA vaccine response compared to the control group (CSP-1 and CSP-2). The results indicate that CSP-1 is effective in delivering FIPV N MRNA into animals and enhancing specific cellular immune function in mice
Example 10: animal toxicity and immunohistochemical analysis
To assess the potential toxicity of mRNA vaccines during animal immunization, changes in body weight of mice were recorded every 4 days, and organ-specific analysis was performed on different groups of mice after immunization was completed. The liver, kidney and spleen of the mice are collected, the samples are fixed for 24 hours through 4% paraformaldehyde fixing solution, the fixed tissue samples are prepared into pathological sections after the steps of dehydration, wax dipping, embedding, slicing, dewaxing, HE staining or immunohistochemistry, and the like, and the pathological sections are subjected to microscopic examination through an optical microscope to observe whether pathological changes occur in important organs of the mice.
The results of fig. 22 demonstrate that mice are vigilantly monitored for changes in body weight every 4 days throughout the animal immunization. The results showed that no significant inflammatory response was elicited at the mouse injection site and that the mice in each group grew normally without variability in body weight. Furthermore, we examined the important organ liver, spleen, kidney histopathological changes of mice after intramuscular injection of CSP-1, CSP-2, CSP-1/FIPV N MRNA vaccine, CSP-2/FIPV N MRNA vaccine, nude FIPV N MRNA. FIG. 23 shows that (CSP-1, CSP-2, CSP-1/FIPV N MRNA vaccine, CSP-2/FIPV N MRNA vaccine compared to control group nude FIPV N MRNA) there was no significant pathological change.
As can be seen from the example, the CSP vector and the CSP/RNA vaccine have no obvious toxic and side effects on cells and animals, and the safety of the CSP-1 vector and the CSP-1/FIPV N MRNA vaccine thereof is proved.
The above examples merely illustrate specific embodiments of the application, which are described in more detail and are not to be construed as limiting the scope of the application. It should be noted that it is possible for a person skilled in the art to make several variants and modifications without departing from the technical idea of the application, which fall within the scope of protection of the application.
Claims (8)
1. Self-assembled short peptide, characterized in that it comprises CSP-1 and/or CSP-2;
the amino acid sequences of CSP-1 are respectively: d (Gln) d (Pro) d (Arg) d (Arg) d (Arg) Lys Lys Arg Arg Lys Lys Arg Gly;
The amino acid sequences of CSP-2 are respectively: d (Asp) d (Gly) d (Arg) Arg Arg Lys Lys Ala Ala Ala Ala Ala Ala.
2. A self-assembling oligopeptide according to claim 1, wherein;
amidation of the carbon end of the self-assembled short peptide, and the sequence after amidation is as follows:
The amino acid sequences of the amidated CSP-1 are respectively as follows: d (Gln) d (Pro) d (Arg) d (Arg) d (Arg) Lys Lys Arg Arg Lys Lys Arg Gly-NH2;
The amino acid sequences of the amidated CSP-2 are respectively: d (Asp) d (Gly) d (Arg) Arg Arg Lys Lys Ala Ala Ala Ala Ala Ala-NH2.
3. Use of the self-assembled short peptide of claim 1 as a delivery vehicle.
4. Use of the self-assembled short peptide of claim 3 as a delivery vehicle for delivery of an mRNA vaccine.
5. The use according to claim 4, wherein: the delivery vehicle is mRNA.
6. The use according to claim 5, wherein: mRNA includes mRNA vaccines and RNA drugs.
7. The use according to claim 5, wherein: after mRNA loading, mRNA/CSP-1 and/or mRNA/CSP-2 forms a nanovesicle structure.
8. The use according to claim 7, wherein: the particle size of the nano vesicle structure is 200-350nm.
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