Polypeptide self-agglomerated particle capable of efficiently delivering mRNA as well as preparation method and application of polypeptide self-agglomerated particle

Abstract

The invention provides a polypeptide self-agglomerated particle capable of efficiently delivering mRNA (messenger ribonucleic acid) as well as a preparation method and application of the polypeptide self-agglomerated particle, and particularly, the invention proposes that polypeptide with the sequence of 'GHGVYGHGVYGHGPYGHGPYGHGLYW' is applied to delivery of mRNA for the first time, and the delivery efficiency in mRNA cells and the expression efficiency of functional protein are improved by utilizing the polypeptide self-agglomerated particle; experiments prove that the polypeptide self-agglomerated particle can be used as a novel mRNA delivery carrier, the transfection efficiency of the polypeptide self-agglomerated particle is remarkably higher than that of a classic commercial transfection reagent, the polypeptide self-agglomerated particle has good biological safety, the transfection operation process is simple and easy to operate, raw materials are easy to synthesize and obtain, and commercial popularization is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to polypeptide self-condensing microparticles for efficiently delivering mRNA, and a preparation method and application thereof. Background Art

[0002] Gene therapy refers to the introduction of exogenously produced nucleic acid substances into cells for the expression of corresponding proteins, which is used for disease-specific treatment or as a vaccine for disease prevention. As a new type of nucleic acid drug, messenger RNA (mRNA) has become a research hotspot in the fields of regenerative medicine, infectious disease vaccines, immunotherapy, etc. in recent years, and has great potential for clinical application. Compared with DNA-based gene therapy, mRNA therapy has many potential advantages: 1) mRNA does not need to be integrated into the cell genome, which can avoid the risk of insertional gene mutations and carcinogenesis; 2) The cell nucleus is the biggest barrier for nucleic acid drugs to enter cells and exert their effects, while mRNA can directly express functional proteins in the cytoplasm without crossing the barrier of the nuclear membrane, especially in non-dividing cells or cells that divide very slowly, greatly improving the efficacy of nucleic acid drugs.

[0003] However, the development, preparation and subsequent systemic administration of mRNA drugs face many difficulties: 1) The negative charge of mRNA itself makes it difficult to pass through the cell membrane and enter the cell, so it relies on the transcellular membrane delivery of the carrier; 2) mRNA entering the cell is easily captured by the endosome and cannot enter the cytoplasm to be translated into protein, so it relies on the endosomal escape function of the delivery carrier. Therefore, it is of great significance to develop efficient delivery mRNA carriers and efficiently express the corresponding functional proteins. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a polypeptide self-condensing microparticle for efficient delivery of mRNA and a preparation method and application thereof. The technical solution adopted by the present invention is as follows: In a first aspect, a polypeptide self-condensing particle for efficiently delivering mRNA is provided, wherein the sequence of the polypeptide is GHGVYGHGVYGHGPYGHGPYGHGLYW.

[0005] In a second aspect, a method for preparing the above-mentioned polypeptide self-condensing microparticles is provided, the steps of which include: S1. Prepare a polypeptide powder having a sequence of "GHGVYGHGVYGHGPYGHGPYGHGLYW"; S2. The polypeptide powder prepared in S1 is dissolved in an acetic acid solution to prepare a polypeptide mother solution, and the polypeptide mother solution is dropped into an RNase-free PBS solution to obtain polypeptide self-condensing microparticles. In a third aspect, the application steps of the above polypeptide self-condensing microparticles are provided, comprising: The solution of the polypeptide self-condensing microparticles is dropped into the mRNA solution expressing a specific protein, and self-condensation occurs to obtain an mRNA / polypeptide transfection complex, which is then co-cultured with cells.

[0006] Preferably, in the application of the above-mentioned polypeptide self-condensing microparticles, 0.5 mg / mL concentration of polypeptide self-condensing microparticles are dropped into the mRNA solution expressing a specific protein, and the solvent of the mRNA solution is PBS; according to the test by the inventors, 0.5 mg / mL polypeptide self-condensing microparticles have good transfection efficiency for different mRNA concentrations.

[0007] In summary, the present invention adopts a solid phase synthesis method to prepare a pure polypeptide containing 26 amino acids. By adjusting the pH of the solvent, the polypeptide can quickly self-condense from a monomer state (under a 10 mM acetic acid environment) into micron-sized particles (under a neutral-weakly alkaline environment, i.e., a pH range of 7.4-9.5), and achieve efficient loading of mRNA; the polypeptide self-condensing particles loaded with mRNA are directly co-cultured with cells, which can effectively deliver mRNA into cells, avoid endosome capture, and ultimately efficiently express the target protein, so that the polypeptide self-condensing particles can be used as a new type of mRNA delivery carrier.

[0008] The beneficial effects of the present invention are as follows: ① The mRNA transfection efficiency is significantly higher than that of the classic commercial transfection reagent Lipofectamine®2000, and has a high commercial application prospect; ② It also has a high transfection efficiency in difficult-to-transfect primary cells, and has the prospect of being developed into an mRNA vaccine or applied to mRNA therapy; ③ Good biological safety; ④ The transfection operation process is simple and easy to operate, and only one-step method is required to achieve the preparation and transfection of mRNA / polypeptide transfection complex, which is conducive to large-scale production; ⑤ The process of delivering mRNA can effectively avoid endosome capture, which helps to avoid the adverse effects of the transfection process on endosome function, and is suitable for the transfection needs of endosomal function-related research; ⑥ The time law of mRNA delivery and the law of cell membrane uptake pathway have been clearly studied; ⑦ The polypeptide contains only 26 amino acids, and the synthesis of raw materials is relatively easy, which is conducive to commercial promotion.

[0009] In summary, the polypeptide self-condensing microparticles provided by the present invention have high scientific research and technological application value and medical technology application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0011] Figure 1 is the mass spectrometry value of the polypeptide described in the present invention; Figure 2 To identify the purity of the polypeptide microparticles by high performance liquid chromatography; Figure 3 The relative turbidity of the polypeptide microparticles was obtained by measuring the absorbance at 600 nm using a microplate reader; Figure 4 To detect the particle size distribution of polypeptide self-agglomerated microparticles using a laser particle size analyzer; Figure 5 The morphology of the peptide self-condensed particles was observed and photographed using an optical microscope; Figure 6 The expression of functional proteins in each group of transfected human embryonic kidney cells (HEK293) was observed using a fluorescence microscope (A) and the statistical results of the transfection efficiency of each group (B, C); Figure 7 Fluorescence microscope observation of functional protein expression of transfected primary human trabecular meshwork cells in each group (A) and statistical transfection efficiency (B); Figure 8 The survival rates of primary human trabecular meshwork cells (A) and human embryonic kidney cells (B) after co-culture of peptide self-aggregating microparticles with cells; Fig. 9 This is a real-time video recording and tracking of the spatial localization of polypeptide self-condensing microparticles in endosomes during mRNA delivery using a spinning disk fluorescence microscope. (A) is the mRNA cell uptake stage within 4 hours of transfection, and (B) is the mRNA expression into protein stage within 24 hours of transfection. Fig.10 The spinning disk fluorescence microscope is used to record and track the delivery of mRNA by polypeptide self-condensing microparticles in real time. Almost all mRNA is delivered into the cell within 4 hours (A), and the expression level of the target protein successfully translated and expressed by the mRNA that enters the cell reaches a peak after 24 hours (B). Fig.11The fluorescence intensity distribution of peptide self-condensing microparticles delivering mRNA after treatment with different cell membrane uptake pathway inhibitors (A) and its corresponding statistical results of average fluorescence intensity (B) were detected by flow cytometry. The functional protein expression of peptide self-condensing microparticles delivering mRNA after treatment with different cell membrane uptake pathway inhibitors (C) and its corresponding statistical results of transfection efficiency (D) were detected by fluorescence microscopy. DETAILED DESCRIPTION In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with embodiments and drawings.

[0012] The present invention applies a polypeptide with a sequence of "GHGVYGHGVYGHGPYGHGPYGHGLYW" to the delivery of mRNA for the first time, and utilizes the polypeptide self-condensing microparticles to improve the mRNA intracellular delivery efficiency and the functional protein expression efficiency.

[0013] Example (1) Synthesis of peptides The pure polypeptide powder was prepared by solid phase synthesis. The polypeptide contained 26 amino acids, and the specific sequence was: glycine-histidine-glycine-valine-tyrosine-glycine-histidine-glycine-valine-tyrosine-glycine-histidine-glycine-proline-tyrosine-glycine-histidine-glycine-proline-tyrosine-glycine-histidine-glycine-leucine-tyrosine-tryptophan (GHGVYGHGVYGHGPYGHGPYGHGLYW). The theoretical molecular weight of this polypeptide was 2782.01, and the actual mass spectrometry value was 2781.95 ( Figure 1 ), the purity of which was identified by high performance liquid chromatography technology was over 95% ( Figure 2 ).

[0014] (2) Preparation of peptide self-aggregating microparticles Different volumes of peptide stock solution (prepared with 10 mM acetic acid solution) were dropped into RNase-free PBS solution to obtain peptide self-aggregated microparticles with final concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, and 5 mg / mL, respectively. The relative turbidity of the peptide microparticles was measured at 600 nm using an ELISA reader and was found to be as high as about 91.30±1.81% ( Figure 3 ), indicating successful self-agglomeration into microparticles. The particle size distribution of the self-agglomerated microparticles of the polypeptide was detected by a laser particle size analyzer and was between 0.96±0.22 μm and 2.10±0.42 μm ( Figure 4 ), the self-condensed peptide particles were observed and photographed under an optical microscope to be uniform spherical particles ( Figure 5 ).

[0015] (3) Efficient delivery of mRNA by peptide self-condensing microparticles The polypeptide solution was dropped into the mRNA solution expressing EGFP protein (EGFP-mRNA, dissolved in PBS), and self-condensation occurred immediately to obtain EGFP-mRNA / polypeptide transfection complex. After the EGFP-mRNA / polypeptide transfection complex was incubated with human embryonic kidney cells (HEK293) for 4 hours, it was replaced with cell culture medium without transfection complex and continued to be cultured for 24 hours. The transfection efficiency was observed and photographed using a fluorescence microscope and the transfection efficiency was calculated. The formulas of the components used in the experiment were: the final concentration of the polypeptide was 0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL, and the mRNA concentration was 1 μg / mL. The experimental results showed that the mRNA transfection efficiency of the polypeptide experimental group could reach 69.83±5.83%, which was significantly higher than that of the classic commercial transfection reagent Lipofectamine®2000 experimental group (59.21±5.19%); in addition, compared with other polypeptide concentrations, the 0.5 mg / mL polypeptide had the highest transfection efficiency ( Figure 6 ).

[0016] (4) Peptide self-aggregating microparticles are suitable for mRNA delivery to difficult-to-transfect primary cells The polypeptide solution was dropped into the mRNA solution expressing EGFP protein (EGFP-mRNA, dissolved in PBS), and self-condensation occurred immediately to obtain EGFP-mRNA / polypeptide transfection complex. After the EGFP-mRNA / polypeptide transfection complex was incubated with primary human trabecular meshwork cells for 4 hours, the culture medium without transfection complex was replaced and cultured for another 24 hours. The transfection efficiency was observed and photographed using a fluorescence microscope and statistically analyzed. The experimental results showed that the polypeptide can efficiently transfect primary human trabecular meshwork cells with a slower division rate (transfection efficiency is 47.97±4.69%) ( Figure 7 ).

[0017] (5) Peptide self-aggregating microparticles have good biosafety After culturing the cells with 0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL peptide self-aggregating microparticles for 4 hours, the culture medium was replaced with a normal cell culture medium for 24 hours, and the cell survival rate was detected using a CCK8 kit. The experimental results showed that the survival rate of primary human trabecular meshwork cells could reach about 100%, and the cell survival rate of human embryonic kidney cells could reach more than 80%, indicating that the peptide self-aggregating microparticles have good biosafety ( Figure 8 ).

[0018] (6) The mRNA delivery system of peptide self-condensing microparticles effectively avoids endosomal capture The fluorescent molecule Cy3-labeled EGFP-mRNA / peptide transfection complex (Cy3-EGFP-mRNA@peptide) was added to the cells, and the mature endosomes (lysosomes) were labeled with Lysosome deep red fluorescent markers. The spatial localization relationship between the peptide self-condensed microparticles and the endosomes during the delivery of mRNA was tracked in real time using a rotating disk fluorescence microscope. The experimental results showed two typical stages of mRNA intracellular delivery: mRNA cell uptake stage (within 4 hours, Fig. 9 A) and target protein expression stage (16-24 hours, Fig. 9 B) There is no co-localization between the EGFP-mRNA / peptide transfection complex and the endosome, indicating that the mRNA delivery system of the polypeptide self-condensing microparticles effectively avoids endosomal capture.

[0019] (7) Temporal pattern of mRNA delivery by peptide self-condensing microparticles The EGFP-mRNA / peptide transfection complex labeled with fluorescent molecule Cy3 (Cy3-EGFP-mRNA@peptide) was added to the cells, and a rotating disk fluorescence microscope was used to capture and track the time pattern of mRNA delivery by peptide self-condensing microparticles in real time. The experimental results showed that peptide self-condensing microparticles could deliver part of the mRNA into the cells within 40 minutes and almost complete the intracellular delivery of all mRNA in 4 hours ( Fig.10 A); Starting from 6 hours, the mRNA delivered into the cell was successfully translated and expressed into a small amount of target protein, and the expression level reached a peak at 24 hours ( Fig.10 B).

[0020] (8) Cell membrane uptake pathways of mRNA delivered by peptide self-condensing microparticles After pre-treating the cells for 1 hour with the cell membrane uptake endocytosis inhibitors methyl-β-cyclodextrin (cholesterol-dependent lipid raft endocytosis pathway), amiloride (pinocytosis pathway), ES9-17 (clathrin-mediated endocytosis pathway), and sodium azide (oxidative phosphorylation-related energy-dependent endocytosis pathway), the fluorescent molecule Cy3-labeled EGFP-mRNA / peptide transfection complex (Cy3-EGFP-mRNA@peptide) was added to the cells for co-culture for 2 hours or cultured at 4°C (energy-dependent endocytosis pathway) for 2 hours. The Cy3 average fluorescence intensity was detected by flow cytometry to characterize the uptake efficiency, and the EGFP target protein expression was photographed using a fluorescence microscope. The experimental results showed that only the 4°C treatment group significantly reduced the uptake of the EGFP-mRNA / peptide transfection complex ( Fig.11 AB) and EGFP target protein expression ( Fig.11 CD), indicating that polypeptide self-condensed particles deliver mRNA across the cell membrane into the cell via an energy-dependent endocytosis pathway.

[0021] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A polypeptide self-condensing microparticle for efficiently delivering mRNA, wherein the polypeptide sequence is: GHGVYGHGVYGHGPYGHGPYGHGLYW.

2. The method for preparing polypeptide self-condensing microparticles for efficient delivery of mRNA according to claim 1, characterized in that: The following steps are involved: S1. Prepare a polypeptide powder having a sequence of "GHGVYGHGVYGHGPYGHGPYGHGLYW"; S2. The polypeptide powder prepared in S1 is dissolved in an acetic acid solution to prepare a polypeptide mother solution, and the polypeptide mother solution is dropped into an RNase-free PBS solution to obtain polypeptide self-condensing microparticles, wherein the pH range of the RNase-free PBS solution is 7.4-9.

5.

3. The use of polypeptide self-condensing microparticles for efficient delivery of mRNA as claimed in claim 1, comprising: The solution of the polypeptide self-condensing microparticles is dropped into the mRNA solution expressing a specific protein, and self-condensation occurs to obtain an mRNA / polypeptide transfection complex, which is then co-cultured with cells.

4. The use of polypeptide self-condensing microparticles for efficient delivery of mRNA according to claim 3, characterized in that: The peptide self-condensing microparticles with a concentration of 0.5 mg / mL were dropped into the mRNA solution expressing a specific protein.

5. Use of the polypeptide self-condensing microparticles for efficient mRNA delivery as claimed in claim 1 in mRNA vaccines.

6. Use of the polypeptide self-condensing microparticles for efficient mRNA delivery as claimed in claim 1 in mRNA pharmaceutical preparations.

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