CN113413467A

CN113413467A - Carrier-free mRNA delivery method

Info

Publication number: CN113413467A
Application number: CN202110746485.9A
Authority: CN
Inventors: 喻国灿; 戚少龙
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2021-07-01
Filing date: 2021-07-01
Publication date: 2021-09-21

Abstract

The invention discloses a carrier-free mRNA delivery method. The method comprises the following steps: synthesizing carrier-free Metal-mRNA nano particles by coordination-driven self-assembly effect of Metal cations and mRNA molecules, and further realizing delivery of the mRNA molecules. The invention relates to a universal carrier-free mRNA delivery technology, which does not need a loading material to wrap mRNA, but completes self-assembly by virtue of a supermolecule coordination effect between metal ions and the mRNA, and has sensitive micro-environment response, good dispersion stability, high-efficiency mRNA delivery efficiency, low biotoxicity and good biological safety. In addition, the technology is simple and feasible, has universality, is suitable for wide metal ion and mRNA coordination assembly and delivery of different nucleic acid molecular weight lengths and different nucleic acid sequences, is easy for clinical popularization, and has wide application prospect in the field of mRNA treatment.

Description

Carrier-free mRNA delivery method

Technical Field

The invention relates to the technical fields of biological medicine technology, nano medicine, supramolecular chemistry and mRNA delivery, in particular to a carrier-free mRNA delivery method, and specifically relates to a preparation method and application of a carrier-free Metal-mRNA nano material synthesized by coordination-driven self-assembly of Metal ions and mRNA molecules.

Background

Similar to DNA gene therapy, RNA-based therapy is the latest initiative for global medical innovation and has great potential. RNA therapy was marketed from the first antisense therapy (antisense therapy) concept proposed in 1978 to the first antisense oligonucleotide (ASO) drug fomivirsen in 1998, to the RNA interference (RNAi) mechanism research in 2006 with a nobel physiology or medical prize, and in 2020, the introduction of the ultra-individualized drug Technology represented by the ASO drug Milasen into MIT Technology Review ten major breakthroughs, the development of RNA therapy is tortuous and lengthy. RNA therapy refers to therapy for regulating the expression of pathogenic genes from the root using nucleic acids having a function of treating diseases. RNA therapy can be further subdivided into the following three major directions: 1. messenger RNA therapy (Messenger RNA, mRNA) encoding a therapeutic protein or antigen; 2. small nucleic acid therapies that target nucleic acids and inhibit pathogenic RNA activity or activate gene activity, including antisense oligonucleotides (ASOs), small interfering RNAs (sirnas), micro RNAs (mi RNAs), and the like; 3. RNA aptamer (aptamer) therapy which takes protein as a target and regulates the activity of the protein.

The principle of mRNA therapy is to introduce exogenous mRNA into target cells, which allows the target cells to self-synthesize target proteins. The mRNA therapy is simple and convenient to design and prepare and high in safety, compared with the traditional gene therapy, the expression of mRNA is safe due to no risk of inserting a genome, and compared with the traditional targeted drug taking protein as a target point, the mRNA therapy is short in development period and rich in candidate target points. More importantly, mRNA drugs are not confined to dividing cells, do not risk integration into the host genome, and are subject to auto-degradation in vivo. At present, the mRNA therapy is widely applied to the treatment of various diseases such as rare diseases, tumors, infectious diseases and the like by virtue of the characteristics of high efficiency, safety and rich treatment.

The research and development of the mRNA therapy generally include the processes of sequencing, target determination, sequence design and optimization, delivery technology research, nucleic acid synthesis, purification, preparation, production and the like, the current nucleic acid design and preparation processes are relatively mature, and the efficient delivery of mRNA to various tissue cells of a human body is a key to the performance of the therapeutic function of mRNA and is a main technical difficulty of the mRNA therapy. Currently, delivery technology difficulties include: nucleic acid is unstable, is easily degraded by ribonuclease (RNase) in blood plasma and tissues, and is quickly removed by liver and kidney; the immunogenicity of the nucleic acid itself and the carrier material, thereby causing a series of side effects; ③ the nucleic acid has negative charge, so that the nucleic acid is not easy to cross over the cell membrane with negative charge and enter the cytoplasm; fourthly, the card can not be released in vivo. In addition, how to achieve targeted delivery is also the focus of current research. The solutions of the above points are focused on three links of mRNA drug preparation, namely in vitro transcription, chemical modification and delivery vectors, and the most difficult, or technical barrier to construct, is the drug delivery system. Therefore, the reasonable design of the nano delivery technology has important significance for improving the delivery of the therapeutic genes and the treatment efficiency.

At present, technologies for delivering mRNA vectors mainly include liposome nanoparticle carrier technology and polymer carrier technology, wherein liposomes are the mainstream carrier materials with the most extensive applications, and mainly include cationic liposome complexes, liposome polymers, Liposome Nanoparticles (LNPs), cationic nanoemulsions, and the like. Cationic polymers have been widely used for nucleic acid delivery, such as poly (L-lysine), PEI, DEAE-dextran, PBAE and chitosan. In the simplest form, the cationic polymer is mixed with the nucleic acid in excess to form an electrostatically bound cationic polymer. Although many polymers have been developed, they are less advanced in nucleic acid delivery than lipid nanoparticles. LNP delivery technology has achieved good clinical results, but LNP vector technology still has great promotion space, for example, LNP has problems of anaphylaxis, easy oxidative degradation, poor preparation reproducibility, etc. In addition, the mRNA preparation prepared by taking LNP as a carrier can be gathered in the liver and spleen, is difficult to target other parts, and has potential toxic and side effects in clinical use. In recent years, researches show that although PEG in liposome nano materials can effectively prolong half-life and improve water solubility, more and more evidences prove that the great application of PEG can possibly cause PEG 'distress', accelerate the in vivo clearance rate of PEG modified nano-drugs and seriously influence the curative effect of the PEG nano-drugs. Therefore, developing a simple, universal and efficient method for synthesizing a nano-scale mRNA nano-delivery system with specific morphology and function has important significance for various applications.

Disclosure of Invention

The invention aims to provide a carrier-free mRNA delivery method.

In a first aspect, the invention claims a method for vector-free mRNA delivery.

The presently claimed vector-free mRNA delivery method may comprise the steps of:

(A1) synthesizing carrier-free Metal-RNA nano particles (hereinafter referred to as Metal-mRNA) by coordination-driven self-assembly effect of Metal cations and mRNA molecules;

(A2) the delivery of the RNA molecule is achieved in the form of the carrier-free Metal-mRNA nanoparticles.

The non-carrier metal-mRNA nanoparticles obtained in step (a1) can be prepared by the method of the second aspect.

In a second aspect, the invention claims a method of making carrier-free Metal-mRNA nanoparticles.

The method for preparing carrier-free Metal-mRNA nano particles, which is claimed by the invention, can comprise the following steps:

(B1) mixing the target mRNA and the metal cation according to the charge ratio of 500/1-1/500;

(B2) standing the treated system (B1) at 37-60 deg.C (such as 55 deg.C), and naturally cooling;

(B3) and (B2) centrifuging the system after treatment, and collecting precipitates to obtain the carrier-free Metal-mRNA nano particles.

Further, in the step (B1), the charge ratio of the mRNA of interest and the metal cation may be 100/1 to 1/10.

Further, the charge ratio of the mRNA of interest and the metal cation can be 1: 2.

further, in the step (B1), the mixing of the mRNA of interest and the metal cation may be achieved by: the aqueous solution containing the metal cation is added dropwise to the aqueous solution of the target mRNA, and stirred for 1 to 10min (e.g., 1 min).

Further, in the step (B2), the standing time may be 1.5 to 4 hours (e.g., 3 hours).

Further, in the step (B2), the temperature after the natural cooling is room temperature.

Further, in the step (B3), a step of washing the treated system of (B2) with deionized water may be further included before the centrifugation.

Further, in the step (B3), the centrifugation is 10000-. The centrifugation may be performed at room temperature.

Further, in the step (B3), the centrifugation may further include a step of dispersing the precipitate in deionized water for storage.

In the first and second aspects above, the valency of the metal cation may be from +2 to +8 (e.g. +2, + 4); the metal cation is any metal cation that can participate in the phosphate coordination reaction of RNA and can be bioavailable.

Further, the metal cation may be selected from the following: ca²⁺、Zr⁴⁺、Zn²⁺、Fe²⁺、Fe³⁺、Mn²⁺、 Cu²⁺、Al³⁺Or/and Hf⁴⁺And the like. The metal cation can be a single metal cation, or different metal cations can be mixed for use.

Further, the mRNA may be a naked mRNA or a modified mRNA. Wherein the modification includes, but is not limited to, phosphorylation and/or glycosylation.

In a specific embodiment of the present invention, the target mRNA is specifically the following mRNA: mouse IL-10, Human EPO, SV40 NLS-Cas9, NLS-Cre, Mouse ECD, EGFP, Luciferase.

In a particular embodiment of the invention, when the metal cation is Ca²⁺When the metal cation-containing aqueous solution is CaCl₂An aqueous solution. More specifically, the CaCl₂CaCl in aqueous solution₂The concentration of (3) was 798 mM. Accordingly, the mRNA of interest is an EGFP mRNA. More specifically, the EGFP mRNAThe concentration of the aqueous solution was 1 mM. In the step (B1), the EGFP mRNA aqueous solution and the CaCl₂The dosage ratio of the aqueous solution is 1000 mu L: 1000 μ L, charge ratio 1: 2.

In a particular embodiment of the invention, when the metal cation is Al³⁺When the "aqueous solution containing the metal cation" is specifically AlCl₃An aqueous solution. More specifically, the AlCl₃AlCl in aqueous solution₃The concentration of (3) was 798 mM. Accordingly, the mRNA of interest is an EGFP mRNA. More specifically, the EGFP mRNA aqueous solution concentration is 1.5 mM. In the step (B1), the EGFP mRNA aqueous solution and the AlCl₃The dosage ratio of the aqueous solution is 1000 mu L: 1000 μ L, charge ratio 1: 2.

In a particular embodiment of the invention, when the metal cation is Zr⁴⁺When the "aqueous solution containing the metal cation" is ZrF₄An aqueous solution. More specifically, the ZrF₄ZrF in aqueous solution₄The concentration of (3) was 798 mM. Accordingly, the mRNA of interest is an EGFP mRNA. More specifically, the concentration of the EGFP mRNA aqueous solution is 2 mM. In the step (B1), the EGFP mRNA aqueous solution and the ZrF₄The dosage ratio of the aqueous solution is 1000 mu L: 1000 μ L, charge ratio 1: 2.

In a specific embodiment of the invention, the optimal charge ratio of the target mRNA to the metal cation is 1:2 and the volume ratio is 1: 1. The mRNA of interest is EGFP mRNA. The charging ratio of the other valence metal ions is similar to the three types: namely, 2-valent metal: mRNA (molar ratio) 798: 1; a 3-valent metal: mRNA 798: 1.5; 4-valent metal: mRNA 798: 2; other cases may be analogized depending on the specific RNA sequence and metal ion species.

In a third aspect, the invention claims an unsupported Metal-mRNA nanoparticle prepared by the method of the second aspect.

The particle size of the carrier-free Metal-mRNA nano particle obtained by the invention is 20-300nm, and the zeta potential is-100 to 40.

In a fourth aspect, the invention claims the use of the method of the first or second aspect above or the carrier-free Metal-mRNA nanoparticles of the third aspect above for carrier-free delivery of an mRNA of interest to a cell or tissue or organism.

The application may be a non-disease diagnostic therapeutic application.

The present invention is applicable to not only mRNA but also siRNA and/or microRNA.

The technology of the invention synthesizes the carrier-free Metal-mRNA nano particles by the coordination-driven self-assembly effect of Metal ions and mRNA molecules, and has the following advantages: 1. can easily penetrate cell membrane and quickly enter cell. 2. The Metal-mRNA nano particles have good biocompatibility and are more stable. 3. mRNA is easier to dissociate in cytoplasm, so that the endosome escape performance is enhanced, the cell delivery efficiency is improved, and the target protein expression is increased. 4. Metal-mRNA nanoparticles can realize carrier-free delivery and immune disturbance of carrier-free materials. 5. The one-pot supermolecule self-assembly strategy is extremely simple and easy to implement, saves the cost and is suitable for large-scale transformation and application. 6. The technology has universality and is suitable for mRNA delivery of different nucleic acid molecular weight lengths and different nucleic acid sequences.

In conclusion, the invention relates to a universal carrier-free mRNA delivery technology, which does not need a loading material to wrap mRNA, completes self-assembly by virtue of a supermolecule coordination effect between metal ions and mRNA, and has sensitive micro-environment response, good dispersion stability, high-efficiency mRNA delivery efficiency, low biological toxicity and good biological safety. In addition, the technology is simple and feasible, has universality, is suitable for wide metal ion and mRNA coordination assembly and delivery of different nucleic acid molecular weight lengths and different nucleic acid sequences, is easy for clinical popularization, and has wide application prospect in the field of mRNA treatment.

Drawings

FIG. 1 is a schematic diagram of the synthesis of Metal-mRNA nanoparticles by coordination-driven self-assembly of Metal cations and mRNA molecules according to the present invention.

FIG. 2 is an electron micrograph of Ca-mRNA (different sequences) nanoparticles

FIG. 3 shows the survival rate of cells in each group measured by MTT method.

FIG. 4 shows the liver function changes in mice of different groups.

FIG. 5 shows the expression of inflammatory factors in mice of different groups.

FIG. 6 shows EGFP mRNA delivery and quantitation of GFP protein expression for different Metal-EGFP mRNAs.

FIG. 7 is a graph showing the effect of Ca-EGFP mRNA and Lipo2000-mRNA protein expression under a fluorescence microscope.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The invention provides a universal technology for synthesizing Metal-mRNA nano particles by coordination-driven self-assembly of Metal cations and mRNA molecules, which is a carrier-free mRNA delivery technology and a synthetic schematic diagram of the technology is shown in figure 1.

Wherein n is an integer between 2 and 8, and represents the valence potential number of the selected metal ion (i.e., the valence of the metal cation may be from +2 to + 8);

m represents a metal ion species including Mn²⁺，Cu²⁺，Zn²⁺，Fe²⁺，Ca²⁺，Zr²⁺，Fe³⁺，Zr³⁺， Zr⁴⁺，Hf⁴⁺And all the biologically available metal ions which can participate in the phosphate coordination reaction of RNA in the nature, and different metal ions can also be mixed for use, thereby achieving similar assembly effect.

mRNA Chain is used for delivering mRNA, and comprises mRNA chains with different nucleic acid molecular weight lengths and different nucleic acid sequences (including mRNA post-modification, such as phosphorylation and glycosylation and the like). Meanwhile, the discovery is also suitable for other RNA systems, such as siRNA, microRNA and the like.

In the invention, the Metal-mRNA nano-particle consists of Metal ions and mRNA fragments, has uniform particle size and stable property, and can be simply and efficiently used for mRNA delivery in organisms.

Example 1 Synthesis of Metal-mRNA nanoparticles (Ca)²⁺Example of the design reside in

In this example, the selected metal ion is Ca²⁺And the mRNA is EGFP mRNA (SEQ ID No. 6). Specifically, 1000. mu.L of CaCl was added₂An aqueous solution (798mM) was added dropwise to 1000. mu.L of an aqueous solution of EGFP mRNA (1mM) so that EGFP mRNA was brought into contact with Ca²⁺Has a charge ratio of 1: 2. Stirring thoroughly for 1min to mix well, standing the mixture at 55 deg.C for 3 hr, and naturally cooling to room temperature. Washing the obtained product with deionized water, centrifuging at 12000 r/min for 5min at room temperature to obtain precipitate as Ca-EGFP mRNA nano particle, and dispersing in deionized water for storage.

The charge ratio of the metal cations and the mRNA sequences adopted in the reaction is determined according to the charge ratio relationship of the mRNA and the metal cations: mRNA^-/M⁺＝500/1～1/500(M⁺Represents the positive charge of the metal cation, as described in table 1: zn (ZnCl)₂)，Fe(FeCl₂)，Mn(MnCl₂)，Cu(CuSO₄)，Ca(CaCl₂)，Fe(FeCl₃)，Al(AlCl₃)， Zr(ZrF₄)，Hf(Hf Cl₄) Etc., mRNA^-Representing the negative charge of different base sequences. The mRNA is specifically Mouse IL-10 (the tail end carries 6 XHis), Human EPO, Mouse ECD (the tail end carries 6 XHis), NLS-Cre, SV40 NLS-Cas9, EGFP and Luciferase (the specific sequences of the mRNA are sequentially shown as SEQ ID No.1 to SEQ ID No. 7). The implementation method of coordination self-assembly of other metal ions and mRNA is similar to the above, according to the optimal charge ratio of the target mRNA and metal cations being 1:2, the mRNA is selected to be EGFP mRNA, the volume ratio of the two substances added is 1:1 as an example, the feeding ratio of the metal ions to the mRNA is as follows: 2-valent metal/mRNA (molar ratio, the same applies hereinafter) 798/1; metal 3/mRNA 798/1.5; 4 valence metal/mRNA 798/2; in specific implementation, the base number of mRNA with different sequences and lengths can be adjusted according to the principle according to the valence potential of the metal ion and the selected mRNA.

The experimental results prove that the delivery technology has universality and is suitable for mRNA with different metal ions and different sequences and lengths (see table 1 and figure 2 for details).

TABLE 1 partial nanoparticle sizes of different metal ions coordinated with mRNA of different lengths and sequences

FIG. 2 shows Ca²⁺Electron micrograph of mRNA (different sequence) nanoparticles. a is Ca-EGFP mRNA; b is Ca-Luciferase mRNA.

As can be seen from Table 1 and FIG. 2, the Metal ions with different valence states and RNAs with different lengths and sequences can be well assembled through coordination, so as to form Metal-mRNA nanoparticles with uniform particle size, controllable morphology, uniform dispersion and stability.

Example 2 Metal-mRNA nanoparticle Dispersion stability evaluation

According to the method described in example 1, the metal ions are Ca²⁺For example, the mRNA is EGFP mRNA. An aqueous solution of metal ions was chosen at a concentration of 798mM and an aqueous solution of EGFP mRNA at a concentration of 1mM, based on the ratio of EGFP mRNA to metal cation charge: 100/1-1/10, calculating the volume of the two materials added respectively. Specifically, when the charge ratio is 100/1, the volume ratio of mRNA/metal is 200/1; when the charge ratio is selected to be 50/1, the volume ratio of mRNA/metal is 100/1; when the charge ratio is selected to be 30/1, the volume ratio of mRNA/metal is 60/1; when the charge ratio is selected to be 20/1, the volume ratio of mRNA/metal is 40/1; when the charge ratio is selected to be 10/1, the volume ratio of mRNA/metal is 20/1; when the charge ratio is selected to be 5/1, the volume ratio of mRNA/metal is 10/1; when the charge ratio is selected to be 2/1, the volume ratio of mRNA/metal is 4/1; when the charge ratio is selected to be 1/1, the volume ratio of mRNA/metal is 2/1; when the charge ratio is selected to be 1/2, the volume ratio of mRNA/metal is 1/1; when the charge ratio is selected 1/5, mRThe volume ratio of NA/metal is 1/2.5; when the charge ratio was chosen to be 1/10, the mRNA/metal volume ratio was 1/5. The other valency metals may be converted according to the relationship given in example 1. Specifically, the desired Metal-mRNA nanoparticles were synthesized as described in example 1. The dispersion stability of Metal-mRNA nanoparticles under different charge ratios is evaluated by a zeta potential method, a light transmittance method (a spectrophotometer) and other methods. (see tables 2 and 3 for details).

TABLE 2 zeta potential changes of Metal-EGFP mRNA nanoparticles at different Metal particles and ratios

Table 3, Metal-EGFP mRNA nanoparticle dispersion stability variation conditions under different Metal particles and ratios

As can be seen from table 2: with the increase of the charge ratio of mRNA/Metal ions, the Metal-EGFP mRNA nano system gradually becomes neutral and weak electropositive. Correspondingly, the nano system can penetrate cell membranes more easily, and can enter the interior of cells more efficiently to finish mRNA delivery.

As can be seen from Table 3, the Metal-mRNA nanosystem is more stable in this range with increasing charge ratio of mRNA/Metal ion, and the system can be kept intact before reaching the target environment.

Example 3 in vitro cytotoxicity assay

HEK293 cells (CRL-1573-293) were seeded in 96-well plates in DMEM medium (10% fetal bovine serum and 1% penicillin). Cells were incubated at 37 ℃ in the presence of 5% CO₂Is incubated in an atmosphere of (2). Cells were incubated for 24 hours and then replaced with fresh medium. Different Metal ion and mRNA assemblies (prepared in example 1, Metal-EGFP mRNA, 15. mu.g), Lipofectamin 2000-EGFP mRNA (15. mu.g) and PEI-EGFP mRNA (15. mu.g) were added as commercial mRNA delivery model controls (see below for specific preparation methods). Incubating the cellsAfter 48 hours, the original medium was replaced with 100. mu.L of medium containing 0.5mg/mL of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium (MTT). After 4 hours of incubation, the medium containing MTT was removed and carefully washed 3 times with PBS. Then, DMSO (100. mu.L) was added, absorbance at a wavelength of 570nm was measured in a BioTek Synergy H4 reader, and cell viability was calculated to verify cytotoxicity of the assembly obtained in the present invention.

Attached: synthesis method of PEI-EGFR mRNA and Lipofectamin 2000-EGFP mRNA

An appropriate amount of PEI/Lipofectamin 2000 (all commercially available) was dissolved in 15ml PBS with EGFP mRNA in a mass ratio of PEI or Lipofectamin 2000/EGFP mRNA of 3/1. Stirring at room temperature overnight, and centrifuging the obtained precipitate to obtain PEI-EGFR mRNA and Lipofectamin 2000-EGFP mRNA.

FIG. 3 shows the survival rate of cells of each group measured by MTT method, a: Zr-EGFP mRNA; Zn-EGFP mRNA; fe (II) -EGFP mRNA; d Fe (III) -EGFP mRNA; e is Mn-EGFP mRNA; f is Cu-EGFP mRNA; Ca-EGFP mRNA; h is Al-EGFP mRNA; Hf-EGFP mRNA; j lipofectamine2000-EGFP mRNA; k is PEI-EGFP mRNA.

As can be seen from FIG. 3, the Metal-EGFP mRNA nanoparticles have significantly reduced cytotoxicity and exhibit good biological safety compared with Lipofectamine2000-EGFP mRNA and PEI-EGFP mRNA.

Example 4 Metal-mRNA nanoparticle biosafety analysis

Balb/c mice were divided into 5 groups of 5 mice each, and the Balb/c mice (4-6 weeks old, female, approximately 18-20g in weight) were fed under normal conditions with a 12 hour light-dark cycle per day. PBS, PEI-EGFP mRNA (50. mu.g), PEI-EGFP mRNA (10. mu.g), Ca-EGFP mRNA (50. mu.g) and Ca-EGFP mRNA (10. mu.g) were injected tail vein separately. The Ca-EGFP mRNA was prepared as described in example 1; see example 3 for PEI-EGFP mRNA preparation. After injection, venous blood of the mice is taken 5 to 7 days, and the supernatant is obtained by centrifugation, and various indexes of liver function are measured.

Mice were divided into groups under the same conditions, and injected into tail vein: LPS (15 μ g); II: r848(15 μ g); III: PBS (15. mu.g); IV: PEI-EGFP mRNA (15. mu.g); v: Ca-EGFP mRNA (15. mu.g). The Ca-EGFP mRNA was prepared as described in example 1; PEI-EGFP mRNA was prepared according to example 3. After injection, venous blood of the mice is taken 5 to 7 days, supernatant fluid is obtained by centrifugation, and the level of inflammatory factors in tissues is determined by enzyme-linked immunosorbent assay (ELISA).

FIG. 4 shows the liver function changes in mice of different groups. Wherein a is ALT (glutamic pyruvic transaminase); b is AST (aspartate aminotransferase); c is LDH (lactate dehydrogenase); d is Total bilirubin (Total bilirubin). I: PBS; II: PEI-EGFP mRNA (50. mu.g); III: PEI-EGFP mRNA (10. mu.g); IV: Ca-EGFP mRNA (50. mu.g); v: Ca-EGFP mRNA (10. mu.g). As can be seen from FIG. 4, the Ca-EGFP mRNA nanoparticles had substantially no effect on liver function, were significantly smaller than PEI-EGFP mRNA, and were substantially equivalent to the PBS group. The results show that Metal-mRNA has little liver metabolic toxicity and good biological safety.

FIG. 5 shows the expression of different inflammatory factors in different groups of mice. Wherein a is IL-1 β; b is IL-10; c is IL-6; d is IL-8. I: LPS (positive control); II: r848 (positive control); III: PBS; IV: PEI-EGFP mRNA; v: Ca-EGFP mRNA. As can be seen in FIG. 5, the expression of inflammatory factors in vivo after the injection of Ca-EGFP mRNA nanoparticles was maintained at a low level, which was substantially equal to that of PBS. The results show that Metal-mRNA has small immunogenicity and good biological safety.

Example 5 intracellular protein expression of Metal-mRNA nanoparticles

In this example, the Luciferase mRNA was selected as the target mRNA and the intracellular protein expression experiment was performed. Specifically, the ratio of Luciferase mRNA to metal cation charge was adjusted as described in example 1: 100/1-1/10. In particular, with Ca²⁺For example, molar concentration ratio: 1/1818, charge ratio: 1/2. The Luciferase mRNA is mixed with CaCl₂The volume ratio of the aqueous solution was 1/1 (in the case of other charge ratios, the volume ratio was calculated by the method in example 2). In particular, with Al³⁺For example, the concentration ratio: 1.5/1818, charge ratio: 1/2. The Luciferase mRNA and AlCl₃The volume ratio of the aqueous solution was 1/1 (in the case of other charge ratios, the volume ratio was calculated by the method in example 2). In particular, with Hf⁴⁺For example, asConcentration ratio of 1/909, charge ratio: 1/2. The Luciferase mRNA and HfCl₄The volume ratio of the aqueous solution was 1/1 (in the case of other charge ratios, the volume ratio was calculated by the method in example 2). The obtained nanoparticles are used for verifying intracellular delivery efficiency and protein expression efficiency of Metal-mRNA. The feasibility and universality of this method were demonstrated by measuring the bioluminescent signal of intracellular luciferase (see table 4 for details).

TABLE 4 luciferase expression levels in Metal-mRNA (luciferase mRNA) cells at different Metal particle and ratio

As can be seen from Table 4, the luciferase protein expression tends to increase as the ratio of metal ions increases. This is due to two reasons: firstly, the metal ion ratio is improved, which is beneficial to the stability of mRNA assembly and the intracellular expression of mRNA; and secondly, the proportion of metal ions is improved, the Zeta potential value of the assembly is close to neutral or electropositive, so that endocytosis of cells is facilitated, and the transfer efficiency is improved. The two cooperate to improve the delivery of the target mRNA and the protein expression.

Example 6 intracellular expression assay of Metal-EGFP mRNA

In this example, EGFP mRNA was selected as the delivery mRNA and Ca was selected²⁺As metal ions, Ca-EGFP mRNA nanoparticles were synthesized according to the method of example 1, then the Ca-EGFP mRNA (0.2. mu.g/ml) nanoparticles were incubated with HEK293 cells for 24h to express green fluorescent protein, the culture medium in the cell culture dish was removed, and washed 3 times with PBS. The expression efficiency of the green fluorescent protein of the Ca-EGFP mRNA delivery system is qualitatively and quantitatively detected by a fluorescence microscope and flow cytometry. The control group comprises Lipo2000-EGFP mRNA nanoparticles and single EGFP mRNA. Other Metal-EGFP mRNA intracellular expression experiment methodsSimilarly.

FIG. 6 shows EGFP mRNA delivery and GFP protein expression for different Metal-EGFP mRNAs, where a: Zr-EGFP mRNA, b: Zn-EGFP mRNA, c: fe (ii) -EGFP mRNA, d: fe (iii) -EGFP mRNA, e: Mn-EGFP mRNA, f: Cu-EGFP mRNA, g: Ca-EGFP mRNA, h: Al-EGFP mRNA, i: Hf-EGFP mRNA, j: lipo2000-EGFP mRNA, k: EGFP mRNA, l: PBS. As can be seen in FIG. 6, Metal-EGFP mRNA has similar high-efficiency mRNA delivery ability and protein expression efficiency compared with Lipo2000-EGFP mRNA.

FIG. 7 is a graph showing the effect of Ca-EGFP mRNA protein expression and Lipo2000-EGFP mRNA protein expression under a fluorescence microscope. As can be seen from FIG. 7, both expressed bright GFP fluorescence in the cells, and both expressed well. In conclusion, the Metal-mRNA has high-efficiency mRNA delivery capability and protein expression efficiency and excellent transformation application prospect.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> Qinghua university

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<213> Artificial sequence

<400> 3

augcuagccc gcgccccucc ucgccgcccg ccgcggcugg ugcugcuccg uuugcuguug 60

cugcaucugc ugcugcucgc ccugcgcgcc cgcugccuga gcgcugagcc gggucagggc 120

gcgcagaccu gggcucgcuu cgcgcgcgcu ccugccccag aggccgcugg ccuccuccac 180

gacaccuucc ccgacgguuu ccucugggcg guaggcagcg ccgccuauca gaccgagggc 240

ggcuggcgac agcacggcaa aggcgcgucc aucugggaca cuuucaccca ucacucuggg 300

gcggccccgu ccgacucccc gaucgucgug gcgccgucgg gugccccguc gccuccccug 360

uccuccacug gagauguggc cagcgauagu uacaacaacg ucuaccgcga cacagagggg 420

cugcgcgaac ugggggucac ccacuaccgc uucuccauau cgugggcgcg ggugcucccc 480

aauggcaccg cgggcacucc caaccgcgag gggcugcgcu acuaccggcg gcugcuggag 540

cggcugcggg agcugggcgu gcagccggug guuacccugu accauuggga ccugccacag 600

cgccugcagg acaccuaugg cggaugggcc aaucgcgccc uggccgacca uuucagggau 660

uaugccgagc ucugcuuccg ccacuucggu ggucagguca aguacuggau caccauugac 720

aaccccuacg ugguggccug gcacggguau gccaccgggc gccuggcccc gggcgugagg 780

ggcagcucca ggcucgggua ccugguugcc cacaaccuac uuuuggcuca ugccaaaguc 840

uggcaucucu acaacaccuc uuuccgcccc acacagggag gccggguguc uaucgccuua 900

agcucccauu ggaucaaucc ucgaagaaug acugacuaua auaucagaga augccagaag 960

ucucuugacu uugugcuagg cugguuugcc aaacccauau uuauugaugg cgacuaccca 1020

gagaguauga agaacaaccu cucgucucuu cugccugauu uuacugaauc ugagaagagg 1080

cucaucagag gaacugcuga cuuuuuugcu cucuccuucg gaccaaccuu gagcuuucag 1140

cuauuggacc cuaacaugaa guuccgccaa uuggagucuc ccaaccugag gcagcuucug 1200

ucuuggauag aucuggaaua uaaccacccu cccauauuua uuguggaaaa uggcugguuu 1260

gucucgggaa ccaccaaaag ggaugaugcc aaauauaugu auuaucucaa gaaguucaua 1320

auggaaaccu uaaaagcaau cagacuggau ggggucgacg ucauugggua caccgcgugg 1380

ucgcucaugg acgguuucga guggcauagg ggcuacagca uccggcgagg acucuucuac 1440

guugacuuuc ugagucagga caaggagcug uugccaaagu cuucggccuu guucuaccaa 1500

aagcugauag aggacaaugg cuuuccuccu uuaccugaaa accagccccu ugaagggaca 1560

uuucccugug acuuugcuug gggaguuguu gacaacuacg uucaagugga cacuacucuc 1620

ucucaguuua cugacccgaa ugucuaucug ugggaugugc aucacaguaa gaggcuuauu 1680

aaaguagacg ggguuguagc caagaagaga aaaccuuacu guguugauuu cucugccauc 1740

cggccucaga uaaccuuacu ucgagaaaug cgggucaccc acuuucgcuu cucccuggac 1800

ugggcccuga ucuugccucu ggguaaccag acccaaguga accacacggu ucugcacuuc 1860

uaccgcugca ugaucagcga gcuggugcac gccaacauca cuccaguggu ggcccugugg 1920

cagccagcag ccccgcacca aggccugcca caugcccuug caaaacaugg ggccugggag 1980

aacccgcaca cugcucuggc guuugcagac uacgcaaacc uguguuuuaa agaguugggu 2040

cacuggguca aucucuggau caccaugaac gagccaaaca cacggaacau gaccuaucgu 2100

gccgggcacc accuccugag agcccaugcc uuggcuuggc aucuguacga ugacaaguuu 2160

agggcggcuc agaaaggcaa aauauccauc gccuugcagg cugacuggau agaaccggcc 2220

ugcccuuucu cucaaaauga caaagaagug gccgagagag uuuuggaauu ugauauaggc 2280

uggcuggcag agccuauuuu ugguuccgga gauuauccac gugugaugag ggacuggcug 2340

aaccaaaaaa acaauuuucu uuugcccuau uucaccgaag augaaaaaaa gcuaguccgg 2400

guuccuuuga cuuccuggcg gugagucauu acaccaccau ucugguagac ugggaaaagg 2460

aggauccgau gaaauacaac gauuacuugg agguacagga gaugacugac aucacauggc 2520

ucaacucucc cagucaggug gcaguggugc cuugggggcu gcgcaaagug cucaacuggc 2580

uaagguucaa guacggagac cucccgaugu augugacagc caauggaauc gaugaugacc 2640

cccacgccga gcaagacuca cugaggaucu auuauauuaa gaauuaugug aaugaggcuc 2700

ugaaagccua cguguuggac gacaucaacc uuuguggcua cuuugcguau ucacuuagug 2760

aucgcucagc ucccaagucu ggcuuuuauc gauaugcugc gaaucaguuu gagcccaaac 2820

caucuaugaa acauuacagg agaauuauug acagcaaugg cuuccugggu ucuggaacac 2880

ugggaagguu uuguccagaa gaauacacug ugugcaccga auguggauuu uuucaaaccc 2940

ggaagggcgg cucucaccac caccaucacc acuaa 2975

<210> 4

<211> 1053

<212> RNA

<213> Artificial sequence

<400> 4

augcccaaga agaagaggaa gguggccaau uuacugaccg uacaccaaaa uuugccugca 60

uuaccggucg augcaacgag ugaugagguu cgcaagaacc ugauggacau guucagggau 120

cgccaggcgu uuucugagca uaccuggaaa augcuucugu ccguuugccg gucgugggcg 180

gcauggugca aguugaauaa ccggaaaugg uuucccgcag aaccugaaga uguucgcgau 240

uaucuucuau aucuucaggc gcgcggucug gcaguaaaaa cuauccagca acauuugggc 300

cagcuaaaca ugcuucaucg ucgguccggg cugccacgac caagugacag caaugcuguu 360

ucacugguua ugcggcguau ccgaaaagaa aacguugaug ccggugaacg ugcaaaacag 420

gcucuagcgu ucgaacgcac ugauuucgac cagguucguu cacucaugga aaauagcgau 480

cgcugccagg auauacguaa ucuggcauuu cuggggauug cuuauaacac ccuguuacgu 540

auagccgaaa uugccaggau caggguuaaa gauaucucac guacugacgg ugggagaaug 600

uuaauccaua uuggcagaac gaaaacgcug guuagcaccg cagguguaga gaaggcacuu 660

agccuggggg uaacuaaacu ggucgagcga uggauuuccg ucucuggugu agcugaugau 720

ccgaauaacu accuguuuug ccgggucaga aaaaauggug uugccgcgcc aucugccacc 780

agccagcuau caacucgcgc ccuggaaggg auuuuugaag caacucaucg auugauuuac 840

ggcgcuaagg augacucugg ucagagauac cuggccuggu cuggacacag ugcccguguc 900

ggagccgcgc gagauauggc ccgcgcugga guuucaauac cggagaucau gcaagcuggu 960

ggcuggacca auguaaauau ugucaugaac uauauccgua accuggauag ugaaacaggg 1020

gcaauggugc gccugcugga agauggcgau uaa 1053

<210> 5

<211> 4203

<212> RNA

<213> Artificial sequence

<400> 5

auggccccaa agaagaagcg gaaggucggu auccacggag ucccagcagc cgacaagaag 60

uacagcaucg gccuggacau cggcaccaac ucugugggcu gggccgugau caccgacgag 120

uacaaggugc ccagcaagaa auucaaggug cugggcaaca ccgaccggca cagcaucaag 180

aagaaccuga ucggagcccu gcuguucgac agcggcgaaa cagccgaggc cacccggcug 240

aagagaaccg ccagaagaag auacaccaga cggaagaacc ggaucugcua ucugcaagag 300

aucuucagca acgagauggc caagguggac gacagcuucu uccacagacu ggaagagucc 360

uuccuggugg aagaggauaa gaagcacgag cggcacccca ucuucggcaa caucguggac 420

gagguggccu accacgagaa guaccccacc aucuaccacc ugagaaagaa acugguggac 480

agcaccgaca aggccgaccu gcggcugauc uaucuggccc uggcccacau gaucaaguuc 540

cggggccacu uccugaucga gggcgaccug aaccccgaca acagcgacgu ggacaagcug 600

uucauccagc uggugcagac cuacaaccag cuguucgagg aaaaccccau caacgccagc 660

ggcguggacg ccaaggccau ccugucugcc agacugagca agagcagacg gcuggaaaau 720

cugaucgccc agcugcccgg cgagaagaag aauggccugu ucggaaaccu gauugcccug 780

agccugggcc ugacccccaa cuucaagagc aacuucgacc uggccgagga ugccaaacug 840

cagcugagca aggacaccua cgacgacgac cuggacaacc ugcuggccca gaucggcgac 900

caguacgccg accuguuucu ggccgccaag aaccuguccg acgccauccu gcugagcgac 960

auccugagag ugaacaccga gaucaccaag gccccccuga gcgccucuau gaucaagaga 1020

uacgacgagc accaccagga ccugacccug cugaaagcuc ucgugcggca gcagcugccu 1080

gagaaguaca aagagauuuu cuucgaccag agcaagaacg gcuacgccgg cuacauugac 1140

ggcggagcca gccaggaaga guucuacaag uucaucaagc ccauccugga aaagauggac 1200

ggcaccgagg aacugcucgu gaagcugaac agagaggacc ugcugcggaa gcagcggacc 1260

uucgacaacg gcagcauccc ccaccagauc caccugggag agcugcacgc cauucugcgg 1320

cggcaggaag auuuuuaccc auuccugaag gacaaccggg aaaagaucga gaagauccug 1380

accuuccgca uccccuacua cgugggcccu cuggccaggg gaaacagcag auucgccugg 1440

augaccagaa agagcgagga aaccaucacc cccuggaacu ucgaggaagu gguggacaag 1500

ggcgcuuccg cccagagcuu caucgagcgg augaccaacu ucgauaagaa ccugcccaac 1560

gagaaggugc ugcccaagca cagccugcug uacgaguacu ucaccgugua uaacgagcug 1620

accaaaguga aauacgugac cgagggaaug agaaagcccg ccuuccugag cggcgagcag 1680

aaaaaggcca ucguggaccu gcuguucaag accaaccgga aagugaccgu gaagcagcug 1740

aaagaggacu acuucaagaa aaucgagugc uucgacuccg uggaaaucuc cggcguggaa 1800

gaucgguuca acgccucccu gggcacauac cacgaucugc ugaaaauuau caaggacaag 1860

gacuuccugg acaaugagga aaacgaggac auucuggaag auaucgugcu gacccugaca 1920

cuguuugagg acagagagau gaucgaggaa cggcugaaaa ccuaugccca ccuguucgac 1980

gacaaaguga ugaagcagcu gaagcggcgg agauacaccg gcuggggcag gcugagccgg 2040

aagcugauca acggcauccg ggacaagcag uccggcaaga caauccugga uuuccugaag 2100

uccgacggcu ucgccaacag aaacuucaug cagcugaucc acgacgacag ccugaccuuu 2160

aaagaggaca uccagaaagc ccaggugucc ggccagggcg auagccugca cgagcacauu 2220

gccaaucugg ccggcagccc cgccauuaag aagggcaucc ugcagacagu gaagguggug 2280

gacgagcucg ugaaagugau gggccggcac aagcccgaga acaucgugau cgaaauggcc 2340

agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaau gaagcggauc 2400

gaagagggca ucaaagagcu gggcagccag auccugaaag aacaccccgu ggaaaacacc 2460

cagcugcaga acgagaagcu guaccuguac uaccugcaga augggcggga uauguacgug 2520

gaccaggaac uggacaucaa ccggcugucc gacuacgaug uggaccauau cgugccucag 2580

agcuuucuga aggacgacuc caucgacaac aaggugcuga ccagaagcga caagaaccgg 2640

ggcaagagcg acaacgugcc cuccgaagag gucgugaaga agaugaagaa cuacuggcgg 2700

cagcugcuga acgccaagcu gauuacccag agaaaguucg acaaucugac caaggccgag 2760

agaggcggcc ugagcgaacu ggauaaggcc ggcuucauca agagacagcu gguggaaacc 2820

cggcagauca caaagcacgu ggcacagauc cuggacuccc ggaugaacac uaaguacgac 2880

gagaaugaca agcugauccg ggaagugaaa gugaucaccc ugaaguccaa gcuggugucc 2940

gauuuccgga aggauuucca guuuuacaaa gugcgcgaga ucaacaacua ccaccacgcc 3000

cacgacgccu accugaacgc cgucguggga accgcccuga ucaaaaagua cccuaagcug 3060

gaaagcgagu ucguguacgg cgacuacaag guguacgacg ugcggaagau gaucgccaag 3120

agcgagcagg aaaucggcaa ggcuaccgcc aaguacuucu ucuacagcaa caucaugaac 3180

uuuuucaaga ccgagauuac ccuggccaac ggcgagaucc ggaagcggcc ucugaucgag 3240

acaaacggcg aaaccgggga gaucgugugg gauaagggcc gggauuuugc caccgugcgg 3300

aaagugcuga gcaugcccca agugaauauc gugaaaaaga ccgaggugca gacaggcggc 3360

uucagcaaag agucuauccu gcccaagagg aacagcgaua agcugaucgc cagaaagaag 3420

gacugggacc cuaagaagua cggcggcuuc gacagcccca ccguggccua uucugugcug 3480

gugguggcca aaguggaaaa gggcaagucc aagaaacuga agagugugaa agagcugcug 3540

gggaucacca ucauggaaag aagcagcuuc gagaagaauc ccaucgacuu ucuggaagcc 3600

aagggcuaca aagaagugaa aaaggaccug aucaucaagc ugccuaagua cucccuguuc 3660

gagcuggaaa acggccggaa gagaaugcug gccucugccg gcgaacugca gaagggaaac 3720

gaacuggccc ugcccuccaa auaugugaac uuccuguacc uggccagcca cuaugagaag 3780

cugaagggcu cccccgagga uaaugagcag aaacagcugu uuguggaaca gcacaagcac 3840

uaccuggacg agaucaucga gcagaucagc gaguucucca agagagugau ccuggccgac 3900

gcuaaucugg acaaagugcu guccgccuac aacaagcacc gggauaagcc caucagagag 3960

caggccgaga auaucaucca ccuguuuacc cugaccaauc ugggagcccc ugccgccuuc 4020

aaguacuuug acaccaccau cgaccggaag agguacacca gcaccaaaga ggugcuggac 4080

gccacccuga uccaccagag caucaccggc cuguacgaga cacggaucga ccugucucag 4140

cugggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag 4200

uaa 4203

<210> 6

<211> 798

<212> RNA

<213> Artificial sequence

<400> 6

auggugagca agggcgagga gcuguucacc gggguggugc ccauccuggu cgagcuggac 60

ggcgacguaa acggccacaa guucagcgug uccggcgagg gcgagggcga ugccaccuac 120

ggcaagcuga cccugaaguu caucugcacc accggcaagc ugcccgugcc cuggcccacc 180

cucgugacca cccugaccua cggcgugcag ugcuucagcc gcuaccccga ccacaugaag 240

cagcacgacu ucuucaaguc cgccaugccc gaaggcuacg uccaggagcg caccaucuuc 300

uucaaggacg acggcaacua caagacccgc gccgagguga aguucgaggg cgacacccug 360

gugaaccgca ucgagcugaa gggcaucgac uucaaggagg acggcaacau ccuggggcac 420

aagcuggagu acaacuacaa cagccacaac gucuauauca uggccgacaa gcagaagaac 480

ggcaucaagg ugaacuucaa gauccgccac aacaucgagg acggcagcgu gcagcucgcc 540

gaccacuacc agcagaacac ccccaucggc gacggccccg ugcugcugcc cgacaaccac 600

uaccugagca cccaguccgc ccugagcaaa gaccccaacg agaagcgcga ucacaugguc 660

cugcuggagu ucgugaccgc cgccgggauc acucucggca uggacgagcu guacaagucc 720

ggagcugcgg ccgcugccgc ugcggcagcg gccgaauucc ccgggcucga gaagcuugga 780

uccaccggau cuagauaa 798

<210> 7

<211> 1818

<212> RNA

<213> Artificial sequence

<400> 7

gaaaccgaca ccaccaugaa gauaauaauu cugucuguua uauuggccua cugugucacc 60

gacaacuguc aagaugcaug uccuguagaa gcggaaccgc caucaaguac accaacaguu 120

ccaacuucuu gugaagcuaa agaaggagaa uguauagaua ccagaugcgc aacauguaaa 180

cgagauauac uaucagaugg acugugugaa aauaaaccag ggaagacaug cuguagaaug 240

ugccaguaug ugauugaaug cagaguagaa gcagcugguu auuuuagaac guuuuacggc 300

aaaagauuua auuuucagga accugguaaa uaugugcugg cuaggggaac caaggguggc 360

gauuggucug uaacccucac cauggagaau cuagauggac agaagggagc ugugcugacu 420

aagacaacac uggagguugc aggagacgua auagacauua cucaagcuac ugcagauccu 480

aucacaguua acggaggagc ugacccaguu aucgcuaacc cguucacaau uggugaggug 540

accauugcug uuguugaaau accgggcuuc aauaucacag ucaucgaauu cuuuaaacua 600

aucgugauug auauucuggg aggaagaucu gugagaauug cuccagacac agcaaacaaa 660

ggacugauau cugguaucug ugguaaucug gagaugaaug acgcugauga cuuuacuaca 720

gaugcagauc agcuggcgau ccaacccaac auaaacaaag aguucgacgg cugcccauuc 780

uauggcaauc cuucugauau cgaauacugc aaaggucuga uggagccaua cagagcugua 840

ugucguaaca auaucaacuu cuacuauuac acucuauccu gugccuucgc uuacuguaug 900

ggaggagaag aaagagcuaa acacguccuu uucgacuaug uugagacaug cgcugcgccg 960

gaaacgagag gaacgugugu uuuaucagga cauacuuucu augacacauu cgacaaagca 1020

agauaucaau uccagggccc augcaaggag auucugaugg ccgcagacug uuacuggaac 1080

acaugggaug uaaagguuuc acauagagac gucgaaucau acacugaggu agagaaagua 1140

acaaucagga aacagucaac uguaguagau cucauugugg auggcaagca ggucaagguu 1200

ggaggagugg auguaucuau cccguacagc ucugagaaca cuuccauaua cuggcaggau 1260

ggagacaucc ugacgacggc cauccuaccu gaagcucuug ucguuaaguu caacuuuaag 1320

cagcuccuug uaguucauau cagagaucca uucgauggaa agacaugcgg cauauguggu 1380

aacuauaauc aagauucaac ugaugauuuc uuugacgcag aaggagcaug cgcucuaacc 1440

cccaaccccc caggauguac agaggaacag aaaccagaag cugagcgacu uugcaauaau 1500

cucuuugauu cuucuaucga cgagaaaugu aaugucugcu acaagccuga ccggauugcc 1560

cgauguaugu acgaguauug ccugagggga caacaaggau uuugugacca ugcuugggag 1620

uucaagaaag aaugcuacau aaaacaugga gacacucuag aaguaccacc ugaaugucaa 1680

uaaacguaca aagauacaga agcuaaggcu acuacagcag aagauaaaaa agaaacugua 1740

guucuucaaa aaccguguau uuuauguacu cauuguuuaa uuagaggaaa auaaauuguu 1800

auuaucauaa cuuaaagu 1818

Claims

1. A method of carrier-free RNA delivery comprising the steps of:

(A1) synthesizing carrier-free Metal-mRNA nanoparticles by coordination-driven self-assembly effect of Metal cations and RNA molecules;

2. A method of making carrier-free Metal-mRNA nanoparticles, comprising the steps of:

(B2) standing the treated system (B1) at 37-60 ℃ and naturally cooling;

3. The method of claim 2, wherein: in the step (B1), the charge ratio of the mRNA of interest and the metal cation is 100/1 to 1/10;

further, the charge ratio of the target mRNA and the metal cation is 1: 2.

4. A method according to claim 2 or 3, characterized in that: in the step (B1), mixing the mRNA of interest and the metal cation is achieved by: and dropwise adding the aqueous solution containing the metal cations into the target mRNA aqueous solution, and stirring for 1-10 min.

5. The method according to any one of claims 2-4, wherein: in the step (B2), the standing time is 1.5-4 h; and/or

In the step (B2), the temperature after the natural cooling is room temperature.

6. The method according to any one of claims 2-5, wherein: in the step (B3), before the centrifugation, a step of washing the treated system of (B2) with deionized water is further included; and/or

In the step (B3), the centrifugation is 10000-; and/or

In the step (B3), the centrifugation further comprises a step of dispersing the precipitate in deionized water for storage.

7. The method according to any one of claims 1-6, wherein: the valence of the metal cation is from +2 to + 8;

the metal cation is any metal cation that can participate in the phosphate coordination reaction of RNA and can be bioavailable.

8. The method according to any one of claims 1-7, wherein: the mRNA is naked mRNA or modified mRNA.

9. Carrier-free Metal-mRNA nanoparticles prepared by the method of any one of claims 2-8.

10. Use of the method of any one of claims 1-8 or the carrier-free Metal-mRNA nanoparticles of claim 8 for carrier-free delivery of an mRNA of interest to a cell or tissue or organism.