Mrna / lipid-polymer hybrid nanoparticle delivery system, and preparation method therefor and use thereof

Abstract

An mRNA / lipid-polymer hybrid nanoparticle delivery system, and a preparation method therefor and the use thereof. The delivery system comprises mRNA, cationic molecules, a polymer and modified amphiphilic molecules, wherein the cationic molecules and the negatively charged mRNA form a compound by means of electrostatic interaction, the polymer is used for accommodating the cationic molecule-mRNA compound, and the three form a stable core structure; and the modified amphiphilic molecules are anchored on the surface of the core structure by means of hydrophobic interaction, thereby forming a sphere-like core-shell structure.

Description

An mRNA polymer-lipid hybrid delivery system and its preparation method and application Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an mRNA polymer-lipid hybrid delivery system and a preparation method and application thereof. Background Art

[0002] Messenger RNA (mRNA) is transcribed from a DNA template, carrying the genetic information encoding the target protein, and then translated into a functional protein under the action of the ribosome. By delivering exogenous mRNA into cells and translating the required functional protein, mRNA technology has become an emerging therapeutic method with great potential. However, individual mRNA shows great instability and immunogenicity in the biological environment, and due to its negatively charged nature, it is difficult to cross the cell membrane, which is also negatively charged. Therefore, it is necessary to develop a powerful delivery system to protect mRNA, slow down its degradation, and enable it to be successfully delivered to the cytoplasm to ultimately translate the target protein and exert its effect.

[0003] The new materials currently developed for mRNA delivery are mainly concentrated in lipid nanoparticles (LNPs), which usually contain ionizable lipid molecules, cholesterol lipid molecules, auxiliary phospholipid molecules, and PEGylated lipid molecules. Among them, the ionizable lipid molecules are positively charged and form complexes with negatively charged mRNA molecules through electrostatic interactions in acidic buffer solutions, and help mRNA molecules escape from endosomes in cells; cholesterol lipid molecules are used to fill the gaps between lipids and increase the stability of nanoparticles; auxiliary phospholipid molecules can regulate the membrane fluidity of nanoparticles to improve their ultimate performance; PEGylated lipid molecules are used to reduce the interaction between nanoparticles and biological macromolecules in the body, increasing the blood circulation and half-life of nanoparticles. However, the current LNPs-based mRNA delivery system still has the following major problems: (1) The components of LNPs are relatively complex. In addition to the functional mRNA component, it must contain at least four other components, and each component has a decisive influence on the final delivery effect of mRNA. Therefore, when developing LNPs delivery systems, it is usually necessary to spend a lot of financial, material and human resources to optimize the LNPs formula. (2) The various lipid molecules in the LNPs components, such as cholesterol and phospholipids, have relatively small molecular weights and stronger fluidity. Therefore, the overall structure of the prepared LNPs is not very stable, resulting in a relatively short half-life in the blood circulation in the body, and ultimately it is difficult to play the expected role. (3) The core components of LNPs - ionizable lipids, as well as lipid structures such as auxiliary phospholipid molecules, are relatively difficult to synthesize and scale up.

[0004] Therefore, there is still an urgent need to develop a new type of mRNA delivery system with fewer key components and more stable structure, so as to improve the translation and expression performance of mRNA and ultimately promote the application and development of mRNA technology.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to disclose an mRNA polymer-lipid hybrid delivery system (Lipid-Polymer Hybrid Nanoparticles, LPNPs).

[0007] The second object of the present invention is to disclose a method for preparing the above-mentioned delivery system.

[0008] The third object of the present invention is to disclose the application of the above delivery system.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] In a first aspect, the present invention provides an mRNA polymer-lipid hybrid delivery system (mRNA-LPNPs delivery system).

[0011] A mRNA polymer-lipid hybrid delivery system, wherein: the components of the delivery system include mRNA, cationic molecules, polymers and modified amphiphilic molecules; wherein the cationic molecules and negatively charged mRNA form a complex through electrostatic interaction; the polymer is used to accommodate the cationic molecule-mRNA complex, and the three form a stable core structure; the modified amphiphilic molecules are anchored to the surface of the core structure through hydrophobic interactions, forming a spherical core-shell structure.

[0012] The mRNA in the mRNA polymer-lipid hybrid delivery system described in the above technical solution can theoretically be any type of mRNA, including but not limited to: self-reporter gene mRNA, genome editing mRNA, antigen mRNA, tumor suppressor gene mRNA, interleukin mRNA or cell chimeric antigen receptor mRNA or other functional mRNA.

[0013] The mRNA polymer-lipid hybrid delivery system described in the above technical solution, wherein: the self-reporter gene mRNA includes but is not limited to GFP, eGFP, Fluc, mCherry, beta gal or renilla Luc;

[0014] The genome editing mRNA includes but is not limited to Cas9, Cas9 Nickase or Cre recombinase;

[0015] The antigen mRNA includes but is not limited to OVA;

[0016] The tumor suppressor gene mRNA includes but is not limited to p53 or PTEN;

[0017] The interleukin mRNA includes but is not limited to IL-10, IL-12 or IL-6.

[0018] The mRNA polymer-lipid hybrid delivery system described in the above technical solution, wherein: the cationic molecules illustratively include but are not limited to: ammonia molecule derivatives, ammonium salts and their derivatives, positively charged amphiphilic lipid compounds, polyamides and their derivatives, polyethyleneimine and its derivatives.

[0019] Figure 39 shows several representative cationic molecules among the above-mentioned cationic molecules: constantly positively charged cationic lipids (such as DOTAP, DOTMA, DMRIE, etc.), unsaturated double-bond ionizable lipids (such as Dlin-MC3-DMA, etc.), multi-tail ionizable lipids (such as C12-200, etc.), and polymer ionizable lipids (such as polyamide dendrimers PAMAM, polyethyleneimine PEI, etc.).

[0020] The mRNA polymer-lipid hybrid delivery system described in the above technical solution, wherein: the polymers illustratively include but are not limited to: aliphatic polyester polymers and their derivatives, such as polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, etc.; polycarbonate and its derivatives; polysaccharides and their derivatives; polyamino acids and their derivatives; polymer polyols and their derivatives, polyacrylic acid and its derivatives. Figure 40 shows several representative polymers among the above polymer molecules:

[0021] The mRNA polymer-lipid hybrid delivery system described in the above technical solution, wherein: the modified amphiphilic molecules illustratively include but are not limited to: PEGylated amphiphilic molecules, such as DSPE-PEG, DMG-PEG, F127, etc.; amphiphilic surfactants, such as CO-520, etc.; amphiphilic proteins, such as albumin; and other modified molecules, such as mannitol, modified chitosan, dextran, carboxydextran, tetraethyl orthosilicate, polyacrylic acid, KH560, KH550, diethylenetriamine pentaacetic acid, meglumine, arginine, polyglutamic acid, polypeptides, etc. Figure 41 shows several representative molecules among the above modified amphiphilic molecules:

[0022] In a second aspect, the present invention provides a method for preparing the mRNA-LPNPs delivery system as described in the first aspect, the preparation method comprising the following steps:

[0023] (a) preparing a cationic molecule solution;

[0024] (b) mRNA and cationic molecules form an electrostatic complex;

[0025] (c) sequentially adding a polymer and a modified amphiphilic molecule to the mRNA / cationic molecule complex formed above;

[0026] (d) Nanoparticles that stably encapsulate mRNA—mRNA polymer-lipid hybrid delivery systems—are obtained through self-assembly;

[0027] The method for preparing the mRNA-LPNPs delivery system described in the above technical solution comprises the following specific steps:

[0028] (a1) dissolving the cationic molecule in a solvent to prepare a solution containing the cationic molecule at a concentration of 0.5 to 5.0 mg / mL;

[0029] The cationic molecules include but are not limited to at least one of ammonia molecule derivatives, ammonium salts and their derivatives, positively charged amphiphilic lipid compounds, polyamides and their derivatives, polyethyleneimine and its derivatives;

[0030] The solvent is but not limited to at least one of methanol, ethanol, ethylene glycol, diethylene glycol, DMF, benzyl alcohol, acetone, dichloromethane and chloroform;

[0031] (b1) uniformly mixing a solution containing mRNA and a solution containing cationic molecules, and reacting for a period of time to obtain an mRNA / cationic molecule electrostatic complex;

[0032] The reaction time is 5s to 30min, and the reaction temperature is 0°C to 37°C;

[0033] The concentration of mRNA in the mRNA-containing solution is 0.1 to 10 mg / mL;

[0034] The mass ratio of the mRNA to the cationic molecule is 1:1 to 1:100;

[0035] The mRNA can theoretically be any type of mRNA, including but not limited to: self-reporter gene mRNA, genome editing mRNA, antigen mRNA, tumor suppressor gene mRNA, interleukin mRNA, cell chimeric antigen receptor mRNA or other functional mRNA;

[0036] The self-reporter gene mRNA includes but is not limited to one of GFP, eGFP, Fluc, mCherry, beta gal or renilla Luc;

[0037] The genome editing mRNA includes but is not limited to one of Cas9, Cas9 Nickase or Cre recombinase;

[0038] The antigen mRNA includes but is not limited to OVA;

[0039] The tumor suppressor gene mRNA includes but is not limited to one of p53 or PTEN;

[0040] The interleukin mRNA includes but is not limited to one of IL-10, IL-12 or IL-6;

[0041] (c1) uniformly mixing the mRNA / cationic molecule electrostatic complex with the polymer solution;

[0042] The reaction time is 5s to 30min, and the reaction temperature is 0°C to 37°C;

[0043] The concentration of the polymer molecules in the solution containing the polymer molecules is 0.1 to 20 mg / mL;

[0044] The polymer molecules mentioned above include but are not limited to at least one of the following polymers: aliphatic polyester polymers and their derivatives, such as polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, etc.; polycarbonate and its derivatives; polysaccharide and its derivatives; polyamino acid and its derivatives; polymer polyols and their derivatives, polyacrylic acid and its derivatives;

[0045] (d1) adding the modified amphiphilic molecule solution to the mRNA / cationic molecule / polymer mixed solution and mixing uniformly;

[0046] The concentration of the modified amphiphilic molecule in the modified amphiphilic molecule solution is 0.1 to 20 mg / mL;

[0047] The modified amphiphilic molecules include, but are not limited to, at least one of the following molecules: PEGylated amphiphilic molecules, such as DSPE-PEG, DMG-PEG, F127, etc.; amphiphilic surfactants, such as CO-520, etc.; amphiphilic proteins, such as albumin; and other modified molecules, such as mannitol, modified chitosan, dextran, carboxydextran, tetraethyl orthosilicate, polyacrylic acid, KH560, KH550, diethylenetriamine pentaacetic acid, meglumine, arginine, polyglutamic acid, polypeptides, etc.;

[0048] (e1) adding a mixture of mRNA / cationic molecule / polymer / modified amphiphilic molecule to an acidic aqueous solution and reacting for a period of time to obtain the mRNA-LPNPs nanodelivery system particles;

[0049] The pH range of acidic solutions is 1 to 6;

[0050] The reaction time is 30s to 30min, and the reaction temperature is 0°C to 37°C;

[0051] The acidic buffer solution includes but is not limited to at least one of citric acid / citrate, acetic acid / acetate, hydrochloric acid / hydrochloride, sulfuric acid / sulfate, and nitric acid / nitrate;

[0052] In steps (b1), (c1), (d1) and (e1), the solution mixing method includes but is not limited to at least one of the following methods: dripping, stirring, standing, microfluidic equipment, peristaltic pump, microinjection pump, etc.

[0053] The mRNA-LPNPs delivery system preparation method described in the above technical solution, wherein: the preparation method is a microfluidic preparation, comprising steps (a1), (b1) and (c1);

[0054] (d1) adding the modified amphiphilic molecule solution to the mRNA / cationic molecule / polymer mixed solution prepared in step (c1) and mixing them uniformly to obtain a DMF organic phase;

[0055] (e1) preparing an aqueous solution;

[0056] (f1) Preparation of mRNA LPNPs under microfluidic conditions with a DMF organic phase flow rate of 1–5 mL / min and an aqueous solution flow rate of 5–25 mL / min.

[0057] The preparation method described in the above technical solution prepares an mRNA-LPNPs delivery system.

[0058] The mRNA-LPNPs delivery system described in the above technical solution, wherein: the encapsulation rate of mRNA in the mRNA-LPNPs delivery system is ≥50%, preferably ≥60%, more preferably ≥70%, more preferably ≥80%, more preferably ≥90%, and optimally ≥95% (such as 99%).

[0059] The mRNA-LPNPs delivery system described in the above technical solution, wherein: the mass ratio of mRNA in the mRNA-LPNPs delivery system to other molecules in the delivery system is 1:100 to 10:1.

[0060] The mRNA-LPNPs delivery system described in the above technical solution, wherein: the mRNA-LPNPs delivery system is a two-dimensional, three-dimensional or layered structure; the nanodelivery system particles are three-dimensional nanocomposite materials with mRNA-cationic molecules-polymer as the core and modified amphiphilic molecules coated on the outside as the shell; the particle size of the nanodelivery system particles is 10nm to 500nm, preferably 20-250nm, more preferably 30-200nm, more preferably 70-130nm, more preferably 100-125nm, and most preferably 110-125nm.

[0061] An mRNA-LPNPs delivery system as described in the above technical solution, wherein: based on the total number of the mRNA-LPNPs delivery systems, 80% (preferably 85%, more preferably 90%, more preferably 95%, and optimally ≥99%) of the particle size of the delivery system materials is within the D50 range of ±15% (preferably ±10%, more preferably ±1%) of the nanocomposite material.

[0062] The mRNA-LPNPs delivery system described in the above technical solution, wherein: the mRNA-LPNPs delivery system can be stably dispersed in aqueous solution and 0.9% physiological saline.

[0063] The mRNA-LPNPs delivery system described in the above technical solution, wherein: the mRNA-LPNPs delivery system is stored in water or 0.9% physiological saline at 4°C, 25°C, 37°C, -20°C, and -80°C for 5-20 days, preferably 20-40 days, and more preferably 40-60 days, and the average particle size of the nanodelivery system particles changes by ≤20%, preferably ≤10%, more preferably ≤5%, and optimally ≤3%.

[0064] An mRNA-LPNPs delivery system as described in the above technical solution, wherein: the cell transfection efficiency of mRNA in the mRNA-LPNPs delivery system is ≥50%, preferably ≥60%, more preferably ≥70%, more preferably ≥80%, more preferably ≥90%, and optimally ≥95% (such as 99%).

[0065] In a third aspect, the present invention provides an application of the mRNA-LPNPs delivery system as described in the first aspect, wherein: the application is an application in the preparation of tumor treatment drugs, an application in the preparation of tumor vaccines, an application in the preparation of tumor targeted drugs, an application in the preparation of in vivo and in vitro tumor diagnostic materials, an application in the preparation of inflammatory-related disease treatments, an application in the preparation of infectious disease treatments, an application in the preparation of infectious disease prevention, an application in the preparation of wound healing treatments, an application in the preparation of cardiovascular and cerebrovascular chronic disease treatments, an application in the preparation of organ fibrosis treatments, an application in the preparation of viral infection treatments, an application in the preparation of viral infection prevention, an application in the preparation of in vivo and in vitro gene editing treatments, an application in the preparation of non-alcoholic fatty liver disease treatments, an application in the preparation of intestinal disease treatments, an application in the preparation of new antigen vaccines, an application in the preparation of cell induced differentiation, an application in the preparation of protein beauty, an application in the preparation of protein deficiency disease treatments, an application in the preparation of aging repair, an application in the preparation of anti-aging treatments, or an application in the preparation of drug carriers.

[0066] The application of the mRNA-LPNPs delivery system obtained in this technical solution varies depending on the function of the delivered mRNA.

[0067] In a fourth aspect, the present invention provides a product containing the mRNA-LPNPs delivery system described in any of the above technical solutions.

[0068] The present invention has the following beneficial effects:

[0069] 1. The polymer-lipid hybrid delivery system for mRNA delivery provided by the present invention comprises mRNA, a cationic molecule, a polymer, and a modified amphiphilic molecule. The introduction of a higher molecular weight polymer reduces the fluidity within the nanoparticles and stabilizes the mRNA-cationic molecule complex, thereby achieving higher mRNA transfection efficiency while reducing the number of key components in the mRNA delivery system.

[0070] 2. The mRNA-LPNPs delivery system preparation method provided by the present invention is to obtain a series of nanomaterials with excellent mRNA transfection ability through a step-by-step self-assembly method. The nanomaterials have suitable particle size, good stability, good water dispersibility, excellent biocompatibility, stable structure, few material components, high mRNA transfection efficiency, and the raw materials are simple and inexpensive to produce.

[0071] 3. The preparation method of the mRNA-LPNPs delivery system provided by the present invention has the advantages of being environmentally friendly, safe and reliable, simple in process, low in cost and high in yield, easy to control in quality, and easy to produce on a large scale.

[0072] 4. The mRNA-LPNPs delivery system provided by the present invention has a high mRNA encapsulation rate, can protect the mRNA structure, reduce the enzymatic degradation rate of mRNA, and reduce immunogenicity.

[0073] 5. The mRNA-LPNPs delivery system provided by the present invention can improve the adaptability of mRNA to temperature storage: compared with simple mRNA, the nanomaterials of the mRNA-LPNPs delivery system can be stored longer at 4°C, 25°C, 37°C, -20°C, and -80°C.

[0074] 6. Compared with the traditional LNPs delivery system, the mRNA-LPNPs delivery system provided by the present invention increases the stability of the overall structure due to the introduction of polymers with larger molecular weight. Therefore, it can reduce the components of the delivery system, reduce the difficulty and cost of large-scale production of raw materials, and provide a new platform for efficient delivery of mRNA. Description of the drawings:

[0075] 1. Figure 1 shows the size and morphology characterization results of mRNA / G0-C14 LPNPs.

[0076] 2. Figure 2 shows the characterization of the mRNA encapsulation efficiency of mRNA / G0-C14 LPNPs.

[0077] 3. Figure 3 shows the size change of mRNA / G0-C14 LPNPs over time when stored in 1× PBS at 4°C.

[0078] 4. Figure 4 shows GFP fluorescence imaging of mRNA / G0-C14 LPNPs (1.0 ng / μL mRNA) after co-incubation with A549 cells for 24 h.

[0079] 5. Figure 5 shows the Luciferase expression levels at different time points after intramuscular injection of mRNA / G0-C14 LPNPs (50 ng / μL mRNA, 50 μL).

[0080] 6. Figure 6 shows the size and morphology characterization results of mRNA / DOTMA LPNPs.

[0081] 7. Figure 7 shows the characterization of mRNA encapsulation efficiency of mRNA / DOTMA LPNPs.

[0082] 8. Figure 8 shows the size change of mRNA / DOTMA LPNPs over time when stored in 1× PBS at 4°C.

[0083] 9. Figure 9 shows GFP fluorescence imaging of mRNA / DOTMA LPNPs (1.0 ng / μL mRNA) after co-incubation with A549 cells for 24 h.

[0084] 10. Figure 10 shows the Luciferase expression at different time points after intramuscular injection of mRNA / DOTMA LPNPs (50 ng / μL mRNA, 50 μL).

[0085] 11. Figure 11 shows the size and morphology characterization results of mRNA / MC3 LPNPs.

[0086] 12. Figure 12 shows the characterization of mRNA encapsulation efficiency of mRNA / MC3 LPNPs.

[0087] 13. Figure 13 shows the size changes of mRNA / MC3 LPNPs over time when stored in 1× PBS at 4°C.

[0088] 14. Figure 14 shows GFP fluorescence imaging of mRNA / MC3 LPNPs (1.0 ng / μL mRNA) after co-incubation with A549 cells for 24 h.

[0089] Figure 15 shows the Luciferase expression levels at different time points after intramuscular injection of mRNA / MC3 LPNPs (50 ng / μL mRNA, 50 μL).

[0090] 16. Figure 16 shows the size and morphology characterization results of mRNA / C12-200 LPNPs.

[0091] 17. Figure 17 shows the mRNA encapsulation efficiency of mRNA / C12-200 LPNPs.

[0092] 18. Figure 18 shows the size change of mRNA / C12-200 LPNPs over time when stored in 1× PBS at 4°C.

[0093] 19. Figure 19 shows GFP fluorescence imaging of mRNA / C12-200 LPNPs (1.0 ng / μL mRNA) after co-incubation with A549 cells for 24 h.

[0094] 20. Figure 20 shows the Luciferase expression levels at different time points after intramuscular injection of mRNA / C12-200 LPNPs (50 ng / μL mRNA, 50 μL).

[0095] 21. Figure 21 shows the size and morphology characterization results of mRNA / SM-102 LPNPs.

[0096] 22. Figure 22 shows the mRNA encapsulation efficiency of mRNA / SM-102 LPNPs.

[0097] 23. Figure 23 shows the size change of mRNA / SM-102 LPNPs over time when stored in 1× PBS at 4°C.

[0098] 24. Figure 24 shows GFP fluorescence imaging of mRNA / SM-102 LPNPs (1.0 ng / μL mRNA) after co-incubation with A549 cells for 24 h.

[0099] 25. Figure 25 shows the Luciferase expression levels at different time points after intramuscular injection of mRNA / SM-102 LPNPs (50 ng / μL mRNA, 50 μL).

[0100] 26. Figure 26 shows the size and stability of mRNA PDSA LPNPs and the encapsulation efficiency.

[0101] 27. Figure 27 shows the EGFP expression ability of mRNA PDSA LPNPs on Hep3B cells.

[0102] 28. Figure 28 shows the cell viability after co-incubation of Hep3B cells with mRNA PDSA LPNPs at different concentrations.

[0103] 29. Figure 29 shows the characterization of the morphology, size, stability, and encapsulation efficiency of mRNA HA LPNPs.

[0104] 30. Figure 30 shows the EGFP expression ability of mRNA HA LPNPs on H1299 cells.

[0105] 31. Figure 31 shows the Luciferase expression levels at different time points after intramuscular injection of mRNA HA LPNPs (50 ng / μL mRNA, 50 μL).

[0106] 32. Figure 32 is a schematic diagram of the microfluidic preparation of mRNA / G0-C14 LPNPs.

[0107] 33. Figure 33 shows the size and morphology characterization of mRNA / G0-C14 LPNPs prepared by microfluidics.

[0108] 34. Figure 34 shows the mRNA encapsulation efficiency characterization of mRNA / G0-C14 LPNPs prepared by microfluidics.

[0109] 35. Figure 35 shows the in vitro cytotoxicity test of mRNA / G0-C14 LPNPs at different concentrations.

[0110] 36. Figure 36 shows the bioluminescence levels of A549 cells transfected with different concentrations of mRNA / G0-C14 LPNPs.

[0111] 37. Figure 37 shows the Luciferase expression levels at different time points after intramuscular injection of mRNA / G0-C14 LPNPs (50 ng / μL mRNA, 50 μL).

[0112] 38. Figure 38 is a schematic diagram of the preparation process of the mRNA polymer-lipid hybrid delivery system of the present invention.

[0113] 39. Figure 39 shows several representative cationic molecules.

[0114] 40. Figure 40 shows several representative polymers among polymer molecules.

[0115] 41. Figure 41 shows several representative modified amphiphilic molecules. Specific implementation method:

[0116] To facilitate understanding of the technical solution of the present invention, an mRNA polymer-lipid hybrid delivery system, its preparation method, and application are further described below in conjunction with specific examples.

[0117] In view of the drawbacks of existing lipid nanoparticle systems for mRNA delivery, the inventors of this case, through research and practice, were able to propose the technical solution of the present invention. The technical solution of the present invention will be clearly and completely described below. Obviously, the embodiments described are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0118] The key general test methods and equipment in this invention include:

[0119] (1) Dynamic light scattering (DLS) characterization:

[0120] The mRNA nanoparticle solution was measured using a Malvern Panalytical Zetasizer at a 90° scattering angle, and the total sample concentration was maintained within 0.1 to 5.0 mg / mL.

[0121] (2) Transmission electron microscopy (TEM) imaging:

[0122] Test instrument: JEOL-2100 transmission electron microscope; test conditions: 200 kV, 101 μA; the nanoparticles to be tested are dispersed in water for testing.

[0123] (3) Agarose gel electrophoresis:

[0124] All samples were analyzed by electrophoresis in 1% agarose gel; electrophoresis conditions: 100 V, 30 min; electrophoresis buffer: 1×TAE buffer.

[0125] (4) Cytotoxicity test:

[0126] 1×10 4 Cells were seeded in 96-well plates at a concentration of 10 cells / well and cultured for 24 h.

[0127] The old culture medium was replaced with complete culture medium containing mRNA nanoparticles and incubated for another 24 h;

[0128] 10 μL of MTT PBS solution (5 mg / mL) was added to each well and incubated for another 4 h;

[0129] Carefully remove the culture medium and add 100 μL of dimethyl sulfoxide to each well;

[0130] The absorbance was measured at a wavelength of 490 nm using a microplate reader, and the cell viability was calculated.

[0131] (5) GFP mRNA expression experiment at the cellular level:

[0132] Cells were seeded into 96-well plates at a density of 1 × 104 The cells were cultured for 24 h. The old culture medium was replaced with complete culture medium containing mRNA nanoparticles and the culture was continued for 24 h.

[0133] Under blue light excitation, fluorescence imaging of GFP mRNA was observed using a fluorescence microscope.

[0134] (6) Luciferase mRNA expression experiment at animal level:

[0135] The hind legs of 4-6 week-old mice were depilated, and then mRNA nanoparticles were injected intramuscularly. The bioluminescence imaging results were observed at different time points.

[0136] Mice were intraperitoneally injected with luciferin sodium salt (150 mg / kg), and bioluminescence imaging was performed 5 minutes later.

[0137] One aspect of the embodiments of the present invention is to provide a method for preparing a polymer-lipid hybrid delivery system for delivering mRNA. The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and the accompanying drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the scope of protection of the present invention is not limited to the following embodiments.

[0138] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0139] The preparation process of the present invention is shown in FIG38 . The following representative examples are presented, mainly including three types of polymers (PLGA, PDSA, HA) and five types of cationic lipids (G0-C14, DOTMA, MC3, C1-200, SM-102), a total of seven types of mRNA-LPNPs, and a representative example of microfluidic preparation of mRNA-LPNPs.

[0140] Example 1: Preparation of polymer PLGA mRNA-LPNPs:

[0141] PLGA (molecular weight 30000-60000) is used as the polymer in the "mRNA lipid-polymer hybrid delivery system", DSPE-PEG 2k (CAS No.: 147867-65-0) is a modified amphiphilic molecule. By changing the type of cationic lipid / ionizable lipid, the following different mRNA lipid-polymer hybrid delivery systems were prepared:

[0142] Example 1.1: mRNA / G0-C14 LPNPs—Polymer-based ionizable lipid-LPNPs:

[0143] (1) Synthesis of cationic molecule G0-C14: 1 mmol of PAMAM dendrimer (ethylenediamine core, G0 generation) methanol solution (CAS No.: 155773-72-1) and 7 mmol of 1,2-epoxytetradecane (CAS No.: 3234-28-4) were added to a 20 mL reaction bottle to form a mixed reaction solution. The methanol solution was removed by vacuum distillation. Then, under argon protection, the reaction was carried out at 90°C and a stirring speed of 800 rpm for 48 hours. After the reaction was completed, the reaction product was diluted with dichloromethane and then separated and purified by silica gel column chromatography to obtain cationic lipid G0-C14;

[0144] (2) Preparation of key component mother solutions: G0-C14, PLGA, and DSPE-PEG were fully dissolved in N,N-dimethylformamide (DMF) to obtain the corresponding component mother solutions: G0-C14 (2.5 mg / mL), PLGA (5.0 mg / mL), and DSPE-PEG (20 mg / mL);

[0145] (3) Prepare 50 mM and 10 mM citrate buffer (pH = 4.0) and treat with DEPC to remove nuclease;

[0146] (4) Preparation of mRNA / G0-C14 complex: 10 μL of mRNA (1 μg / μL) was taken and 2.5 μL of citric acid buffer (50 mM) was added thereto. After mixing evenly, the mixture was quickly added to 50 μL of G0-C14 DMF solution under vortexing and continued to vortex for 20 s to obtain the mRNA / G0-C14 complex.

[0147] (5) Add 50 μL of PLGA DMF solution to the above mRNA / G0-C14 complex and mix well for 10 seconds;

[0148] (6) Add 25 μL of DSPE-PEG DMF solution to the rising solution and mix for 10 seconds to obtain solution A.

[0149] (7) While vortexing, quickly add solution A to 625 μL of 10 mM citrate buffer and continue vortexing for 50 s.

[0150] (8) Slowly add 625 μL of 1× PBS dropwise to the mixed solution while vortexing;

[0151] (9) The organic solvent was removed by ultrafiltration centrifugation (molecular weight cutoff 100 kDa), repeated three times; centrifugation conditions: 3000 g, 8 min;

[0152] (10) Finally, the volume was adjusted to 500 μL mRNA / G0-C14 LPNPs (mRNA concentration: 20 ng / μL) with 1× PBS.

[0153] Results and performance characterization:

[0154] 1. Size and morphology of mRNA / G0-C14 LPNPs:

[0155] Figure 1 shows the size and morphology of mRNA / G0-C14 LPNPs. Figure 1-a shows the DLS size distribution, which demonstrates uniform size distribution (d.size = 103 ± 3 nm, PDI = 0.19 ± 0.06). Figure 1-b shows TEM images, which reveal the spherical shape of the prepared mRNA nanoparticles. Figure 1-c shows the size and PDI distribution of different batches.

[0156] 2. mRNA encapsulation efficiency of mRNA / G0-C14 LPNPs:

[0157] Figure 2 shows the mRNA encapsulation efficiency of mRNA / G0-C14 LPNPs. Figure 2-a shows a representative agarose gel electrophoresis image of mRNA / G0-C14 LPNPs, and Figure 2-b shows the mRNA encapsulation efficiency of mRNA / G0-C14 LPNPs prepared from different batches. Figure 2 demonstrates that the prepared mRNA / G0-C14 LPNPs can achieve an mRNA encapsulation efficiency of ~95%.

[0158] 3. Size stability of mRNA / G0-C14 LPNPs:

[0159] Figure 3 shows the size change of mRNA / G0-C14 LPNPs over time when stored in 1×PBS at 4°C. Figure 3 shows that the prepared mRNA / G0-C14 LPNPs have good size stability in 1×PBS at 4°C.

[0160] 4. GFP expression of mRNA / G0-C14 LPNPs at the cellular level:

[0161] Figure 4 shows GFP fluorescence imaging of mRNA / G0-C14 LPNPs (1.0 ng / μL mRNA) after 24 hours of incubation with A549 cells. Figure 4 demonstrates that mRNA / G0-C14 LPNPs prepared using GFP mRNA as a model exhibit efficient protein expression at the cellular level.

[0162] 5. Luciferase expression of mRNA / G0-C14 LPNPs in animals:

[0163] Figure 5 shows Luciferase expression levels at different time points after intramuscular injection of mRNA / G0-C14 LPNPs (50 ng / μL mRNA, 50 μL). Figure 5-a shows bioluminescence imaging of mice at different time points after intramuscular injection of mRNA / G0-C14 LPNPs; Figure 5-b shows quantitative analysis of mouse bioluminescence. Figure 5 demonstrates that mRNA / G0-C14 LPNPs prepared using Luciferase mRNA as a model also exhibit efficient protein expression in animals.

[0164] Example 1.2: mRNA / DOTMA LPNPs—Constantly Positively Charged Cationic Lipid-LPNPs:

[0165] Compared with Example 1.1, the only difference is that the cationic lipid in Example 1.1 is replaced with DOTMA. The other preparation processes remain the same to obtain mRNA / DOTMA LPNPs, which are also subjected to the following structural and performance characterization.

[0166] 1. Size and morphology of mRNA / DOTMA LPNPs:

[0167] Figure 6 shows the size and morphology characterization results of mRNA / DOTMA LPNPs. Figure 6-a shows the DLS size distribution, which demonstrates uniform size distribution (d.size = 105 ± 1 nm, PDI = 0.23 ± 0.08). Figure 6-b shows TEM images, which demonstrate the spherical structure of the prepared mRNA nanoparticles. Figure 6-c shows the size and PDI distribution of different batches.

[0168] 2. mRNA encapsulation efficiency of mRNA / DOTMA LPNPs:

[0169] Figure 7 shows the mRNA encapsulation efficiency of mRNA / DOTMA LPNPs. Figure 7-a shows a representative agarose gel electrophoresis image of mRNA / DOTMA LPNPs; Figure 7-b shows the mRNA encapsulation efficiency of mRNA / DOTMA LPNPs prepared from different batches. Figure 7 demonstrates that the prepared mRNA / DOTMA LPNPs can achieve an mRNA encapsulation efficiency of ~85%.

[0170] 3. Size stability of mRNA / DOTMA LPNPs:

[0171] Figure 8 shows the size changes of mRNA / DOTMA LPNPs over time when stored in 1× PBS at 4°C. Figure 8 shows that the prepared mRNA / DOTMA LPNPs have good size stability in 1× PBS at 4°C.

[0172] 4. GFP expression of mRNA / DOTMA LPNPs at the cellular level:

[0173] Figure 9 shows GFP fluorescence imaging of mRNA / DOTMA LPNPs (1.0 ng / μL mRNA) after 24 hours of incubation with A549 cells. Figure 9 demonstrates that mRNA / DOTMA LPNPs prepared using GFP mRNA as a model exhibit efficient protein expression at the cellular level.

[0174] 5. Luciferase expression of mRNA / DOTMA LPNPs in animals:

[0175] Figure 10 shows Luciferase expression at different time points after intramuscular injection of mRNA / DOTMA LPNPs (50 ng / μL mRNA, 50 μL). Figure 10-a shows bioluminescence imaging of mice at different time points after intramuscular injection of mRNA / DOTMA LPNPs, and Figure 10-b shows quantitative analysis of mouse bioluminescence. Figure 10 demonstrates that mRNA / DOTMA LPNPs prepared using Luciferase mRNA as a model also exhibit efficient protein expression in animals.

[0176] Example 1.3: mRNA / MC3 LPNPs—Unsaturated Double-Bond Ionizable Lipid-LPNPs

[0177] Compared with Example 1.1, the only difference is that the cationic lipid in Example 1.1 is replaced with MC3. The other preparation processes remain the same to obtain mRNA / MC3 LPNPs, which are also subjected to the following structural and performance characterization.

[0178] 1. Size and morphology of mRNA / MC3 LPNPs:

[0179] Figure 11 shows the size and morphology of mRNA / MC3 LPNPs. Figure 11a shows the DLS size distribution, which demonstrates uniform size distribution (d.size = 118 ± 2 nm, PDI = 0.19 ± 0.08). Figure 11b shows TEM images, which demonstrate the spherical shape of the prepared mRNA nanoparticles. Figure 11c shows the size and PDI distribution of different batches.

[0180] 2. mRNA encapsulation efficiency of mRNA / MC3 LPNPs:

[0181] Figure 12 shows the mRNA encapsulation efficiency of mRNA / MC3 LPNPs. Figure 12-a shows a representative agarose gel electrophoresis image of mRNA / MC3 LPNPs, and Figure 12-b shows the mRNA encapsulation efficiency of mRNA / MC3 LPNPs prepared from different batches. Figure 12 demonstrates that the prepared mRNA / MC3 LPNPs can achieve an mRNA encapsulation efficiency of ~43%.

[0182] 3. Size stability of mRNA / MC3 LPNPs:

[0183] Figure 13 shows the size changes of mRNA / MC3 LPNPs over time when stored in 1×PBS at 4°C. Figure 13 shows that the prepared mRNA / MC3 LPNPs have good size stability in 1×PBS at 4°C.

[0184] 4. GFP expression of mRNA / MC3 LPNPs at the cellular level:

[0185] Figure 14 shows GFP fluorescence imaging of mRNA / MC3 LPNPs (1.0 ng / μL mRNA) after 24 hours of incubation with A549 cells. Figure 14 demonstrates that mRNA / MC3 LPNPs prepared using GFP mRNA as a model achieve protein expression at the cellular level.

[0186] 5. Luciferase expression of mRNA / MC3 LPNPs in animals:

[0187] Figure 15 shows the Luciferase expression levels at different time points after intramuscular injection of mRNA / MC3 LPNPs (50 ng / μL mRNA, 50 μL). Figure 15 demonstrates that mRNA / MC3 LPNPs prepared using Luciferase mRNA as a model can also achieve protein expression in animals.

[0188] Example 1.4: mRNA / C12-200 LPNPs—Multi-tailed ionizable lipid-LPNPs

[0189] Compared with Example 1.1, the only difference is that the cationic lipid in Example 1.1 is replaced with C12-200. The other preparation processes remain the same to obtain mRNA / C12-200 LPNPs, which are also subjected to the following structural and performance characterization.

[0190] 1. Size and morphology of mRNA / C12-200 LPNPs:

[0191] Figure 16 shows the size and morphology characterization results of mRNA / C12-200 LPNPs; Figure 16-a shows the DLS size distribution, and the DLS results show that its size distribution is uniform (d.size = 128 ± 6 nm, PDI = 0.25 ± 0.06); Figure 16-b is TEM imaging, and the TEM image shows that the prepared mRNA nanoparticles have a spherical structure; Figure 16-c shows the size and PDI distribution of different batches.

[0192] 2. mRNA encapsulation efficiency of mRNA / C12-200 LPNPs:

[0193] Figure 17 shows the mRNA encapsulation efficiency of mRNA / C12-200 LPNPs. Figure 17-a shows a representative agarose gel electrophoresis image of mRNA / C12-200 LPNPs, while Figure 17-b shows the mRNA encapsulation efficiency of mRNA / C12-200 LPNPs prepared from different batches. Figure 17 shows that the prepared mRNA / C12-200 LPNPs can achieve an mRNA encapsulation efficiency of ~71%.

[0194] 3. Size stability of mRNA / C12-200 LPNPs:

[0195] FIG18 shows the size change of mRNA / C12-200 LPNPs over time when stored in 1×PBS at 4°C. FIG18 shows that the prepared mRNA / C12-200 LPNPs have good size stability in 1×PBS at 4°C.

[0196] 4. GFP expression of mRNA / C12-200 LPNPs at the cellular level:

[0197] Figure 19 is a GFP fluorescence imaging image of mRNA / C12-200 LPNPs (1.0 ng / μL mRNA) after co-incubation with A549 cells for 24 hours; Figure 19 shows that the mRNA / C12-200 LPNPs prepared using GFP mRNA as a model exhibit efficient protein expression performance at the cellular level.

[0198] 5. Luciferase expression of mRNA / C12-200 LPNPs in animals:

[0199] Figure 20 shows Luciferase expression levels at different time points after intramuscular injection of mRNA / C12-200 LPNPs (50 ng / μL mRNA, 50 μL). Figure 20-a shows bioluminescence imaging of mice at different time points after intramuscular injection of mRNA / C12-200 LPNPs; Figure 20-b shows quantitative analysis of mouse bioluminescence. Figure 20 demonstrates that mRNAC12-200 LPNPs prepared using Luciferase mRNA as a model also exhibit efficient protein expression in animals.

[0200] Example 1.5: mRNA / SM-102 LPNPs—Multi-tailed ionizable lipid-LPNPs (Moderna's ionizable lipids)

[0201] Compared with Example 1.1, the only difference is that the cationic lipid in Example 1.1 is replaced with SM-102. The other preparation processes remain the same to obtain mRNA / SM-102 LPNPs, which are also subjected to the following structural and performance characterization.

[0202] 1. Size and morphology of mRNA / SM-102 LPNPs:

[0203] Figure 21 shows the size and morphology characterization results of mRNA / SM-102 LPNPs; Figure 21-a shows the DLS size distribution, and the DLS results show that its size distribution is uniform (d.size = 116 ± 2 nm, PDI = 0.17 ± 0.02); Figure 21-b is TEM imaging, and the TEM image shows that the prepared mRNA nanoparticles have a spherical structure; Figure 21-c shows the size and PDI distribution of different batches.

[0204] 2. mRNA encapsulation efficiency of mRNA / SM-102 LPNPs:

[0205] Figure 22 shows the mRNA encapsulation efficiency of mRNA / SM-102 LPNPs. Figure 22-a shows a representative agarose gel electrophoresis image of mRNA / SM-102 LPNPs, while Figure 22-b shows the mRNA encapsulation efficiency of mRNA / SM-102 LPNPs prepared from different batches. Figure 22 demonstrates that the prepared mRNA / SM-102 LPNPs achieve nearly complete mRNA encapsulation, with an encapsulation efficiency approaching ~94%.

[0206] 3. Size stability of mRNA / SM-102 LPNPs:

[0207] Figure 23 shows the size change of mRNA / SM-102 LPNPs over time when stored in 1×PBS at 4°C. Figure 23 shows that the prepared mRNA / SM-102 LPNPs have good size stability in 1×PBS at 4°C.

[0208] 4. GFP expression of mRNA / SM-102 LPNPs at the cellular level:

[0209] Figure 24 shows GFP fluorescence imaging of mRNA / SM-102 LPNPs (1.0 ng / μL mRNA) after 24 hours of incubation with A549 cells. Figure 24 demonstrates that mRNA / SM-102 LPNPs prepared using GFP mRNA as a model exhibit efficient protein expression at the cellular level.

[0210] 5. Luciferase expression of mRNA / SM-102 LPNPs in animals:

[0211] Figure 25 shows Luciferase expression levels at different time points after intramuscular injection of mRNA / SM-102 LPNPs (50 ng / μL mRNA, 50 μL). Figure 25-a shows bioluminescence imaging of mice at different time points after intramuscular injection of mRNA / SM-102 LPNPs; Figure 25-b shows quantitative analysis of mouse bioluminescence. Figure 25 demonstrates that mRNA / SM-102 LPNPs prepared using Luciferase mRNA as a model also exhibit efficient protein expression in animals.

[0212] Example 2: Preparation of mRNA PDSA LPNPs (polymer PDSA mRNA-LPNPs):

[0213] mRNA PDSA LPNPs were prepared using PDSA as the polymer in the "mRNA lipid-polymer hybrid delivery system," G0-C14 as the cationic lipid, and DSPE-PEG (CAS No. 147867-65-0) as the modified amphiphilic molecule. The specific steps are as follows:

[0214] (1) Hydrophobic PDSA polymer is synthesized by one-step polycondensation of (H-Cys-OMe)2·2HCl and fatty acid dichloride, and then 1 Characterization was performed by H NMR.

[0215] (2) Preparation of the mother liquor of key components: G0-C14, PDSA, and DSPE-PEG were fully dissolved in DMF to a concentration of 2.5 mg / mL, 20 mg / mL, and 20 mg / mL, respectively.

[0216] (3) 24 μg of mRNA (1.0 μg / μL) and 360 μg of G0-C14 (144 μL of the G0-C14-containing solution prepared in step (2)) were mixed to obtain an electrostatic complex of G0-C14 / mRNA.

[0217] (4) 4 mg of PDSA (200 μL of the PDSA-containing solution prepared in step (2)) and 2.8 mg of DSPE-PEG (140 μL of the DSPE-PEG-containing solution prepared in step (2)) were added to the above mixture in sequence to obtain solution A.

[0218] (5) Under magnetic stirring (800 rpm), solution A was added dropwise into 10 mL of ultrapure water and stirring was continued for 30 min.

[0219] (6) Remove the organic solvent using ultrafiltration centrifugation (molecular weight cutoff 100 kDa), repeat 3 times; centrifugation conditions: 3000 g, 8 min.

[0220] Finally, the volume was adjusted to 600 μL mRNA / G0-C14 LPNPs (mRNA concentration: 40 ng / μL) with 1×PBS.

[0221] Structure and performance characterization:

[0222] 1. Morphology, size and stability of mRNA PDSA LPNPs:

[0223] Figure 26 is a diagram illustrating the size and stability of mRNA PDSA LPNPs and the encapsulation efficiency; Figure 26-a shows the size change of mRNA PDSA LPNPs in PBS containing 10% fetal bovine serum. Figure 26-a shows that the prepared mRNA PDSA LPNPs have good size stability in PBS containing serum.

[0224] 2. mRNA encapsulation efficiency of mRNA PDSA LPNPs:

[0225] Here, the fluorescence quantification method was used to measure the mRNA encapsulation efficiency. Specifically, Cy5-labeled mRNA was used to quantify the mRNA fluorescence. The Cy5-mRNA fluorescence intensity encapsulated in the mRNA nanoparticles was measured by a microplate reader (TECAN, Infinite M200 Pro). After testing, the mRNA encapsulation efficiency of the synthesized mRNA PDSA LPNPs was determined to be ~50%, as shown in Figure 26-b.

[0226] 3. EGFP expression of mRNA PDSA LPNPs at the cellular level:

[0227] Figure 27 shows the EGFP expression ability of mRNA PDSA LPNPs in Hep3B cells. Specifically, Figure 27 shows the EGFP-positive rate after Hep3B cells were co-incubated with mRNA nanoparticles at different concentrations. Figure 27 demonstrates that mRNA PDSA LPNPs prepared using EGFP mRNA as a model exhibit efficient protein expression at the cellular level.

[0228] 4. Cytotoxicity evaluation of mRNA PDSA LPNPs:

[0229] Figure 28 shows the cell viability after co-incubation of Hep3B cells with mRNA PDSA LPNPs at different concentrations. Figure 28 shows that EGFP mRNA PDSA LPNPs exhibit good cell safety.

[0230] Example 3: Preparation of mRNA HA LPNPs (polymer HA mRNA-LPNPs):

[0231] mRNA HA LPNPs were prepared using hyaluronic acid (HA) (molecular weight 1.01-1.80 MDa) as the polymer in the "mRNA lipid-polymer hybrid delivery system," G0-C14 as the cationic lipid, and DSPE-PEG (CAS No. 147867-65-0) as the modifying amphiphilic molecule. The specific steps are as follows:

[0232] (1) Preparation of the mother liquor of key components: G0-C14, HA, and DSPE-PEG were fully dissolved in DMF, ultrapure water, and ultrapure water, respectively, with concentrations of 0.5 mg / mL, 0.5 mg / mL, and 5.0 mg / mL, respectively.

[0233] (2) 20 μg of mRNA (1.0 μg / μL) and 250 μg of G0-C14 (500 μL) were mixed to obtain an electrostatic complex of G0-C14 / mRNA.

[0234] (3) 250 μg HA (500 μL) was quickly added to the above electrostatic complex and mixed evenly to obtain solution A.

[0235] (4) Solution A was added dropwise to ultrapure water (10 mL) containing 1 mg of DSPE-PEG under magnetic stirring (1200 rpm) and stirring was continued for 30 min.

[0236] (5) Remove the organic solvent using ultrafiltration centrifugation (molecular weight cutoff 100 kDa), repeat 3 times; centrifugation conditions: 3000 g, 8 min.

[0237] (6) Finally, the volume was adjusted to 500 μL mRNA / G0-C14 LPNPs (mRNA concentration: 40 ng / μL) with 1× PBS.

[0238] Structure and performance characterization:

[0239] 1. Size, morphology and stability of mRNA HA LPNPs:

[0240] Figure 29 shows the characterization of the morphology, size, and stability of mRNA HA LPNPs, as well as an illustration of the encapsulation efficiency; Figure 29-a is a TEM image, which shows that the prepared mRNA nanoparticles have a spherical structure with a size of approximately 200 nm; Figure 29-b shows the size change of mRNA HA LPNPs in PBS, and Figure 29-b shows that the prepared mRNA HA LPNPs have good size stability during storage in PBS.

[0241] 2. mRNA encapsulation efficiency of mRNA HA LPNPs:

[0242] Here, the mRNA encapsulation efficiency was measured using a fluorescence quantification method. Specifically, Cy5-labeled mRNA was used to quantify mRNA fluorescence. The Cy5-mRNA fluorescence intensity encapsulated in the mRNA nanoparticles was measured using a microplate reader (TECAN, Infinite M200 Pro). The mRNA encapsulation efficiency of the synthesized mRNA HA LPNPs was determined to be ~50%, as shown in Figure 29-c.

[0243] 3. EGFP expression of mRNA HA LPNPs at the cellular level:

[0244] Figure 30 shows the EGFP expression ability of mRNA HA LPNPs in H1299 cells; Figure 30-a shows EGFP fluorescence imaging after 24 hours of co-incubation with H1299 cells; Figure 30-b shows flow cytometric analysis of H1299 cells co-incubated with different mRNA nanoparticles. Figure 30 demonstrates that mRNA HA LPNPs prepared using EGFP mRNA as a model exhibit efficient protein expression at the cellular level.

[0245] 4. Luciferase expression of mRNA HA LPNPs in animals:

[0246] Figure 31 shows Luciferase expression levels at different time points after intramuscular injection of mRNA HA LPNPs (50 ng / μL mRNA, 50 μL); Figure 31-a shows bioluminescence imaging of mice at different time points after intramuscular injection of mRNA HA LPNPs; Figure 31-b shows quantitative analysis of mouse bioluminescence. Figure 31 demonstrates that mRNA HA LPNPs prepared using Luciferase mRNA as a model also exhibit efficient protein expression in animals.

[0247] Example 4: A microfluidic preparation of mRNA / G0-C14 LPNPs:

[0248] The present invention provides a dual-channel microfluidic method for preparing mRNA-LPNPs. mRNA and cationic lipids are first complexed, and then a polymer and a modified amphiphilic molecule are sequentially added to the mixture to form a uniform DMF organic phase solution. The two phases are mixed in a dual-channel microfluidic device (as shown in Figure 32), and the organic solvent is removed from the effluent by ultrafiltration to obtain mRNA-LPNPs. The microfluidic operating conditions are as follows: DMF organic phase flow rate: 2 mL / min; aqueous solution flow rate: 5 mL / min; synthesis volume: 500 μL to 20 mL.

[0249] Taking "mRNA / G0-C14 LPNPs" as an example, the specific steps for preparing mRNA / G0-C14 LPNPs using microfluidics are as follows:

[0250] (1) Preparation of key component mother liquor: G0-C14, PLGA, and DSPE-PEG were fully dissolved in N,N-dimethylformamide (DMF) to obtain the corresponding component mother liquors - G0-C14 (2.5 mg / mL), PLGA (5.0 mg / mL), and DSPE-PEG (20 mg / mL).

[0251] (2) Prepare 50 mM and 10 mM citric acid buffer (pH = 4.0) and treat with DEPC to remove nuclease.

[0252] (3) Preparation of mRNA / G0-C14 complex: Take 50 μL of mRNA (1 μg / μL) and add 12.5 μL of citric acid buffer (50 mM) to it. After mixing evenly, quickly add it to 250 μL of G0-C14 DMF solution under vortexing and continue vortexing for 20 seconds to obtain mRNA / G0-C14 complex.

[0253] (4) 250 μL of PLGA DMF solution and 125 μL of DSPE-PEG DMF solution were added to the above mRNA / G0-C14 complex in sequence and mixed evenly to obtain a DMF organic phase.

[0254] (5) Aqueous phase solution: 3125 μL of citric acid buffer (10 mM).

[0255] (6) Under microfluidic conditions (Y-type chip / Miana), the DMF organic phase flow rate was 2 mL / min and the aqueous solution flow rate was 5 mL / min to prepare mRNA LPNPs.

[0256] (7) Slowly add 3125 μL of 1× PBS dropwise to the above mixed solution under vortexing.

[0257] (8) Remove the organic solvent using ultrafiltration centrifugation (molecular weight cutoff 100 kDa), repeat 3 times; centrifugation conditions: 3000 g, 8 min.

[0258] (9) Finally, the volume was adjusted to 1000 μL mRNA / G0-C14 LPNPs (mRNA concentration: 50 ng / μL) with 1× PBS.

[0259] Structure and performance characterization:

[0260] 1. Characterization of size and morphology of mRNA / G0-C14 LPNPs prepared by microfluidics:

[0261] Figure 33 shows the size and morphology of microfluidic-prepared mRNA / G0-C14 LPNPs. Figure 33-a shows the DLS size distribution; Figure 33-b shows TEM imaging; and Figure 33-c shows the size and PDI distributions of different batches. Figure 33 shows that the microfluidic-prepared mRNA / G0-C14 LPNPs are approximately 89 nm in size and have a PDI of approximately 0.114, demonstrating smaller size and greater polydispersity.

[0262] 2. mRNA encapsulation efficiency of microfluidic prepared mRNA / G0-C14 LPNPs:

[0263] Figure 34 shows the mRNA encapsulation efficiency of microfluidically prepared mRNA / G0-C14 LPNPs. Figure 34-a shows agarose gel electrophoresis imaging of a representative microfluidically prepared mRNA / G0-C14 LPNP; Figure 34-b shows the mRNA encapsulation efficiency of different batches of microfluidically prepared mRNA / G0-C14 LPNPs. Figure 34 demonstrates that microfluidically prepared mRNA / G0-C14 LPNPs achieve nearly complete mRNA encapsulation, with an encapsulation efficiency approaching ~100%.

[0264] 3. Cytotoxicity evaluation of mRNA / G0-C14 LPNPs:

[0265] Figure 35 shows the in vitro cytotoxicity test of mRNA / G0-C14 LPNPs at different concentrations. Figure 35 shows that EGFP mRNA / G0-C14 LPNPs exhibit good cellular safety.

[0266] 4. Luci-mRNA expression capability of microfluidic prepared mRNA / G0-C14 LPNPs:

[0267] Figure 36 shows the bioluminescence levels of A549 cells transfected with different concentrations of mRNA / G0-C14 LPNPs. Figure 37 shows the luciferase expression levels at different time points after intramuscular injection of mRNA / G0-C14 LPNPs (50 ng / μL mRNA, 50 μL). Figure 37-a shows bioluminescence imaging of mice at different time points after intramuscular injection of mRNA / G0-C14 LPNPs. Figure 37-b shows the quantitative analysis of bioluminescence in mice.

[0268] Figures 36 and 37 show that the mRNA LPNPs prepared by microfluidics exhibited efficient protein expression capabilities at both the cellular and animal levels.

[0269] The inventors of this case also referred to the aforementioned embodiments and conducted experiments using other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0270] The various aspects, embodiments, features and examples of the present invention should be considered as illustrative in all respects and are not intended to limit the present invention, the scope of which is defined solely by the claims. Other embodiments, modifications and uses will be apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0271] The use of headings and sections in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the disclosure.

[0272] Throughout this disclosure, where compositions are described as having, containing, or comprising particular components, or where processes are described as having or comprising particular process steps, it is contemplated that the compositions taught by this disclosure also consist essentially of, or consist of, the recited components, and that the processes taught by this disclosure also consist essentially of, or consist of, the recited process steps.

[0273] Should be understood that, the order of each step or the order in which specific action is performed is not very important, as long as the present invention teachings remain operable.In addition, two or more steps or actions can be performed simultaneously.

[0274] The above description is only a preferred embodiment of the present invention and does not impose any formal or substantial limitation on the present invention. Any technician familiar with the present profession can make use of the technical content disclosed above without departing from the scope of the technical solution of the present invention, and any equivalent changes, modifications and evolutions made by them are equivalent embodiments of the present invention. At the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. An mRNA polymer-lipid hybrid delivery system, characterized in that: The components of the delivery system include mRNA, cationic molecules, polymers and modified amphiphilic molecules; wherein the cationic molecules and the negatively charged mRNA form a complex through electrostatic interaction; the polymer is used to accommodate the cationic molecule-mRNA complex, and the three form a stable inner core structure; the modified amphiphilic molecules are anchored on the surface of the inner core structure through hydrophobic interaction to form a spherical core-shell structure.

2. The mRNA polymer-lipid hybrid delivery system according to claim 1, characterized in that: The mRNA is selected from at least one of the following mRNAs: self-reporter gene mRNA, genome editing mRNA, antigen mRNA, tumor suppressor gene mRNA, interleukin mRNA or cell chimeric antigen receptor mRNA or other functional mRNA.

3. The mRNA polymer-lipid hybrid delivery system according to claim 2, characterized in that: The self-reporter gene mRNA is selected from one of GFP, eGFP, Fluc, mCherry, beta gal or renilla Luc; The genome editing mRNA is selected from one of Cas9, Cas9 Nickase or Cre recombinase; The antigen mRNA is selected from OVA; The tumor suppressor gene mRNA is selected from one of p53 and PTEN; The interleukin mRNA is selected from one of IL-10, IL-12 or IL-6.

4. The mRNA polymer-lipid hybrid delivery system according to claim 1, characterized in that: The cationic molecule is selected from at least one of ammonia molecule derivatives, ammonium salts and their derivatives, positively charged amphiphilic lipid compounds, polyamides and their derivatives, polyethyleneimine and their derivatives.

5. The mRNA polymer-lipid hybrid delivery system according to claim 1, characterized in that: The polymer is selected from at least one of the following polymers: aliphatic polyester polymers and their derivatives, polycarbonate and its derivatives, polysaccharides and their derivatives, polyamino acids and their derivatives, polymer polyols and their derivatives, polyacrylic acid and its derivatives.

6. The mRNA polymer-lipid hybrid delivery system according to claim 5, characterized in that: The aliphatic polyester polymer and its derivatives are selected from one of polylactic acid, polycaprolactone and polylactic acid-glycolic acid copolymer.

7. The mRNA polymer-lipid hybrid delivery system according to claim 1, characterized in that: The modified amphiphilic molecule is selected from at least one of the following molecules: PEGylated amphiphilic molecules, amphiphilic surfactants, amphiphilic proteins or other modified molecules.

8. The mRNA polymer-lipid hybrid delivery system according to claim 7, characterized in that: The PEGylated amphiphilic molecule is selected from one of DSPE-PEG, DMG-PEG, and F127; The amphiphilic surfactant is CO-520; The amphipathic protein is albumin; The other modifying molecules are selected from one of mannitol, modified chitosan, dextran, carboxydextran, tetraethyl orthosilicate, polyacrylic acid, KH560, KH550, diethylenetriaminepentaacetic acid, meglumine, arginine, polyglutamic acid or polypeptide.

9. A method for preparing an mRNA polymer-lipid hybrid delivery system, comprising the following steps: (a) preparing a cationic molecule solution; (b) mRNA and cationic molecules form an electrostatic complex; (c) adding a polymer and a modified amphiphilic molecule to the mRNA / cationic molecule complex formed above in sequence; (d) Nanoparticles that stably encapsulate mRNA, namely mRNA polymer-lipid hybrid delivery system, were obtained by self-assembly method.

10. The preparation method according to claim 9, characterized in that: The specific steps are: (a1) dissolving the cationic molecule in a solvent to prepare a solution containing the cationic molecule at a concentration of 0.5 to 5.0 mg / mL; The cationic molecule is selected from at least one of ammonia molecule derivatives, ammonium salts and their derivatives, positively charged amphiphilic lipid compounds, polyamides and their derivatives, polyethyleneimine and their derivatives; The solvent is selected from at least one of methanol, ethanol, ethylene glycol, diethylene glycol, DMF, benzyl alcohol, acetone, dichloromethane and chloroform; (b1) mixing the solution containing mRNA and the solution containing cationic molecules uniformly, and reacting for a period of time to obtain an mRNA / cationic molecule electrostatic complex; The reaction time is 5s to 30min, and the reaction temperature is 0°C to 37°C; The concentration of mRNA in the mRNA-containing solution is 0.1 to 10 mg / mL; The mass ratio of the mRNA to the cationic molecule is 1:1 to 1:100; The mRNA is selected from at least one of the following mRNAs: self-reporter gene mRNA, genome editing mRNA, antigen mRNA, tumor suppressor gene mRNA, interleukin mRNA or cell chimeric antigen receptor mRNA or other functional mRNA; The self-reporter gene mRNA is selected from one of GFP, eGFP, Fluc, mCherry, beta gal or renilla Luc; The genome editing mRNA is selected from one of Cas9, Cas9 Nickase or Cre recombinase; The antigen mRNA is selected from OVA; The tumor suppressor gene mRNA is selected from one of p53 and PTEN; The interleukin mRNA is selected from one of IL-10, IL-12 or IL-6; (c1) uniformly mixing the mRNA / cationic molecule electrostatic complex and the polymer solution; The reaction time is 5s to 30min, and the reaction temperature is 0°C to 37°C; The concentration of the polymer molecules in the solution containing the polymer molecules is 0.1 to 20 mg / mL; The polymer is selected from at least one of the following polymers: aliphatic polyester polymers and their derivatives, polycarbonate and its derivatives, polysaccharides and their derivatives, polyamino acids and their derivatives, polymer polyols and their derivatives, polyacrylic acid and its derivatives; (d1) adding the modified amphiphilic molecule solution to the mRNA / cationic molecule / polymer mixed solution and mixing well; The concentration of the modified amphiphilic molecule in the modified amphiphilic molecule solution is 0.1 to 20 mg / mL; The modified amphiphilic molecule is selected from at least one of the following molecules: PEGylated amphiphilic molecules, amphiphilic surfactants, amphiphilic proteins or other modified molecules; (e1) adding the mRNA / cationic molecule / polymer / modified amphiphilic molecule mixture to the acidic aqueous phase solution, and reacting for a period of time to obtain the mRNA-LPNPs nano delivery system particles; The pH range of acidic solutions is 1 to 6; The reaction time is 30s to 30min, and the reaction temperature is 0°C to 37°C; The acidic buffer solution includes but is not limited to at least one of citric acid / citrate, acetic acid / acetate, hydrochloric acid / hydrochloride, sulfuric acid / sulfate, and nitric acid / nitrate; In steps (b1), (c1), (d1) and (e1), the solution mixing method includes but is not limited to at least one of the following methods: dripping, stirring, standing, microfluidic equipment, peristaltic pump, microinjection pump, etc.

11. The preparation method according to claim 10, characterized in that: The aliphatic polyester polymer and its derivatives in step (c1) are selected from one of polylactic acid, polycaprolactone, and polylactic acid-glycolic acid copolymer.

12. The preparation method according to claim 10, characterized in that: The PEGylated amphiphilic molecule in step (d1) is selected from one of DSPE-PEG, DMG-PEG, and F127; The amphiphilic surfactant is CO-520; The amphipathic protein is albumin; The other modifying molecules are selected from one of mannitol, modified chitosan, dextran, carboxydextran, tetraethyl orthosilicate, polyacrylic acid, KH560, KH550, diethylenetriaminepentaacetic acid, meglumine, arginine, polyglutamic acid or polypeptide.

13. The preparation method according to any one of claims 10 to 12, characterized in that: The preparation method is a microfluidic preparation, comprising steps (a1), (b1) and (c1); (d1) adding the modified amphiphilic molecule solution to the mRNA / cationic molecule / polymer mixed solution prepared in step (c1), mixing well to obtain a DMF organic phase; (e1) preparing an aqueous solution; (f1) Under microfluidic conditions, the DMF organic phase flow rate was 1-5 mL / min, and the aqueous solution flow rate was 5-25 mL / min to prepare mRNA LPNPs.

14. An mRNA polymer-lipid hybrid delivery system prepared by the preparation method according to any one of claims 9 to 13.

15. An mRNA polymer-lipid hybrid delivery system according to any one of claims 1 to 8 or claim 14, characterized in that: The encapsulation rate of mRNA in the mRNA-LPNPs delivery system is ≥50%, preferably ≥60%, more preferably ≥70%, more preferably ≥80%, more preferably ≥90%, and most preferably ≥95%.

16. An mRNA polymer-lipid hybrid delivery system according to any one of claims 1 to 8 or claim 14, characterized in that: The mass ratio of mRNA in the mRNA-LPNPs delivery system to other molecules in the delivery system is 1:100 to 10:

1.

17. An mRNA polymer-lipid hybrid delivery system according to any one of claims 1 to 8 or claim 14, characterized in that: The mRNA-LPNPs delivery system is a two-dimensional, three-dimensional or layered structure; the nano-delivery system particles are three-dimensional nano-composite materials with mRNA-cationic molecules-polymers as the core and modified amphiphilic molecules coated on the outside as the shell; the particle size of the nano-delivery system particles is 10nm to 500nm, preferably 20-250nm, more preferably 30-200nm, more preferably 70-130nm, more preferably 100-125nm, and optimally 110-125nm.

18. An mRNA polymer-lipid hybrid delivery system according to any one of claims 1 to 8 or claim 14, characterized in that: Based on the total number of the mRNA-LPNPs delivery system, 80%, preferably 85%, more preferably 90%, more preferably 95%, and optimally ≥99% of the particle sizes of the delivery system materials are within the D50 range of ±15%, preferably ±10%, and more preferably ±1% of the nanocomposite material.

19. An mRNA polymer-lipid hybrid delivery system according to any one of claims 1 to 8 or claim 14, characterized in that: The mRNA-LPNPs delivery system can be stably dispersed in aqueous solution and 0.9% saline.

20. An mRNA polymer-lipid hybrid delivery system according to any one of claims 1 to 8 or claim 14, characterized in that: When the mRNA-LPNPs delivery system is stored in water or 0.9% physiological saline at 4°C, 25°C, 37°C, -20°C, or -80°C for 5-20 days, preferably 20-40 days, and more preferably 40-60 days, the average particle size change of the nanodelivery system particles is ≤20%, preferably ≤10%, more preferably ≤5%, and optimally ≤3%.

21. An mRNA polymer-lipid hybrid delivery system according to any one of claims 1 to 8 or claim 14, characterized in that: The cell transfection efficiency of mRNA in the mRNA-LPNPs delivery system is ≥50%, preferably ≥60%, more preferably ≥70%, more preferably ≥80%, more preferably ≥90%, and most preferably ≥95%.

22. Use of an mRNA polymer-lipid hybrid delivery system according to any one of claims 1 to 8 or claim 14, characterized in that: The applications include applications in preparing tumor therapeutic drugs, applications in preparing tumor vaccines, applications in preparing tumor targeted drugs, applications in preparing materials for in vivo and in vitro diagnosis of tumors, applications in preparing treatments for inflammation-related diseases, applications in preparing treatments for infectious diseases, applications in preparing treatments for the prevention of infectious diseases, applications in preparing treatments for wound healing, applications in preparing treatments for chronic cardiovascular and cerebrovascular diseases, applications in preparing treatments for organ fibrosis, applications in preparing treatments for viral infections, applications in preparing treatments for the prevention of viral infections, applications in preparing treatments for in vivo and in vitro gene editing, applications in preparing treatments for non-alcoholic fatty liver disease, applications in preparing treatments for intestinal diseases, applications in preparing new antigen vaccines, applications in preparing cell induced differentiation, applications in preparing protein beauty, applications in preparing treatments for protein defective diseases, applications in preparing aging repair, applications in preparing anti-aging products, or applications in preparing drug carriers.

23. A preparation comprising the mRNA polymer-lipid hybrid delivery system according to any one of claims 1 to 8 or claim 14.

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