Nano-preparation for promoting tissue repair and use thereof

Abstract

A nano-preparation for promoting tissue repair and use thereof. The nano-preparation comprises a circular RNA encoding a growth factor, a circular RNA encoding a cytokine, and a nano-vector. The nano-preparation can accurately express and release a growth factor and a cytokine in a cell, regulate the immune environment, inhibit inflammation, and promote wound tissue repair.

Description

A nano-formulation for promoting tissue repair and its application Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nano-formulation that promotes tissue repair and its application. Background Technology

[0002] Difficult-to-heal wounds (ulcers) include diabetic foot ulcers, pressure ulcers, vascular ulcers, radiation ulcers, tophi ulcers, iatrogenic wounds, traumatic ulcers, infected ulcers, drug-induced ulcers, neurotrophic ulcers, snake bite ulcers, post-burn scar ulcers, fistulas, sinus tracts, etc., characterized by their inability to heal completely despite long-term treatment. Diabetic foot ulcer (DFU) is one of the most serious, common, and costly complications in diabetic patients. It is caused by neuropathy and poor blood supply, leading to inflammation and damage to the skin and soft tissues. The local immune microenvironment disorder, characterized by an imbalance in macrophage phenotypic polarization, is the main reason for the prolonged non-healing of DFU wounds, manifested as persistent inflammation, high levels of inflammatory cytokines, matrix metalloproteinases, and low levels of growth factors. Effective revascularization is one of the most promising treatments for promoting wound healing and functional recovery in diabetic patients. Growth factors, such as human vascular endothelial growth factor a (VEGFa), play a crucial role in promoting increased vascular permeability, extracellular matrix degeneration, vascular endothelial cell migration, proliferation, and angiogenesis. Therefore, wound supplementation with VEGFa, platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF2), and nerve growth factor (NGF) can promote capillary angiogenesis, repair cell recruitment and proliferation, granulation tissue formation, and nerve repair. Growth factors can be administered in recombinant protein form to treat diabetic fibrosis (DFU). However, protein-based growth factors degrade rapidly in wounds, resulting in short half-lives, requiring multiple doses, leading to poor patient compliance and hindering wound healing. Macrophages, as innate immune cells, play a crucial role in resisting pathogen infection and regulating inflammation. M1 macrophages have pro-inflammatory effects, while M2 macrophages primarily inhibit inflammation and promote tissue repair. In DFU (disease-induced fibrosis) chronic wound tissue, macrophage M2 polarization is inhibited, leading to persistent inflammation and suppressing the initiation of proliferative states, subsequent extracellular matrix deposition, and tissue remodeling. Therefore, regulating macrophage polarization and inhibiting inflammation are prerequisites for promoting DFU healing. The mechanism of immune dysregulation lies in cytokine signaling imbalance. Administering cytokines to the wound can regulate immune function, achieving immunotherapy. Interleukin-4 (IL4), by inducing STAT6 phosphorylation, activates M2 tag gene transcription, causing changes in target cell phenotype. This transforms macrophage polarization from a pro-inflammatory M1 phenotype to an anti-inflammatory, tissue-healing M2 phenotype, promoting extracellular matrix deposition and tissue regeneration. However, administering cytokines in recombinant protein form also suffers from drawbacks such as rapid degradation at the wound site, short half-life, inconvenience in clinical use, and difficulty in achieving sustained pharmacological activity and high therapeutic efficacy. mRNA therapy is one of the important treatment methods that has emerged in recent years, especially mRNA-based protein replacement therapy, which has developed rapidly due to the high specificity and flexibility of mRNA, resulting in fewer side effects, lower production costs, and shorter treatment times.However, linear mRNAs have poor stability and short expression time of proteins within cells. The amount of growth factors expressed is insufficient to meet the goals of tissue repair and disease treatment. Therefore, there is an urgent need to invent an agent that can express and release cytokines and growth factors for a long time to continuously control inflammation and promote wound tissue repair. Summary of the Invention

[0003] Technical Problem to be Solved: To address the aforementioned technical problems, the present invention aims to provide a nano-formulation for promoting tissue repair and its application. This nano-formulation comprises circular RNA encoding growth factors and circular RNA encoding cytokines, along with a nanocarrier. Its preparation method includes the preparation of a lipid carrier material, the preparation of a solution of the circular RNA encoding growth factors and cytokines, and microfluidic mixing. This invention belongs to the field of biomedical technology. The nanocarrier prepared by this invention can simultaneously and precisely express and release growth factors and cytokines within cells, exerting a long-term effect to promote wound tissue repair. It is characterized by non-toxicity, high safety, ease of use, simple preparation, and excellent efficacy, providing a new therapeutic approach for tissue repair and regeneration, and has clinical application value.

[0004] Technical solution: A nano-formulation for promoting tissue repair, wherein the nano-formulation is composed of three components: component 1 is a circular RNA encoding a growth factor or a growth factor fusion protein; component 2 is a circular RNA encoding a cytokine or a cytokine fusion protein; and component 3 is a nanocarrier; wherein the content of component 1 is 0.5-7.5%, the content of component 2 is 0.5-7.5%, and the content of component 3 is 85-99%. Further, component 1 is composed of spacer region 1, exon 1, exon 2, spacer region 2, ribosome insertion site, Kozak sequence, and coding region connected sequentially; the sequence of spacer region 1 is the sequence shown in SEQ ID No. 1 or a sequence with at least 75% identity to it; the sequence of exon 1 is the sequence shown in SEQ ID No. 2 or a sequence with at least 75% identity to it; the sequence of exon 2 is the sequence shown in SEQ ID No. 3 or a sequence with at least 75% identity to it; the sequence of spacer region 2 is the sequence shown in SEQ ID No. 4 or a sequence with at least 75% identity to it; the sequence of the ribosome insertion site is the sequence shown in any one of SEQ ID No. 5, 6, 7, or 8 or a sequence with at least 75% identity to it; the sequence of Kozak is the sequence shown in SEQ ID No. 9; the sequence of the coding region is the sequence shown in any one of growth factor SEQ ID Nos. 10-22, or a growth factor fusion protein sequence or a sequence with at least 75% identity to it; the growth factor fusion protein sequence is derived from the growth factor sequence SEQ ID No. 10-22. It consists of any one of the sequences shown in ID No. 10-22, the linker peptide sequence shown in SEQ ID No. 36, and any one of the following sequences: human serum albumin SEQ ID No. 32, human serum albumin antibody SEQ ID No. 33, human immunoglobulin antibody constant region SEQ ID No. 34, and placental growth factor heparin binding domain SEQ ID No. 35. Further, component 2 is formed by sequentially connecting spacer region 1, exon 1, exon 2, spacer region 2, ribosome insertion site, Kozak sequence, and coding region; the sequence of spacer region 1 is the sequence shown in SEQ ID No. 1 or a sequence with at least 75% identity to it; the sequence of exon 1 is the sequence shown in SEQ ID No. 2 or a sequence with at least 75% identity to it; the sequence of exon 2 is the sequence shown in SEQ ID No. 3 or a sequence with at least 75% identity to it; the sequence of spacer region 2 is the sequence shown in SEQ ID No. 4 or a sequence with at least 75% identity to it; the sequence of ribosome insertion site is the sequence shown in any one of SEQ ID No. 5, 6, 7, or 8 or a sequence with at least 75% identity to it; the sequence of Kozak is the sequence shown in SEQ ID No. 9.The sequence of the coding region is any one of the sequences shown in SEQ ID No. 23-31 of the cytokine sequence, or a growth factor fusion protein sequence or a sequence having at least 75% identity with it; the cytokine fusion protein sequence is composed of any one of the sequences shown in SEQ ID No. 23-31 of the cytokine sequence, the sequence shown in SEQ ID No. 36 of the linker peptide sequence, and any one of the sequences shown in SEQ ID No. 32 of human serum albumin, SEQ ID No. 33 of human serum albumin antibody, SEQ ID No. 34 of the constant region of human immunoglobulin antibody, and SEQ ID No. 35 of the placental growth factor heparin binding domain. Furthermore, the growth factor is platelet-derived growth factor (PDGF), epidermal growth factor (EGF), acidic fibroblast growth factor (FGF1), basic fibroblast growth factor (FGF2), nerve growth factor (NGF), insulin-like growth factor 1 (IGF1), insulin-like growth factor 2 (IGF2), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGFa), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT3), or transforming growth factor β1 (TGFβ1); the growth factor fusion protein is composed of component A, a linker peptide, and component B, wherein component A is a growth factor, and component B is one or more of human serum albumin, human serum albumin antibody, human immunoglobulin antibody constant region protein, and placental growth factor heparin binding domain (PlGF2-123-144) protein. Furthermore, the cytokines are one or more of the following: interleukin-4 (IL4), interleukin-10 (IL10), interleukin-13 (IL13), interleukin-22 (IL22), macrophage colony-stimulating factor (MCSF), chemokine 12 (CXCL12), interferon regulatory factor 4 (IRF4), islet regeneration protein (REG3A), and tumor necrosis factor α-stimulating gene / inducible protein-6 (TSG-6);The cytokine fusion protein is composed of component C, a linker peptide, and component D, wherein component C is a cytokine, and component D is one or more of human serum albumin, human serum albumin antibody, human immunoglobulin antibody constant region protein, and placental growth factor heparin binding domain (PlGF2-123-144) protein. Further, the nanoformulation consists of three components: component 1 is one or more of the following: circular RNA encoding platelet-derived growth factor or its fusion protein; circular RNA encoding epidermal growth factor or its fusion protein; circular RNA encoding basic fibroblast growth factor or its fusion protein; circular RNA encoding nerve growth factor or its fusion protein; circular RNA encoding vascular endothelial growth factor α or its fusion protein; and circular RNA encoding transforming growth factor β1 or its fusion protein; component 2 is a circular RNA encoding interleukin-4 or its fusion protein and a circular RNA encoding macrophage colony-stimulating factor or its fusion protein; and component 3 is a nanocarrier. Furthermore, the preparation method of the nano-formulation includes the following steps: S1. Adding ethanol to lipid raw material 1 and lipid raw material 2 and mixing evenly to obtain an alcohol phase containing lipids; S2. Adding citrate buffer to component 1 and mixing evenly to obtain an aqueous phase A; S3. Adding citrate buffer to component 2 and mixing evenly to obtain an aqueous phase B; S4. Microfluidic mixing to remove ethanol and obtain the nano-formulation.The lipid raw material 1 is one or more of ionizable lipids, cationic lipids, and thiourea lipids, wherein the ionizable lipid is O,O'-bis(1-octylnonyl)-7,7'-((2-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate (U-101), O,O'-bis(2-hexyldecyl)-7,7'-((2-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate (U-102), and O-2-hexyldecyl-O'-dodecyl-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate (U-103). O-2-hexyldecyl-O'-dodecyl-6,6'-((3-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate (U-104), O,O'-bis(1-octylnonyl)-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate (U-105), O,O'-bis(2-hexyldecyl)-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate (U-106), O-1-octylnonyl-O'-dodecyl-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl) Azadiyl)dihexanoate (U-107), O-1-octylnonyl-O'-dodecyl-7,7'-((2-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate (U-108), O,O'-bis(1-heptyloctyl)-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate (U-109), O,O'-bis(1-heptyloctyl)-7,7'-((2-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate (U-110), O-1-heptyloctyl-O'-dodecyl-6,6'-(( Any one of the following: 3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate (U-111), O-1-heptyloctyl-O'-dodecyl-7,7'-((2-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate (U-112), 6-((2-hexyldecanoyl)oxy)-N-6-(((2-ethyldecanoyl)oxy)hexyl)-N(4-hydroxybutyl)hex-1-amine (ALC-0315), heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecanoyl)hexyl)amino)octanoate (SM-102);The lipid raw material 2 is one or more of sterols, phospholipids, glycerides, and polyethylene glycol-modified lipids, wherein the sterol is any one of cholesterol, ergosterol, 7-dehydrocholesterol, and β-sitosterol; the phospholipid is any one of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, ceramide, and cardiolipin; and the polyethylene glycol-modified lipid is any one of polyethylene glycol (PEG)-modified phosphatidylethanolamine, polyethylene glycol (PEG)-modified ceramide, polyethylene glycol (PEG)-modified diacylglycerol, polyethylene glycol (PEG)-modified phosphatidic acid, polyethylene glycol (PEG)-modified dialkylamine, and PEG-modified dialkylglycerol. The microfluidic mixing method includes mixing a three-phase system containing lipids (alcohol phase, aqueous phase A, and aqueous phase B); mixing a two-phase system containing lipids (alcohol phase and aqueous phase A); and mixing a two-phase system containing lipids (alcohol phase and aqueous phase B). The application of the tissue-repair-promoting nano-formulations described above in the preparation of drug nano-formulations for treating refractory wounds, myocardial infarction, stroke, arthritis, acute lung injury, and tendinitis. Further, the drug nano-formulation comprises pharmaceutically acceptable adjuvants, including porous polymer scaffolds and hydrogels. Further, the dosage forms of the drug nano-formulation include, but are not limited to, powders, granules, patches, ointments, lyophilized powders, dressings, gels, sprays, liniments, liquid drops, and injections; the routes of administration include, but are not limited to, local administration, intravenous administration, subcutaneous administration, and intramuscular injection. Further, the refractory wounds are diabetic foot ulcers, pressure ulcers, vascular ulcers, radiation ulcers, tophi ulcers, iatrogenic wounds, traumatic ulcers, infected ulcers, drug-induced ulcers, neurotrophic ulcers, snake bite ulcers, post-burn scar ulcers, fistulas, and chronic refractory wounds caused by sinuses. Beneficial effects: 1. The nano-formulation in this invention can, on the one hand, regulate the RNA expression encoding growth factors or growth factor fusion proteins, promote capillary angiogenesis, recruit and proliferate various repair cells, and promote granulation tissue formation; on the other hand, it can regulate the RNA expression encoding cytokines and cytokine fusion proteins, regulate the internal immune microenvironment, promote macrophage polarization and proliferation, induce macrophages to change from a pro-inflammatory M1 phenotype to a pro-tissue-healing M2 phenotype, inhibit macrophage inflammation, promote extracellular matrix deposition and tissue remodeling and regeneration, and work synergistically to achieve efficient repair of difficult-to-heal wounds. 2. The nano-formulation prepared in this invention contains PDGF circRNA, IL4 circRNA, and MCSF circRNA, which can express and secrete PDGF, IL4, and MCSF in cells at the wound site. Among them, PDGF can stimulate the division and proliferation of various cells such as vascular smooth muscle cells, fibroblasts, and nerve cells, and promote the formation of granulation tissue; IL4 has an immunomodulatory effect on macrophages, can induce macrophage M2 polarization, promote tissue remodeling, and inhibit inflammation; MCSF can promote monocyte survival, monocyte transformation into macrophages, and macrophage proliferation, enhance the killing effect of macrophages on microorganisms, and efficiently achieve rapid healing of difficult-to-heal wounds. 3. The nano-formulation prepared in this invention contains growth factor (FGF2, EGF, VEGFa or NGF) circRNA, IL4 circRNA, and MCSF circRNA, which can express and secrete growth factors, IL4, and MCSF in cells at the wound site. Among them, growth factors (FGF2, EGF, VEGFa or NGF) can stimulate the division and proliferation of various cells such as vascular smooth muscle cells, fibroblasts, and nerve cells, and promote the formation of epithelial tissue and granulation tissue; IL4 has an immunomodulatory effect on macrophages, can induce macrophage M2 polarization, promote tissue remodeling, and inhibit inflammation; MCSF can promote monocyte survival, monocyte transformation into macrophages, and macrophage proliferation, enhance the killing effect of macrophages on microorganisms, and efficiently achieve rapid healing of difficult-to-heal wounds. 4. The nano-formulation in this invention is safe and non-toxic, with high safety. It can simultaneously and continuously express and release growth factors and cytokines within cells, resulting in a long-lasting effect. It can be applied locally at the wound site, making it convenient to use. It can also be used in combination with pharmaceutically acceptable adjuvants to achieve efficient and rapid repair and healing of wounded tissues. It has a good effect on chronic and difficult-to-heal wounds caused by various ulcers, and can provide a new approach for tissue repair and regeneration, thus having clinical application value. Attached Figure Description Figure 1 is a schematic diagram of the sequence structure of circular RNA; Figure 2 shows the relationship between protein expression levels and time after co-incubation of the comparative 2-13 nanoparticle formulation with cells, where a represents PDGF protein concentration (1 represents U-LNP / PDGF circRNA, 2 represents A-LNP / PDGF circRNA, 3 represents U-LNP / PDGF mRNA, and 4 represents A-LNP / PDGF mRNA), b represents IL4 protein concentration (1 represents U-LNP / IL4 circRNA, 2 represents A-LNP / IL4 circRNA, 3 represents U-LNP / IL4 mRNA, and 4 represents A-LNP / IL4 mRNA), and c represents MCSF protein concentration (1 represents U-LNP / MCSF circRNA, 2 represents A-LNP / MCSF circRNA, 3 represents U-LNP / MCSF mRNA, and 4 represents A-LNP / MCSF mRNA). Figure 3 shows the wound healing status of diabetic mice on days 0, 4, 7, and 10 after single-dose administration of different nanoparticles. In the figure, a represents PBS, b represents U-LNP / eGFP circRNA, c represents U-LNP / IL4 mRNA, d represents U-LNP / PDGF mRNA, e represents U-LNP / MCSF mRNA, f represents U-LNP / IL4 circRNA, g represents U-LNP / PDGF circRNA, h represents U-LNP / MCSF circRNA, i represents U-LNP / PDGF circRNA / U-LNP / IL4 circRNA, j represents U-LNP / PDGF circRNA / U-LNP / MCSF circRNA, k represents U-LNP / PDGF circRNA / U-LNP / IL4 circRNA / U-LNP / MCSF circRNA, l represents U-LNP / PDGF circRNA / IL4 circRNA, and m represents U-LNP / PDGF circRNA / MCSF. circRNA, n represents U-LNP / PDGF circRNA / IL4 circRNA / MCSF circRNA; Figure 4 shows the changes in wound area on days 0, 4, 7, and 10 after administration of different nanoparticles to the wounds of diabetic mice. In the figure, a represents PBS, b represents U-LNP / eGFP circRNA, c represents U-LNP / IL4 mRNA, d represents U-LNP / PDGF mRNA, e represents U-LNP / MCSF mRNA, f represents U-LNP / IL4 circRNA, g represents U-LNP / PDGF circRNA, and h represents U-LNP / MCSF circRNA. Figure 5 shows the changes in wound area on days 0, 4, 7, and 10 after administration of different nanoparticles to the wounds of diabetic mice. In the figure, i represents U-LNP / PDGF circRNA / U-LNP / IL4 circRNA, j represents U-LNP / PDGF circRNA / U-LNP / MCSF circRNA, k represents U-LNP / PDGF circRNA / U-LNP / IL4 circRNA / U-LNP / MCSF circRNA, l represents U-LNP / PDGF circRNA / IL4 circRNA, m represents U-LNP / PDGF circRNA / MCSF circRNA, and n represents U-LNP / PDGF circRNA / IL4 circRNA / MCSF circRNA. Figure 6 shows microscopic images of wound tissues from diabetic mice treated with different nanoparticles after H&E staining. In the figures, a represents PBS, b represents U-LNP / eGFP circRNA, c represents U-LNP / IL4 mRNA, d represents U-LNP / PDGF mRNA, e represents U-LNP / MCSF mRNA, f represents U-LNP / IL4 circRNA, g represents U-LNP / PDGF circRNA, h represents U-LNP / MCSF circRNA, i represents U-LNP / PDGF circRNA / U-LNP / IL4 circRNA, j represents U-LNP / PDGF circRNA / U-LNP / MCSF circRNA, k represents U-LNP / PDGF circRNA / U-LNP / IL4 circRNA / U-LNP / MCSF circRNA, and l represents U-LNP / PDGF circRNA / IL4 circRNA. Figure 7 shows microscopic images of wound tissues from diabetic mice after Masson staining following treatment with different nanoparticles. In the figures, a represents PBS, b represents U-LNP / eGFP circRNA, c represents U-LNP / IL4 mRNA, d represents U-LNP / PDGF mRNA, e represents U-LNP / MCSF mRNA, f represents U-LNP / IL4 circRNA, g represents U-LNP / PDGF circRNA, h represents U-LNP / MCSF circRNA, i represents U-LNP / PDGF circRNA / U-LNP / IL4 circRNA, j represents U-LNP / PDGF circRNA / U-LNP / MCSF circRNA, k represents U-LNP / PDGF circRNA / U-LNP / IL4 circRNA / U-LNP / MCSF circRNA, and l represents U-LNP / PDGF circRNA / IL4 circRNA. Figure 8 shows microscopic images of CD31 immunohistochemical staining of wound tissues from diabetic mice treated with different nanoparticles. In the figures, a represents PBS, b represents U-LNP / eGFP circRNA, c represents U-LNP / IL4 mRNA, d represents U-LNP / PDGF mRNA, e represents U-LNP / MCSF mRNA, f represents U-LNP / IL4 circRNA, g represents U-LNP / PDGF circRNA, h represents U-LNP / MCSF circRNA, i represents U-LNP / PDGF circRNA / U-LNP / IL4 circRNA, j represents U-LNP / PDGF circRNA / U-LNP / MCSF circRNA, k represents U-LNP / PDGF circRNA / U-LNP / IL4 circRNA / U-LNP / MCSF circRNA, and l represents U-LNP / PDGF circRNA / IL4 circRNA. Figure 9 shows the changes in wound area on days 0, 4, 7, and 10 after administration of different nanoparticles to the wounds of diabetic mice. In the figure, a represents the PBS group, b represents the U-LNP / FGF2 circRNA group, c represents the U-LNP / FGF2 circRNA / IL4 circRNAc group, d represents the U-LNP / FGF2 circRNA / MCSF circRNA group, e represents the U-LNP / FGF2 circRNA / IL4 circRNA / MCSF circRNA group, f represents the U-LNP / PDGF-PlGF circRNA / IL4-PlGF circRNA group, g represents the U-LNP / PDGF-PlGF circRNA / MCSF-PlGF circRNA group, h represents the U-LNP / PDGF-PlGF circRNA / IL4-PlGF circRNA / MCSF-PlGF circRNA group, i represents the U6-LNP / PDGF-Fc circRNA / IL4-Fc circRNA group, and j represents the U6-LNP / PDGF-Fc circRNA group. circRNA / MCSF-Fc circRNA group, k represents U6-LNP / PDGF-Fc circRNA / IL4-Fc circRNA / MCSF-Fc circRNA group. Detailed Implementation The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments: The materials used in this invention: 6-((2-hexyldecanoyl)oxy)-N-(6-(((2-ethyldecanoyl)oxy)hexyl)-N(4-hydroxybutyl)hex-1-amine (ALC-0315), O,O'-bis(1-octylnonyl)-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate (U-105), O,O'-bis(2-hexyldecanoyl)-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate (U-106), dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000), distearate phosphatidylcholine (DSPC), were all purchased from Aivit Shanghai Pharmaceutical Technology Co., Ltd.; PDGF mRNA (encoding platelet-derived growth factor mRNA), FGF2 mRNA (encoding basic fibroblast growth factor), IL4 mRNA (encoding interleukin-4), and MCSF mRNA (encoding macrophage colony-stimulating factor) were all purchased from Kaituo Biotechnology (Suzhou) Co., Ltd.; PDGF circRNA (platelet-derived growth factor circular RNA), EGF circRNA (epidermal growth factor circular RNA), FGF2 circRNA (basic fibroblast growth factor circular RNA), IL4 circRNA (interleukin-4 circular RNA), IL22 circRNA (interleukin-22 circular RNA), MCSF circRNA (macrophage colony-stimulating factor circular RNA), PDGF-Fc circRNA (platelet-derived growth factor-human immunoglobulin antibody constant region fusion protein circular RNA), IL4-Fc circRNA (interleukin-4-human immunoglobulin antibody constant region fusion protein circular RNA), and MCSF-Fc circRNA (macrophage colony-stimulating factor-human immunoglobulin antibody constant region fusion protein circular RNA), PDGF-PlGF circRNA (platelet-derived growth factor-placental growth factor heparin-binding domain (PlGF2-123-144) fusion protein circular RNA), IL4-PlGF circRNA (interleukin-4-placental growth factor heparin-binding domain (PlGF2-123-144) fusion protein circular RNA), MCSF-PlGF circRNA (macrophage colony-stimulating factor-placental growth factor heparin-binding domain (PlGF2-123-144) fusion protein circular RNA), and eGFP circRNA (enhanced green fluorescent protein circRNA) were all purchased from Kaituo Biotechnology (Suzhou) Co., Ltd. or synthesized and provided by the Southeast University laboratory, with a purity of over 90%; TE buffer and TE-Triton buffer were purchased from Anhui Baisha Biotechnology Co., Ltd.PBS buffer was purchased from Wuhan Saive Biotechnology Co., Ltd.; Ribogreen reagents and microplate readers were purchased from Thermo Fisher Scientific, Inc.; Amicon Ultra centrifuge filters were purchased from Guangzhou Jet Biofiltration Co., Ltd. Example 1 A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Dissolve 25 μg IL4 circRNA and 25 μg PDGF circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL. S3. Mix 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL using a microfluidic system, and then purify the mixture with 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with IL4 circRNA and PDGF circRNA, denoted as U-LNP / PDGF circRNA / IL4 circRNA nanoparticles. Example 2 A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Dissolve 25 μg MCSF circRNA and 25 μg PDGF circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL. S3. 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by Amicon Ultra centrifugation filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle formulation loaded with MCSF circRNA and PDGF circRNA, denoted as U-LNP / PDGF circRNA / MCSF circRNA nanoparticles. Example 3 A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 17 μg PDGF circRNA, 17 μg IL4 circRNA and 17 μg MCSF circRNA, dissolve them in 350 μL of 20 mM citrate buffer (pH 4.0), mix well to prepare an aqueous phase with a concentration of 142 μg / mL; S3. 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by Amicon Ultra centrifugation filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle formulation loaded with PDGF circRNA, IL4 circRNA and MCSF circRNA, denoted as U-LNP / PDGF circRNA / IL4 circRNA / MCSF circRNA nanoparticles. Example 4 A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Dissolve 50 μg of PDGF circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase of PDGF circRNA with a concentration of 142 μg / mL; S3. 150 μL of ethanol phase with a concentration of 25.7 mg / mL and 350 μL of aqueous phase of PDGF circRNA with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by Amicon Ultra centrifugation filter with 5 mL of PBS buffer (pH 7.4) to prepare 500 μL of U-LNP / PDGF circRNA nanoparticles. S4. Dissolve 50 μg of IL4 circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase of IL4 circRNA with a concentration of 142 μg / mL. S5. 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase of IL4 circRNA with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by Amicon Ultra centrifugation filter with 5 mL of PBS buffer (pH 7.4) to prepare 500 μL of U-LNP / IL4 circRNA nanoparticles. S6. Take 250 μL of U-LNP / PDGF circRNA nanoparticles and 250 μL of U-LNP / IL4 circRNA nanoparticles, mix them evenly, and prepare U-LNP / PDGF circRNA / U-LNP / IL4 circRNA nanoparticles. Example 5 A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Dissolve 50 μg of PDGF circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase of PDGF circRNA with a concentration of 142 μg / mL; S3. 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase of PDGF circRNA with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by Amicon Ultra centrifugation filter with 5 mL of PBS buffer (pH 7.4) to prepare 500 μL of U-LNP / PDGF circRNA nanoparticles. S4. Dissolve 50 μg of MCSF circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase of MCSF circRNA with a concentration of 142 μg / mL; S5. 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase of MCSF circRNA with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by Amicon Ultra centrifugation filter with 5 mL of PBS buffer (pH 7.4) to prepare 500 μL of U-LNP / MCSF circRNA nanoparticles. S6.S1. Take 250 μL of U-LNP / PDGF circRNA nanoparticles and 250 μL of U-LNP / MCSF circRNA nanoparticles, mix them evenly, and prepare U-LNP / PDGF circRNA / U-LNP / MCSF circRNA nanoparticles. Example 6 A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Dissolve 50 μg of PDGF circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase of PDGF circRNA with a concentration of 142 μg / mL; S3. 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase of PDGF circRNA with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by Amicon Ultra centrifugation filter with 5 mL of PBS buffer (pH 7.4) to prepare 500 μL of U-LNP / PDGF circRNA nanoparticles. S4. Dissolve 50 μg of IL4 circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase of IL4 circRNA with a concentration of 142 μg / mL. S5. 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase of IL4 circRNA with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by Amicon Ultra centrifugation filter with 5 mL of PBS buffer (pH 7.4) to prepare 500 μL LU-LNP / IL4 circRNA nanoparticles. S6. Dissolve 50 μg of MCSF circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase of MCSF circRNA with a concentration of 142 μg / mL; S7. 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase of MCSF circRNA with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by Amicon Ultra centrifugation filter with 5 mL of PBS buffer (pH 7.4) to prepare 500 μL of U-LNP / MCSF circRNA nanoparticles. S8. Take 250 μL of U-LNP / PDGF circRNA nanoparticles, 250 μL of U-LNP / IL4 circRNA nanoparticles, and 250 μL of U-LNP / MCSF circRNA nanoparticles, mix them evenly, and prepare U-LNP / PDGF circRNA / / U-LNP / IL4 circRNA / U-LNP / MCSF circRNA nanoparticles. Comparative Example 1 The difference between this comparative example and Example 2 is that IL4 circRNA and PDGF circRNA are replaced with eGFP circRNA. A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Dissolve 50 μg of eGFP circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. Mix 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL using a microfluidic system, and then purify the mixture with 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with eGFP circRNA, denoted as U-LNP / eGFP circRNA nanoparticles. Comparative Example 2 The difference between this comparative example and Example 2 is that IL4 circRNA is replaced with PDGF circRNA. A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Dissolve 50 μg of PDGF circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. Mix 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL using a microfluidic system, and then purify the mixture with 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with PDGF circRNA, denoted as U-LNP / PDGF circRNA nanoparticles. Comparative Example 3 The difference between this comparative example and Example 2 is that PDGF circRNA is replaced with IL4 circRNA. A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Dissolve 50 μg of IL4 circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. Mix 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL using a microfluidic system, and then purify the mixture with 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with IL4 circRNA, denoted as U-LNP / IL4 circRNA nanoparticles. Comparative Example 4 The difference between this comparative example and Example 2 is that PDGF circRNA and IL4 circRNA are replaced with MCSF circRNA. A method for preparing a nano-formulation to promote tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Dissolve 50 μg of MCSF circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. Mix 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL using a microfluidic system, and then purify the mixture with 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with MCSF circRNA, denoted as U-LNP / MCSF circRNA nanoparticles. Comparative Example 5 The difference between this comparative example and Example 2 is that IL4 circRNA is replaced with PDGF circRNA, and ionizable lipid U-105 is replaced with ionizable lipid ALC-0315. A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid ALC-0315 2.26 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.3 mg / mL; S2. Dissolve 50 μg of PDGF circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. Mix 150 μL of the alcohol phase with a concentration of 25.3 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL using a microfluidic system, and then purify the mixture with 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with PDGF circRNA, denoted as A-LNP / PDGF circRNA nanoparticles. Comparative Example 6 The difference between this comparative example and Example 2 is that PDGF circRNA is replaced with IL4 circRNA, and ionizable lipid U-105 is replaced with ionizable lipid ALC-0315. A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid ALC-0315 2.26 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.3 mg / mL; S2. Dissolve 50 μg of IL4 circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. Mix 150 μL of the alcohol phase with a concentration of 25.3 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL using a microfluidic system, and then purify the mixture with 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with IL4 circRNA, denoted as A-LNP / IL4 circRNA nanoparticles. Comparative Example 7 The difference between this comparative example and Example 2 is that PDGF circRNA and IL4 circRNA were both replaced with MCSF circRNA, and ionizable lipid U-105 was replaced with ionizable lipid ALC-0315. A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid ALC-0315 2.26 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.3 mg / mL; S2. Dissolve 50 μg of MCSF circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. Mix 150 μL of the alcohol phase with a concentration of 25.3 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL using a microfluidic system, and then purify the mixture with 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with MCSF circRNA, denoted as A-LNP / MCSF circRNA nanoparticles. Comparative Example 8 The difference between this comparative example and Example 2 is that IL4 circRNA and PDGF circRNA are replaced with PDGF mRNA. A method for preparing a nano-formulation to promote tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Dissolve 50 μg of PDGF mRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL. S3. Mix 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL using a microfluidic system, and then purify the mixture with 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with PDGF mRNA, denoted as U-LNP / PDGF mRNA nanoparticles. Comparative Example 9 The difference between this comparative example and Example 2 is that IL4 circRNA and PDGF circRNA are replaced with IL4 mRNA. A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Dissolve 50 μg of IL4 mRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL. S3. Mix 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL using a microfluidic system, and then purify the mixture with 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with IL4 mRNA, denoted as U-LNP / IL4 mRNA nanoparticles. Comparative Example 10 The difference between this comparative example and Example 2 is that IL4 circRNA and PDGF circRNA are replaced with MCSF mRNA. A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Dissolve 50 μg MCSF mRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. Mix 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL using a microfluidic system, and then purify the mixture with 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with MCSF mRNA, denoted as U-LNP / MCSF mRNA nanoparticles. Comparative Example 11 The difference between this comparative example and Example 2 is that IL4 circRNA and PDGF circRNA are replaced with PDGF mRNA, and ionizable lipid U-105 is replaced with ionizable lipid ALC-0315. A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid ALC-0315 2.26 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.3 mg / mL; S2. Dissolve 50 μg of PDGF mRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL. S3. Mix 150 μL of the alcohol phase with a concentration of 25.3 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL using a microfluidic system, and then purify the mixture with 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with PDGF mRNA, denoted as A-LNP / PDGF mRNA nanoparticles. Comparative Example 12 The difference between this comparative example and Example 2 is that IL4 circRNA and PDGF circRNA are replaced with IL4 mRNA, and ionizable lipid U-105 is replaced with ionizable lipid ALC-0315. A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid ALC-0315 2.26 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.3 mg / mL; S2. Dissolve 50 μg of IL4 mRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL. S3. Mix 150 μL of the alcohol phase with a concentration of 25.3 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL using a microfluidic system, and then purify the mixture with 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with IL4 mRNA, denoted as A-LNP / IL4 mRNA nanoparticles. Comparative Example 13 The difference between this comparative example and Example 2 is that IL4 circRNA and PDGF circRNA are replaced with MCSF mRNA, and ionizable lipid U-105 is replaced with ionizable lipid ALC-0315. A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid ALC-0315 2.26 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.3 mg / mL; S2. Dissolve 50 μg MCSF mRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. Mix 150 μL of the alcohol phase with a concentration of 25.3 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL using a microfluidic system, and then purify the mixture with 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with MCSF mRNA, denoted as A-LNP / MCSF mRNA nanoparticles. Performance testing (1) Particle size, polydispersity index (PDI) and zeta potential The particle size, polydispersity index (PDI), and zeta potential of Examples 1-6 and Comparative Examples 1-13 were measured using a Zetasizer Nano ZS dynamic light scattering instrument. The results are shown in Table 1. (2) Packet efficiency The encapsulation efficiency of Examples 1-6 and Comparative Examples 1-13 was measured using the Ribogreen reagent method. Specifically, the samples were treated with TE buffer and 2% TE-Triton buffer, respectively, Ribogreen reagent was added, and fluorescence intensity was measured using a microplate reader. The amount of dissociated circRNA (or mRNA) and the total amount of circRNA (or mRNA) were obtained and calculated using the following formula: Packing efficiency (%) = [(Amount of dissociated circRNA (or mRNA)) / Total amount of circRNA (or mRNA)] × 100%. (3) Cell viability HUVECs cells were seeded into 96-well plates (2 × 10⁶ cells per well). 3 HUVECs were cultured until 85% confluence was achieved. Then, Opti-MEMI Reduced Serum Medium was added instead of the cell culture medium. The nanoparticles prepared in Examples 1-6 and Comparative Examples 1-13 (all nanoparticles were 10 μg circRNA or mRNA dissolved in 100 μL PBS buffer) were co-cultured with HUVECs for 24 h, with a blank control included. The cells were then treated with a cell counting kit-8, and OD was measured using a microplate reader. 450nm Cell viability was calculated, and the results are shown in Table 1. Table 1. Particle size, PDI, Zeta potential, loading efficiency, and cell viability of Examples 1-6 and Comparative Examples 1-13 As shown in Table 1, the particle size of Examples 1-6 and Comparative Examples 1-13 is around 100 nm, the PDI is around 0.2, the Zeta potential is in the range of -6 to -3 mV, the encapsulation efficiency is in the range of 87-92%, and the cell viability reaches 100-126%. They all have suitable size, good encapsulation efficiency, and are non-toxic, have high safety, and do not cause much damage to cells. This indicates that there is no significant difference in the physicochemical properties of nano-formulations prepared based on circRNA or mRNA of growth factors or cytokines. (4) Long-term in vitro expression assay With 1×10 per hole 6 HUVECs were seeded at a density of 100 cells per well in 6-well plates until 85% confluence was achieved. Then, HUVECs were co-cultured with nanoparticles prepared in Comparative Examples 2-13 (all nanoparticles were 10 μg circRNA or mRNA dissolved in 100 μL PBS) for 6 h. The expression of PDGF, IL4, and MCSF proteins was detected by ELISA. As shown in Figure 2, in Comparative Examples 2-13, the concentrations of growth factors or cytokines expressed by the circRNA-loaded nanoparticles prepared in Comparative Examples 2-7 were much higher than those of the linear mRNA-loaded nanoparticles prepared in Comparative Examples 8-13. Furthermore, the former's expression lasted for about 14 days, while the latter's lasted for only about 3 days. On the 14th day, the former still expressed protein at a concentration of about 10 ng / mL, indicating that compared to linear mRNA, circRNA can express active proteins for a long time, and the protein concentration expressed by the circRNA loaded with U-LNP was higher than that of the circRNA loaded with A-LNP. (3) Diabetic wound healing test Establishment of a diabetic mouse model: All mice (C57BL / 6, male) were fed a high-sugar, high-fat diet for the first 4 weeks. In the 5th week, the mice were injected intraperitoneally with streptozotocin (STZ, Sigma-Aldrich) dissolved in citrate buffer and fasted for 12 hours. The mice treated with STZ were then fed a high-sugar, high-fat diet. Blood glucose levels in the tail vein of the mice were measured using a glucometer. Two weeks after STZ injection, the fasting blood glucose level exceeded 16.7 mmol / L, and the diabetic mouse model was successfully established. Wound surface construction: All diabetic mice were divided into 14 groups: PBS (group a), U-LNP / eGFP circRNA (group b), U-LNP / IL4 mRNA (group c), U-LNP / PDGF mRNA (group d), U-LNP / MCSF mRNA (group e), U-LNP / IL4 circRNA (group f), U-LNP / PDGF circRNA (group g), U-LNP / MCSF circRNA (group h), U-LNP / PDGF circRNA / U-LNP / IL4 circRNA (group i), U-LNP / PDGF circRNA / U-LNP / MCSF circRNA (group j), U-LNP / PDGF circRNA / U-LNP / IL4 circRNA / U-LNP / MCSF circRNA (group k), U-LNP / PDGF circRNA / IL4 circRNA (group l), and U-LNP / PDGF circRNA / MCSF circRNA (group 2). circRNA (m group), U-LNP / PDGF circRNA / IL4 circRNA / MCSF circRNA (n group). In the experiment, all mice were anesthetized with isoflurane, and their back hair was removed. A full-thickness skin wound was created on the shaved back of each mouse using a sterile dermal biopsy punch with a diameter of 8 mm. Nanoparticle administration: According to the grouping, mice in the control PBS group were instilled with 20 μL of PBS solution, and single-dose U-LNP / eGFP circRNA nanoparticles (Comparative Example 1), U-LNP / IL4 mRNA nanoparticles (Comparative Example 9), U-LNP / PDGF mRNA nanoparticles (Comparative Example 8), U-LNP / MCSF mRNA nanoparticles (Comparative Example 10), U-LNP / IL4 circRNA nanoparticles (Comparative Example 3), U-LNP / PDGF circRNA nanoparticles (Comparative Example 2), U-LNP / MCSF circRNA nanoparticles (Comparative Example 4), U-LNP / PDGF circRNA / U-LNP / IL4 circRNA nanoparticles (Example 4), U-LNP / PDGF circRNA / U-LNP / MCSF circRNA nanoparticles (Example 5), and U-LNP / PDGF circRNA / / U-LNP / IL4 circRNA / U-LNP / MCSF nanoparticles were instilled into the wounds of diabetic mice, respectively. 100 μL each of the following nanoparticles were prepared: circRNA nanoparticle (Example 6), U-LNP / PDGF circRNA / IL4 circRNA nanoparticle (Example 1), U-LNP / PDGF circRNA / MCSF circRNA nanoparticle (Example 2), and U-LNP / PDGF circRNA / IL4 circRNA / MCSF circRNA nanoparticle (Example 3). All nanoparticles were prepared by pipetting them onto mouse wounds and fixing the mice for 30 min to promote absorption. The wounds were then rinsed three times with PBS buffer to remove any residual preparations. Results analysis: Wound conditions were recorded using smartphones at 0, 4, 7 and 10 days after nano-treatment, and the data were processed using ImageJ software. To accurately record wound size, a circular black card with a diameter of 14 mm was used as a reference. The results are shown in Figure 3. The wound area ratio was calculated, and the results are shown in Figures 4 and 5. As shown in Figures 3, 4, and 5, compared with group a, group b could only express fluorescent proteins without any pharmacological activity and had no effect on promoting wound healing; while the wounds of mice treated with groups c, d, and e did not show significant shrinkage, indicating that the linear mRNA loaded with the nano-formulation prepared in proportion 8-10 could only express growth factors or cytokines for a short time and had no significant effect on promoting wound healing. The wounds of mice treated in groups f, g, and h were significantly smaller than those in group ce, indicating that the circular mRNA loaded with the nanoparticles prepared in Comparative Examples 2-4 can persistently express growth factors or cytokines, which is beneficial for wound healing and reduces the wound area. The wounds of mice treated in groups i and j showed significant reduction in area after day 7 and almost complete healing after day 10, indicating that the combination of circRNA loaded with one growth factor and one cytokine prepared in Examples 4 and 5 is more effective in wound healing. Group k (Example 6) is a combination of nanoparticles containing one growth factor and two cytokine circRNAs, which also showed excellent wound repair effects after treating mouse wounds, with almost complete healing after 10 days. After 7 days, the wounds treated with groups l, m, and n showed significant shrinkage, and after 10 days, the wounds were almost completely healed. This indicates that there was no significant difference in the wound healing process in mice after treatment with the nano-formulations of Examples 1-3 and Examples 4-6 obtained by different preparation methods, proving that different preparation methods have no effect on wound healing. Therefore, while Comparative Examples 2-4, containing only one growth factor or one cytokine circRNA, showed some wound repair effects, the combination of at least one growth factor and at least one cytokine circRNA in Examples 1-6 was more beneficial for wound healing, with the most significant correlation in healing effects. This is because growth factor circRNAs, after being delivered to wound cells via LNPs, express growth factors for a prolonged period, promoting the growth of skin fibroblasts and vascular endothelial cells. Cytokine circRNAs, after being delivered to wound cells via LNPs, express cytokines for a prolonged period, inhibiting inflammation and regulating the wound's immune microenvironment. These two effects synergistically promote wound healing to the greatest extent, especially the combination of one growth factor and two cytokines, IL4 and MCSF, which is most beneficial for wound healing. (3) Tissue sectioning test at the wound site Histological assessments of granulation tissue formation, collagen deposition, and capillary density in the experimental group A1 wound were performed using hematoxylin and eosin (H&E) staining, Masson's trichrome staining, and CD31 histochemical staining. On day 10, wound tissue from diabetic mice was isolated, fixed with 4% paraformaldehyde, paraffin-coated, and cut into 4 μm thick sections. For CD31 immunohistochemical analysis, rabbit anti-CD31 primary antibody diluted in PBS buffer (1:200) was used, followed by incubation with biotin-labeled secondary antibody. The stained sections were observed and recorded using a Leica DM750 microscope for CD31 immunohistochemical and histological analysis. As shown in Figure 6, after day 10, tissue regeneration and biosafety were analyzed from wound tissues of diabetic mice. The epidermis of the PBS control group (group a) and the U-LNP / eGFP circRNA group (group b) was very thin and discontinuous, indicating that the wound had not healed. The epidermis of the U-LNP / IL4 mRNA group (group c), U-LNP / PDGF mRNA group (group d), and U-LNP / MCSF mRNA group (group e) was continuous and showed a clear dermis. The healed skin layer was significantly thicker than that of groups a and b, indicating that the nano-formulations prepared in Comparative Examples 8-10 loaded with linear IL4 mRNA, PDGF mRNA, and MCSF mRNA had certain growth factor or cytokine active protein expression on the wound surface, promoting wound repair and skin thickening. The U-LNP / IL4 circRNA (group f), U-LNP / PDGF circRNA (group g), and U-LNP / MCSF The epidermal and dermal thickness of the circRNA (h group) was significantly better than that of the ce group, and the healing effect was more than twice that of the ce group. This indicates that the nano-formulations prepared in comparative examples 2-4 loaded with circRNA can express growth factor or cytokine active proteins on the wound surface for a long time, which can more effectively promote wound repair and significantly increase the thickness of the epidermis and dermis. The epidermal and dermal thicknesses of the U-LNP / PDGF circRNA / U-LNP / IL4 circRNA (group i), U-LNP / PDGF circRNA / U-LNP / MCSF circRNA (group j), and U-LNP / PDGF circRNA / U-LNP / IL4 circRNA / U-LNP / MCSF circRNA (group k) were significantly greater than those of the ce and fh groups. This indicates that the nano-formulations prepared in Examples 4-6, containing combinations of two or more growth factors and cytokine circRNAs, are more conducive to promoting epidermal growth and wound healing. In particular, the epidermal thickness of the U-LNP / PDGF circRNA / U-LNP / IL4 circRNA / U-LNP / MCSF circRNA group (group k) was even greater, indicating that the combination of PDGF, IL4, and MCSF circRNAs in the nano-formulations prepared in Example 6 is most beneficial to epidermal and dermal growth. Furthermore, the epidermal thickness of the U-LNP / PDGF circRNA / IL4 circRNA group (group l) was comparable to that of the U-LNP / PDGF circRNA / U-LNP / IL4 circRNA group (group i), indicating that although the preparation methods of the nano-formulations were different, there was no significant difference in their ability to promote epidermal growth and wound healing. As shown in Figure 7, collagen deposition was minimal in the PBS control group (group a) and the U-LNP / eGFP circRNA group (group b), which was not conducive to wound repair. Collagen deposition was more significant in the U-LNP / IL4 mRNA (group c), U-LNP / PDGF mRNA (group d), and U-LNP / MCSF mRNA (group e), which were superior to groups a and b. This indicates that the linear IL4 mRNA, PDGF mRNA, and MCSF mRNA loaded with nanoparticles prepared in Comparative Examples 8-10 expressed certain growth factor or cytokine active proteins on their surface, promoting collagen deposition and wound repair. Collagen deposition was even more significant in the U-LNP / IL4 circRNA (group f), U-LNP / PDGF circRNA (group g), and U-LNP / MCSF circRNA (group h), indicating that the circRNA loaded with nanoparticles prepared in Comparative Examples 2-4 expressed growth factor or cytokine active proteins on the wound surface for a longer period of time, promoting collagen production more efficiently and thus benefiting wound repair. Collagen deposition was more pronounced in the three groups: U-LNP / PDGF circRNA / U-LNP / IL4 circRNA (group i), U-LNP / PDGF circRNA / U-LNP / MCSF circRNA (group j), and U-LNP / PDGF circRNA / U-LNP / IL4 circRNA / U-LNP / MCSF circRNA (group k). This indicates that the nano-formulations prepared in Examples 4-6 containing two or more growth factors and cytokine circRNAs are more conducive to promoting collagen deposition. In particular, the collagen deposition was most significant in the U-LNP / PDGF circRNA / U-LNP / IL4 circRNA / U-LNP / MCSF circRNA group (group k), indicating that the combination of PDGF, IL4, and MCSF circRNAs is most beneficial to wound healing. Furthermore, the collagen deposition in the U-LNP / PDGF circRNA / IL4 circRNA group (group l) was comparable to that in the U-LNP / PDGF circRNA / U-LNP / IL4 circRNA group (group i), indicating that although the preparation procedures of the nano-formulations were different, the degree of collagen deposition was similar, and there was no significant difference in the degree of wound healing promotion. As shown in Figure 8, no significant angiogenesis was observed in the PBS control group (group a) and the U-LNP / eGFP circRNA group (group b), which was not conducive to wound repair. A very small amount of angiogenesis was observed in the U-LNP / IL4 mRNA (group c), U-LNP / PDGF mRNA (group d), and U-LNP / MCSF mRNA (group e), which was superior to groups a and b. This indicates that the linear IL4 mRNA, PDGF mRNA, and MCSF mRNA loaded with nanoparticles prepared in Comparative Examples 8-10 expressed certain growth factor or cytokine active proteins on their surface, but angiogenesis was very limited. Significant angiogenesis was observed in the U-LNP / IL4 circRNA (group f), U-LNP / PDGF circRNA (group g), and U-LNP / MCSF circRNA (group h), indicating that the circRNA loaded with nanoparticles prepared in Comparative Examples 2-4 expressed growth factor or cytokine active proteins on the wound surface for a longer period, promoting angiogenesis more efficiently and thus benefiting wound repair. The high vascular density of the U-LNP / PDGF circRNA / U-LNP / IL4 circRNA (group i), U-LNP / PDGF circRNA / U-LNP / MCSF circRNA (group j), and U-LNP / PDGF circRNA / U-LNP / IL4 circRNA / U-LNP / MCSF circRNA (group k) indicates that the nano-formulations prepared in Examples 4-6 contain a combination of two or more growth factors and cytokine circRNAs, which is more conducive to angiogenesis. The formation of new blood vessels is beneficial to the supply of nutrients and oxygen, and promotes wound healing. In particular, the U-LNP / PDGF circRNA / U-LNP / IL4 circRNA / U-LNP / MCSF circRNA group (group k) has the highest vascular density, indicating that the combination of PDGF, IL4, and MCSF circRNAs in the nano-formulations prepared in Example 6 is most conducive to angiogenesis and effectively promotes wound healing. Furthermore, the vascular density of the U-LNP / PDGF circRNA / IL4 circRNA group (group l) was comparable to that of the U-LNP / PDGF circRNA / U-LNP / IL4 circRNA group (group i), indicating that although the preparation procedures for the nano-formulations were different, the vascular density was similar and there was no significant difference in the degree of wound healing promotion. The above results indicate that the nano-formulations prepared in Examples 1-6 containing circRNAs of at least one growth factor and at least one cytokine significantly promoted the formation of granulation tissue, collagen, and blood vessels. In particular, the combination of PDGF circRNA, IL4 circRNA, and MCSF circRNA nano-formulations was most conducive to the formation of granulation tissue, collagen deposition, and angiogenesis, and was most conducive to wound healing, enabling the wound to achieve complete healing. Example 7 A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 25 μg FGF2 circRNA and 25 μg IL4 circRNA, dissolve them in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with FGF2 circRNA and IL4 circRNA, denoted as U-LNP / FGF2 circRNA / IL4 circRNA nanoparticles. Example 8 A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 25 μg FGF2 circRNA and 25 μg MCSF circRNA, dissolve them in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with FGF2 circRNA and MCSF circRNA, denoted as U-LNP / FGF2 circRNA / MCSF circRNA nanoparticles. Example 9 A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 17 μg FGF2 circRNA, 17 μg MCSF circRNA and 17 μg IL4 circRNA, dissolve them in 350 μL of 20 mM citrate buffer (pH 4.0), mix well to prepare an aqueous phase with a concentration of 142 μg / mL; S3. 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with FGF2 circRNA, IL4 circRNA and MCSF circRNA, denoted as U-LNP / FGF2 circRNA / IL4 circRNA / MCSF circRNA nanoparticles. Example 10 A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 25 μg of PDGF-PlGF circRNA and 25 μg of IL4-PlGF circRNA, dissolve them in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifugal filter to obtain a lipid nanoparticle formulation loaded with PDGF-PlGF circRNA and IL4-PlGF circRNA, denoted as U-LNP / PDGF-PlGF circRNA / IL4-PlGF circRNA nanoparticles. Example 11 A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 25 μg of PDGF-PlGF circRNA and 25 μg of MCSF-PlGF circRNA, dissolve them in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifugal filter to obtain a lipid nanoparticle formulation loaded with PDGF-PlGF circRNA and MCSF-PlGF circRNA, denoted as U-LNP / PDGF-PlGF circRNA / MCSF-PlGF circRNA nanoparticles. Example 12 A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 17 μg PDGF-PlGF circRNA, 17 μg IL4-PlGF circRNA and 17 μg MCSF-PlGF circRNA, dissolve them in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifugal filter to obtain a lipid nanoparticle formulation loaded with PDGF-PlGF circRNA, IL4-PlGF circRNA and MCSF-PlGF circRNA, denoted as U-LNP / PDGF-PlGF circRNA / IL4-PlGF circRNA / MCSF-PlGF circRNA nanoparticles. Example 13 A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-106 2.28mg, DMG-PEG2000 0.18mg, DSPC 0.44mg and cholesterol 0.89mg were dissolved in 150μL of ethanol to prepare an alcohol phase with a concentration of 25.3mg / mL; S2. Take 25 μg PDGF-Fc circRNA and 25 μg IL4-Fc circRNA, dissolve them in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. 150 μL of the alcohol phase with a concentration of 25.3 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with PDGF-Fc circRNA and IL4-Fc circRNA, denoted as U6-LNP / PDGF-Fc circRNA / IL4-Fc circRNA nanoparticles. Example 14 A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-106 2.28mg, DMG-PEG2000 0.18mg, DSPC 0.44mg and cholesterol 0.89mg were dissolved in 150μL of ethanol to prepare an alcohol phase with a concentration of 25.3mg / mL; S2. Take 25 μg PDGF-Fc circRNA and 25 μg MCSF-Fc circRNA, dissolve them in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. 150 μL of the alcohol phase with a concentration of 25.3 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with PDGF-Fc circRNA and MCSF-Fc circRNA, denoted as U6-LNP / PDGF-Fc circRNA / MCSF-Fc circRNA nanoparticles. Example 15 A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-106 2.28mg, DMG-PEG2000 0.18mg, DSPC 0.44mg and cholesterol 0.89mg were dissolved in 150μL of ethanol to prepare an alcohol phase with a concentration of 25.3mg / mL; S2. Take 17 μg PDGF-Fc circRNA, 17 μg IL4-Fc circRNA and 17 μg MCSF-Fc circRNA, dissolve them in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL. S3. 150 μL of ethanol phase with a concentration of 25.3 mg / mL and 350 μL of aqueous phase with a concentration of 142 μg / mL were mixed by microfluidic mixing, and then purified by 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with PDGF-Fc circRNA, IL4-Fc circRNA and MCSF-Fc circRNA, denoted as U6-LNP / PDGF-Fc circRNA / IL4-Fc circRNA / MCSF-Fc circRNA nanoparticles. Comparative Example 14 The difference between this comparative example and Example 8 is that IL4 circRNA is replaced with FGF2 circRNA. A method for preparing a nano-formulation that promotes tissue repair includes the following steps: S1. Ionizable lipid U-105 2.32 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg were dissolved in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Dissolve 50 μg of FGF2 circRNA in 350 μL of 20 mM citrate buffer (pH 4.0), mix well, and prepare an aqueous phase with a concentration of 142 μg / mL. S3. Mix 150 μL of the alcohol phase with a concentration of 25.7 mg / mL and 350 μL of the aqueous phase with a concentration of 142 μg / mL using a microfluidic system, and then purify the mixture with 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifuge filter to obtain a lipid nanoparticle formulation loaded with FGF2 circRNA, denoted as U-LNP / FGF2 circRNA nanoparticles. Performance testing (1) Particle size, polydispersity index (PDI) and zeta potential The particle size, polydispersity index (PDI), and zeta potential of Examples 7-15 and Comparative Example 14 were measured using a Zetasizer Nano ZS dynamic light scattering instrument. The results are shown in Table 2. (2) Packet efficiency The encapsulation efficiency of Examples 7-15 and Comparative Example 14 was measured using the Ribogreen reagent method. Specifically, the samples were treated with TE buffer and 2% TE-Triton buffer, respectively, Ribogreen reagent was added, and fluorescence intensity was measured using a microplate reader. The amount of dissociated circRNA (or mRNA) and the total amount of circRNA (or mRNA) were obtained and calculated using the following formula: Packing efficiency (%) = [(Amount of dissociated circRNA (or mRNA)) / Total amount of circRNA (or mRNA)] × 100%. (5) Cell viability HUVECs cells were seeded into 96-well plates (2 × 10⁶ cells per well). 3 HUVECs were cultured until 85% confluence was achieved. Then, Opti-MEMI Reduced Serum Medium was added instead of the cell culture medium. The nanoparticles prepared in Examples 7-15 and Comparative Example 14 (all nanoparticles were 10 μg circRNA or mRNA dissolved in 100 μL PBS buffer) were co-cultured with HUVECs for 24 h, with a blank control included. The cells were then treated with a cell counting kit-8, and OD was measured using a microplate reader. 450nm Cell viability was calculated, and the results are shown in Table 2. Table 2. Particle size, PDI, Zeta potential, loading efficiency, and cell viability of Examples 7-15 and Comparative Example 14. As shown in Table 2, the particle sizes of Examples 7-15 and Comparative Example 14 are all around 100 nm, the PDI is around 0.2, the Zeta potential is in the range of -6 to -3 mV, the encapsulation efficiency is in the range of 87-92%, and the cell viability reaches 100-130%. They all have suitable size, good encapsulation efficiency, and are non-toxic, have high safety, and do not cause much damage to cells. This indicates that there is no significant difference in the physicochemical properties of the nano-formulations prepared based on circRNA of growth factors or cytokines. (2) Diabetic wound healing test Establishment of a diabetic mouse model: All mice (C57BL / 6, male) were fed a high-sugar, high-fat diet for the first 4 weeks. In the 5th week, the mice were injected intraperitoneally with streptozotocin (STZ, Sigma-Aldrich) dissolved in citrate buffer and fasted for 12 hours. The mice treated with STZ were then fed a high-sugar, high-fat diet. Blood glucose levels in the tail vein of the mice were measured using a glucometer. Two weeks after STZ injection, the fasting blood glucose level exceeded 16.7 mmol / L, and the diabetic mouse model was successfully established. Wound surface construction: All diabetic mice were divided into 14 groups: PBS (group a), U-LNP / FGF2 circRNA (group b, comparative example 14), U-LNP / FGF2 circRNA / IL4 circRNA (group c, example 7), U-LNP / FGF2 circRNA / MCSF circRNA (group d, example 8), U-LNP / FGF2 circRNA / IL4 circRNA / MCSF circRNA (group e, example 9), U-LNP / PDGF-PlGF circRNA / IL4-PlGF circRNA (group f, example 10), U-LNP / PDGF-PlGF circRNA / MCSF-PlGF circRNA (group g, example 11), U-LNP / PDGF-PlGF circRNA / IL4-PlGF circRNA / MCSF-PlGF circRNA (group h, example 12), and U6-LNP / PDGF-Fc circRNA / IL4-Fc circRNA. circRNA (group i, Example 13), U6-LNP / PDGF-Fc circRNA / MCSF-Fc circRNA (group j, Example 14), U6-LNP / PDGF-Fc circRNA / IL4-Fc circRNA / MCSF-Fc circRNA (group k, Example 15). In the experiment, all mice were anesthetized with isoflurane, their back hair was removed, and a full-thickness skin wound was created on the shaved back of each mouse using a sterile dermal biopsy punch with a diameter of 8 mm. Nanoparticle administration: According to the grouping, mice in the control PBS group were instilled with 20 μL of PBS solution, and diabetic mice were instilled with single doses of U-LNP / FGF2 circRNA nanoparticles, U-LNP / FGF2 circRNA / IL4 circRNA nanoparticles, U-LNP / FGF2 circRNA / MCSF circRNA nanoparticles, U-LNP / FGF2 circRNA / IL4 circRNA / MCSF circRNA nanoparticles, U-LNP / PDGF-PlGF circRNA / IL4-PlGF circRNA, U-LNP / PDGF-PlGF circRNA / MCSF-PlGF circRNA, U-LNP / PDGF-PlGF circRNA / IL4-PlGF circRNA / MCSF-PlGF circRNA, U6-LNP / PDGF-Fc circRNA / IL4-Fc circRNA, and U6-LNP / PDGF-Fc circRNA / MCSF-Fc circRNA, respectively. 100 μL each of circRNA, U6-LNP / PDGF-Fc circRNA / IL4-Fc circRNA / MCSF-Fc circRNA nanoparticles (each nanoparticle contained 10 μg circRNA in 100 μL PBS buffer) were used for accurate measurement. All nanoparticles were applied to mouse wounds using a pipette, and the mice were fixed for 30 min to promote absorption of the nanoparticles. The wounds were then carefully rinsed three times with PBS buffer to remove any residual formulations. Results analysis: Wound size was precisely recorded at 0, 4, 7 and 10 days after nano-treatment of the wound. A circular black card with a diameter of 14 mm was used as a reference to calculate the change in the wound area ratio with healing time. As shown in Figure 9, compared with the PBS control group (group a), the wound area of ​​mice treated with the U-LNP / FGF2 circRNA group (group b) was reduced, indicating that the circular mRNA can persistently express the growth factor FGF2, which is beneficial to wound healing and reduces the wound area. The following groups were also treated: U-LNP / FGF2 circRNA / IL4 circRNA (group c), U-LNP / FGF2 circRNA / MCSF circRNA (group d), U-LNP / FGF2 circRNA / IL4 circRNA / MCSF circRNA (group e), U-LNP / PDGF-PlGF circRNA / IL4-PlGF circRNA (group f), U-LNP / PDGF-PlGF circRNA / MCSF-PlGF circRNA (group g), U-LNP / PDGF-PlGF circRNA / IL4-PlGF circRNA / MCSF-PlGF circRNA (group h), and U6-LNP / PDGF-Fc circRNA / IL4-Fc circRNA. Mice treated with nanoparticles containing circRNA (group i), U6-LNP / PDGF-Fc circRNA / MCSF-Fc circRNA (group j), and U6-LNP / PDGF-Fc circRNA / IL4-Fc circRNA / MCSF-Fc circRNA (group k) showed more significant wound area reduction after day 7 and almost complete healing after day 10. This indicates that nanoparticles combining circRNAs of one growth factor FGF2 and one or two cytokines (IL4 and MCSF) are more effective in wound healing, demonstrating that nanoparticles containing circRNAs of at least one growth factor and at least one cytokine are most beneficial for wound healing. This is because after FGF2 circRNA is delivered to wound cells via LNP, it expresses FGF2 for a prolonged period, promoting the growth of skin fibroblasts and vascular endothelial cells. Simultaneously, after IL4 and MCSF circRNAs are delivered to wound cells via LNP, they express cytokines for a prolonged period, inhibiting inflammation and regulating the wound's immune microenvironment. These two effects synergistically promote wound healing to the greatest extent. It is particularly noteworthy that, when comparing healing effects based on the ratio of final wound area, groups f, g, and h were superior to groups i, j, and k, which in turn were superior to groups c, d, and e. This indicates that the circRNA combination nanoparticles loaded with PlGF-binding fusion protein are superior to the circRNA combination nanoparticles loaded with Fc-binding fusion protein, and superior to the non-fusion protein circRNA combination nanoparticles. This is because the circRNA combination in the nanoparticles expresses the PlGF fusion protein of growth factor and cytokine, which binds strongly to the extracellular matrix, allowing for the sustained release of growth factors and cytokines to exert pharmacological effects. While the other nanoparticle combination, expressing the Fc fusion protein of growth factor and cytokine, also delays the release of growth factors and cytokines to exert pharmacological effects, it is less potent than the former. The non-fusion protein circRNA combination nanoparticles only express growth factors and cytokines, and their pharmacological effects are inferior to those of the fusion protein circRNA combination. The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A nanofomulation for promoting tissue repair, characterized in that, The nanoformulation consists of three components: component 1 is a circular RNA encoding a growth factor or a growth factor fusion protein, component 2 is a circular RNA encoding a cytokine or a cytokine fusion protein, and component 3 is a nanocarrier. The content of component 1 is 0.5-7.5%, the content of component 2 is 0.5-7.5%, and the content of component 3 is 85-99%.

2. The nanoformulation for promoting tissue repair as claimed in claim 1, wherein: Component 1 is composed of spacer region 1, exon 1, exon 2, spacer region 2, ribosome insertion site, Kozak sequence and coding region connected sequentially; the sequence of spacer region 1 is the sequence shown in SEQ ID No. 1 or a sequence that has at least 75% identity with it; The sequence of exon 1 is the sequence shown in SEQ ID No. 2 or a sequence that has at least 75% identity with it; The sequence of exon 2 is the sequence shown in SEQ ID No. 3 or a sequence that has at least 75% identity with it; The sequence of the spacer region 2 is the sequence shown in SEQ ID No. 4 or a sequence that has at least 75% identity with it; The sequence of the ribosome insertion site is the sequence shown in any one of SEQ ID No. 5, 6, 7 or 8 or a sequence that has at least 75% identity with it; The sequence of Kozak is the sequence shown in SEQ ID No. 9; The sequence of the coding region is any one of the sequences shown in SEQ ID No. 10-22 of the growth factor, or a growth factor fusion protein sequence or a sequence that has at least 75% identity with it; The growth factor fusion protein sequence comprises any one of the sequences shown in any of the growth factor sequences SEQ ID No. 10-22, the linker peptide sequence shown in SEQ ID No. 36, and any one of the sequences shown in the human serum albumin sequence SEQ ID No. 32, the human serum albumin antibody sequence SEQ ID No. 33, the human immunoglobulin antibody constant region sequence SEQ ID No. 34, and the placental growth factor heparin binding domain sequence SEQ ID No.

35.

3. The nanoformulation for promoting tissue repair as claimed in claim 1, wherein: Component 2 is composed of spacer region 1, exon 1, exon 2, spacer region 2, ribosome insertion site, Kozak sequence and coding region connected sequentially; the sequence of spacer region 1 is the sequence shown in SEQ ID No. 1 or a sequence that has at least 75% identity with it; The sequence of exon 1 is the sequence shown in SEQ ID No. 2 or a sequence that has at least 75% identity with it; The sequence of exon 2 is the sequence shown in SEQ ID No. 3 or a sequence that has at least 75% identity with it; The sequence of the spacer region 2 is the sequence shown in SEQ ID No. 4 or a sequence that has at least 75% identity with it; The sequence of the ribosome insertion site is the sequence shown in any one of SEQ ID No. 5, 6, 7 or 8 or a sequence that has at least 75% identity with it; The sequence of Kozak is the sequence shown in SEQ ID No. 9; The sequence of the coding region is any one of the sequences shown in SEQ ID No. 23-31 of the cytokines, or a cytokine fusion protein sequence or a sequence that has at least 75% identity with it; The cytokine fusion protein sequence comprises any one of the sequences shown in SEQ ID No. 23-31 (cytokine sequence), the sequence shown in SEQ ID No. 36 (linking peptide sequence), and any one of the sequences shown in SEQ ID No. 32 (human serum albumin), SEQ ID No. 33 (human serum albumin antibody), SEQ ID No. 34 (human immunoglobulin antibody constant region), and SEQ ID No. 35 (placental growth factor heparin binding domain).

4. The nanoformulation to promote tissue repair as claimed in claim 1, wherein: The growth factor is one or more of platelet-derived growth factor, epidermal growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, nerve growth factor, insulin-like growth factor 1, insulin-like growth factor 2, hepatocyte growth factor, keratinocyte growth factor, vascular endothelial growth factor α, brain-derived neurotrophic factor, neurotrophic factor 3, and transforming growth factor β1; the growth factor fusion protein is composed of component A, a linker peptide, and component B, wherein component A is a growth factor, and component B is one or more of human serum albumin, human serum albumin antibody, human immunoglobulin antibody constant region protein, and placental growth factor heparin binding domain protein.

5. The nanoformulation to promote tissue repair as claimed in claim 1, wherein: The cytokines are one or more of the following: interleukin-4, interleukin-10, interleukin-13, interleukin-22, macrophage colony-stimulating factor, chemokine 12, interferon regulatory factor 4, pancreatic islet regeneration protein, and tumor necrosis factor α stimulating gene / inducible protein-6; the cytokine fusion protein is composed of component C, a linker peptide, and component D, wherein component C is a cytokine, and component D is one or more of the following: human serum albumin, human serum albumin antibody, human immunoglobulin antibody constant region protein, and placental growth factor heparin binding domain protein.

6. The nanofonnulation to promote tissue repair as claimed in claim 1, wherein: The nanoformulation consists of three components: component 1 is one or more of the following: circular RNA encoding platelet-derived growth factor or its fusion protein; circular RNA encoding epidermal growth factor or its fusion protein; circular RNA encoding basic fibroblast growth factor or its fusion protein; circular RNA encoding nerve growth factor or its fusion protein; circular RNA encoding vascular endothelial growth factor a or its fusion protein; and circular RNA encoding transforming growth factor β1 or its fusion protein; component 2 is a combination of circular RNA encoding interleukin-4 or its fusion protein and circular RNA encoding macrophage colony-stimulating factor or its fusion protein; and component 3 is a nanocarrier.

7. The nanofonnulation to promote tissue repair as claimed in claim 1 wherein, The preparation method of nano-formulations includes the following steps: S1. Add ethanol to lipid raw material 1 and lipid raw material 2 and mix evenly to obtain an alcohol phase containing lipids; S2. Component 1 is added to citrate buffer and mixed evenly to obtain aqueous phase A; S3. Component 2 is added to citrate buffer and mixed evenly to obtain aqueous phase B; S4. Microfluidic mixing to remove ethanol, yielding nano-formulations; The lipid raw material 1 is one or more of ionizable lipids, cationic lipids, and thiourea lipids; The lipid raw material 2 is one or more of sterols, phospholipids, glycerides, and polyethylene glycol-modified lipids; The microfluidic mixing method includes mixing a three-phase system containing lipids (alcohol phase, aqueous phase A, and aqueous phase B); mixing a two-phase system containing lipids (alcohol phase and aqueous phase A); and mixing a two-phase system containing lipids (alcohol phase and aqueous phase B).

8. The use of the tissue-repair-promoting nano-formulation according to any one of claims 1-7 in the preparation of drug nano-formulations for treating refractory wounds, myocardial infarction, stroke, arthritis, acute lung injury, and tendinitis.

9. Use of a nanoformulation for promoting tissue repair according to claim 8, characterized in that: The drug nanoformulation contains pharmaceutically acceptable adjuvants, including porous polymer scaffolds and hydrogels; the dosage forms of the drug nanoformulation include, but are not limited to, powders, granules, patches, ointments, lyophilized powders, dressings, gels, sprays, liniments, liquid drops, and injections; and the administration methods of the drug nanoformulation include, but are not limited to, local administration, intravenous administration, subcutaneous administration, and intramuscular injection.

10. Use of a nanoformulation for promoting tissue repair according to claim 7, characterized in that: The refractory wounds mentioned include diabetic foot ulcers, pressure ulcers, vascular ulcers, radiation ulcers, tophi ulcers, iatrogenic wounds, traumatic ulcers, infected ulcers, drug-induced ulcers, neurotrophic ulcers, snake bite ulcers, post-burn scar ulcers, fistulas, and chronic refractory wounds caused by sinus tracts.

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