Nanometer preparation for promoting tissue repair and application thereof

Abstract

The invention provides a nano preparation for promoting tissue repair and application thereof, the nano preparation comprises circular RNA for coding growth factors, circular RNA for coding cytokines and a nano carrier, and the preparation method comprises the following steps: preparation of a lipid carrier material, preparation of a solution of the circular RNA for coding the growth factors and the cytokines, and microfluidic mixing. Belongs to the technical field of biological medicine. The nano preparation prepared by the invention can simultaneously and accurately express and release growth factors and cell factors in cells, plays a role for a long time, regulates an immune environment, inhibits inflammation and promotes wound tissue repair, has the characteristics of no toxicity, high safety, convenience in use, simplicity in preparation and excellent effect, and has clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a nano preparation for promoting tissue repair and application thereof. Background Art

[0002] Refractory wounds (ulcers) include diabetic foot ulcers, pressure ulcers, vascular ulcers, radiation ulcers, tophi ulcers, iatrogenic wounds, traumatic ulcers, infectious ulcers, drug-induced ulcers, neurodystrophy ulcers, snake bite ulcers, post-burn scar ulcers, fistulas, sinus tracts, etc., which are characterized by the inability to completely heal after 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 circulation, leading to inflammation and damage of skin and soft tissues. The local immune microenvironment disorder characterized by imbalance of phenotypic polarization of macrophages is the main reason for the delayed healing of DFU wounds, which is manifested by 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 diabetes. Growth factors, such as human vascular endothelial growth factor a (VEGFa), have the effects of promoting increased vascular permeability, extracellular matrix degeneration, endothelial cell migration, proliferation and angiogenesis. Therefore, supplementing the wound with VEGFa, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF2), nerve growth factor (NGF), etc., can promote capillary formation, repair cell recruitment and proliferation, granulation tissue formation, and nerve repair. Growth factors can be administered in the form of recombinant proteins to treat DFU. Due to the rapid degradation of protein growth factors in the wound, it is difficult to exert long-term efficacy. They have the disadvantages of short half-life, multiple doses are required in clinical practice, poor patient compliance, and are very unfavorable to wound healing. As innate immune cells, macrophages play a key role in resisting pathogen infection and regulating inflammation. Among them, M1 macrophages have proinflammatory effects, while M2 macrophages mainly inhibit inflammation and promote tissue repair. In chronic wound tissues of DFU, macrophage M2 polarization is blocked, the wound surface continues to be inflamed, and the initiation of the proliferation state, subsequent extracellular matrix deposition and tissue reconstruction are inhibited. Therefore, regulating macrophage polarization and inhibiting inflammation are the prerequisites for promoting DFU healing. The mechanism of immune disorder lies in the dysregulation of cytokine signaling. Administration of cytokines to wounds can regulate immune function and achieve immunotherapy. Among them, interleukin 4 (IL4) triggers STAT6 phosphorylation, activates M2 tag gene transcription, causes target cell phenotype changes, and changes macrophage polarization from pro-inflammatory M1 phenotype to anti-inflammatory, tissue healing M2 phenotype, promotes extracellular matrix deposition and tissue regeneration. However, cytokines administered in the form of recombinant proteins also have the disadvantages of rapid degradation in wounds, short half-life, very inconvenient clinical use, and difficulty in achieving sustained pharmacological activity and efficient treatment. mRNA therapy is one of the important treatment methods that has emerged in recent years, especially protein replacement therapy based on mRNA has developed rapidly, because the high specificity and flexibility of mRNA give it fewer side effects, lower production costs and time. However, linear mRNA has poor stability, short protein expression time in cells, and the amount of growth factors expressed is insufficient to meet the goals of tissue repair and disease treatment. Therefore, it is urgent to invent a drug that can express and release cytokines and growth factors for a long time, continuously control inflammation, and promote wound tissue repair. Summary of the invention

[0003] Technical problems to be solved: In view of the above technical problems, the purpose of the present invention is to provide a nano preparation for promoting tissue repair and its application, which comprises a circular RNA encoding a growth factor and a circular RNA encoding a cytokine and a nano carrier, and its preparation method comprises the preparation of a lipid carrier material, the preparation of a solution of the circular RNA encoding the growth factor and the cytokine, and microfluidic mixing, which belongs to the field of biomedical technology. The nano carrier prepared by the present invention can simultaneously and accurately express and release growth factors and cytokines in cells, exert effects for a long time, and promote wound tissue repair. It has the characteristics of non-toxicity, high safety, convenient use, simple preparation, and excellent effect, and can provide a new treatment approach for tissue repair and regeneration, and has clinical application value.

[0004] Technical solution: A nano preparation for promoting tissue repair, the nano preparation 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 nano carrier; 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, the component 1 is formed by sequentially connecting spacer 1, exon 1, exon 2, spacer 2, ribosome insertion site, Kozak sequence and coding region; The sequence of the spacer 1 is the sequence shown in SEQ ID No. 1 or a sequence having at least 75% identity thereto; The sequence of exon 1 is the sequence shown in SEQ ID No. 2 or a sequence having at least 75% identity thereto; The sequence of exon 2 is the sequence shown in SEQ ID No. 3 or a sequence having at least 75% identity thereto; The sequence of the spacer 2 is the sequence shown in SEQ ID No. 4 or a sequence having at least 75% identity thereto; The sequence of the ribosome insertion site is any one of SEQ ID No. 5, 6, 7 or 8, or a sequence having at least 75% identity thereto; The Kozak sequence is the sequence shown in SEQ ID No.9; The sequence of the coding region is a sequence shown in any one of SEQ ID No. 10-22 of a growth factor, or a growth factor fusion protein sequence or a sequence having at least 75% identity therewith; The growth factor fusion protein sequence is composed of any one of the growth factor sequences SEQ ID No.10-22, the connecting peptide sequence SEQ ID No.36, and any one of 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 placenta growth factor heparin binding domain sequence SEQ ID No.35. Further, the component 2 is formed by sequentially connecting the spacer 1, exon 1, exon 2, spacer 2, ribosome insertion site, Kozak sequence and coding region; The sequence of the spacer 1 is the sequence shown in SEQ ID No. 1 or a sequence having at least 75% identity thereto; The sequence of exon 1 is the sequence shown in SEQ ID No. 2 or a sequence having at least 75% identity thereto; The sequence of exon 2 is the sequence shown in SEQ ID No. 3 or a sequence having at least 75% identity thereto; The sequence of the spacer 2 is the sequence shown in SEQ ID No. 4 or a sequence having at least 75% identity thereto; The sequence of the ribosome insertion site is any one of SEQ ID No. 5, 6, 7 or 8, or a sequence having at least 75% identity thereto; The Kozak sequence is the sequence shown in SEQ ID No.9; The sequence of the coding region is a sequence shown in any one of cytokine SEQ ID No. 23-31, or a growth factor fusion protein sequence or a sequence having at least 75% identity therewith; The cytokine fusion protein sequence is composed of any one of the cytokine sequences SEQ ID No.23-31, the connecting peptide sequence SEQ ID No.36, and any one of 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 placenta growth factor heparin binding domain sequence SEQ ID No.35. 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), neurotrophin 3 (NT3) or transforming growth factor β1 (TGFβ1); the growth factor fusion protein is composed of component A, a connecting 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 cytokine is one or more of 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 source protein (REG3A), and tumor necrosis factor α-stimulated gene / induced protein-6 (TSG-6); the cytokine fusion protein is composed of component C, a connecting 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. Furthermore, the nanoformulation is composed of three components, component 1 is one or more of 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 alpha or its fusion protein, and circular RNA encoding transforming growth factor β1 or its fusion protein, component 2 is 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. Furthermore, the preparation method of the nano preparation comprises the following steps: S1. Add ethanol to lipid raw material 1 and lipid raw material 2 and mix them evenly to obtain a lipid-containing alcohol phase; S2. Add citric acid buffer to component 1 and mix well to obtain aqueous phase A; S3. Add citric acid buffer to component 2 and mix well to obtain aqueous phase B; S4. Microfluidic mixing, removal of ethanol, and preparation of nanoformulation; The lipid raw material 1 is one or more of an ionizable lipid, a cationic lipid, and a thiourea lipid, 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), 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) 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'-(( 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)hexan-1-amine (ALC-0315), heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)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 of a lipid-containing alcohol phase, an aqueous phase A and an aqueous phase B; mixing a two-phase system of a lipid-containing alcohol phase and an aqueous phase A; and mixing a two-phase system of a lipid-containing alcohol phase and an aqueous phase B. The use of any of the above-mentioned nanoformulations for promoting tissue repair in the preparation of pharmaceutical nanoformulations for treating refractory wounds, myocardial infarction, stroke, arthritis, acute lung injury, and tendinitis. Furthermore, the drug nanoformulation contains pharmaceutically acceptable adjuvants, and the adjuvants include polymer porous scaffolds and hydrogels. Furthermore, the dosage form of the drug nanoformulation includes but is not limited to powder, 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 topical administration, intravenous administration, subcutaneous administration, and intramuscular injection. Furthermore, the refractory wound is a chronic refractory wound caused by diabetic foot ulcer, pressure ulcer, vascular ulcer, radiation ulcer, tophi ulcer, iatrogenic wound, traumatic ulcer, infectious ulcer, drug-induced ulcer, neurodystrophy ulcer, snake bite ulcer, scar ulcer after burn, fistula, and sinus tract. Beneficial effects: 1. The nanoformulation of the present invention can, on the one hand, regulate the expression of RNA encoding growth factors or growth factor fusion proteins, promote capillary formation, recruitment and proliferation of various repair cells, and promote granulation tissue formation. On the other hand, it can regulate the expression of RNA encoding cytokines and cytokine fusion proteins, regulate the immune microenvironment inside the body, promote macrophage polarization and proliferation, induce macrophages to change from the pro-inflammatory M1 phenotype to the M2 phenotype that promotes tissue healing, inhibit the inflammatory nature of macrophages, promote extracellular matrix deposition and tissue reconstruction and regeneration, and work synergistically to achieve efficient repair of difficult-to-heal wounds. 2. The nanoformulation prepared by the present invention contains PDGF circRNA, IL4 circRNA and MCSF circRNA, which can express and secrete PDGF, IL4 and MCSF in cells at the wound site, wherein PDGF can stimulate the division and proliferation of various cells such as vascular smooth muscle cells, fibroblasts, and neurons, and promote the formation of granulation tissue; IL4 has an immunomodulatory effect on macrophages, etc., can induce macrophage M2 polarization, promote tissue reconstruction, and inhibit inflammation; MCSF can promote monocyte survival, monocyte transformation to macrophages and macrophage proliferation, enhance the killing effect of macrophages on microorganisms, and efficiently achieve rapid healing of difficult-to-heal wounds. 3. The nanoformulation prepared by the present 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, wherein the growth factor (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, etc., can induce macrophage M2 polarization, promote tissue reconstruction, and inhibit inflammation; MCSF can promote monocyte survival, monocyte transformation to macrophages and macrophage proliferation, enhance the killing effect of macrophages on microorganisms, and efficiently achieve rapid healing of difficult-to-heal wounds. 4. The nanoformulation of the present invention is safe and non-toxic, with high safety. It can simultaneously and continuously express and release growth factors and cytokines in cells, exerting a long-lasting effect. It can be applied locally at the site of injury, is easy to use, and can be used in combination with pharmaceutically acceptable adjuvants to achieve efficient and rapid repair and healing of the body's injured tissues. It has a good effect on chronic and refractory wounds caused by various ulcers, can provide a new way for tissue repair and regeneration, and has clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Schematic diagram of the sequence structure of circular RNA; Figure 2It is a graph showing the relationship between protein expression and time after the nanoformulations of Comparative Examples 2-13 were co-incubated with cells, wherein a represents the 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 the 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 the 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 The wound healing status of diabetic mice on days 0, 4, 7, and 10 after a single dose of different nanoagents was administered to the wound. 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 / PDGFcircRNA / U-LNP / MCSF circRNA, k represents U-LNP / PDGF circRNA / U-LNP / IL4 circ RNA / U-LNP / MCSF circRNA, l represents U-LNP / PDGF circRNA / IL4 circRNA, and m represents U-LNP / PDGF circRNA / MCSFcircRNA, n represents U-LNP / PDGF circRNA / IL4 circRNA / MCSF circRNA; Figure 4 The changes in wound area on days 0, 4, 7, and 10 after administration of different nanoagents to the wounds of diabetic mice, where 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 The changes in wound area on days 0, 4, 7, and 10 after administration of different nanoagents to the wounds of diabetic mice, where i represents U-LNP / PDGF circRNA / U-LNP / IL4 circRNA, j represents U-LNP / PDGF circRNA / U-LNP / MCSFcircRNA, 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 The following are microscopic photographs of wound tissues of diabetic mice treated with different nanoagents after H&E staining, where 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 / PDGFcircRNA / U-LNP / MCSF circRNA, k represents U-LNP / PDGF circRNA / U-LNP / IL4circRNA / U-LNP / MCSF circRNA, and l represents U-LNP / PDGF circRNA / IL4 circRNA; Figure 7The following are photos of Masson staining of wound tissues of diabetic mice treated with different nanoagents. 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 / PDGFcircRNA / U-LNP / MCSF circRNA, k represents U-LNP / PDGF circRNA / U-LNP / IL4circRNA / U-LNP / MCSF circRNA, and l represents U-LNP / PDGF circRNA / IL4 circRNA; Figure 8 The following are microscopic photos of CD31 immunohistochemical staining of wound tissues of diabetic mice treated with different nanoagents, where 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 / IL4circRNA / U-LNP / MCSF circRNA, and l represents U-LNP / PDGF circRNA / IL4 circRNA; Fig. 9Figure 2 Changes in wound area of ​​diabetic mice on days 0, 4, 7, and 10 after administration of different nanoagents to the wounds, where a represents PBS group, b represents U-LNP / FGF2 circRNA group, c represents U-LNP / FGF2 circRNA / IL4 circRNAc group, d represents U-LNP / FGF2 circRNA / MCSF circRNA group, e represents U-LNP / FGF2 circRNA / IL4 circRNA / MCSFcircRNA group, f represents U-LNP / PDGF-PlGF circRNA / IL4-PlGF circRNA group, g represents U-LNP / PDGF-PlGF circRNA / MCSF-PlGF circRNA group, h represents U-LNP / PDGF-PlGF circRNA / IL4-PlGFcircRNA / MCSF-PlGF circRNA group, i represents U6-LNP / PDGF-Fc circRNA / IL4-Fc circRNA group, and j represents U6-LNP / PDGF-Fc circRNA / MCSF-Fc circRNA group, k represents U6-LNP / PDGF-Fc circRNA / IL4-FccircRNA / MCSF-Fc circRNA group. DETAILED DESCRIPTION The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: The materials used in the present invention: 6-((2-hexyldecanoyl)oxy)-N-(6-(((2-ethyldecanoyl)oxy)hexyl)-N(4-hydroxybutyl)hexan-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-hexyldecyl)-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate (U-106), dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000), distearoylphosphatidylcholine (DSPC), were purchased from Avituo Shanghai Pharmaceutical Technology Co., Ltd.; PDGF mRNA (mRNA encoding platelet-derived growth factor), FGF2 mRNA (mRNA encoding basic fibroblast growth factor), IL4mRNA (mRNA encoding interleukin 4), and MCSF mRNA (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), IL4circRNA (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), 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), eGFP circRNA (enhanced green fluorescent protein circRNA) were purchased from Kaituo Biotechnology (Suzhou) Co., Ltd. or synthesized and provided by the laboratory of Southeast University, with a content of more than 90%; TE buffer and TE-Triton buffer were purchased from Anhui White Shark Biotechnology Co., Ltd.;PBS buffer was purchased from Wuhan Sevier Biotechnology Co., Ltd.; Ribogreen reagent and microplate reader were purchased from Thermo Fisher Scientific, USA; Amicon Ultra centrifugal filter was purchased from Guangzhou Jiete Biofiltration Co., Ltd.; Example 1 A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Dissolve 25 μg of IL4 circRNA and 25 μg of PDGF circRNA in 350 μL of 20 mM citric acid 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with IL4 circRNA and PDGF circRNA, which was recorded as U-LNP / PDGF circRNA / IL4 circRNA nanoparticle. Example 2 A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 25 μg MCSF circRNA and 25 μg PDGF circRNA and dissolve them in 350 μL citric acid buffer (pH 4.0) with a concentration of 20 mM, mix them evenly, 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with MCSFcircRNA and PDGFcircRNA, which was recorded as U-LNP / PDGFcircRNA / MCSFcircRNA nanoparticle. Example 3 A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 17 μg of PDGF circRNA, 17 μg of IL4 circRNA and 17 μg of MCSF circRNA, dissolve them in 350 μL of 20 mM citric acid buffer (pH 4.0), mix them evenly, 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with PDGFcircRNA, IL4circRNA and MCSFcircRNA, which was recorded as U-LNP / PDGFcircRNA / IL4circRNA / MCSFcircRNA nanoagent. Example 4 A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 50 μg of PDGF circRNA and dissolve it in 350 μL of citric acid buffer (pH 4.0) with a concentration of 20 mM, mix well, and prepare a PDGF circRNA aqueous phase with a concentration of 142 μg / mL; S3. 150 μL of 25.7 mg / mL alcohol phase and 350 μL of 142 μg / mL PDGF circRNA aqueous phase were mixed by microfluidics, and then purified by Amicon Ultra centrifugal filter with 5 mL PBS buffer (pH 7.4) to obtain 500 μL U-LNP / PDGF circRNA nanoparticles; S4. Take 50 μg of IL4 circRNA and dissolve it in 350 μL of citric acid buffer (pH 4.0) with a concentration of 20 mM, mix well, and prepare an IL4 circRNA aqueous phase 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 IL4 circRNA aqueous phase with a concentration of 142 μg / mL were mixed by microfluidics, and then purified by Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain 500 μL U-LNP / IL4 circRNA nanoparticles; S6. Take 250 μL U-LNP / PDGF circRNA nanoparticle and 250 μL U-LNP / IL4 circRNA nanoparticle, mix them evenly, and prepare U-LNP / PDGF circRNA / U-LNP / IL4 circRNA nanoparticle. Example 5 A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 50 μg of PDGF circRNA and dissolve it in 350 μL of citric acid buffer (pH 4.0) with a concentration of 20 mM, mix well, and prepare a PDGF circRNA aqueous phase with a concentration of 142 μg / mL; S3. 150 μL of 25.7 mg / mL alcohol phase and 350 μL of 142 μg / mL PDGF circRNA aqueous phase were mixed by microfluidics, and then purified by Amicon Ultra centrifugal filter with 5 mL PBS buffer (pH 7.4) to obtain 500 μL U-LNP / PDGF circRNA nanoparticles; S4. Take 50 μg of MCSF circRNA and dissolve it in 350 μL of citric acid buffer (pH 4.0) with a concentration of 20 mM, mix well, and prepare an MCSF circRNA aqueous phase 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 MCSF circRNA aqueous phase with a concentration of 142 μg / mL were mixed by microfluidics, and then purified by Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain 500 μL U-LNP / MCSF circRNA nanoparticles; S6.S1. Take 250 μL U-LNP / PDGF circRNA nanoagent and 250 μL U-LNP / MCSF circRNA nanoagent, mix them evenly, and prepare U-LNP / PDGF circRNA / U-LNP / MCSF circRNA nanoagent. Example 6 A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 50 μg of PDGF circRNA and dissolve it in 350 μL of citric acid buffer (pH 4.0) with a concentration of 20 mM, mix well, and prepare a PDGF circRNA aqueous phase with a concentration of 142 μg / mL; S3. 150 μL of 25.7 mg / mL alcohol phase and 350 μL of 142 μg / mL PDGF circRNA aqueous phase were mixed by microfluidics, and then purified by Amicon Ultra centrifugal filter with 5 mL PBS buffer (pH 7.4) to obtain 500 μL U-LNP / PDGF circRNA nanoparticles; S4. Take 50 μg of IL4 circRNA and dissolve it in 350 μL of citric acid buffer (pH 4.0) with a concentration of 20 mM, mix well, and prepare an IL4 circRNA aqueous phase 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 IL4 circRNA aqueous phase with a concentration of 142 μg / mL were mixed by microfluidics, and then purified by Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain 500 μL U-LNP / IL4 circRNA nanoparticles; S6. Take 50 μg of MCSF circRNA and dissolve it in 350 μL of citric acid buffer (pH 4.0) with a concentration of 20 mM, mix well, and prepare an MCSF circRNA aqueous phase 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 MCSF circRNA aqueous phase with a concentration of 142 μg / mL were mixed by microfluidics, and then purified by Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain 500 μL U-LNP / MCSF circRNA nanoparticles; S8. Take 250 μL of U-LNP / PDGF circRNA nanoagent, 250 μL of U-LNP / IL4 circRNA nanoagent, and 250 μL of U-LNP / MCSF circRNA nanoagent, mix them evenly, and prepare U-LNP / PDGF circRNA / / U-LNP / IL4circRNA / U-LNP / MCSF circRNA nanoagent. Comparative Example 1 The difference between this comparative example and Example 2 is that IL4 circRNA and PDGF circRNA are replaced by eGFP circRNA. A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg 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 citric acid 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with eGFP circRNA, which was recorded as U-LNP / eGFP circRNA nanoparticle. Comparative Example 2 The difference between this comparative example and Example 2 is that IL4 circRNA is replaced by PDGF circRNA. A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg 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 citric acid 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with PDGF circRNA, which was recorded as U-LNP / PDGF circRNA nanoparticle. Comparative Example 3 The difference between this comparative example and Example 2 is that PDGF circRNA is replaced by IL4 circRNA. A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg 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 citric acid 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with IL4 circRNA, which was recorded as U-LNP / IL4 circRNA nanoparticle. Comparative Example 4 The difference between this comparative example and Example 2 is that PDGF circRNA and IL4 circRNA are replaced by MCSF circRNA. A method for preparing a nanoformulation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 50 μg of MCSF circRNA and dissolve it in 350 μL of citric acid buffer (pH 4.0) with a concentration of 20 mM, 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with MCSFcircRNA, which was recorded as U-LNP / MCSF circRNA nanoparticle. Comparative Example 5 The difference between this comparative example and Example 2 is that IL4 circRNA is replaced by PDGF circRNA, and the ionizable lipid U-105 is replaced by ionizable lipid ALC-0315. A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid ALC-0315 2.26 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.3 mg / mL; S2. Take 50 μg of PDGF circRNA and dissolve it in 350 μL of citric acid buffer (pH 4.0) with a concentration of 20 mM, 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with PDGF circRNA, which was recorded as A-LNP / PDGF circRNA nanoparticle. Comparative Example 6 The difference between this comparative example and Example 2 is that PDGF circRNA is replaced by IL4 circRNA, and the ionizable lipid U-105 is replaced by ionizable lipid ALC-0315. A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid ALC-0315 2.26 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg 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 citric acid 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with IL4 circRNA, which was recorded as A-LNP / IL4 circRNA nanoparticle. Comparative Example 7 The difference between this comparative example and Example 2 is that both PDGF circRNA and IL4 circRNA are replaced by MCSF circRNA, and the ionizable lipid U-105 is replaced by ionizable lipid ALC-0315. A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid ALC-0315 2.26 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg 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 citric acid 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with MCSFcircRNA, which was recorded as A-LNP / MCSF circRNA nanoparticle. Comparative Example 8 The difference between this comparative example and Example 2 is that IL4 circRNA and PDGF circRNA are replaced by PDGF mRNA. A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. 50 μg of PDGF mRNA was dissolved in 350 μL of 20 mM citric acid buffer (pH 4.0), mixed evenly, and the aqueous phase was prepared 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with PDGF mRNA, which was recorded as U-LNP / PDGF mRNA nanoparticle. Comparative Example 9 The difference between this comparative example and Example 2 is that IL4 circRNA and PDGF circRNA are replaced by IL4 mRNA. A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. 50 μg of IL4 mRNA was dissolved in 350 μL of 20 mM citric acid buffer (pH 4.0), mixed evenly, and an aqueous phase with a concentration of 142 μg / mL was prepared; 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with IL4 mRNA, which was recorded as U-LNP / IL4 mRNA nanoparticle. Comparative Example 10 The difference between this comparative example and Example 2 is that IL4 circRNA and PDGF circRNA are replaced by MCSFmRNA. A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. 50 μg of MCSF mRNA was dissolved in 350 μL of 20 mM citric acid buffer (pH 4.0), mixed evenly, and an aqueous phase with a concentration of 142 μg / mL was prepared; 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with MCSF mRNA, which was recorded as U-LNP / MCSF mRNA nanoparticle. Comparative Example 11 The difference between this comparative example and Example 2 is that IL4 circRNA and PDGF circRNA are replaced by PDGF mRNA, and the ionizable lipid U-105 is replaced by ionizable lipid ALC-0315. A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid ALC-0315 2.26 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.3 mg / mL; S2. 50 μg of PDGF mRNA was dissolved in 350 μL of 20 mM citric acid buffer (pH 4.0), mixed evenly, and the aqueous phase was prepared 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with PDGF mRNA, which was recorded as A-LNP / PDGF mRNA nanoparticle. Comparative Example 12 The difference between this comparative example and Example 2 is that IL4 circRNA and PDGF circRNA are replaced by IL4 mRNA, and the ionizable lipid U-105 is replaced by ionizable lipid ALC-0315. A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid ALC-0315 2.26 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.3 mg / mL; S2. 50 μg of IL4 mRNA was dissolved in 350 μL of 20 mM citric acid buffer (pH 4.0), mixed evenly, and an aqueous phase with a concentration of 142 μg / mL was prepared; 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with IL4 mRNA, which was recorded as A-LNP / IL4 mRNA nanoparticle. Comparative Example 13 The difference between this comparative example and Example 2 is that IL4 circRNA and PDGF circRNA are replaced by MCSFmRNA, and the ionizable lipid U-105 is replaced by ionizable lipid ALC-0315. A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid ALC-0315 2.26 mg, DMG-PEG2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.3 mg / mL; S2. 50 μg of MCSF mRNA was dissolved in 350 μL of 20 mM citric acid buffer (pH 4.0), mixed evenly, and an aqueous phase with a concentration of 142 μg / mL was prepared; 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with MCSF mRNA, which was recorded as A-LNP / MCSF mRNA nanoparticle. 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) Packaging efficiency The encapsulation efficiency of Examples 1-6 and Comparative Examples 1-13 was measured using the Ribogreen reagent method. The specific method was to treat the samples to be tested with TE buffer and 2% TE-Triton buffer, respectively, add Ribogreen reagent, and use a microplate reader to measure the fluorescence intensity to obtain the amount of dissociated circRNA (or mRNA) and the amount of total circRNA (or mRNA), and calculate according to the following formula: Encapsulation efficiency (%) = [(amount of dissociated circRNA (or mRNA)) / total amount of circRNA (or mRNA)] × 100%. (3) Cell survival rate HUVECs cells were seeded into 96-well plates (2×10 3 Cells were cultured until the confluence reached 85%, and then Opti-MEMI Reduced Serum Medium was added to replace the cell culture medium. The nanoagents prepared in Examples 1-6 and Comparative Examples 1-13 (all nanoagents were 10 μg circRNA or mRNA dissolved in 100 μL PBS buffer) were co-cultured with HUVECs for 24 h, and a blank control was set. The cells were then treated with a cell counting kit-8, and the OD was measured using a microplate reader. 450nm , the cell survival rate was calculated and the results are shown in Table 1. Table 1 Particle size, PDI, Zeta potential, encapsulation efficiency and cell viability of Examples 1-6 and Comparative Examples 1-13 Particle size (nm) PDI Zeta potential (mV) Packaging efficiency (%) Cell survival rate (%) Example 1 118.2 0.17 -3.16 91.3 112.3 Example 2 113.3 0.21 -3.68 90.5 118.1 Example 3 110.9 0.19 -3.42 88.7 125.3 Example 4 102.5 0.16 -3.54 89.2 118.6 Example 5 108.1 0.20 -3.12 88.7 124.1 Example 6 111.7 0.18 -3.69 91.2 120.6 Comparative Example 1 103.8 0.20 -3.25 90.3 108.3 Comparative Example 2 110.6 0.22 -3.65 88.6 122.3 Comparative Example 3 112.5 0.19 -3.74 89.2 115.5 Comparative Example 4 107.6 0.22 -4.12 90.2 117.6 Comparative Example 5 103.2 0.19 -3.24 89.2 125.4 Comparative Example 6 111.3 0.16 -3.15 87.6 109.8 Comparative Example 7 112.8 0.20 -4.45 88.3 121.8 Comparative Example 8 108.5 0.18 -3.61 91.1 113.3 Comparative Example 9 112.3 0.23 -4.53 90.5 116.1 Comparative Example 10 106.5 0.21 -3.20 88.4 126.5 Comparative Example 11 98.8 0.15 -5.11 90.5 119.1 Comparative Example 12 104.3 0.21 -3.65 88.3 110.1 Comparative Example 13 101.5 0.24 -4.30 87.4 123.8 As can be seen from Table 1, the particle sizes of Examples 1-6 and Comparative Examples 1-13 are all around 100 nm, the PDI is all around 0.2, the Zeta potential is all in the range of -6--3 mV, the encapsulation efficiency is in the range of 87-92%, and the cell survival rate reaches 100-126%. They all have suitable sizes, good encapsulation efficiency, are non-toxic, have high safety, and do not cause high damage to cells, indicating that there is no significant difference in the physical and chemical indicators of the nanoformulations prepared based on circRNA or mRNA of growth factors or cytokines. (4) In vitro long-term expression test 1×10 per well 6 HUVECs cells were inoculated on a 6-well plate at a density of 10 cells until the confluence was 85%, and then the nanoagents prepared in Comparative Examples 2-13 (all nanoagents were 10 μg circRNA or mRNA dissolved in 100 μL PBS) were co-cultured with HUVECs cells for 6 h, and the expression of PDGF, IL4, and MCSF proteins was detected by Elisa method. Depend on Figure 2It can be seen that in Comparative Examples 2-13, the concentration of growth factors or cytokines expressed by the circRNA loaded prepared in Comparative Examples 2-7 is much higher than that of the nanoagents loaded with linear mRNA prepared in Comparative Examples 8-13, and the expression time of the former lasts for about 14 days, while the latter only lasts for about 3 days. On the 14th day, the former still has protein expression, and its concentration reaches about 10 ng / mL, indicating that compared with linear mRNA, circRNA can express active proteins for a long time, and the protein concentration expressed by the circRNA loaded by U-LNP is higher than that of the circRNA loaded by A-LNP. (3) Diabetes wound healing test Preparation of diabetic mouse model: All mice (C57BL / 6, male) were fed a high-sugar and high-fat diet for the first 4 weeks. In the 5th week, the mice were intraperitoneally injected with streptozotocin (STZ, Sigma-Aldrich) dissolved in citrate buffer and fasted for 12 hours. Then, the mice treated with STZ were still fed a high-sugar and high-fat diet. The blood glucose level in the tail vein blood of the mice was measured using a blood glucose meter. Two weeks after the injection of STZ, the fasting blood glucose level exceeded 16.7mmol / L, and the diabetic mouse model was successfully established. Wound surface construction: All diabetic mice were divided into 14 groups, namely 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 / IL4circRNA (group f), U-LNP / PDGF circRNA (group g), U-LNP / MCSF circRNA (group h), U-LNP / PDGFcircRNA / U-LNP / IL4 circRNA (group i), U-LNP / PDGF circRNA / U-LNP / MCSF circRNA (group j), U-LNP / PDGF circRNA / U-LNP / IL4circRNA / U-LNP / MCSF circRNA (group k), U-LNP / PDGF circRNA / IL4 circRNA (group l), U-LNP / PDGF circRNA / MCSF circRNA (group m), U-LNP / PDGF circRNA / IL4circRNA / MCSFcircRNA (n group), all mice were anesthetized with isoflurane in the experiment, and then the dorsal 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; Nanoagent administration: According to the grouping, the mice in the control PBS group were instilled with 20 μL PBS solution, and the wounds of diabetic mice were instilled with a single dose of U-LNP / eGFP circRNA nanoagent (Comparative Example 1), U-LNP / IL4mRNA nanoagent (Comparative Example 9), U-LNP / PDGF mRNA nanoagent (Comparative Example 8), U-LNP / MCSF mRNA nanoagent (Comparative Example 10), U-LNP / IL4 circRNA nanoagent (Comparative Example 3), U-LNP / PDGF circRNA nanoagent (Comparative Example 2), U-LNP / MCSF circRNA nanoagent (Comparative Example 4), U-LNP / PDGF circRNA / U-LNP / IL4circRNA nanoagent (Example 4), U-LNP / PDGF circRNA / U-LNP / MCSF circRNA nanoagent (Example 5), U-LNP / PDGFcircRNA / / U-LNP / IL4 circRNA / U-LNP / MCSF 100 μL of each circRNA nanoagent (Example 6), U-LNP / PDGFcircRNA / IL4 circRNA nanoagent (Example 1), U-LNP / PDGFcircRNA / MCSFcircRNA nanoagent (Example 2), and U-LNP / PDGFcircRNA / IL4circRNA / MCSFcircRNA nanoagent (Example 3) (all nanoagents contain 10 μg of circRNA or mRNA in 100 μL PBS buffer) for accurate measurement, and all nanoagents are dropped on the mouse wound with a pipette, and the mouse is fixed for 30 minutes to promote the absorption of the nanoagent, and then the wound is carefully rinsed 3 times with PBS buffer to remove the residual preparation; Results: At 0, 4, 7 and 10 days after the nano-agent treatment, the wound condition was recorded with a smartphone and the data was processed with ImageJ software. In order to accurately record the wound size, a round black card with a diameter of 14 mm was used as a reference. The results are shown in Figure 3 , and calculated the wound area ratio. The results are shown in Figure 4 and Figure 5 . Depend on Figure 3 , Figure 4 , Figure 5 It can be seen that compared with group a, group b can only express fluorescent protein without any pharmacological activity, and has no promoting effect on wound healing; while the wound surface of mice treated with groups c, d and e did not show significant reduction, indicating that the linear mRNA loaded by the nanoformulations prepared in comparative examples 8-10 can only express growth factors or cytokines for a short time, and has no obvious promoting effect on wound healing. The wound surfaces of mice treated with groups F, G and H were significantly smaller than those of group CE, indicating that the circular mRNA loaded by the nanoformulations prepared in comparative examples 2-4 can express growth factors or cytokines for a long time, which is beneficial to wound healing and the reduction of wound area; the wound area of ​​mice treated with groups I and J was significantly reduced after the 7th day, and almost completely healed after the 10th day, indicating that the synergistic effect of the nanoformulations prepared in Examples 4 and 5 loaded with a circRNA combination of a growth factor and a cytokine is more effective for wound healing; group K (Example 6) is a nanoformulation combination of a growth factor and two cytokine circRNAs, which also showed excellent wound repair effects after treating the wounds of mice, and the wounds were almost completely healed after 10 days. The wounds treated in groups L, M and N showed significant shrinkage after 7 days, and were almost completely healed after 10 days, indicating that there was no significant difference in the wound healing process of mice after treatment with the nanoformulations 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, the comparative examples 2-4 only contain one growth factor or one cytokine circRNA, which has a certain wound repair effect, while the circRNA combination of at least one growth factor and at least one cytokine in Examples 1-6 is more beneficial to wound healing, and the healing effect is most significant. Because the growth factor circRNA is delivered to the wound cells by LNP and expresses the growth factor for a long time, it promotes the growth of skin fibroblasts, vascular endothelial cells, etc., and the cytokine circRNA is delivered to the wound cells by LNP and expresses the cytokine for a long time, which inhibits inflammation and regulates the immune microenvironment of the wound. The two effects cooperate with each other to promote wound healing to the greatest extent, especially the circRNA combination of one growth factor and two cytokines IL4 and MCSF is most beneficial to wound healing. (3) Tissue section test at the wound site Hematoxylin and eosin (H&E) staining, Masson's trichrome staining, and CD31 histochemical staining were used to histologically evaluate the granulation tissue formation, collagen deposition, and capillary density of the wound surface in the experimental group al. On the 10th day, the wound tissues of diabetic mice were isolated and fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4-μm-thick sections. For CD31 immunohistochemical analysis, rabbit anti-CD31 primary antibody (1:200) diluted in PBS buffer was used, followed by incubation with biotin-labeled secondary antibody. The stained sections were observed and recorded using a Leica microscope DM750 for CD31 immunohistochemical and histological analysis. Depend on Figure 6It can be seen that after the 10th day, the wound tissue of diabetic mice was taken to analyze the tissue regeneration process and biosafety. 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 was not repaired; the epidermis of the U-LNP / IL4 mRNA group (group c), the U-LNP / PDGF mRNA group (group d) and the U-LNP / MCSF mRNA group (group e) was continuous and showed an obvious dermis, and the healing skin layer was significantly thicker than that of the a group and the b group, indicating that the nanoformulations prepared in comparative examples 8-10 loaded with linear IL4 mRNA, PDGF mRNA and MCSF mRNA had certain growth factors or cytokine active protein expression on the wound surface, which promoted wound repair and skin thickening; U-LNP / IL4 circRNA (group f), U-LNP / PDGF circRNA (group g) and U-LNP / MCSF The thickness of the epidermis and dermis 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, indicating that the nanoformulations prepared in comparative examples 2-4 loaded with circRNA expressed growth factors or cytokine active proteins on the wound surface for a long time, promoted wound repair more efficiently, and significantly increased the thickness of the epidermis. The epidermis thickness of 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) was much thicker than that of ce group and fh group, indicating that the nanoformulations prepared in Examples 4-6 containing two or more growth factor and cytokine circRNA combinations are more conducive to promoting epidermal growth and wound healing; in particular, the epidermis thickness of U-LNP / PDGF circRNA / U-LNP / IL4 circRNA / U-LNP / MCSF circRNA group (group k) was thicker, indicating that the nanoformulation prepared in Example 6 containing a combination of PDGF, IL4 and MCSF circRNAs is most conducive to epidermal growth. In addition, 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 nanoformulations were different, there was no significant difference in the degree of promoting epidermal growth and wound healing. Depend on Figure 7It can be seen that the collagen deposition in the PBS control group (group a) and the U-LNP / eGFP circRNA group (group b) is very small, which is not conducive to wound repair; the collagen deposition of U-LNP / IL4 mRNA (group c), U-LNP / PDGF mRNA (group d) and U-LNP / MCSF mRNA (group e) is more obvious, better than that of group a and group b, indicating that the linear IL4 mRNA, PDGF mRNA and MCSF mRNA loaded by the nanoformulations prepared in comparative examples 8-10 have certain growth factor or cytokine active protein expression on the surface, which promotes collagen deposition and promotes wound repair; the collagen deposition of U-LNP / IL4 circRNA (group f), U-LNP / PDGF circRNA (group g) and U-LNP / MCSFcircRNA (group h) is more significant, indicating that the nanoformulations prepared in comparative examples 2-4 loaded with circRNA express growth factor or cytokine active protein on the wound for a long time, more efficiently promote collagen production, and are conducive to wound repair. The collagen deposition of the three groups was more prominent, including 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), indicating that the nanoformulations prepared in Examples 4-6 containing two or more growth factor and cytokine circRNA combinations are more conducive to promoting collagen deposition; in particular, the collagen deposition of the U-LNP / PDGF circRNA / U-LNP / IL4circRNA / U-LNP / MCSF circRNA group (group k) was the most significant, indicating that the combination of PDGF, IL4 and MCSFcircRNAs is most conducive to wound healing. In addition, 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 nanoformulations were different, the extent of collagen deposition was similar and there was no significant difference in the extent of promoting wound healing. Depend on Figure 8It can be seen that there is no obvious angiogenesis in the PBS control group (group a) and the U-LNP / eGFP circRNA group (group b), which is not conducive to wound repair; U-LNP / IL4 mRNA (group c), U-LNP / PDGF mRNA (group d) and U-LNP / MCSF mRNA (group e) have very little angiogenesis, which is better than groups a and b, indicating that the linear IL4 mRNA, PDGF mRNA and MCSF mRNA loaded by the nanoformulations prepared in comparative examples 8-10 have certain growth factor or cytokine active protein expression on the surface, but the angiogenesis is very limited; U-LNP / IL4 circRNA (group f), U-LNP / PDGF circRNA (group g) and U-LNP / MCSF circRNA (group h) have obvious angiogenesis, indicating that the nanoformulations prepared in comparative examples 2-4 loaded with circRNA express growth factor or cytokine active protein on the wound for a long time, more efficiently promote angiogenesis, and are conducive to wound repair. The vascular density of 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) was high, indicating that the nanoformulations prepared in Examples 4-6 containing two or more combinations of growth factors and cytokine circRNAs are more conducive to angiogenesis, and the formation of new blood vessels is beneficial to the supply of nutrition and oxygen, thereby promoting wound healing; in particular, the vascular density of the U-LNP / PDGF circRNA / U-LNP / IL4 circRNA / U-LNP / MCSF circRNA group (group k) was the highest, indicating that the nanoformulation prepared in Example 6 containing a combination of PDGF, IL4 and MCSF circRNAs is most conducive to angiogenesis and efficiently promotes wound healing. In addition, 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 of the nanoformulations were different, the vascular densities were similar and there was no significant difference in the degree of promoting wound healing. The above results show that the nanoformulations prepared in Examples 1-6 containing circRNA of at least one growth factor and at least one cytokine significantly promoted the formation of granulation tissue, collagen and blood vessels, especially the combination of PDGFcircRNA, IL4circRNA and MCSFcircRNA nanoformulation was most conducive to the formation of granulation tissue, collagen deposition and angiogenesis, and was most conducive to wound healing, so that the wound reached the level of complete healing. Example 7 A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 25 μg of FGF2 circRNA and 25 μg of IL4 circRNA, dissolve them in 350 μL of 20 mM citric acid buffer (pH 4.0), mix them evenly, 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with FGF2 circRNA and IL4 circRNA, which was recorded as U-LNP / FGF2 circRNA / IL4 circRNA nanoparticle. Example 8 A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 25 μg of FGF2 circRNA and 25 μg of MCSF circRNA, dissolve them in 350 μL of 20 mM citric acid buffer (pH 4.0), mix them evenly, 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 microfluidics, and then purified by 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifugal filter to obtain a lipid nanoparticle preparation loaded with FGF2 circRNA and MCSF circRNA, which was recorded as U-LNP / FGF2 circRNA / MCSF circRNA nanoparticle. Example 9 A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.7 mg / mL; S2. Take 17 μg of FGF2 circRNA, 17 μg of MCSF circRNA and 17 μg of IL4 circRNA, dissolve them in 350 μL of 20 mM citric acid buffer (pH 4.0), mix them evenly, 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with FGF2circRNA, IL4circRNA and MCSFcircRNA, which was recorded as U-LNP / FGF2circRNA / IL4circRNA / MCSFcircRNA nanoagent. Example 10 A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg 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 citric acid buffer (pH 4.0) with a concentration of 20 mM, mix them evenly, 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with PDGF-PlGFcircRNA and IL4-PlGFcircRNA, which was recorded as U-LNP / PDGF-PlGFcircRNA / IL4-PlGFcircRNA nanoagent. Embodiment 11 A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg 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 citric acid buffer (pH 4.0), mix them evenly, 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with PDGF-PlGFcircRNA and MCSF-PlGFcircRNA, which was recorded as U-LNP / PDGF-PlGFcircRNA / MCSF-PlGFcircRNA nanoagent. Example 12 A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg 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-PlGFcircRNA, dissolve them in 350 μL citric acid buffer (pH 4.0) with a concentration of 20 mM, mix them well, and prepare an aqueous phase with a concentration of 142 μg / mL; S3. Mix 150 μL of alcohol phase with a concentration of 25.7 mg / mL and 350 μL of aqueous phase with a concentration of 142 μg / mL by microfluidics, and then purify them through an Amicon Ultra centrifugal filter with 5 mL PBS buffer (pH 7.4) to prepare a lipid nanoparticle preparation loaded with PDGF-PlGFcircRNA, IL4-PlGFcircRNA and MCSF-PlGFcircRNA, recorded as U-LNP / PDGF-PlGFcircRNA / IL4-PlGFcircRNA / MCSF-PlGFcircRNA nanoagent. Example 13 A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-106 2.28 mg, DMG-PEG 2000 0.18 mg, DSPC 0.44 mg and cholesterol 0.89 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.3 mg / mL; S2. Take 25 μg of PDGF-Fc circRNA and 25 μg of IL4-Fc circRNA, dissolve them in 350 μL of 20 mM citric acid buffer (pH 4.0), mix them evenly, 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with PDGF-FccircRNA and IL4-FccircRNA, which was recorded as U6-LNP / PDGF-FccircRNA / IL4-FccircRNA nanoagent. Embodiment 14 A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-106 2.28 mg, DMG-PEG 2000 0.18 mg, DSPC 0.44 mg and cholesterol 0.89 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.3 mg / mL; S2. Take 25 μg of PDGF-Fc circRNA and 25 μg of MCSF-Fc circRNA, dissolve them in 350 μL of 20 mM citric acid buffer (pH 4.0), mix them 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 microfluidics, and then purified by 5 mL of PBS buffer (pH 7.4) through an Amicon Ultra centrifugal filter to obtain a lipid nanoparticle preparation loaded with PDGF-FccircRNA and MCSF-FccircRNA, which was recorded as U6-LNP / PDGF-FccircRNA / MCSF-FccircRNA nanoparticle. Embodiment 15 A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-106 2.28 mg, DMG-PEG 2000 0.18 mg, DSPC 0.44 mg and cholesterol 0.89 mg in 150 μL of ethanol to prepare an alcohol phase with a concentration of 25.3 mg / mL; S2. Take 17 μg PDGF-Fc circRNA, 17 μg IL4-Fc circRNA and 17 μg MCSF-Fc circRNA, dissolve them in 350 μL citric acid buffer (pH 4.0) with a concentration of 20 mM, mix them 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with PDGF-FccircRNA, IL4-Fc circRNA and MCSF-Fc circRNA, which was recorded as U6-LNP / PDGF-FccircRNA / IL4-Fc circRNA / MCSF-Fc circRNA nanoagent. Comparative Example 14 The difference between this comparative example and Example 8 is that IL4 circRNA is replaced by FGF2 circRNA. A method for preparing a nano preparation for promoting tissue repair comprises the following steps: S1. Dissolve ionizable lipid U-105 2.32 mg, DMG-PEG 2000 0.22 mg, DSPC 0.43 mg and cholesterol 0.88 mg 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 citric acid 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 microfluidics, and then purified by an Amicon Ultra centrifugal filter with 5 mL of PBS buffer (pH 7.4) to obtain a lipid nanoparticle preparation loaded with FGF2 circRNA, which was recorded as U-LNP / FGF2 circRNA nanoparticle. 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) Packaging efficiency The encapsulation efficiency of Examples 7-15 and Comparative Example 14 was measured using the Ribogreen reagent method. The specific method was to treat the samples to be tested with TE buffer and 2% TE-Triton buffer, respectively, add Ribogreen reagent, and use a microplate reader to measure the fluorescence intensity to obtain the amount of dissociated circRNA (or mRNA) and the amount of total circRNA (or mRNA), and calculate according to the following formula: Encapsulation efficiency (%) = [(amount of dissociated circRNA (or mRNA)) / total amount of circRNA (or mRNA)] × 100%. (5) Cell survival rate HUVECs cells were seeded into 96-well plates (2×10 3 Cells were cultured until the confluence reached 85%, and then Opti-MEMI Reduced Serum Medium was added to replace the cell culture medium. The nanoagents prepared in Examples 7-15 and Comparative Example 14 (all nanoagents were 10 μg circRNA or mRNA dissolved in 100 μL PBS buffer) were co-cultured with HUVECs for 24 h, and a blank control was set. The cells were then treated with a cell counting kit-8, and the OD was measured using a microplate reader. 450nm , the cell survival rate was calculated and the results are shown in Table 2. Table 2 Particle size, PDI, Zeta potential, encapsulation efficiency and cell viability of Examples 7-15 and Comparative Example 14 Particle size (nm) PDI Zeta potential (mV) Packaging efficiency (%) Cell survival rate (%) Example 7 104.3 0.21 -3.56 89.3 112 Example 8 112.1 0.16 -5.21 88.5 116 Example 9 106.2 0.18 -3.45 90.1 114 Example 10 104.6 0.17 -3.12 91.4 108 Embodiment 11 98.6 0.16 -4.10 89.7 119 Example 12 103.7 0.20 -3.78 90.4 113 Example 13 108.2 0.17 -3.65 88.7 120 Embodiment 14 112.3 0.19 -3.02 89.2 114 Embodiment 15 104.1 0.21 -3.24 91.3 113 Comparative Example 14 103.8 0.17 -3.87 90.4 118 As can be seen from 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--3 mV, the encapsulation efficiency is in the range of 87-92%, and the cell survival rate reaches 100-130%. They all have suitable sizes, good encapsulation efficiency, are non-toxic, have high safety, and do not cause high damage to cells, indicating that there is no significant difference in the physical and chemical indicators of the nanoformulations prepared based on circRNA of growth factors or cytokines. (2) Diabetes wound healing test Preparation of diabetic mouse model: All mice (C57BL / 6, male) were fed a high-sugar and high-fat diet for the first 4 weeks. In the 5th week, the mice were intraperitoneally injected with streptozotocin (STZ, Sigma-Aldrich) dissolved in citrate buffer and fasted for 12 hours. Then, the mice treated with STZ continued to eat a high-sugar and high-fat diet. The blood glucose level in the tail vein blood of the mice was measured using a blood glucose meter. Two weeks after the injection of STZ, the fasting blood glucose level exceeded 16.7mmol / L, and the diabetic mouse model was successfully established. Wound surface construction: All diabetic mice were divided into 14 groups, namely 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-PlGFcircRNA / MCSF-PlGF circRNA (group g, Example 11), U-LNP / PDGF-PlGF circRNA / IL4-PlGFcircRNA / MCSF-PlGF circRNA (group h, Example 12), U6-LNP / PDGF-Fc circRNA / IL4-Fc 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), all mice were anesthetized with isoflurane, and then the dorsal 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; Nanoagent administration: According to the grouping, the mice in the control PBS group were instilled with 20 μL PBS solution, and the wounds of diabetic mice were instilled with a single dose of U-LNP / FGF2 circRNA nanoagent, U-LNP / FGF2 circRNA / IL4 circRNA nanoagent, U-LNP / FGF2 circRNA / MCSF circRNA nanoagent, U-LNP / FGF2 circRNA / IL4 circRNA / MCSFcircRNA nanoagent, U-LNP / PDGF-PlGF circRNA / IL4-PlGF circRNA, U-LNP / PDGF-PlGFcircRNA / MCSF-PlGF circRNA, U-LNP / PDGF-PlGF circRNA / IL4-PlGF circRNA / MCSF-PlGFcircRNA, U6-LNP / PDGF-Fc circRNA / IL4-Fc circRNA, U6-LNP / PDGF-Fc circRNA / MCSF-FccircRNA, and U6-LNP / PDGF-Fc 100 μL of each circRNA / IL4-Fc circRNA / MCSF-Fc circRNA nanoagent (all nanoagents contained 10 μg of circRNA in 100 μL PBS buffer) was added for accurate measurement, and all nanoagents were dropped onto the mouse wounds with a pipette, and the mice were fixed for 30 min to promote the absorption of the nanoagents, and then the wounds were carefully rinsed 3 times with PBS buffer to remove the residual agents; Analysis of results: On days 0, 4, 7 and 10 after the wound was treated with the nanoagent, the wound size was accurately recorded, and a round black card with a diameter of 14 mm was used as a reference to calculate the change in wound area ratio with healing time. Depend on Fig. 9It can be seen that compared with the PBS control group (group a), the wound area of ​​mice treated with U-LNP / FGF2 circRNA group (group b) was reduced, indicating that circular mRNA can express growth factor FGF2 for a long time, which is beneficial to wound healing and reduces the wound area; U-LNP / FGF2 circRNA / IL4 circRNA group (group c), U-LNP / FGF2 circRNA / MCSFcircRNA group (group d), U-LNP / FGF2 circRNA / IL4 circRNA / MCSF circRNA group (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), U6-LNP / PDGF-Fc circRNA / IL4-Fc The wound area of ​​mice treated with 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) nanoagents was significantly reduced after the 7th day, and almost completely healed after the 10th day, indicating that the circRNA combination nanoagent of a growth factor FGF2 and one or two cytokines (IL4 and MCSF) is more effective for wound healing, proving that the circRNA nanoagent of at least one growth factor and at least one cytokine is most beneficial for wound healing. This is because the growth factor FGF2 circRNA is delivered to the wound cells by LNP and the growth factor FGF2 is expressed for a long time, which promotes the growth of skin fibroblasts, vascular endothelial cells, etc. At the same time, the cytokine IL4 and MCSF circRNA are delivered to the wound cells by LNP and the cytokines are expressed for a long time, which inhibits inflammation and regulates the immune microenvironment of the wound. The two effects synergize with each other to promote wound healing to the greatest extent. It is particularly pointed out that according to the final wound area ratio, the healing effect of groups f, g, and h is better than that of groups i, j, and k, and the latter is better than that of groups c, d, and e, indicating that the fusion protein circRNA combination nanoformulation loaded with PlGF is better than the fusion protein circRNA combination nanoformulation combined with Fc, and better than the non-fusion protein circRNA combination nanoformulation. This is because the circRNA combination in the nanoformulation expresses the growth factor and cytokine PlGF fusion protein and strongly binds to the extracellular matrix, and the growth factor and cytokine are released to exert pharmacological effects; while the other nanoformulation circRNA combination expresses the growth factor and cytokine Fc fusion protein, although it also delays the release of growth factors and cytokines to exert pharmacological effects, it is not as good as the former; the non-fusion protein circRNA combination nanoformulation only expresses growth factors and cytokines, and the pharmacological effect is not as good as the fusion protein circRNA combination. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A nanoparticle preparation for promoting tissue repair, characterized in that: The nanoformulation 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; 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 nanoparticle preparation for promoting tissue repair according to claim 1, characterized in that: The component 1 is composed of spacer 1, exon 1, exon 2, spacer 2, ribosome insertion site, Kozak sequence and coding region connected in sequence; the sequence of spacer 1 is the sequence shown in SEQ ID No. 1 or a sequence with at least 75% identity thereto; The sequence of exon 1 is the sequence shown in SEQ ID No. 2 or a sequence having at least 75% identity thereto; The sequence of exon 2 is the sequence shown in SEQ ID No. 3 or a sequence having at least 75% identity thereto; The sequence of the spacer 2 is the sequence shown in SEQ ID No. 4 or a sequence having at least 75% identity thereto; The sequence of the ribosome insertion site is any one of SEQ ID No. 5, 6, 7 or 8, or a sequence having at least 75% identity thereto; The Kozak sequence is the sequence shown in SEQ ID No.9; The sequence of the coding region is a sequence shown in any one of SEQ ID No. 10-22 of a growth factor, or a growth factor fusion protein sequence or a sequence having at least 75% identity therewith; The growth factor fusion protein sequence is composed of any one of the growth factor sequences SEQ ID No.10-22, the connecting peptide sequence SEQ ID No.36, and any one of 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 placenta growth factor heparin binding domain sequence SEQ ID No.

35.

3. The nanoparticle preparation for promoting tissue repair according to claim 1, characterized in that: The component 2 is formed by sequentially connecting the spacer 1, exon 1, exon 2, spacer 2, ribosome insertion site, Kozak sequence and coding region; the sequence of the spacer 1 is the sequence shown in SEQ ID No. 1 or a sequence having at least 75% identity thereto; The sequence of exon 1 is the sequence shown in SEQ ID No. 2 or a sequence having at least 75% identity thereto; The sequence of exon 2 is the sequence shown in SEQ ID No. 3 or a sequence having at least 75% identity thereto; The sequence of the spacer 2 is the sequence shown in SEQ ID No. 4 or a sequence having at least 75% identity thereto; The sequence of the ribosome insertion site is any one of SEQ ID No. 5, 6, 7 or 8, or a sequence having at least 75% identity thereto; The Kozak sequence is the sequence shown in SEQ ID No.9; The sequence of the coding region is a sequence shown in any one of cytokine SEQ ID No. 23-31, or a cytokine fusion protein sequence or a sequence having at least 75% identity thereto; The cytokine fusion protein sequence is composed of any one of the cytokine sequences SEQ ID No.23-31, the connecting peptide sequence SEQ ID No.36, and any one of 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 placenta growth factor heparin binding domain sequence SEQ ID No.

35.

4. The nanoparticle preparation for promoting tissue repair according to claim 1, characterized in that: 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 a, brain-derived neurotrophic factor, neurotrophic factor 3, and transforming growth factor β1; the growth factor fusion protein is composed of component A, a connecting 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 nanoparticle preparation for promoting tissue repair according to claim 1, characterized in that: The cytokine is one or more of interleukin 4, interleukin 10, interleukin 13, interleukin 22, macrophage colony stimulating factor, chemokine 12, interferon regulatory factor 4, islet regeneration source protein, tumor necrosis factor α stimulating gene / inducible protein-6; the cytokine fusion protein is composed of component C, connecting 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 protein.

6. The nanoparticle preparation for promoting tissue repair according to claim 1, characterized in that: The nano preparation consists of three components, component 1 is one or more of 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 alpha 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 nano carrier.

7. The nano preparation for promoting tissue repair according to claim 1, characterized in that: The method for preparing the nano preparation comprises the following steps: S1. Add ethanol to lipid raw material 1 and lipid raw material 2 and mix them evenly to obtain a lipid-containing alcohol phase; S2. Add citric acid buffer to component 1 and mix well to obtain aqueous phase A; S3. Add citric acid buffer to component 2 and mix well to obtain aqueous phase B; S4. Microfluidic mixing, removal of ethanol, and preparation of nanoformulation; 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 of a lipid-containing alcohol phase, an aqueous phase A and an aqueous phase B; mixing a two-phase system of a lipid-containing alcohol phase and an aqueous phase A; and mixing a two-phase system of a lipid-containing alcohol phase and an aqueous phase B.

8. Use of the nanoformulation for promoting tissue repair according to any one of claims 1 to 7 in the preparation of a pharmaceutical nanoformulation for treating refractory wounds, myocardial infarction, stroke, arthritis, acute lung injury, and tendinitis.

9. The use of a nano preparation for promoting tissue repair according to claim 8, characterized in that: The drug nanoformulation contains pharmaceutically acceptable adjuvants, which include polymer porous 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; the administration methods of the drug nanoformulation include but are not limited to topical administration, intravenous administration, subcutaneous administration, and intramuscular injection.

10. The use of a nano preparation for promoting tissue repair according to claim 7, characterized in that: The refractory wounds are diabetic foot ulcers, pressure ulcers, vascular ulcers, radiation ulcers, tophi ulcers, iatrogenic wounds, traumatic ulcers, infectious 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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