Gene delivery ionizable lipid-based injectable microsphere as well as preparation method and application thereof

Abstract

The invention provides a gene delivery ionizable lipid-based injectable microsphere as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention synthesizes ionizable lipid based on a vitamin E derivative, replaces a traditional lipid component, and successfully constructs an injectable lipid nanoparticle (iLNP) / microsphere compound (iLMP) with an endoplasmic reticulum stress (ERS) relieving function by combining with hydrogel microspheres. On one hand, the novel ionizable lipid has physicochemical properties similar to those of the traditional ionizable lipid D-Lin-MC3-DMA, can be directly replaced and used for construction of lipid nanoparticles (LNP), and has stable loading capacity and transfection efficiency similar to those of mRNA (messenger ribonucleic acid). On the other hand, under an in-vitro high ERS cell model, the iLMP can significantly reduce ERS and PERK pathway activation caused by the ERS, reduce eIF2alpha phosphorylation and significantly improve the translation efficiency of mRNA. In-vitro and in-vivo results show that after the iLMP is used as the carrier to load the BMP-2 mRNA, the BMP-2 translation efficiency and the osteogenesis promoting function are obviously improved.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicine technology, and particularly relates to an ionizable lipid-based injectable microsphere for gene delivery, its preparation method and application. Background Art

[0002] Currently, mRNA therapy is a disruptive technology in the medical field including regenerative medicine. Once exogenous mRNA enters the cell, it directly initiates protein translation in the cytoplasm without the need for nuclear import and reverse transcription processes, thereby regulating cell fate. However, during the delayed repair of inflamed tissues, cells usually undergo upregulated endoplasmic reticulum stress (ERS) and actively initiate the unfolded protein response (UPR), which significantly reduces the translation of mRNA in ribosomes. Unfortunately, current mRNA therapies do not fully account for this ERS-related mRNA translation limitation. Therefore, it is necessary to develop a strategy that can alleviate ERS to enhance mRNA therapy by accelerating ribosome translation and overall protein synthesis.

[0003] Normally, UPR (including the downregulation of mRNA translation) is a protective mechanism for cells to respond to elevated pathological ERS. During tissue regeneration, cells face various external stresses, such as inflammatory and hypoxic microenvironments, which may disrupt the intracellular protein homeostasis. The endoplasmic reticulum (ER) is the main site for protein folding and maturation, and the accumulation of unfolded or misfolded proteins in the ER further exacerbates ERS. Under ERS conditions, cells can initiate UPR through multiple pathways to reduce the ER load. Among them, downregulating mRNA translation is an active strategy to reduce the synthesis of new proteins to reduce the accumulation of unfolded or misfolded proteins. In addition, by downregulating mRNA translation, cells can reduce the damage and apoptosis risk associated with ERS, thereby increasing the survival rate under stress conditions.

[0004] Although some stress response proteins that contribute to restoring endoplasmic reticulum homeostasis (such as ATF4 and XBP1) show selective upregulation, the global downregulation of intracellular mRNA translation is indisputable. The process of mRNA translation downregulation involves multiple UPR-activated signaling pathways, among which the protein kinase R-like ER kinase (PERK) pathway plays the most crucial role. Here, PERK can be regarded as a key sensor of the UPR. When ERS is activated, PERK undergoes autophosphorylation and activates its kinase activity. The activated PERK phosphorylates the Ser51 site of eukaryotic translation initiation factor 2α (eIF2α). Finally, the phosphorylation of eIF2α directly inhibits the translation level of whole-cell mRNA. In addition, the inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) pathways are also involved in the related mechanism, but they have no direct relationship with the mRNA translation level. Therefore, to increase the global mRNA translation level, it is necessary to relieve ERS at the source and reduce the cell's dependence on the UPR protection mechanism.

[0005] Lipid nanoparticle (LNP) gene transfection tools have enabled meaningful mRNA therapies to move from the laboratory to clinical applications. Currently, typical LNP formulations reported include ionizable lipids, polyethylene glycolylated lipids, cholesterol, and helper lipids. Among them, ionizable lipids are the most critical component, which can achieve stable mRNA loading and efficient lysosomal escape through pH-dependent reversible protonation, significantly affecting the delivery efficiency of LNP mRNA. Based on this, developing novel ionizable lipid LNP systems is a key way to expand mRNA therapies. For example, researchers constructed ionizable lipids for LNP preparation by lipid-modified guanidinium cation polysulfides, and improved the transfection efficiency of mRNA in dendritic cells by endowing dendritic cells with the ability to scavenge inflammation-related reactive oxygen species (ROS). In addition, researchers designed a single-component ionizable cationic lipid rich in secondary amines for selectively delivering mRNA to the spleen and T cells, solving the extrahepatic delivery challenge faced by traditional LNPs. Therefore, the design of ionizable lipids can further endow the mRNA LNP system with additional capabilities.

[0006] In previous studies, it has been reported that various lipophilic drug molecules are directly used to replace traditional phospholipids and lipid alkyl chains to construct amphiphilic conjugates as formulation components, thereby fabricating functionalized liposomes and vesicles. For example, existing studies have reported a series of artemisinin-ether-phospholipid and cationic lipid conjugates, which not only maintain the drug activity of artemisinin-ether when constructing liposome drug / gene delivery systems but also completely replace traditional phospholipids. These data fully demonstrate that amphiphilic conjugates have physicochemical properties similar to those of traditional phospholipids and lipids and can be completely replaced in terms of structural assembly.

[0007] In addition, patent document CN 118078773 A discloses an mRNA polymer-lipid hybrid delivery system. The components of this delivery system consist of mRNA, cationic molecules, polymers, and modified amphiphilic molecules. Compared with the traditional lipid nanoparticle system, the introduction of polymers reduces the components of the delivery system, increases the stability of the overall structure, and reduces the difficulty and cost of large-scale production of raw materials. Patent document CN 116970652 A discloses a CD19-CAR-mRNA-LNP particle and its preparation method and application. It prepares CAR-mRNA-LNP by filtering a lipid-ethanol solution and an mRNA-citrate buffer solution through a microfiltration membrane, which solves to a certain extent the problems of low transfection efficiency and poor stability existing in existing transfection particles. When existing gene transfection lipid nanoparticles deliver mRNA, they can improve the transfection efficiency compared with traditional lipids. However, existing gene transfection tools are unable to reduce intracellular reactive oxygen species (ROS) in MC3T3-E1 cells and do not have the function of alleviating endoplasmic reticulum stress (ERS). Therefore, they cannot improve the translation efficiency of mRNA in a high-ERS intracellular environment and have poor effects in promoting cell osteogenesis.

[0008] As a natural fat-soluble active molecule, vitamin E has been reported to have the effects of alleviating the endoplasmic reticulum of cells and enhancing translation-related pathways. Therefore, introducing vitamin-based ionizable lipids into the LNP formulation is a feasible method, which is expected to enable LNPs to have the ability to release ERS, thereby improving the cellular translation level of mRNA therapy. However, related research is still blank and a large number of studies need to be carried out.

[0009] Injectable hydrogel microspheres are well-known for their high biocompatibility and microenvironment remodeling ability and have been widely used in tissue repair. Some researchers have explored the feasibility of using hydrogel microsphere-based microplexes as gene delivery vectors. However, how to develop a vitamin E-based lipid based on vitamin E and successfully combine it with hydrogel microspheres to construct an ionizable lipid that can replace traditional lipids, in order to achieve the sustained release of VES, so as to achieve enhanced ribosomal translation and mRNA therapy, still faces many difficulties and has not been studied before. Therefore, how to design a new lipid component to effectively improve the translation efficiency of mRNA in a high-ERS intracellular environment and significantly promote cell osteogenesis, so as to better cope with the ribosomal translation limitation caused by the UPR effect induced by ERS and promote tissue regeneration, has become an urgent technical problem to be solved. Summary of the Invention

[0010] The present invention is to solve the above technical problems, and thus provides an ionizable lipid-based injectable microsphere for gene delivery, a preparation method and an application thereof. The technical object of the present invention is to solve the problems that existing lipid materials have a low translation efficiency of mRNA under the intracellular high ERS environment during mRNA delivery, and a poor osteogenic effect of cells due to the existence of high ERS in tissue regeneration, so as to provide a gene delivery lipid composite material with high translation efficiency of exogenous mRNA and excellent osteogenic effect of cells under the intracellular high ERS environment.

[0011] In order to achieve the above technical object, the technical solution adopted by the present invention is as follows:

[0012] The present invention first provides a preparation method of an ionizable lipid-based injectable microsphere for gene delivery, comprising the following steps:

[0013] (1) Synthesize an ionizable vitamin E-based lipid by an esterification reaction of vitamin E succinate and an ionizable structure; the ionizable structure includes an ionizable amino group, a guanidine group and a heterocyclic group;

[0014] (2) Mix a composition of a traditional ionizable lipid replaced or partially replaced by the ionizable vitamin E-based lipid obtained in step (1) with phospholipids, cholesterol and a PEG component, dissolve the mixture in ethanol as an organic phase, use a gene and a citrate buffer solution as an aqueous phase, and prepare ionizable lipid nanoparticles (iLNPs) loaded with the gene by a microfluidic method; the traditional ionizable lipid includes but is not limited to: DLin-MC3-DMA, ALC-0315 or SM-102;

[0015] (3) Prepare a hydrogel microsphere by a microfluidic method, perform freeze-drying, and place it in the ionizable lipid nanoparticle solution obtained in step (2). After complete adsorption, the ionizable lipid-based injectable microsphere for gene delivery is obtained.

[0016] The present invention synthesizes a completely new vitamin E (VE)-derived ionizable lipid (iLNP) to replace the traditional lipid component, and combines it with hydrogel microspheres to construct an injectable lipid nanoparticle (iLNP) / microsphere complex (iLMP) with endoplasmic reticulum stress (ERS) alleviating function, thereby improving the translation efficiency of exogenous mRNA. On the one hand, the newly synthesized iLNP has physicochemical properties similar to those of the traditional ionizable lipid D-Lin-MC3-DMA, and can directly replace the construction of lipid nanoparticles (LNPs), with similar mRNA stable loading capacity and transfection efficiency. On the other hand, under an in vitro high-ERS cell model, iLMP can significantly reduce the activation of the PERK pathway, reduce eIF2α phosphorylation, and significantly improve the mRNA translation efficiency. Both in vitro and in vivo results show that iLMP as a carrier loaded with BMP-2 mRNA (B / iLMP) exhibits significantly improved BMP-2 translation efficiency and osteogenic function. Therefore, the ionizable lipid / microsphere complex system based on VE constructed in the present invention improves the global translation level of cells by reducing intracellular ERS, and can optimize the application of mRNA therapy in the field of tissue regeneration.

[0017] The above method provided by the present invention, first, synthesizes a dimer with an ionizable tertiary amine structure through an esterification reaction. The novel ionizable lipid (iVES) has physicochemical properties similar to those of the classic D-Lin-MC3-DMA ionizable lipid, including the closed acid dissociation constant (pKa) value. Then, using the traditional LNPs and hydrogel microsphere microfluidic system, mRNA LNPs (iLNP) and iLMPs based on iVES are prepared in sequence. Physical adsorption enables the LNPs to continuously release after in situ injection. In an in vitro ERS-positive cell model, iLMPs significantly reduce the activation of the PERK pathway and reduce eIF2α phosphorylation (p-eIF2α). The O-propargyl-puromycin (OPP) click chemistry method can significantly observe the improvement of mRNA translation efficiency in ERS-positive cells. Finally, an animal model of steroid-induced avascular necrosis of the femoral head (SANFH) with typical ERS-positive pathological characteristics is used to verify the transfection effect of iLMPs. iLMPs significantly alleviate SANFH and improve the translation of BMP-2 mRNA. Therefore, the present invention provides an injectable iLMPs, which is expected to become an enhanced mRNA therapeutic tool for tissue regeneration by synergistically improving cell mRNA translation.

[0018] The lipid nanoparticles developed in existing patents, as shown in the comparative examples of the present invention, cannot achieve the effect of more effectively reducing intracellular reactive oxygen species (ROS) in MC3T3-E1 cells and have a more significant osteogenic effect on MC3T3-E1 cells as compared with the present invention. Therefore, the therapeutic effect of the ionizable lipids mentioned in the existing methods under high ERS conditions cannot reach the level of the present invention.

[0019] Further, the ionizable amino groups in step (1) include primary amines, secondary amines or tertiary amines.

[0020] Further, the ionizable structure in step (1) includes, but is not limited to, 3-dimethylamino-1,2-propanediol.

[0021] Further, the esterification reaction in step (1) includes, but is not limited to, the esterification reaction catalyzed by N,N'-dicyclohexylcarbodiimide.

[0022] Further, the molar ratio of the vitamin E-based lipid, phospholipid, cholesterol and PEG component in step (2) is 40-60:10-20:30-40:1-5; wherein, the dosage of the vitamin E-based lipid can be replaced by traditional ionizable lipids by 0-50 mol%.

[0023] Further, the phospholipids in step (2) include, but are not limited to, lecithin, phosphatidylcholine, phosphatidylethanolamine, diacylglycerol phosphate;

[0024] Further, the PEG components in step (2) include, but are not limited to, DSPE-PEG, DSG-PEG.

[0025] Further, the concentration of the citrate buffer solution in step (2) is 10-30 mM and the pH is 3-5.

[0026] Further, the genes in step (2) include, but are not limited to, mRNA, siRNA or microRNA.

[0027] Further, the volume ratio of the organic phase to the aqueous phase in step (2) is 1:2-1:4.

[0028] Further, the hydrogel microspheres in step (3) are prepared by using a methacrylated polymer and a photoinitiator as the aqueous phase and an oily solvent as the oil phase through microfluidic devices and ultraviolet crosslinking; the methacrylated polymer includes, but is not limited to, methacrylated hyaluronic acid, methacrylated gelatin, methacrylated sodium alginate, methacrylated chitosan, and the oily solvent includes, but is not limited to, a mixture of paraffin oil and span 80.

[0029] A second object of the present invention is to provide a gene delivery ionizable lipid-based injectable microsphere prepared by the method described above.

[0030] A third object of the present invention is to provide the application of the gene delivery ionizable lipid-based injectable microsphere described above in the preparation of gene delivery drugs.

[0031] The beneficial effects of the present invention are as follows:

[0032] (1) The present invention synthesizes an ionizable lipid (iVES) for the first time, which can be used to replace traditional ionizable lipids. The novel iVES has physicochemical properties similar to those of the classic D-Lin-MC3-DMA ionizable lipids, including the closed acid dissociation constant (pKa) value; it can efficiently release VES drugs in a high ERS environment, promote the lysosomal escape of the entire LNP, and enable mRNA to be delivered to the cytoplasm faster;

[0033] (2) The LNP complex prepared based on iVES of the present invention can effectively relieve intracellular ERS and inhibit the activation of the protein translation-related pathway Bip-PERK-eIF2α. At the same time, the OPP method detected a significant increase in the expression of nascent proteins, clearly indicating an upregulation of the ribosomal translation level. Finally, the upregulation of the translation level led to a similar increase in the expression of exogenous mRNA;

[0034] (3) The B / iLMP prepared by the present invention has an excellent effect of promoting osteogenic differentiation in vitro. Compared with B / LMP, this acceleration of osteogenic differentiation is significantly enhanced. And the ribosomal translation level is significantly upregulated, accelerating the translation level of the drug BMP-2mRNA, which can be well used for osteogenic mRNA therapy under bone defect conditions. Description of the Drawings

[0035] Figure 1 For the synthesis of iVES and the preparation of LNPs (iLNPs) based on iVES; (A) The synthetic route for synthesizing iVES from VES; (B) The HRMS spectrum of iVES; (C) The 1 1H-NMR of iVES; (D) The HPLC elution curves of iVES and VES, and iVES under acidic conditions (iVES + H +HPLC elution curve of ( ); (E) Quantitative analysis of VES released from iVES; (F) pKa value of iLNPs, defined as the pH value at half-maximum fluorescence intensity; (G) Different formulations (A1 - A4) of iLNPs; (H) Schematic diagram for the preparation of five-component BMP2@iLNPs; Zeta potential (I), size (J), and encapsulation efficiency (EE%) (K) of optimized iLNPs prepared by a microfluidic device at high pump speed; (L) mRNA binding analysis of iLNPs by agarose gel electrophoresis; (M) Transmission electron microscopy image of iLNPs stained with uranyl acetate solution (scale bar, 200 nm); The experiments were performed in triplicate, and the data are presented as mean ± SD.

[0036] Figure 2 For the preparation and characterization of iLMP; (A) Microfluidic preparation of injectable HAMA microspheres; (B) Preparation of iLMP from HAMA microspheres adsorbed with iLNPs on the surface; (C) Optical microscope images of microspheres after freeze-drying and swelling; (D) Swelling rate of iLMP; (E, F) SEM images of iLMP; (G) LSCM images of Dil-stained LNPs loaded with iLMP and schematic diagram of iLMP; (H) Morphological changes of iLMP observed in the degradation test; (I) Degradation profile of iLMP; (J) Release curve of iLNPs in iLMP; The experiments were performed 3 times, and the data are presented as mean ± SD.

[0037] Figure 3 For the in vitro biocompatibility evaluation of iLMP; (A) Analysis of the live / dead situation of MC3T3-E1 after different treatments for 24 and 72 h by Calcein-AM / PI staining method, scale bar, 50 μm; (B) Representative pictures of different groups in the scratch experiment, showing the migration images of MC3T3-E1 after different treatments, scale bar, 50 μm; (C) Semi-quantitative analysis of the scratch experiment; (D) Cytotoxicity of B / iLMP detected by CCK8 method; *ns: not significant (p > 0.05).

[0038] Figure 4In vitro evaluation of ERS attenuation by iLMP; (A) Mechanism of ERS downregulating mRNA translation efficiency; (B) Western Blot analysis of the protein expression of Bip, p-PERK, and p-eIF2α in MC3T3-E1 cells after different treatments; (C-E) Semi-quantitative analysis of the expression of Bip, p-PERK, and p-eIF2α by Western Blot; (F) Schematic diagram of detecting ribosome translation level by O-propargyl-puromycin (OPP); (G) Fluorescence images of OPP detection in MC3T3-E1 cells after iLMP transfection and immunofluorescence staining of ROS generation in MC3T3-E1 cells, scale bar, 50 μm; (H, I) Fluorescence images of positive Mcherry results shown by flow cytometry in MC3T3-E1 cells after iLMP transfection; scale bar, 50 μm; **p < 0.01.

[0039] Figure 5 In vitro osteogenic evaluation of iLMP; (A) Western Blot analysis of the protein expression of BMP-2 and RUNX2 in MC3T3-E1 cells; (B, C) Semi-quantitative analysis of the expression of BMP-2 and RUNX2 by Western Blot; (D-F) Evaluation of osteogenic differentiation by ALP staining and semi-quantitative analysis, scale bar, 50 μm; (G-I) Evaluation of osteogenic differentiation by alizarin red staining and semi-analysis, scale bar, 50 μm; **p < 0.01, n.s: not significant (p > 0.05), ARS: alizarin red; ALP: alkaline phosphatase.

[0040] Figure 6 Establishment of animal model and pathological analysis; (A) Schematic diagram, administration method, and treatment time of establishing SANFH with methylprednisolone sodium (MPA); (B) Representative photos of femoral head by HE staining, scale bar (upper) is 1 mm, scale bar (lower) is 100 μm; (C) Representative photos of trichrome Masson staining in different groups, scale bar (upper) is 1 mm, scale bar (lower) is 100 μm; (D) Micro-CT images of the sagittal and transverse sections of the femoral head; (E) Different stages of femoral head necrosis by different treatment methods; (F-J) Bone mineral density (BMD), trabecular number (Tb.N), bone volume fraction (BV / TV), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th) of partial femoral head; *p < 0.05, **p < 0.01, n.s: not significant (p > 0.05). Figure 7 In vivo evaluation of ERS attenuation by B / iLMP; (A) Mechanism of ERS downregulating mRNA translation efficiency; (B-D) Immunohistochemical staining of ERS-related proteins (Bip, p-PERK, and p-eIF2α) in femoral head samples; (E-G) Semi-quantitative analysis of Bip, p-PERK, and p-eIF2α in immunohistochemical staining; *p < 0.05, **p < 0.01.Figure 8 In vivo bone regeneration evaluation of B / iLMP; (A) Schematic diagram of bone tissue regeneration; (B, C) Immunohistochemical staining of BMP-2 and RUNX2 in femoral head specimens; (D, E) Semi-quantitative analysis of immunohistochemical staining of BMP-2 and RUNX2; *p<0.05, **p<0.01, n.s: not significant (p>0.05).

[0041] Figure 9 ERS weakens iLMP to promote exogenous mRNA translation and promote in-situ tissue regeneration; (A) Chemical structure of iVES and the effect of alleviating ERS; (B) Schematic diagram of preparing iLNP by mixing with water and organic phase through microfluidics; (C, D) Microfluidic preparation of in-situ injectable iLMP; (E) ERS attenuation and translational improvement mechanism of iLMP.

[0042] Figure 10 For (A), compared with groups A, B, and C, the B / iLMP group was more effective in reducing intracellular oxygen free radicals (ROS) in MC3T3-E1 cells; (B): Compared with groups A, B, and C, alizarin red staining (ARS) showed that the osteogenic effect of MC3T3-E1 cells in the B / iLMP group was more significant.

[0043] Figure 11 1H nuclear magnetic resonance spectrum of iVES.

[0044] Figure 12 13C nuclear magnetic resonance spectrum of iVES.

[0045] Figure 13 FT-IR transmission spectrum of iVES.

[0046] Figure 14 For (A, B) Optical image and particle size distribution of iLMP, scale bar: 100 μm.

[0047] Figure 15 Counting the number of live cells through live / dead staining experiment, *n.s.: no significant difference.

[0048] Figure 16 Evaluating the protein synthesis efficiency in MC3T3-E1 cells using O-propargyl-puromycin, **p<0.01.

[0049] Figure 17 Detecting the intracellular red fluorescence intensity by fluorescence microscopy, **p<0.01.

[0050] Figure 18 Detecting the average fluorescence intensity of mCherry cells inside the cells by fluorescence microscopy.

[0051] Figure 19It is a three-dimensional reconstruction diagram of the femoral head.

[0052] Figure 20 It is a sagittal plane scan and X-ray perspective view of the femoral head. Specific implementation mode

[0053] In order to make the purpose, technical solution and advantages of the present invention clearer and more understandable, the present invention will be specifically described below in combination with embodiments. It should be noted that the following embodiments are only used to explain and illustrate the present invention and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still fall within the protection scope of the present invention.

[0054] Embodiment 1

[0055] I. Experimental materials and methods

[0056] 1. Preparation of iVES

[0057] Dissolve vitamin E succinate (VES, 2.65 g, 5.0 mmol) in 30 mL of dichloromethane, and slowly add N,N'-dicyclohexylcarbodiimide (DCC, 1.13 g, 5.5 mmol) to it for activation. Subsequently, add 3-dimethylamino-1,2-propanediol (0.24 g, 2.0 mmol) and 4-dimethylaminopyridine (DMAP, 0.25 g, 2.0 mmol), and stir the reaction at room temperature for 24 hours. Finally, obtain a colorless oily product through column chromatography separation, which is the ionizable VES, denoted as iVES.

[0058] 2. Preparation of LNPs

[0059] Prepare LNPs by the microfluidic method. The specific method is as follows: First, dissolve 50 μg of BMP-2 mRNA in 450 μL of citrate buffer (20 mM, pH = 4) to form an aqueous solution; then, weigh iVES, DSPC (distearylphosphatidylcholine), cholesterol, DSPE-PEG-RGB according to a molar ratio of 50:10:38.5:1.5:0.75 respectively, and dissolve them in 150 μL of absolute ethanol to form an ethanol solution. Use a microfluidic device to mix the ethanol phase and the aqueous phase at a volume ratio of 1:3, and transfer the obtained mixture to an ultracentrifuge tube containing 10 times the volume of PBS (pH = 7.4) for centrifugation to remove the ethanol phase, and prepare LNPs loaded with BMP-2 mRNA, denoted as LNPs.

[0060] 3. Characterization of LNPs

[0061] To observe and characterize LNPs, the particle size and Zeta potential of LNPs were first detected by a dynamic light scattering instrument (DLS) (Zetasizer Nano S, Malvern, UK). Briefly, the prepared LNPs were diluted with PBS respectively, and the diluted suspension was used to detect the Zeta potential of LNP with a Malvern sample cell. The prepared LNPs were taken and diluted with PBS, and the diluted LNP suspension was put into a quartz cuvette to observe the particle size distribution of LNPs. Next, the encapsulation efficiency of LNPs with different N / P ratios was measured by an RNA Quant quantification kit (DD3511, Nanjing Vazyme Biotech Co., Ltd, China). The morphology of LNPs was observed by transmission electron microscopy (TEM) (Hitachi HT 7800, Japan). Finally, the stability of LNPs in PBS at pH = 7.4 in an environment of 4°C was tested. The prepared LNPs were placed on a shaker at 4°C, and the particle size of LNPs was measured at 1 day, 3 days, 5 days, 7 days and 14 days respectively.

[0062] 4. Transfection efficiency of LNPs

[0063] To clarify the transfection effect of mRNA-LNPs on osteoblast precursor cells (MC3T3-E1), we selected mCherry mRNA as a model. After preparing mRNA-LNPs, they were co-incubated with MC3T3-E1, and the transfection effect was observed by a fluorescence microscope (Nikon ECLIPSE Ts2R, Japan). Briefly, 24 hours before transfection, MC3T3-E1 cells were seeded in a 24-well plate at a cell density of approximately 1×10 5 . After 24 hours of adhesion, when the cell confluence was approximately 70% - 80%, the cells were transfected. 1 μg of BMP-2 mRNA LNPs was added to the corresponding wells, and the transfection of MC3T3-E1 was observed under a fluorescence microscope after incubation for 24 h, 48 h and 72 h.

[0064] 5. Preparation of LNP microcomplexes (iLMP)

[0065] First, synthesize HAMA microspheres: React 5 g of HA (hyaluronic acid, molecular weight ≈ 74 kDa, Bloomage Freda Biopharmaceutical Co., Ltd., China) with 10.4 g of methacrylic anhydride, dialyze for 4 days and then lyophilize, and store at -80 °C. Then, use a microfluidic device to prepare hydrogel microspheres. Aqueous phase: Mix the hydrogel solution (4 wt% HAMA and 0.4 wt% photoinitiator); oil phase: Mix paraffin oil with 5 wt% Span 80; inject them into the inlets of the microfluidic device from syringes respectively, adjust the flow rate ratio between the aqueous phase and the oil phase. After the generated droplets are frozen at -30 °C, crosslink them under ultraviolet light to obtain HAMA microspheres. Then remove the excess paraffin oil, lyophilize at -80 °C and reserve. Place the lyophilized microspheres in the LNPs solution, and adsorb LNPs onto the microspheres through physical electrostatic / hydrogen bond adsorption to prepare LNPs microcomplexes, denoted as iLMP.

[0066] 6. Characterization of iLMP

[0067] Measure the particle size and degradation of iLMP by an inverted microscope (Nikon, Tokyo, Japan). Place 10 mg of iLMP microspheres in PBS with a pH of 7.4 containing 0.2 U / mL collagenase, and then place them on a shaker at 37 °C. Replace the PBS solution containing fresh hyaluronidase every two days. Observe the morphological changes of the hydrogel microspheres and measure the remaining weight of the microspheres at specific time points, and compare it with the initial weight to calculate the degradation percentage of the microspheres. Take 3 mg of lyophilized LNPs, add 1 mL of PBS, and measure the weights of iLMP and the Eppendorf tube. Measure once every 30 minutes for 3 hours in total, and drain the water before each weighing. In addition, observe the morphology of the lyophilized microspheres by a scanning electron microscope (ZEISS, Germany).

[0068] 7. Loading and release of LNPs in microspheres

[0069] To clarify the loading and release of LNPs in microspheres, confocal microscopy and a multi-functional microplate reader were used for measurement respectively. To facilitate the observation of LNPs, a trace amount of DiL dye was introduced during the preparation of LNPs, and its distribution was observed by confocal microscopy. First, LNPs were stained with DiL (Beyotime Co., Ltd., China), and then the freeze-dried HAMA microspheres were placed in the LNPs suspension. LNPs were adsorbed onto the freeze-dried HAMA microspheres by physical electrostatic / hydrogen bond adsorption. After the microspheres precipitated, the absorbance value of the LNPs that were not loaded onto the microspheres in the supernatant was measured, and the loading rate of LNPs on the microspheres was determined by calculation using the standard curve. The adsorption of LNPs was observed by confocal microscopy. Next, the LNP microcomplex was placed in PBS with pH = 7.4 and on a shaker at 37 °C. The absorbance value of LNPs in the supernatant at different time points was measured by a multi-functional microplate reader, and the release curve of LNPs was calculated using the standard curve.

[0070] 8. Biocompatibility evaluation

[0071] To clarify the biosafety of the LNP microcomplex, we used a Cell Counting Kit-8 (CCK-8) and a Calcein Acetoxymethyl Ester / Propidium Iodide (Calcein AM / PI) Cell Viability / Cytotoxicity Assay Kit to test the hydrogel microspheres. In this experiment, a Control group, an MS group, an LM group, a BLM group, and a BdLM were set up. MC3T3-E1 cells were seeded in 24-well plates and co-cultured with the corresponding extracts at 37 °C and 5% CO2. At 24 hours and 72 hours, the cells were incubated with Calcein AM / PI buffer (Beyotime Co., Ltd., China) for 15 min and then observed under a fluorescence microscope. MC3T3-E1 cells were seeded in 96-well plates and co-cultured with the corresponding extracts at 37 °C and 5% CO2. After 24 hours and 72 hours of culture, they were incubated in a medium containing 10% CCK-8 for 1 hour, and then the absorbance at 450 nm was measured by a microplate reader (Molecular Devices, Japan).

[0072] 9. Scratch assay

[0073] MC3T3-E1 cells in the logarithmic growth phase were seeded in 6-well plates (2×10 6(number / per well). When the growth rate of each group reached 70%, the control group, MS group, LM group, BLM group, and BdLM group were added respectively. After 20 h of treatment, a scratch was made on the cell monolayer with the tip of a 200 μL pipette. The wells were washed 3 times with PBS to remove floating cells, and 2 mL of serum-free medium was added to each group. Then, the plates were placed in an incubator at 37 °C and 5% CO2. After 24 h, the cell migration was observed under an inverted optical microscope, and the scratch width was measured by taking pictures with Image J software.

[0074] 10. Evaluation of the in vitro ROS scavenging, endoplasmic reticulum alleviation and protein transfection efficiency improvement of BELMP

[0075] The effect of BdLM on endoplasmic reticulum stress in MC3T3-E1 was analyzed by fluorescence method. MC3T3-E1 cells were seeded in confocal dishes respectively, and the Control group, Pos group, MS group, LM group, BLM group, and BdLM group were set up. Except for the Control group, all were induced with 400 μmol / L H2O2 for 24 h. After 24 h, the Control group and Pos group were not treated, and the remaining groups were cultured with the corresponding leachate at 37 °C. The induced cells were washed 3 times with PBS solution, and DCFH-DA reagent and O-propargyl-puromycin reagent were added respectively to detect the ROS level and protein translation efficiency in MC3T3-E1 cells.

[0076] 11. Western blot analysis

[0077] The expressions of Bip, p-PERK, p-eIF2α, and BMP-2 in MC3T3-E1 cells of each group were detected by Western blot analysis. After scraping the treated MC3T3-E1 cells, the cells were lysed on ice for 0.5 h with RIPA lysis buffer (Servicebio Co., Ltd., China) containing protease inhibitor, and the supernatant was collected by centrifugation. After separation by SDS-PAGE gel electrophoresis, the supernatant was transferred to a PVDF membrane (Millipore, Billerica, MA), then blocked with 5% BSA (Servicebio Co., Ltd., China) at room temperature for 1 h, and incubated with the primary antibody overnight at 4 °C. After incubation with the corresponding horseradish peroxidase-labeled secondary antibody (Protein Technology Inc) for 1 h, the blot was detected by chemiluminescence system, and the optical density value was measured using Image J software.

[0078] 12. Establishment of a rat model of steroid-induced osteonecrosis of the femoral head and in-situ bone regeneration treatment

[0079] All animal experiments were approved by the Animal Ethics Committee of Shanghai Shengchang Biotechnology Co., Ltd. (2023-09-RJYY-CYY-076). An animal model was established, and the modeling method was as follows. After 1 week of adaptive feeding of 8-week-old male SD rats, methylprednisolone sodium succinate (ChongQing Huapontpharm.Co., Ltd, China) was intramuscularly injected into the gluteus maximus at a dose of 40 mg / (kg·d) every 3 days for a total of 6 times. Rats in the blank control group were injected with an equal volume of normal saline. Then, after a one-week interval, microspheres loaded with LNP were injected in situ into the femoral head for treatment. At the 8th week after treatment, the rats were sacrificed under isoflurane anesthesia, and the femurs were taken and fixed with 4wt% paraformaldehyde solution. Experimental groups: Control group, Sham group, MS group, LM group, BLM group, and BdLM group.

[0080] 13. Micro-CT evaluation

[0081] The bone tissue parameters of the femoral head of rats were analyzed by X-ray and Micro-CT. Anteroposterior and lateral X-ray films were taken using the American Faxitron X-ray system (voltage of 59 kV, exposure time of 5 s). The femoral head was scanned and analyzed using Micro-CT (Skyscan, Belgium) with a resolution of 5 μm, a voltage of 50 kV, and a source current of 500 mA. After selecting the region of interest in the femoral head, bone mineral density (BMD), tissue volume / total tissue volume (BV / TV), trabecular bone thickness (Tb.Th), trabecular bone spacing (Tb.Sp), and trabecular bone number (Tb.N) were measured.

[0082] 14. H&E staining, Masson staining, immunofluorescence analysis

[0083] The femoral head specimens were fixed in 4wt% paraformaldehyde solution for 48 hours, and then decalcified in 10wt% ethylenediaminetetraacetic acid (EDTA) solution for 4 weeks. Then, they were dehydrated, paraffin-embedded, sectioned, H&E stained, and Masson stained in sequence. Before immunofluorescence staining, the bone sections were blocked with 5wt% BSA at room temperature for 0.5 h. Then, the tissue sections were incubated with primary antibodies against Bip, BMP-2, and RUNX2 overnight, and then incubated with secondary antibodies for 1 hour. The images were observed under a confocal microscope, and Image J software was used to analyze the immunofluorescence.

[0084] 15. Statistical analysis

[0085] Each experiment in this part was repeated at least 3 times, and data with a normal distribution were expressed as mean and standard deviation (SD). The t-test and Mann-Whitney test were used to compare the differences between the two groups. One-way or two-way ANOVA was used for comparisons among multiple groups, and p < 0.05 was considered statistically significant.

[0086] II. Experimental Results and Discussion

[0087] 1. Preparation and Characterization of iVES

[0088] The present invention first synthesized an ionizable VES (iVES), which can be used to replace traditional ionizable lipids. As shown in A below, under the catalysis of DCC and DMAP, vitamin E succinate undergoes an esterification reaction with 3-dimethylamino-1,2-propanediol to synthesize iVES. High-resolution mass spectrometry (HRMS), as shown in B below, observed molecular ion peaks at m / z 1144.8739 and 1166.8558, corresponding to the calculated values (1144.87, [M+H] Figure 1 ; 1166.86, [M+Na] Figure 1 ). In addition, the + H- and + C-NMR data are as shown in C below, as well as 1 and 13 shown. All chemical shift peaks can be attributed to the chemical structure of iVES. As shown in C below, the peak corresponding to the methyl group in the phospholipid head appears as a characteristic singlet at about 2.3 ppm. At the same time, the peaks corresponding to the methylene group on vitamin E and the polymethyl groups on the alkyl chain are clearly observed at about 1.3 ppm and 0.8 ppm, respectively. Fourier transform infrared spectroscopy (FT-IR) further confirmed the structure of iVES ( Figure 1 ). Figure 11 and Figure 12 ). The release characteristics of VES from iVES in a weakly acidic medium were studied by high performance liquid chromatography. Here, iVES is not only an ionizable lipid but also a prodrug of VES. The cleavable ester bond endows it with the ability to activate the drug under mild acidic conditions, thus promoting the lysosomal escape process. As shown in D below, the retention time values of iVES and VES on the HPLC elution curve are 3.7 min and 9.0 min, respectively (the blue and red in the curve are the peaks). Under weakly acidic conditions with a pH value of 5.0, an obvious VES peak appears in the elution curve of iVES (green curve). The appearance of the new VES peak means the cleavage of the ester bond to release the iVES prodrug. Further quantitative analysis was carried out to record its VES release behavior under acidic conditions. As shown in E below, VES is released from iVES at an almost constant rate, and the release rate is about 60% after 30 min, and then the release rate slows down. In addition, the pKa value, which is crucial for its ionization characteristics, was measured by fluorescence labeling method. The fluorescence curve is as shown in Figure 1 below. The Figure 13 )

[0089] The release characteristics of VES from iVES in a weakly acidic medium were studied by high performance liquid chromatography. Here, iVES is not only an ionizable lipid but also a prodrug of VES. The cleavable ester bond endows it with the ability to activate the drug under mild acidic conditions, thus promoting the lysosomal escape process. As shown in D below, the retention time values of iVES and VES on the HPLC elution curve are 3.7 min and 9.0 min, respectively (the blue and red in the curve are the peaks). Under weakly acidic conditions with a pH value of 5.0, an obvious VES peak appears in the elution curve of iVES (green curve). The appearance of the new VES peak means the cleavage of the ester bond to release the iVES prodrug. Further quantitative analysis was carried out to record its VES release behavior under acidic conditions. As shown in E below, VES is released from iVES at an almost constant rate, and the release rate is about 60% after 30 min, and then the release rate slows down. In addition, the pKa value, which is crucial for its ionization characteristics, was measured by fluorescence labeling method. The fluorescence curve is as shown in Figure 1 below. As shown in D below, the retention time values of iVES and VES on the HPLC elution curve are 3.7 min and 9.0 min, respectively (the blue and red in the curve are the peaks). Under weakly acidic conditions with a pH value of 5.0, an obvious VES peak appears in the elution curve of iVES (green curve). The appearance of the new VES peak means the cleavage of the ester bond to release the iVES prodrug. Further quantitative analysis was carried out to record its VES release behavior under acidic conditions. As shown in E below, VES is released from iVES at an almost constant rate, and the release rate is about 60% after 30 min, and then the release rate slows down. In addition, the pKa value, which is crucial for its ionization characteristics, was measured by fluorescence labeling method. The fluorescence curve is as shown in Figure 1 below. As shown in E below, VES is released from iVES at an almost constant rate, and the release rate is about 60% after 30 min, and then the release rate slows down. In addition, the pKa value, which is crucial for its ionization characteristics, was measured by fluorescence labeling method. The fluorescence curve is as shown in Figure 1As shown in Figure F, the measured pKa value of iVES is approximately 6.36, which is very close to that of commercially available ionizable lipids such as DLin-MC3-DMA. This provides strong evidence for iVES as a potential alternative to traditional ionizable lipids.

[0090] The above data indicate that most covalently conjugated VESs can be degraded and released within a short period of time. Therefore, it can be inferred that in the lysosomal environment, iVES not only undergoes significant protonation but also accelerates degradation. This not only promotes the release of VES drugs but also promotes the lysosomal escape of the entire LNP, enabling faster delivery of mRNA into the cytoplasm.

[0091] 2. Preparation and Characterization of iLNPs

[0092] Since the structure and pKa value of iVES are similar to those of traditional ionizable lipids, the present invention attempts to directly replace traditional lipids with it to prepare LNPs. Based on the classic four-component formulation, four different formulation systems based on iVES (Formulations A1 - A4, as Figure 1 shown in Figure G) were tested. Since iVES is a novel ionizable lipid, we focused on adjusting the ratio of iVES to DSPC to achieve a balance between LNP formation and mRNA encapsulation efficiency. The iLNPs in the present invention were prepared using a microfluidic device, and the schematic diagram is as Figure 1 shown in Figure H. Briefly, BMP-2 mRNA was dissolved in a citrate buffer (pH = 4) to form an aqueous phase, and iVES, DSPC, cholesterol, and DSPE-PEG-RGB were dissolved in anhydrous ethanol to form an ethanol phase (organic phase). To increase the probability of LNPs being recognized by integrin receptors and improve the targeting of LNPs to specific cells, the traditional PEG component was further modified to DSPE-PEG-RGD. This is different from traditional systemic delivery systems, where effective evasion of clearance by the mononuclear phagocyte system (MPS) is crucial. In systemic delivery, polyethylene glycolylation is used to achieve a balance between extended circulation and targeted delivery. However, in the in-situ delivery system, extended circulation is not a priority, and LNPs can tolerate partial phagocytosis by macrophages. At the same time, BMP-2 mRNA was used as a model gene for encapsulation during the LNP preparation process.

[0093] The Zeta potential and particle size of the LNPs were measured, and the results are as Figure 1 shown in Figures I and J. Overall, there were no significant differences in the Zeta potential and particle size of different formulations. The Zeta potential was approximately between -1 mV and -2 mV, and the particle size was approximately 200 nm. In addition, the encapsulation efficiencies of different formulations were also similar ( Figure 1 shown in Figure K). The results of agarose gel electrophoresis showed that the mRNA was almost completely encapsulated (Figure 1 in L). Under transmission electron microscopy (TEM) Figure 1 in M), the LNPs were clearly spherical, and the particle size information was consistent with the results obtained by DLS Figure 1 in J). Although the relatively low Zeta potential increased the risk of LNP aggregation, subsequent applications did not entirely rely on the LNP suspension. In the composite microsphere system, this Zeta potential was very suitable and was expected to improve the cell uptake efficiency. All these results indicated that the replacement of iVES had significant flexibility. All four formulations showed high mRNA encapsulation efficiency and successful formation of LNPs.

[0094] 3. Preparation and Characterization of iLMP

[0095] Hydrogel microspheres are ideal tools for loading LNPs, significantly improving the in-situ delivery efficiency. On the one hand, the microspheres improve the stability of LNPs (including storage stability and in-vivo stability); on the other hand, the porous microsphere system can quickly fill the defect sites, effectively reshape the physical environment of the extracellular matrix (ECM), and provide adhesion sites for cells.

[0096] Hyaluronic acid methacrylate (HAMA) is a classic microsphere raw material and has been developed for drug and cell loading in previous studies. First, the microspheres were prepared according to the existing literature methods, such as Figure 2 shown in A. HAMA was dissolved in the aqueous phase, and liquid paraffin was used as the oil phase. Microspheres with consistent particle size (150.2 ± 8.0 μm) and uniform dispersion were observed under an optical microscope, and the "oil-shear-water" method was adjusted to ensure the roundness of the microspheres Figure 14 ). For long-term storage and improved adsorption capacity, the microspheres were freeze-dried. Due to dehydration, the freeze-dried microspheres shrank into almost spherical shapes and appeared as black opaque solid particles under an optical microscope Figure 2 in C). It can be seen that the freeze-dried HAMA microspheres had a mesoporous network structure, enabling them to quickly absorb liquids and expand Figure 2 in D).

[0097] Based on these blank HAMA microspheres, lipid microgels (LMPs) were further prepared. The LMPs involving LNP (iLNP) based on iVES were called iLMPs. The preparation of iLMPs was directly achieved by the method of physical adsorption, such as Figure 2As shown in B. First, the HAMA microspheres were completely resuspended in deionized water. Then, BMP-2-loaded iLNPs (BMP-2@iLNPs) were added. Due to RGD modification, physical adsorption interactions such as hydrogen bonds and van der Waals forces occurred between the iLNPs and the HAMA microspheres, ensuring the effective loading of LNPs and other nanoparticles. The relatively low Zeta potential prevented strong electrostatic repulsion and maintained the stability of the system. The iLMP loaded with BMP2@iLNPs was named B / iLMP.

[0098] In addition, the morphology of the microsphere surface was observed by scanning electron microscopy (SEM). The microspheres presented regular, porous, near-spherical microparticles, probably due to shrinkage after dehydration during the freeze-drying process. After magnification, the mesoporous structure on the microsphere surface could be observed, which was beneficial to the adsorption and release of iLNPs. Through non-covalent interactions and physical mesh retardation, the nanoscale lipid nanoparticles could be evenly adsorbed into the mesopores of the microspheres. In addition, after labeling the iLNP with Dil dye, under a confocal microscope, the iLMP showed a uniform red coating, indicating the effective adsorption of iLNP on the microspheres ( Figure 2 as shown in G). Finally, the iLMP was incubated in PBS containing hyaluronidase at 37 °C, and the HAMA was gradually degraded due to the action of the enzyme. An inverted microscope showed that the degradation period of the HAMA hydrogel microspheres was 8 weeks, and the weight measurement results were in good agreement with the degradation observed under the inverted microscope ( Figure 2 as shown in H-J).

[0099] 3. In vitro cytotoxicity and cell migration inhibition of iLMP

[0100] Since the iLMP will be directly injected into the lesion site, the cytotoxicity of the iLMP is crucial. First, the live / dead staining method was used to evaluate the cytotoxicity of the iLMP. Taking the field of bone defect as an example, the pre-osteoblast cell line MC3T3-E1 was selected as the model. Briefly, the MC3T3-E1 cells were co-cultured with the extracts of microspheres (MS), LNP microspheres (LMP), BMP2@LMP (B / LMP), and BMP2@iLMP (B / iLMP) for 24 and 72 h. The live / dead staining results are as Figure 3 shown in A. Obviously, obvious cell proliferation could be observed in all groups, and the cell survival rate was very high ( Figure 15 ). At the same time, the scratch method was used to evaluate the cell migration inhibition effect of B / iLMP on the MC3T3-E1 cell line. After co-culturing B / iLMP with the microsphere extract for 24, 48, and 72 h, there was no significant difference in the migration area of the MC3T3-E1 cells ( Figure 3 as shown in B and C). Finally, the CCK8 method was used to further evaluate the proliferation of the co-cultured MC3T3-E1 cells. As Figure 3As shown in Fig. D, after 3 days of co-culture, the cells in each group proliferated nearly 3-fold compared to the first day, and there was no significant difference between groups (p > 0.05).

[0101] It can be clearly seen from the above results that neither the microspheres nor the complexes of microspheres and LNPs (including iLMP and B / iLMP) showed obvious cytotoxicity or cell proliferation inhibitory effects. This result is consistent with expectations. Among these materials, only iVES is a newly synthesized compound. Although positively charged molecules usually exhibit a certain degree of toxicity, iVES is biologically safe because its pKa is lower than the normal physiological level, which enables it to present a neutral charge under physiological conditions. The safety of other related ionizable lipids has also been strictly evaluated.

[0102] 4. In vitro evaluation of ERS attenuation of iLMP

[0103] Under certain pathological conditions, misfolded proteins in cells can induce the occurrence of ERS. In response to this phenomenon, cells spontaneously activate the unfolded protein response (UPR) to restore protein homeostasis. During the UPR, the overall downregulation of ribosomal mRNA translation is inevitable. Usually, cells encounter various environmental stresses, such as inflammation, ischemia, and hypoxic microenvironments, which disrupt intracellular protein homeostasis and lead to ERS. In the present invention, in order to simulate cells with active ERS under conditions such as inflammation, we selected hydrogen peroxide-treated MC3T3-E1 (300 μmol / L) as the cell model. According to previous studies, the activation of the Bip-PERK-eIF2α pathway in the UPR directly inhibits the translation level of downstream proteins, such as Figure 4 shown in Fig. A. Obviously, this is not desirable for mRNA-based therapeutic research because this ribosomal translation inhibition inevitably affects the efficacy of mRNA therapy.

[0104] To clarify the inhibitory effect of B / iLMP on ERS and its mechanism, we first detected the expression of key proteins in the Bip-PERK-eIF2α pathway by Western Blot( Figure 16 ). The results showed that the reduction of the three proteins (Bip, p-PERK, and p-eIF2α) in the B / iLMP group was the largest, very close to that of the control group( Figure 4 shown in Fig. B - D). Even compared with the B / LMP group, there was still a significant difference in the B / iLMP group (p < 0.05). This result preliminarily confirmed that iLMP has the potential to inhibit the activation of the Bip-PERK-eIF2α pathway. It should be noted that the reduction of p-eIF2α directly indicates the upregulation of ribosomal translation, which is considered the most closely related indicator.

[0105] O-propargyl-puromycin (OPP) assay is a new method for detecting ribosomal translation levels ( Figure 4 in F), which avoids the use of traditional radioactive labels, making it highly safe and efficient. OPP is a derivative of puromycin, and the end of puromycin contains an alkyne group that can enter the ribosomal acceptor site and integrate into the nascent polypeptide chain. After copper-catalyzed azide-alkyne cycloaddition reaction, it emits red fluorescence, allowing for the quantitative determination of protein synthesis in cells. Many previous experiments have confirmed that OPP analysis can accurately measure translation levels. Here, we used the OPP method to evaluate protein translation levels. As Figure 4 shown in G, the red fluorescence associated with OPP can directly indicate protein translation levels. In each experimental group, the red fluorescence in the B / iLMP group was significantly enhanced. Flow cytometry was further used to quantitatively analyze the red fluorescence in cells, as shown in Figure 17 . Meanwhile, the ROS level was significantly negatively correlated with OPP, as Figure 4 shown in G.

[0106] Generally speaking, upregulation of cellular translation levels leads to a simultaneous increase in exogenous mRNA expression. To verify this, we additionally introduced mCherry mRNA to detect the enhanced translation of this exogenous mRNA. Compared with LMP, iLMP significantly showed higher mCherry expression, as Figure 4 shown in H, I, and Figure 18 . This result is consistent with the previously observed upregulation of cellular translation levels.

[0107] The above results indicate that B / iLMP can effectively alleviate intracellular ERS and inhibit the activation of the protein translation-related pathway Bip-PERK-eIF2α. Meanwhile, the OPP assay detected a significant increase in the expression of nascent proteins, clearly indicating an upregulation of ribosomal translation levels. Finally, the upregulation of translation levels led to a similar increase in exogenous mRNA expression. This provides the most compelling experimental evidence for the enhanced therapeutic effect of iLMP on mRNA-based drugs.

[0108] 5. In vitro osteogenesis evaluation of iLMP

[0109] In the case of inflammatory bone defects and other tissue injuries, cells usually exhibit elevated intracellular ROS levels, ERS activation, and PERK pathway activation. More importantly, the phosphorylation level of eIF-2α is generally increased, which inevitably leads to the inhibition of ribosomal translation. Undoubtedly, the mRNA delivery of related cells will be affected. BMP-2 is the most common bone-inducing protein and is most widely used in bone repair. Here, we used iLMP as a nucleic acid delivery tool to load BMP-2 mRNA and attempted to evaluate its effectiveness in bone repair.

[0110] The MC3T3-E1 cell line was co-incubated with drugs such as B / iLMP. The transfection and translation efficiency of BMP-2 mRNA jointly determine its osteogenic process. Therefore, by detecting osteogenic markers, the significance of iLMP regulating ribosomal translation levels can be indirectly reflected.

[0111] First, Western Blot was used to detect the expression of two important osteogenic proteins, BMP-2 and RUNX2, in MC3T3-E1 cells after treatment with B / iLMP. As Figure 5 shown in A-C below, the expression of BMP-2 and RUNX2 in the B / iLMP group was significantly higher than that in other groups (p<0.05). Here, the expression of BMP-2 is partly attributed to the transfection of exogenous mRNA. After inducing osteogenic differentiation of MC3T3-E1 cells, the expression of endogenous BMP-2 is upregulated accordingly. Therefore, the observed BMP-2 may represent a cumulative effect. And RUNX2 is the result of this combined osteogenic differentiation induction. In summary, the high expression of BMP-2 and RUNX2 directly reflects the molecular biological process of cell osteogenic differentiation.

[0112] Meanwhile, the osteogenic differentiation level of MC3T3-E1 cells was further characterized by alkaline phosphatase (ALP) activity and alizarin red (ARS) staining. After 7 days of induction, compared with other groups such as the B / LMP group, ALP staining in the B / iLMP group increased significantly ( Figure 5 shown in D-F below). In addition, ARS staining also showed similar results, indicating an increase in intracellular calcium salt deposition after B / iLMP treatment ( Figure 5 shown in G-I below). Usually, ALP and ARS staining represent the early and late stages of osteogenic differentiation respectively. Therefore, it can be inferred from the above results that B / iLMP rapidly affects MC3T3-E1 cells in the early stage of osteogenesis. In addition, this differentiation induction persists and ultimately accelerates the generation of osteogenic mineralization.

[0113] These data indicate that B / iLMP has a role in promoting osteogenic differentiation in vitro. It is worth noting that compared with B / LMP, this acceleration of osteogenic differentiation is significantly enhanced. This is obviously related to the unique biological activity of iLMP. The previously confirmed upregulation of ribosomal translation levels significantly accelerates the translation of the gene drug BMP-2 mRNA. This regulation of translation level is crucial for osteogenic mRNA therapy under bone defect conditions.

[0114] 6. Establishment of animal model

[0115] In vivo osteogenic function evaluation using animal models will more intuitively demonstrate the therapeutic value of the novel in-situ injection of B / iLMP. SANFH is a typical inflammatory tissue injury disease characterized by high intracellular ERS. Long-term use of glucocorticoids is the main cause of SANFH induction because glucocorticoids can cause oxidative stress in bone metabolism-related cells (such as osteoblasts, mesenchymal stem cells, and osteocytes), ultimately leading to abnormal bone tissue homeostasis. Currently, there is a lack of mRNA treatment strategies for SANFH, which may also be related to the limitations of cellular translation levels under ERS. Therefore, SANFH is a very suitable animal model for evaluating the therapeutic potential of B / iLMP. All animal experiments were approved by the Animal Ethics Committee of Shanghai Ruixing Biotechnology Co., Ltd., approval number 2023-09-RJYY-CYY-076.

[0116] Before B / iLMP treatment, a rat SANFH model was established using methylprednisolone sodium succinate (MPA) ( Figure 6 as shown in A). The SANFH model was established 2 weeks after treatment, and two in-situ injections into the femoral head were performed at the 3rd and 4th weeks, with each injection of 30 μL (the concentration of the microcomplex was 0.1 mg / mL). All animals were sacrificed 8 weeks after MPA injection, and the femoral heads were removed for further analysis.

[0117] 7. In vivo pathological analysis

[0118] First, histopathological analysis of the femoral head tissue was performed using sectioning and micro-CT techniques. The femoral head specimens were fixed, decalcified, embedded, sectioned, and subjected to HE staining and Masson staining for pathological analysis. Through HE staining ( Figure 6 as shown in B), it was found that compared with the B / LMP group, B / iLMP treatment led to a significant increase in new bone tissue and femoral head trabeculae in the femoral head, a reduction in adipocyte infiltration, and a significant decrease in the degree of femoral head necrosis (p < 0.05). In addition, Masson staining ( Figure 6 as shown in C) showed that the bone tissue repair effect in the femoral head of the B / iLMP group was significant, and the number and area of the collagen fiber network increased significantly, indicating a better bone tissue repair effect in the B / iLMP group. At the same time, micro-CT analysis was applied to further evaluate the pathological structure and bone tissue parameters of the femoral head specimens. The three-dimensional reconstructed structure profile diagram is as shown in Figure 6 D. The results showed that 8 weeks after modeling, the femoral head trabeculae of the rats in the model group were sparse, fractured, missing, and the bone marrow cavity appeared. Compared with the model group, the B / iLMP group had increased new bone formation, increased trabecular bone structure, and dense bone tissue.

[0119] According to the established scoring criteria, we analyzed the stages of femoral head necrosis in each group. As shown in Figure 6As shown in E, the necrosis stage of the femoral head in the B / ilmp treatment group was significantly shortened. The results of the Micro-CT bone tissue parameter analysis are as Figure 6 shown in F-J. In the CT data, the bone mineral density (BMD) value, trabecular number (Tb.N), bone volume fraction (BV / TV), and trabecular separation (Tb.Sp) were significantly increased compared with the MS, LMP, and B / LMP groups, indicating obvious improvement in bone destruction and osteoporosis (p<0.05). All pathological examination results showed obvious improvement in bone pathological structure.

[0120] 8. In vivo osteogenic molecular biology evaluation

[0121] Further studies were conducted on whether the improvement of the pathological state of SANFH in bone tissue was attributed to the enhancement of ribosomal translation and the corresponding improvement in the transfection efficiency of BMP-2mRNA.

[0122] First, considering the importance of the ERS, UPR, and Bip-PERK-eIF2α pathways in mRNA translation, which directly determines the efficacy of exogenous BMP-2mRNA at the lesion site ( Figure 7 shown in A). In this process, high oxidative stress damages the endoplasmic reticulum function, leading to the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum and the imbalance of calcium homeostasis, ultimately resulting in the activation of the endoplasmic reticulum and UPR responses. From the in vitro data, iLMP containing the novel iVES showed excellent ERS alleviation ability and enhanced mRNA translation ability. We hypothesized that the same effect also occurred in vivo.

[0123] Based on the pathological sections of the femoral head, further immunofluorescence analysis was performed. Three key proteins (Bip, p-PERK, and p-eIF2α) were stained separately. Among them, phosphorylated proteins (p-PERK and p-eIF2α) are key markers of the activity of this UPR-related pathway. As Figure 7 shown in B, B / iLMP showed extremely low Bip expression (p<0.05), which was very close to that of the control group. At the same time, the Bip expression levels in other groups, including the B / LMP group, were significantly higher than those of the control group, but the difference was not statistically significant (p>0.05). As Figure 7 shown in B-D, the expression of p-PERK and p-eIF2α was similar. The expression levels of the characteristic proteins in the B / iLMP group were generally low (p<0.05). The results of the semi-quantitative analysis are as Figure 7 shown in E-G. These data indicate that B / iLMP can significantly down-regulate the activation of the Bip-PERK-eIF2α pathway. Obviously, this will be beneficial to the UPR of ERS-related femoral head lesions.

[0124] With the attenuation of ERS and UPR, ribosomal translation is restored, leading to increased expression of exogenous mRNA. B / iLMP treatment can effectively increase the expression level of BMP2 in transfected cells. BMP-2 is the most critical secreted osteogenic factor, which can significantly improve the osteogenic state of the entire microenvironment. BMP-2 enters receptor cells such as mesenchymal stem cells through its corresponding receptor, translocates into the nucleus, and induces phosphorylation of proteins such as Smad1 / 5 / 8. This process subsequently affects the expression of related DNA, ultimately enhancing the expression of various other osteogenic markers. Among these markers, Runx2 is one of the most important osteogenic indicators ( Figure 8 in A).

[0125] We further analyzed the femoral head section samples by immunofluorescence staining to detect the expression of BMP-2 and Runx2. As Figure 8 shown in B, compared with other experimental groups including the B / LMP group, the expression of BMP-2 protein in the femoral head of the B / iLMP group was significantly increased. In addition, we observed the expression of Runx2 protein, as Figure 8 shown in C. The semi-quantitative results are as Figure 8 shown in D-E. The three-dimensional reconstruction diagram of the femoral head is as Figure 19 shown, and the sagittal scan and X-ray perspective view of the femoral head are as Figure 20 shown. The results indicate that the B / iLMP group has the best osteogenic effect compared with other groups.

[0126] Since the expression of BMP-2 in B / iLMP is significantly higher than that in B / LMP, this indicates that although BMP-2 can be effectively expressed without the participation of iLMP, its expression level is still highly restricted. Only by using iLMP can the ribosomal translation inhibited by ERS be restored, thus releasing the true efficiency of exogenous mRNA expression. These data clearly demonstrate the key role of iVES and iLMP in mRNA expression under such pathological-like conditions. The expression of Runx2 is significantly positively correlated with BMP-2. The pathway by which BMP-2 affects Runx2 is relatively complex, and this effect partially overlaps with other osteogenic induction effects. Generally speaking, iLMP and iVES play a crucial role in promoting BMP-2 mRNA expression and osteogenic induction, especially in the complex environment of the SANFH pathological condition.

[0127] There is no doubt that the novel ERS-attenuating lipid gel microclusters will serve as a powerful in-situ delivery tool for therapeutic mRNA. By significantly improving the efficacy of mRNA drugs in pathological states where protein homeostasis is disrupted, iLMP has broad application prospects in the treatment of various chronic diseases.

[0128] III. Conclusions

[0129] The present invention synthesizes a new VES-derived ionizable lipid (iVES) for preparing lipid / microsphere complexes (iLMP) based on LNP, aiming to endow an injectable mRNA delivery system with ERS alleviating effect, and ultimately improve the ribosomal translation efficiency of exogenous mRNA. Figure 9 ) Here, iVES has physicochemical properties similar to those of commercial ionizable lipids. As a substitute, the LNP system prepared with iVES still has a comparable mRNA encapsulation efficiency. The subsequently prepared iLMP, as an in-situ delivery tool for mRNA, effectively solves the ribosomal translation limitation caused by the UPR effect induced by ERS. In in-vitro and in-vivo characterizations, both the ERS and ribosomal translation levels are significantly increased. In addition, after loading BMP-2 mRNA, the osteogenic induction of cells under pathological conditions is also significantly enhanced. In summary, iLMP, as an in-situ delivery tool, is of great significance in mRNA therapy for chronic diseases.

[0130] Comparative Example 1

[0131] The iLMP obtained in Example 1 was compared with the schemes reported in existing patents for the efficacy investigation of the model in the present invention, and the following control experiments were constructed:

[0132] Control Group A: An mRNA polymer-lipid hybrid delivery system constructed in the examples of Patent CN 118078773 A.

[0133] Control Group B: A transfection vector based on CD19CAR-mRNA lipid nanoparticles (LNP) constructed in the examples of Patent CN 116970652 A.

[0134] Control Group C: An LNP preparation prepared in the examples of Patent CN 117486738 A.

[0135] According to the method of Example 1, the effects of each control group and the LNP preparation prepared in Example 1 on reducing intracellular reactive oxygen species (ROS) in MC3T3-E1 cells and the osteogenic effect of MC3T3-E1 cells were examined, and the results are as Figure 10 shown. As can be seen from Figure 10 A, compared with Groups A, B, and C, the B / iLMP group prepared in Example 1 is more effective in reducing intracellular reactive oxygen species (ROS) in MC3T3-E1 cells; as can be seen from Figure 10 B, compared with Groups A, B, and C, alizarin red staining (ARS) shows that the osteogenic effect of MC3T3-E1 cells in the B / iLMP group prepared in Example 1 is more significant.

Claims

1. A method for preparing gene delivery ionizable lipid-based injectable microspheres, characterized in that, Comprising the following steps: (1) Synthesizing ionizable vitamin E-based lipids by esterification of vitamin E succinate with an ionizable structure; the ionizable structure includes an ionizable amino group, guanidine group, and heterocyclic group; (2) Mixing a composition of traditional ionizable lipids replaced or partially replaced by the ionizable vitamin E-based lipids obtained in step (1) with phospholipids, cholesterol, and a PEG component, dissolving them in ethanol as the organic phase, using genes and citrate buffer as the aqueous phase, and preparing gene-loaded ionizable lipid nanoparticles by a microfluidic method; the traditional ionizable lipids include, but are not limited to: DLin-MC3-DMA, ALC-0315, or SM-102; (3) Preparing hydrogel microspheres by a microfluidic method, freeze-drying them, and placing them in the ionizable lipid nanoparticle solution obtained in step (2). After complete adsorption, the gene delivery ionizable lipid-based injectable microspheres are obtained.

2. The method according to claim 1, wherein The ionizable amino group in step (1) includes primary amine, secondary amine, or tertiary amine; preferably, the ionizable structure includes, but is not limited to, 3-dimethylamino-1,2-propanediol; more preferably, the esterification reaction includes, but is not limited to, an esterification reaction catalyzed by N,N'-dicyclohexylcarbodiimide.

3. The method according to claim 1 or 2, characterized in that, In step (2), the molar ratio of the vitamin E-based lipid, phospholipid, cholesterol, and PEG component is 40-60:10-20:30-40:1-5; wherein, the amount of the vitamin E-based lipid can be partially replaced by traditional ionizable lipids, and the replacement range is 0-50 mol%.

4. The method according to claim 1 or 2, characterized in that, The phospholipid in step (2) includes, but is not limited to, lecithin, phosphatidylcholine, phosphatidylethanolamine, diacylglycerol phosphate; preferably, the PEG component includes, but is not limited to, DSPE-PEG, DSG-PEG.

5. The method according to claim 1 or 2, characterized in that, In step (2), the concentration of the citrate buffer is 10-30 mM, and the pH is 3-5.

6. The method according to claim 1 or 2, characterized in that, The gene in step (2) includes, but is not limited to, mRNA, siRNA, or microRNA.

7. The method according to claim 1 or 2, characterized in that, In step (2), the volume ratio of the organic phase to the aqueous phase is 1:2-1:

4.

8. The method according to claim 1 or 2, characterized in that, In step (3), the hydrogel microspheres are prepared by using a methacrylated polymer and a photoinitiator as the aqueous phase, an oily solvent as the oil phase, and crosslinking through a microfluidic device and ultraviolet light; the methacrylated polymer includes, but is not limited to, methacrylated hyaluronic acid, methacrylated gelatin, methacrylated sodium alginate, methacrylated chitosan, and the oily solvent includes, but is not limited to, a mixture of paraffin oil and Span 80.

9. Gene delivery ionizable lipid-based injectable microspheres prepared by the method according to any one of claims 1-8.

10. Use of the gene delivery ionizable lipid-based injectable microspheres according to claim 9 in the preparation of gene delivery drugs.

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