构建载免疫调节肽/miR-26a复合物微球缓释体系的体内成骨

Construction and in vivo osteogenesis of microspheres loaded with immunomodulatory peptide/miR-26a complexes for slow release

背景:课题组前期研究证明,免疫调节肽DP7-C具有良好的免疫调节功能,并可高效转染递送miRNA进入细胞发挥调控功能。目的:制备并筛选不同尺寸的多孔聚乳酸-羟基乙酸共聚物微球,装载免疫调节肽DP7-C/miR-26a复合物,构建并优化缓释体系,验证其生物相容性及体内成骨能力。方法:(1)微球制备:以碳酸氢铵作为造孔剂,通过双乳化法制备多孔聚乳酸-羟基乙酸共聚物微球,通过调整转速制备3种不同尺寸的多孔微球,将微球分散于免疫调节肽DP7-C/miR-26a复合物溶液中,制备载药微球,通过微球物理性能表征、投入产出比、包封率及缓释性能,筛选出最佳载药多孔聚乳酸-羟基乙酸共聚物微球,用于后续实验。(2)体外细胞实验:将不同质量浓度的载药与未载药微球溶液分别与大鼠骨髓间充质干细胞共培养,采用CCK-8法检测微球的生物相容性。(3)动物体内实验:取9只成年SD大鼠,按照随机数字表法分为空白组、对照组、实验组,每组3只。3组均建立直径5 mm颅骨缺损模型,空白组不进行干预,对照组植入未载药微球,实验组植入载药微球,术后8周,分别进行主要脏器及颅骨缺损区病理形态、碱性磷酸酶免疫组化染色检测。结果与结论:(1)微球制备:通过相关检测结果得出,相较于转速500,1200 r/min,转速1000 r/min制备的载药微球具有均匀的尺寸、更高的产率及良好的缓释性能,可用于后续实验。(2)体外细胞实验:CCK-8检测结果显示,载药与未载药微球溶液对骨髓间充质干细胞的增殖无影响,无明显的细胞毒性。(3)动物体内实验:苏木精-伊红染色显示,各组大鼠内脏未发生与实验干预相关的病理学改变。苏木精-伊红及Masson染色显示,空白组缺损部位主要被纤维结缔组织增生占据,可见少量血管生成,未见明显新骨生成;对照组缺损部位可见少量新骨生成及不同程度纤维组织增生,可见新生毛细血管;实验组缺损区域有明显新骨生成及不同程度纤维组织增生,可见新生毛细血管。免疫组化染色显示,实验组缺损部位碱性磷酸酶表达高于对照组、空白组(P<0.05)。(4)结果显示,多孔聚乳酸-羟基乙酸共聚物微球/免疫调节肽miR-26a复合物缓释体系具有良好的生物相容性及体内成骨性能,可促进大鼠颅骨临界骨缺损的再生修复。

BACKGROUND:Our group has demonstrated that the immunomodulatory peptide DP7-C has good immunomodulatory functions and can efficiently transfect and deliver miRNAs into cells to exert regulatory functions.OBJECTIVE:To prepare and screen porous poly(lactic-co-glycolic acid)microspheres of different sizes loaded with immunomodulatory peptide miR-26a complexes,construct and optimize the sustained release system,and verify its biocompatibility and osteogenic ability in vivo.METHODS:(1)Microsphere preparation:Porous poly(lactic-co-glycolic acid)microspheres were prepared by double emulsification with NH4HCO3 as the poreforming agent.Three different sizes of porous poly(lactic-co-glycolic acid)microspheres were prepared by adjusting the rotational speed.The microspheres were dispersed in the solution of immunomodulatory peptide DP7-C/miR-26a complex to prepare drug-loaded microspheres.The physical properties of the microspheres were characterized.The best drug-loaded porous poly(lactic-co-glycolic acid)microspheres were selected by comparing productivity,encapsulation rate and release performance for further use.(2)In vitro cell experiment:Drug-loaded and drug-free microsphere solutions with different mass concentrations were co-cultured with rat bone marrow mesenchymal stem cells.CCK-8 assay was performed to verify biocompatibility.(3)Animal in vivo experiment:Nine adult Sprague-Dawley rats were randomly divided into blank group,control group and experimental group,with three rats in each group.Cranial defect models with a diameter of 5 mm were established in all three groups.The rats of the blank group were not implanted with the material.The rats of the control group were implanted with blank microspheres.The rats of the experimental group were implanted with microspheres loaded with DP7-C/miR-26a complex.At 8 weeks after operation,the pathological morphology and immunohistochemical staining for alkaline phosphatase of the main organs and skull defects were performed.RESULTS AND CONCLUSION:(1)Microsphere preparation:According to the relevant detection results,the drug-loaded microspheres prepared at 1000 r/min compared with 500 and 1200 r/min had uniform size,higher yield and better sustained release performance,which could be used in subsequent experiments.(2)In vitro cell experiment:CCK-8 assay results exhibited that drug-loaded and drug-free microsphere solutions had no effect on the proliferation of bone marrow mesenchymal stem cells and had no obvious cytotoxicity.(3)In vivo experiments:There were no pathological changes in the viscera on hematoxylineosin staining related to the treatment with the intervention.Hematoxylin-eosin staining and Masson staining demonstrated that in the blank group,the defect site was mainly filled by fibrous connective tissue,with a small amount of angiogenesis,but without obvious new bone formation.In the control group,a small amount of new bone formation,fibrous tissue hyperplasia and new capillaries could be visible at the defect site.In the experimental group,there was obvious new bone formation,different degrees of fibrous tissue hyperplasia and new capillaries in the defect area.Immunohistochemical results displayed that alkaline phosphatase was highly expressed in the experimental group compared to the blank group and the control group(P<0.05).(4)These findings have concluded that the porous poly(lactic-co-glycolic acid)/DP7-C/miR-26a composite system has good biocompatibility and in vivo osteogenic properties and can promote bone regeneration and repair of critical bone defects in the rat skull.