Abstract: 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 pore-forming 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 1 000 r/min compared with 500 and 1 200 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 hematoxylin-eosin 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. 

Key words: poly(lactic-co-glycolic acid), slow-release microsphere, immunomodulatory peptide, miR-26a, skull defect, bone regeneration repair