Objective·To explore the effects of hydrogel microsphere(MP)loaded with the immunoregulatory factor interleukin-4(IL-4)on the regeneration and repair after stroke injury.Methods·IL-4-loaded nanoparticles were prepared using an emulsification method and subsequently incorporated into polyvinyl alcohol(PVA)hydrogel microspheres via an airflow technique to obtain IL-4-loaded PVA hydrogel microspheres(MP@IL-4).The morphology of MP@IL-4 was characterized by optical microscopy.BV2 microglia were cultured in vitro and treated with MP@IL-4.Cell viability and survival were detected by the CCK-8 assay and live/dead cell staining,respectively.Subsequently,BV2 microglia were further treated with lipopolysaccharide(LPS),and the expression levels of the pro-inflammatory marker inducible nitric oxide synthase(iNOS)and the anti-inflammatory marker arginase 1(Arg-1)were detected by immunofluorescence staining.Meanwhile,a mouse stroke model was constructed using the middle cerebral artery occlusion method,and the mice were randomly divided into the sham operation group(Sham group),the ischemic stroke(IS)group and the microsphere treatment group(IS-MP@IL-4 group).The neurobehavioral functions of mice in the three groups were detected by the modified Neurological Severity Score,elevated body swing test,and hanging wire test.The brain atrophy volume was detected by cresyl violet staining.Immunofluorescence staining was used to assess the expression levels of the pro-inflammatory factor CD86 and the anti-inflammatory factor CD206 in microglia.Results·Optical microscopy confirmed the successful fabrication of MP@IL-4 with a particle size of 200 μm,which did not affect the cell viability and survival of BV2 microglia.Both in vitro and in vivo experiments demonstrated that MP@IL-4 downregulated the expression of pro-inflammatory markers and upregulated the expression of anti-inflammatory markers in microglia.Moreover,compared with the IS group,the neurobehavioral function of mice in the IS-MP@IL-4 group was significantly improved(P<0.05),and the brain atrophy volume was reduced(P<0.001).Conclusion·MP@IL-4 can exert a therapeutic effect on post-stroke neurofunctional injury by improving the immune microenvironment.

Objective·To explore the effects of hydrogel microsphere(MP)loaded with the immunoregulatory factor interleukin-4(IL-4)on the regeneration and repair after stroke injury.Methods·IL-4-loaded nanoparticles were prepared using an emulsification method and subsequently incorporated into polyvinyl alcohol(PVA)hydrogel microspheres via an airflow technique to obtain IL-4-loaded PVA hydrogel microspheres(MP@IL-4).The morphology of MP@IL-4 was characterized by optical microscopy.BV2 microglia were cultured in vitro and treated with MP@IL-4.Cell viability and survival were detected by the CCK-8 assay and live/dead cell staining,respectively.Subsequently,BV2 microglia were further treated with lipopolysaccharide(LPS),and the expression levels of the pro-inflammatory marker inducible nitric oxide synthase(iNOS)and the anti-inflammatory marker arginase 1(Arg-1)were detected by immunofluorescence staining.Meanwhile,a mouse stroke model was constructed using the middle cerebral artery occlusion method,and the mice were randomly divided into the sham operation group(Sham group),the ischemic stroke(IS)group and the microsphere treatment group(IS-MP@IL-4 group).The neurobehavioral functions of mice in the three groups were detected by the modified Neurological Severity Score,elevated body swing test,and hanging wire test.The brain atrophy volume was detected by cresyl violet staining.Immunofluorescence staining was used to assess the expression levels of the pro-inflammatory factor CD86 and the anti-inflammatory factor CD206 in microglia.Results·Optical microscopy confirmed the successful fabrication of MP@IL-4 with a particle size of 200 μm,which did not affect the cell viability and survival of BV2 microglia.Both in vitro and in vivo experiments demonstrated that MP@IL-4 downregulated the expression of pro-inflammatory markers and upregulated the expression of anti-inflammatory markers in microglia.Moreover,compared with the IS group,the neurobehavioral function of mice in the IS-MP@IL-4 group was significantly improved(P<0.05),and the brain atrophy volume was reduced(P<0.001).Conclusion·MP@IL-4 can exert a therapeutic effect on post-stroke neurofunctional injury by improving the immune microenvironment.

Central nervous system (CNS) injuries, including stroke, traumatic brain injury, and spinal cord injury, are essential causes of death and long-term disability and are difficult to cure, mainly due to the limited neuron regeneration and the glial scar formation. Herein, we apply extracellular vesicles (EVs) secreted by M2 microglia to improve the differentiation of neural stem cells (NSCs) at the injured site, and simultaneously modify them with the injured vascular targeting peptide (DA7R) and the stem cell recruiting factor (SDF-1) on their surface via copper-free click chemistry to recruit NSCs, inducing their neuronal differentiation, and serving as the nanocarriers at the injured site (Dual-EV). Results prove that the Dual-EV could target human umbilical vascular endothelial cells (HUVECs), recruit NSCs, and promote the neuronal differentiation of NSCs in vitro. Furthermore, 10 miRNAs are found to be upregulated in Dual-M2-EVs compared to Dual-M0-EVs via bioinformatic analysis, and further NSC differentiation experiment by flow cytometry reveals that among these miRNAs, miR30b-3p, miR-222-3p, miR-129-5p, and miR-155-5p may exert effect of inducing NSC to differentiate into neurons. In vivo experiments show that Dual-EV nanocarriers achieve improved accumulation in the ischemic area of stroke model mice, potentiate NSCs recruitment, and increase neurogenesis. This work provides new insights for the treatment of neuronal regeneration after CNS injuries as well as endogenous stem cells, and the click chemistry EV/peptide/chemokine and related nanocarriers for improving human health.