Study On Reversing The Impaired Diabetic ADSC Theraputic Capacity And The Application Of PPy-NPs Based Photothermal Therapy On Artery Stenosis

Objectives:To clarify the deleterious effect of high glucose condition on the biological function of diabetic adipose-derived stem cells and to ivestigate effective approaches to improve their theraputic efficiency in treating ischemic limbs.To study the effectiveness of PPy nanoparticles-based photothermal therapy in the arterial stenotic diseases.Methods:Adipose-derived stem cells?ADSCs?and diabetic ADSCs?D-ADSCs?were isolated from the inguinal adipose tissues of C57BL/6 and BKS.Cg-m+/+Lepr^db,seperately.The comparement of the cell yield,proliferation,migration,multi-differentiation,and proangiogenic capacity between the D-ADSCs and the ADSCs were compared by CCK-8,flow cytometry,transwell,ELISA,RT-qPCR.GLO1 transfection of D-ADSCs was then achieved using lentiviral vectors and D-ADSC spheroids were obtained by a liquid overlay technique.The comparement of the intracellular ROS,proliferation,apoptosis,migration,multi-differentiation,and proangiogenic capacity between the GLO1 overpressing D-ADSC?G-D-ADSC?or the D-ADSC spheroids and parental D-ADSC were compared by CCK-8,flow cytometry,transwell,ELISA,RT-qPCR,and western blot analysis.Diatectic BALB/C nu-nu mice hindlimb ischemia models were then established and transplanted with the either G-D-ADSCs,D-ADSC spheroids,D-ADSCs,or PBS.The theraputic effect was evaluated by laser doppler blood perfusion,limb salvage rate,and microvessel densities.Biodegradable polypyrrole nanoparticles?PPy-NPs?were synthesized and characterized,including their size distribution,UV-vis-NIR absorbance,molar extinction coefficients,and photothermal properties.The effectiveness of PPy-NPs incubation followed by915 nm near-infrared?NIR?laser irradiation on ablating inflammatory macrophages in vitro were analyzed by TEM,CCK-8,and live/dead cell stainning.Further,we also detected the combination of local PPy-NPs injection with the 915 nm NIR laser irradiation on alleviating arterial inflammation by eliminating infiltrating macrophages and further ameliorating artery stenosis in ApoE-/-mouse model using IF and HE stainning.The in vivo biocompability was evaluated by HE stainning of major organs and blood test.Results:The D-ADSC number form per gram adipose tissue was higher in wild-type mice than in the BKS.Cg-m+/+Lepr^db.Restricted proliferation,migration,differentiation,and proangiogenic capacity were detected in the D-ADSCs compared to the ADSCs.While culturing under high glucose condition,G-D-ADSCs showed higher cell viabilities,lower apoptosis under either normoxia or hypoxia,and higher tubular formation and angiogenic genes expression compared to the D-ADSCs.Better in vivo theraputic efficiency was also observed in the G-D-ADSCs group compared to the D-ADSCs group,showing higher ischemic limb blood perfusion rate and limb salvage rate.Histological analysis revealed higher microvessel densities and expression of angiogenic factors,lower expression of HIF-1?in the ischemic muscle in the G-D-ADSCs group compared to the D-ADSCs group after treatment for 28 d.Notebly,significantly more G-D-ADSCs were integrated into the newly formed vessels than D-ADSCs.All these indicated that GLO1 overexpression could effectively improve the theraputic effeciency of the D-ADSCs.While culturing under high glucose condition,the D-ADSC spheroids showed higher reservation of extracellular matrix,including laminin and fibronectin,compared to D-ADSC monolayer.D-ADSC spheroids also showed higher apoptosis resistance to high glucose and hypoxia,with an higher expression of pAKT and lower expression of apoptotic proteins,including BAD,Caspase 9,and FOXO3,compared to the D-ADSC monolayer.Moreover,the conditioned medium from D-ADSC spheroids induced higher tubular formation and angiogenic proteins release compared to the one from D-ADSC monolayer.Better in vivo theraputic efficiency was also observed in the D-ADSC spheroid group compared to the monolayer group,showing higher ischemic limb blood perfusion rate and limb salvage rate.Histological analysis revealed higher microvessel densities and expression of angiogenic factors,including VEGF,HGF,and FGF2,in the ischemic muscle of the spheroids group compared to the monolayer group after treatment for 28 d.All these indicated that using D-ADSCs as spheroids could effectively elevate the biological performance and improve the theraputic effeciency of the D-ADSCs.Biodegradable polypyrrole nanoparticles?PPy-NPs?were synthesized and characterized,including their size distribution,UV-vis-NIR absorbance,molar extinction coefficients,and photothermal properties.We then verified that the PPy-NPs incubation followed by 915 nm near-infrared?NIR?laser irradiation could effectively ablate inflammatory macrophages in vitro,leading to significant cell apoptosis and cell death.Further,it was found that combination of local PPy-NPs injection with the 915 nm NIR laser irradiation could significantly alleviate arterial inflammation by eliminating infiltrating macrophages and further ameliorate artery stenosis in ApoE^-/-mouse model,without showing any obvious toxic side effects.Conclusions:Diabetes impairs the biological function of ADSCs.Overexpressing GLO1 or cultivating as spheroids could effectively protect the D-ADSCs from the deleterious effect of the high glucose and increasing their theraputic efficay in treating diabetic ischemic limbs.Application of PPy-NPs with 915 nm NIR laser irradiation could effectively ablate macrophages and ameliorate arterial stenosis progression.These studies bring new insights into the treatment of hindlimb ischemia with diabetes and restenosis after angioplasty.