Effects Of MSCs Loaded With Hypoxia-inducible Factor 1α Small Interfering RNA Wrapped In Nanoparticles On The Biological Characteristics And HIF-1α Expression Of RPE Cells In Hypoxia And High Glucose Microenvironment

Purposes: To explore effects of MSCs as carriers loaded with HIF-1α si RNA wrapped in Nanoparticles on RPE cells in hypoxia and hyperglycemia microenvironment.(1) Firstly, biological characteristics of MSCs were investigated under hypoxia and hyperglycemia microenvironment after being cultured and identified.(2) HIF-1α si RNA wrapped in PLGA NPs as a gene vector was constructed for MSCs. Additionally, the related indicators of HIF-1α si RNA were evaluated,(3) The silence efficacy of PLGA NPs on RPE cells was assessed.(4) Finally, effects of MSCs loaded with HIF-1α si RNA wrapped in Nanoparticles on the biological characteristics and HIF-1α expression of the RPE cells were investigated in hypoxia and hyperglycemia microenvironment.Methods:(1) Isolation of MSCs was followed by standard method of density gradient centrifugation. Oil red O staining and Alizarin Red S staining were used to identify MSCs after adipogenic and osteoblastic induction. Flow cytometry confirmed the markers ofMSCs. The experiment was divided into four groups by different concentration of glucose levels and oxygen: A. normal oxygen and glucose(21% O2 + 5.56 mmol/L glucose DMEM), B. normal oxygen and hyperglycemia(21% O2 + 30 mmol/L glucose DMEM), C. hypoxia and normal glucose(5% O2 + 5.56 mmol/L glucose DMEM), D. hypoxia and hyperglycemia(5% O2 + 30 mmol/L glucose DMEM). CCK-8 kit was used to evaluate MSCs proliferation at 12 h, 24 h and 48 h. Apoptosis and migration of MSCs were examined by flow cytometry and transwell culture system at 24 h.(2) Plasmid and lentivirus targeting the human HIF-1α gene were designed and constructed by Genechem company. PLGA loaded with HIF-1α si RNA was produced through the water-in-oil-in-water(W/O/W) multiple emulsion technique. And we evaluated the average particle size, size distribution and zeta potential of the PLGA by laser light scattering. Transmission electron microscopy was used to observe the surface characteristics of PLGA and flow cytometry was performed to access the transfection efficacy of PLGA.(3) Cells were divided into five groups: A. negative control(with no treatment), B. Lentivirus group(using constructed lentivirus to transfect the cells), C. plasmid group(only using constructed plasmid to transfect the cells); D. empty PLGA group(using gamma-sterilized PLGA with no plasmid to transfect cells); E. PLGA loaded with si RNA group(using gamma-sterilized PLGA loaded with constructed plasmid to transfect cells); Real time PCR was used to examine the relative expression of HIF-1α m RNA level of RPE cells at the time point 1 d, 3 d and 7 d.(4) A transwell co-culture system of MSCs and RPE was constructed and cells under hypoxia and hyperglycemia microenvironment were divided into three groups: A. RPE cells(only); B. coculture of MSCs and RPE cells; C. coculture of cells loaded with PLGA. CCK-8 kit was used to evaluate the proliferation of MSCs at 12 h, 24 h and 48 h. Flow cytometry and transwell were performed to observe the apoptosis and migration of RPE respectively at 24 h. Real time PCR was used to access the relative expression level of HIF-1α m RNA in RPE cells at the time point 1 d, 3 d and 7 d.Results:(1) MSCs isolated from human bone were able to grow adhering to the wall andto differentiate into adipogenic and osteogenic cells. Cells surface phenotype of MSCs was positive for CD29 but negative for CD34 and CD45. In hypoxia and hyperglycemia group, both proliferation and migration of cells were increased compared with normoxia and normal glucose group, while there were no difference in apoptosis rate between the two groups(P>0.05).(2) Average size of the PLGA NPs which were narrow distributed was 314.1nm and its zeta potential was-0.36 m V measured by laser light scattering. PLGA NPs had spherical shape and smooth surface as observed by transmission electron microscopy. The encapsulation efficiency of PLGA NPs was(67.3±5.2)% measured by flow cytometry.(3) Inhibitory efficacy of PLGA on the HIF-1α m RNA expression in RPE cells were prolonged(7 d) compared with lentivirus group(< 3 d, P < 0.05). There were no differences between other groups.(4) In a coculture system, the abilities of proliferation, apoptosis and migration between si RNA group and other groups had no differences(P > 0.05). At the point of 1 d, 3 d and 7 d, MSCs loaded with PLGA nano controlled-release system as vectors could effectively inhibit the expression of HIF-1αm RNA in RPE cells for a prolonged period(P < 0.05). Conclusions: Application of MSCs with HIF-1α si RNA wrapped in PLGA NPs as cell vectors significantly reduced the expression of HIF-1α m RNA in RPE cells in hypoxia and hyperglycemia microenvironment. This strategy may be a new treatment for CNV.