| Spermatogonial stem cells（SSCs）,the primitive male germ cells able to strike a balance between self-renewal and differentiation,are the cornerstone of spermatogenesis and thus perpetual male fertility.SSCs have broad application prospects in the preservation of genetic resources,the treatment of male infertility and genetic diseases.However,studies and potential applications of SSCs have been greatly hampered due to low transfection efficiency.Previous reports have shown that SSCs are refractory to calcium phosphate and lipofection-mediated transfection.Poly（amidoamine）（PAMAM）dendrimers have the unique three-dimensional architecture,surface charge and high density of surface groups that are suitable for ligand attachment,thereby facilitating target delivery.C18-4 cells（a mouse SSC line）and primary SSC were selected as the model.We introduced cyclic arginine-glycine-aspartic acid（cRGD）peptides to the fifth generation of PAMAM dendrimers（G5）to generate PAMAM-cRGD dendrimers（G5-cRGD）.The goal of this study was to elucidate whether G5-cRGD can efficiently deliver siRNAs to SSCs.This study is to evaluate the physicochemical properties,cell toxicity,endosomal escape and gene silencing efficiency induced by G5-cRGD,facilitating the future research and clinical application.The results showed as follows:1.G5-cRGD are able to efficiently bind and load siRNAs.Firstly,the weak characteristic peak of sulfhydryl group（–SH）appeared at 2500 cm-1 indicates the successfully modification of cRGD on the surface of G5.The G5 nanoparticles were irregular in shapes.Upon addition of siRNA to G5 or G5-cRGD,these nano-siRNA complexes self-assembled into spherical structures with the average particle sizes 70 and 80nm,respectively.To determine the load efficiency of siRNA in the nano-vectors,the agarose gel retardation assay was performed.With the increase of N/P ratios,siRNA bands gradually decreased and completely disappeared when the N/P ratio was higher than 10 in either G5-siRNA or G5-cRGD-siRNA complexes,indicating that G5-cRGD are able to efficiently bind and load siRNAs.2.Cell viability in G5-cRGD group was over 90%with a 10:1 ratio.Cell viability in the control,a commercial reagent Lipo2000,was 60%.These results demonstrate that G5-cRGD lead to less cytotoxicity.3.G5-cRGD-siRNA complexes showed high transfection efficiency in C18-4 cells and were effective for siRNA to escape from endosomes.G5-siRNA and G5-cRGD-siRNA complexes showed high transfection efficiency in C18-4 cells examined by flow cytometry and confocal microscopy.To detect the endosomal escaping,we used FAM-labeled siRNA（green）and stained endosome/lysosomes with Lyso-Tracker Red.After 24 h,a small number of green spots separated from Lyso-tracker Red,indicating that siRNA began to escape from the endosomes.More green fluorescence was separated from the red fluorescence at 36 h.These results indicated that G5-cRGD-siRNA complexes were more effective for siRNA to escape from endosomes.4.Cells were treated with sodium azide（NaN3）and under low temperature condition,respectively.The results suggest that G5-cRGD-siRNA complexes internalization is chiefly energy-dependent,but a passive translocation mechanism is not completely excluded.5.The transfection efficiency in G5-cRGD was prominently improved in comparison with Lipo2000 in mouse and pig primary SSCs increased to 35%and 51%,respectively.Treatment with siRNAs against Cdk1 and Cdk2 using G5-cRGD as a vector could trigger potent gene（Cdk1 and Cdk2）silencing.The average number of colonies was observed remarkable reduction in G5-cRGD group.EdU assay showed that 31%of the cells were positive for EdU staining in G5-cRGD group,while 58%and 51%EdU+cells in the Mock and NC control group,respectively.These results demonstrate that G5-cRGD efficiently triggered gene silencing and significantly decreased SSC colony formation and EdU-positive cells in primary SSCs.In conclusion,these results demonstrated that an efficient delivery vector（G5-cRGD）for SSCs has been successfully established,which pave the way towards SSC biology and their potential applications to the clinics and animal production. |
