Abstract
BackgroundSpermatogonial stem cells (SSCs) are the cornerstone of sperm production and thus perpetual male fertility. In clinics, transplantation of patient’s own SSCs into testes is a promising technique to restore fertility when male germ cells have been depleted by gonadotoxic therapies. Auto-transplantation of genetically modified SSCs even has the potential to treat male infertility caused by genetic mutations. However, SSCs are refractory to transfection approaches. 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. The goal of this study was to elucidate whether PAMAM dendrimers can efficiently deliver short interfering RNAs (siRNAs) to SSCs.Methods and resultsWe introduced cyclic arginine-glycine-aspartic acid (cRGD) peptides to the fifth generation of PAMAM dendrimers (G5) to generate PAMAM-cRGD dendrimers (G5-cRGD). The characterization of G5-cRGD was detected by Fourier transform infrared spectroscope (FTIR), transmission electron microscope (TEM), and the Cell Counting Kit-8 (CCK-8) assay. Confocal microscopy and flow cytometry were used to evaluate the delivery efficiency of siRNA by G5-cRGD to SSCs. The results showed that G5-cRGD encompassing siRNA could self-assemble into spherical structures with nanoscale size and possess high transfection efficiency, excellent endosomal escape ability, and low cytotoxicity, superior to a commercial transfection reagent Lipofectamine® 2000. Moreover, we demonstrated that G5-cRGD efficiently delivered siRNAs and triggered gene silencing.ConclusionsThis study thus provides a promising nanovector for siRNA delivery in SSCs, facilitating the future clinical application of SSC auto-transplantation with genetically modified cells with a hope to cure male infertility that is caused by genetic disorders.
Highlights
Spermatogonial stem cells (SSCs) are the cornerstone of sperm production and perpetual male fertility
This study provides a promising nanovector for short interfering RNAs (siRNAs) delivery in SSCs, facilitating the future clinical application of SSC auto-transplantation with genetically modified cells with a hope to cure male infertility that is caused by genetic disorders
Synthesis and characterization of The fifth generation of PAMAM dendrimers (G5)-cyclic arginine-glycine-aspartic acid (cRGD) and cRGDdendriplexes Based on the previous report [25], the cRGD was modified on the surface of the fifth generation of PAMAM dendrimers (G5)
Summary
Spermatogonial stem cells (SSCs) are the cornerstone of sperm production and perpetual male fertility. Transplantation of patient’s own SSCs into testes is a promising technique to restore fertility when male germ cells have been depleted by gonadotoxic therapies. Auto-transplantation of genetically modified SSCs even has the potential to treat male infertility caused by genetic mutations. The goal of this study was to elucidate whether PAMAM dendrimers can efficiently deliver short interfering RNAs (siRNAs) to SSCs. Spermatogonial stem cells (SSCs) are unique adult stem cells that transmit genetic information to subsequent generations. Auto-transplantation of genetically modified SSCs has the potential to treat male. Studies and potential applications of SSCs have been greatly hampered due to low transfection efficiency. In case of viral transduction, viral sequences may integrate into the recipient genome, raising safety concerns in clinics [3, 11]
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