BACKGROUND Spermatogonial 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 RESULTS 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 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. CONCLUSIONS This 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.

中文翻译：

---

PAMAM-cRGD介导有效的siRNA传递到精原干细胞。

背景技术精原干细胞（SSCs）是精子生产的基石，因此是永续生育的基石。在临床中，当男性生殖细胞已被性腺毒性疗法耗尽后，将患者自身的SSCs移植到睾丸中是恢复生殖能力的一种有前途的技术。自动移植转基因的SSC甚至具有治疗由基因突变引起的男性不育的潜力。但是，SSC对转染方法不具抵抗力。聚（酰胺基胺）（PAMAM）树状聚合物具有独特的三维结构，表面电荷和高密度的表面基团，适合配体连接，从而促进了靶标的传递。这项研究的目的是阐明PAMAM树状大分子是否可以有效地将短干扰RNA（siRNA）传递给SSC。方法和结果我们将环状精氨酸-甘氨酸-天冬氨酸（cRGD）肽引入了第五代PAMAM树状聚合物（G5），以生成PAMAM-cRGD树状聚合物（G5-cRGD）。通过傅立叶变换红外光谱仪（FTIR），透射电子显微镜（TEM）和细胞计数试剂盒8（CCK-8）分析来检测G5-cRGD的特征。共聚焦显微镜和流式细胞仪用于评估G5-cRGD将siRNA递送至SSC的效率。结果表明，包含siRNA的G5-cRGD可以自组装成纳米级的球形结构，具有高转染效率，优异的内体逃逸能力和低细胞毒性，优于商业转染试剂Lipofectamine®2000。此外，我们证明了G5 -cRGD有效递送siRNA并触发基因沉默。