Key Laboratory for Animal Genetics, Breeding and Reproduction of Shaanxi Province, Key Laboratory for Animal Biotechnology, Ministry of Agriculture of China, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Stem Cell Res Ther. 2019 Dec 18;10(1):399. doi: 10.1186/s13287-019-1506-4.
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.
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.
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.
精原干细胞（SSCs）是精子发生的基石，因此是男性生育力的持久保障。在临床上，将患者自身的 SSCs 移植到睾丸中是一种很有前途的技术，可以在男性生殖细胞因性腺毒性疗法而耗竭时恢复生育能力。经过基因修饰的自体 SSCs 移植甚至有可能治疗由基因突变引起的男性不育症。然而，SSCs 对转染方法具有抗性。聚（酰胺-胺）（PAMAM）树枝状大分子具有独特的三维结构、表面电荷和高密度的表面基团，适合配体附着，从而促进了靶标的传递。本研究的目的是阐明 PAMAM 树枝状大分子是否可以有效地将短干扰 RNA（siRNA）递送到 SSCs。
我们将环状精氨酸-甘氨酸-天冬氨酸（cRGD）肽引入到第五代 PAMAM 树枝状大分子（G5）中，生成 PAMAM-cRGD 树枝状大分子（G5-cRGD）。通过傅里叶变换红外光谱仪（FTIR）、透射电子显微镜（TEM）和细胞计数试剂盒-8（CCK-8）检测 G5-cRGD 的特性。共聚焦显微镜和流式细胞术用于评估 G5-cRGD 向 SSCs 递送 siRNA 的效率。结果表明，包含 siRNA 的 G5-cRGD 可以自组装成具有纳米级尺寸的球形结构，具有高转染效率、出色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 可以有效地递送 siRNA 并触发基因沉默。
因此，本研究为 SSCs 中的 siRNA 递供提供了一种很有前途的纳米载体，为未来 SSC 自体移植与经过基因修饰的细胞的临床应用提供了便利，有望治愈由遗传疾病引起的男性不育症。
