Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University , Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
ERATO Bio-nanotransporter Project, Japan Science and Technology Agency (JST), Kyoto University , Katsura, Nishikyo-ku, Kyoto 615-8530, Japan.
Biomacromolecules. 2017 Dec 11;18(12):3913-3923. doi: 10.1021/acs.biomac.7b00937. Epub 2017 Nov 13.
Nanometer-size gel particles, or nanogels, have potential for delivering therapeutic macromolecules. A cationic surface promotes cellular internalization of nanogels, but undesired electrostatic interactions, such as with blood components, cause instability and toxicities. Poly(ethylene glycol) coating has been used to shield charges, but this decreases delivery efficiency. Technical difficulties in synthesis and controlling molecular weights make it unfeasible to, instead, coat with biodegradable polymers. Our proposed solution is cationized nanogels enzymatically functionalized with branched polysaccharide chains, forming a shell to shield charges and increase stability. Biodegradation of the polysaccharides by an endogenous enzyme would then expose the cationic charges, allowing cellular internalization and cargo delivery. We tested this concept, preparing maltopentaose functionalized cholesteryl poly(l-lysine) nanogel and using tandem enzymatic polymerization with glycogen phosphorylase and glycogen branching enzyme, to add branched amylose moieties, forming a CbAmyPL nanogel. We characterized CbAmyPL nanogels and investigated their suitability as small interfering RNA (siRNA) carriers in murine renal carcinoma (Renca) cells. The nanogels had neutral ζ potential values that became positive after degradation by α-amylase. Foster resonance energy transfer demonstrated that the nanogels formed stable complexes with siRNA, even in the presence of bovine serum albumin and after α-amylase exposure. The nanogels, with or without α-amylase, were not cytotoxic. Complexes of CbAmyPL with siRNA against vascular endothelial growth factor (VEGF), when incubated alone with Renca cells decreased VEGF mRNA levels by only 20%. With α-amylase added, however, VEGF mRNA knockdown by the siRNA/nanogels complexes was 50%. Our findings strongly supported the hypothesis that enzyme-responsive nanogels are promising as a therapeutic siRNA delivery platform.
纳米尺寸的凝胶颗粒，或纳米凝胶，具有递送治疗性大分子的潜力。阳离子表面促进纳米凝胶的细胞内化，但与血液成分等不期望的静电相互作用会导致不稳定性和毒性。聚乙二醇（PEG）涂层已被用于屏蔽电荷，但这会降低递送效率。合成和控制分子量的技术困难使得用可生物降解的聚合物进行涂层变得不可行。我们提出的解决方案是通过酶功能化的支化多糖链对阳离子纳米凝胶进行修饰，形成一个外壳来屏蔽电荷并增加稳定性。然后，内源性酶降解多糖会暴露出阳离子电荷，从而允许细胞内化和货物递送。我们测试了这个概念，制备了麦芽五糖功能化的胆固醇聚（L-赖氨酸）纳米凝胶，并使用糖原磷酸化酶和糖原分支酶的串联酶聚合反应，添加支化的淀粉样物质，形成 CbAmyPL 纳米凝胶。我们对 CbAmyPL 纳米凝胶进行了表征，并研究了它们作为小干扰 RNA（siRNA）载体在小鼠肾癌细胞（Renca）中的适用性。纳米凝胶的 ζ 电位值为中性，在被α-淀粉酶降解后变为正。荧光共振能量转移（FRET）表明，纳米凝胶即使在存在牛血清白蛋白（BSA）和暴露于α-淀粉酶后，也能与 siRNA 形成稳定的复合物。纳米凝胶（有或没有α-淀粉酶）本身没有细胞毒性。与血管内皮生长因子（VEGF）的 siRNA 形成的 CbAmyPL 复合物单独孵育时，与 Renca 细胞孵育时，VEGF mRNA 水平仅降低 20%。然而，加入α-淀粉酶后，siRNA/纳米凝胶复合物对 VEGF mRNA 的敲低率为 50%。我们的研究结果有力地支持了这样一种假设，即酶响应纳米凝胶作为一种有前途的治疗性 siRNA 递送平台。
