ROS-Response-Induced Zwitterionic Dendrimer for Gene Delivery
ROS-Response-Induced Zwitterionic Dendrimer for Gene Delivery
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用于基因传递的 ROS 响应诱导两性离子树枝状聚合物
DOI：
10.1021/acs.langmuir.8b03758
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发表时间：
2019
期刊：
影响因子：
3.9
通讯作者：
Yu Xifei
中科院分区：
文献类型：
--
作者：
Li Shengran;Chen Binggang;Qu Yangchun;Yan Xinxin;Wang Wenliang;Ma Xiaojing;Wang Bo;Liu Sanrong;Yu Xifei
基因治疗作为最有前途的治疗手段之一，以其超高的治疗效率在疾病治疗中发挥着越来越重要的作用。即使以聚阳离子、脂质体、树枝状聚合物、两性离子材料为代表的非病毒基因载体在基因复合、低免疫原性、生物相容性等方面取得了很大进展，但细胞内基因的低毒性释放仍然是制约基因治疗临床应用的瓶颈。我们设计并合成了一种活性氧（ROS）响应性树状聚合物-N-（4-硼苄基）-N，N-二乙基-2-（丙酰氧基）乙烷-1-胺（PAMAM-（B-DEAEP）16）作为基因载体，其在癌细胞中的ROS（H2 O2）升高时，其电位可以从正变为负。动态光散射结果表明，PAMAM-（B-DEAEP）16在80 mM H2 O2的PBS缓冲液中的zeta电位从+12.3 mV降至-5 mV。1H NMR结果表明，PAMAM-（B-DEAEP）16在酯键完全水解前，同时以正的季铵离子和负的羧酸离子的形式存在，在16 h内呈现两性离子的中间状态。凝胶阻滞实验表明，PAMAM-（B-DEAEP）16在N/P = 1以上可以凝聚DNA;然后，PAMAM-（B-DEAEP）16转移到两性离子，随着正电荷的减少和负电荷的增加，开始不断释放DNA，最终在80 mM H2 O2中形成带负电荷的聚（酰胺胺）-丙酸（PAMAM-PAc 16）。荧光标记的Cy-5 DNA表明PAMAM-（B-DEAEP）16在14 h内可完全进入细胞内。实验结果表明，该化合物具有较高的基因转染效率和较低的细胞毒性。这是首次在H2 O2的刺激下将带正电荷的树枝状聚合物转化为两性离子树枝状聚合物，并成功地应用于基因传递。与以往的报道不同，我们没有在高基因转染和低毒性之间寻求妥协，而是通过在带正电荷的树枝状聚合物中引入刺激敏感基团，使其能够根据微环境调节电荷性质，从而找到一种新的途径，使基因载体不仅具有更高的基因转染效率，而且具有更低的毒性。本研究不仅为基因载体材料的设计提供了一种良好的方法，而且为理解基因载体的过程提供了一个新的视角。
As one of the most promising therapeutic methods, gene therapy has been playing a more and more important role in treating disease due to its ultra-high therapy efficiency. Even if nonviral gene vectors represented by polycation, liposomal, dendrimers, and zwitterionic materials have made great progress in gene complexation, low immunogenicity, and biocompatibility, intracellular gene release with low toxicity is effectively still a bottleneck restricting the clinical application of gene therapy. We designed and synthesized a reactive oxygen species (ROS)-responsive dendrimer poly(amido amine)-N-(4-boronobenzyl)-N,N-diethyl-2-(propionyloxy)ethan-1-aminium (PAMAM-(B-DEAEP)16) as a gene vector whose potential can vary from positive to negative under the elevated ROS (H2O2) in cancerous cells. Dynamic light scattering results showed that the zeta potential of PAMAM-(B-DEAEP)16decreased from +12.3 to −5 mV under 80 mM H2O2in PBS buffer. The1H NMR results demonstrated that the intermediate status of PAMAM-(B-DEAEP)16was zwitterionic in ∼6 h because it consisted of the positive quaternary ammonium and negative carboxylic acid simultaneously before the ester bond was completely hydrolyzed. Gel retardation assay showed that PAMAM-(B-DEAEP)16can condense DNA at above N/P = 1; then, PAMAM-(B-DEAEP)16transfers to zwitterionic, which begins to continuously release DNA with the decrease in the positive charges and increase in the negative charges, and finally to negatively charged poly(amido amine)-propionic acid (PAMAM-PAc16) in the 80 mM H2O2. Fluorescence-labeled Cy-5 DNA indicated that PAMAM-(B-DEAEP)16can enter into the cell completely in ∼4 h. The results showed that this compound we designed exhibited higher gene transfection efficiency and lower cytotoxicity than commercial PEI. This is the first time that the positively charged dendrimer was transferred to zwitterionic dendrimer under the stimuli of H2O2and was successfully applied to gene delivery. Unlike all of the previous reports, we did not seek a compromise between the high gene transfection and low toxicity but find a new avenue to make the gene carrier not only have higher gene transfection efficiency but also exhibit lower toxicity by introducing stimuli-sensitive groups into the positively charged dendrimer to make it capable of adjusting the charge property according to the microenvironment. This study not only provides a good method to design materials for gene delivery but also opens a new perspective to understand the process of gene delivery.