Cavallaro Gennara, Campisi Monica, Licciardi Mariano, Ogris Manfred, Giammona Gaetano
Dipartimento di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo Via Archirafi 32, 90123, Palermo - Italy.
J Control Release. 2006 Oct 27;115(3):322-34. doi: 10.1016/j.jconrel.2006.07.027. Epub 2006 Aug 5.
Novel polyaspartamide non-viral carriers for gene therapy were synthesized by introducing, on the same polymer backbone, positively charged groups, for electrostatic interactions with DNA, and thiol groups for the formation of disulfide bridges between polymer chains. The introduction of thiols was aimed to have a vector with low redox potential sensitivity: disulfide crosslinking in fact, being stable in extracellular environment, allowed either to have stable complexes in plasma, that can protect DNA from metabolism, or to be reduced inside the cell, where the excess of glutathion in reduced form maintains a low redox potential. The consequent destabilization of the complex after disulfide cleavage can release DNA selectively inside the cells. Alpha,beta-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) was used as starting polymer being a highly water-soluble synthetic polymer, already proposed with success as therapeutic carrier by our group. In this study, PHEA was firstly functionalised with ethylendiamine, obtaining a well defined copolymer with pendant primary amine groups (PHEA-EDA), to which N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and 3-(carboxypropyl)trimethyl-ammonium chloride (CPTA) were linked in two subsequent steps, allowing the introduction of thiol and cationic groups respectively. Finally DTT treatment lead to the final PHEA-EDA-SH-CPTA thiopolycation, named PESC. The present work describes the synthesis and characterization of the thiopolycation PESC. 1H NMR spectroscopy detected the derivatization molar degrees in SPDP and CPTA; the formation of DNA complexes (thiopolyplexes), their stability in the presence of polyanions and the ability to release DNA under reductive conditions were studied by agarose gel electrophoresis. DNase II degradation study was carried out to detect the ability of thiopolyplex to stabilize DNA towards enzymatic metabolism. Thiopolyplexes were then characterized by Dynamic Light Scattering (DLS) and Zeta Potential analysis. Finally, in vitro toxicity profile (MTT) and gene transfer efficiency (Luciferase assay) were carried out to evaluate thiopolyplex biocompatibility, safety and efficacy to be used as gene delivery system.
通过在同一聚合物主链上引入带正电荷的基团用于与DNA进行静电相互作用，以及引入硫醇基团用于在聚合物链之间形成二硫键，合成了用于基因治疗的新型聚天冬酰胺非病毒载体。引入硫醇的目的是获得一种对氧化还原电位敏感性较低的载体：事实上，二硫键交联在细胞外环境中是稳定的，这使得复合物在血浆中能够稳定存在，从而保护DNA不被代谢，或者在细胞内被还原，因为细胞内过量的还原型谷胱甘肽维持着较低的氧化还原电位。二硫键断裂后复合物随之发生的不稳定能够使DNA选择性地在细胞内释放。α,β-聚（N-2-羟乙基）-D,L-天冬酰胺（PHEA）被用作起始聚合物，它是一种高度水溶性的合成聚合物，我们小组已经成功地将其作为治疗载体提出。在本研究中，首先用乙二胺对PHEA进行功能化，得到一种带有侧链伯胺基团的明确共聚物（PHEA-EDA），然后在随后的两个步骤中分别将N-琥珀酰亚胺基3-（2-吡啶二硫基）丙酸酯（SPDP）和3-（羧丙基）三甲基氯化铵（CPTA）连接到该共聚物上，从而分别引入硫醇基团和阳离子基团。最后，经过二硫苏糖醇（DTT）处理得到最终的PHEA-EDA-SH-CPTA硫代聚阳离子，命名为PESC。本工作描述了硫代聚阳离子PESC的合成与表征。1H核磁共振光谱检测了SPDP和CPTA中的衍生化摩尔度；通过琼脂糖凝胶电泳研究了DNA复合物（硫代多聚体）的形成、它们在聚阴离子存在下的稳定性以及在还原条件下释放DNA的能力。进行了DNase II降解研究以检测硫代多聚体使DNA对酶促代谢稳定的能力。然后通过动态光散射（DLS）和zeta电位分析对硫代多聚体进行表征。最后，进行了体外毒性分析（MTT）和基因转移效率分析（荧光素酶测定）以评估硫代多聚体作为基因递送系统的生物相容性、安全性和有效性。
