iMed.UL-Research Institute for Medicines and Pharmaceutical Sciences, Faculdade de Farmácia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
Eur J Pharm Sci. 2012 Mar 12;45(4):451-8. doi: 10.1016/j.ejps.2011.09.018. Epub 2011 Oct 1.
Chitosan is one of the most promising polymers for drug delivery through the mucosal routes because of its polycationic, biocompatible, and biodegradable nature, and particularly due to its mucoadhesive and permeation-enhancing properties. Bile salts are known to interact with lipid membranes, increasing their permeability. The addition of bile salts to chitosan matrices may improve the delivery characteristics of the system, making it suitable for mucosal administration of bioactive substances. In the present study we have developed chitosan nanoparticles using sodium deoxycholate as a counter ion and evaluated their potential as gene delivery carriers. Chitosan-sodium deoxycholate nanoparticles (CS/DS) obtained via a mild ionic gelation procedure using different weight ratios were used to encapsulate plasmid DNA (pDNA) expressing a "humanized" secreted Gaussia Luciferase as reporter gene (pGLuc, 5.7 kDa). Mean particle size, polydispersity index and zeta potential were evaluated in order to select the best formulation for further in vitro studies. The nanoparticles presented an average size of 153-403 nm and a positive zeta potential ranging from +33.0 to +56.9 mV, for nanoparticles produced with CS/DS ratios from 1:4 to 1:0.6 (w:w), respectively. The pDNA was efficiently encapsulated and AFM studies showed that pDNA-loaded nanoparticles presented a more irregular surface due to the interaction between cationic chitosan and negatively charged pDNA which results in a more compact structure when compared to empty nanoparticles. Transfection efficiency of CS/DS-pDNA nanoparticles into moderately (AGS) and well differentiated (N87) gastric adenocarcinoma cell lines was determined by measuring the expression of luciferase, while cell viability was assessed using the MTT reduction. The CS/DS nanoparticles containing encapsulated pDNA were able to transfect both AGS and N87 cell lines, being more effective with AGS cells, the less differentiated cell line. The highest enzymatic activity was achieved with 20% pDNA encapsulated and after 24 h of transfection time. Low cytotoxicity was observed for the CS/DS nanoparticles either with or without pDNA, suggesting this could be a new potential vehicle for mucosal delivery of pDNA.
壳聚糖是通过粘膜途径给药最有前途的聚合物之一，因为它具有聚阳离子、生物相容性和可生物降解性，特别是由于其粘膜粘附性和渗透增强特性。胆汁盐已知与脂质膜相互作用，增加其通透性。向壳聚糖基质中添加胆汁盐可以改善系统的传递特性，使其适合于生物活性物质的粘膜给药。在本研究中，我们使用脱氧胆酸钠作为抗衡离子开发了壳聚糖纳米粒子，并评估了它们作为基因传递载体的潜力。通过使用不同重量比的温和离子凝胶化程序获得的壳聚糖-脱氧胆酸钠纳米粒子（CS / DS）用于包封表达“人源化”分泌型 Gaussia 荧光素酶作为报告基因（pGLuc，5.7 kDa）的质粒 DNA（pDNA）。为了选择用于进一步体外研究的最佳配方，评估了平均粒径、多分散指数和 Zeta 电位。纳米粒子的平均粒径为 153-403nm，Zeta 电位为+33.0 至+56.9mV，对于 CS / DS 比例为 1：4 至 1：0.6（w / w）的纳米粒子分别为。pDNA 被有效地包封，AFM 研究表明，负载 pDNA 的纳米粒子由于阳离子壳聚糖与带负电荷的 pDNA 之间的相互作用，表面呈现更不规则的形态，与空纳米粒子相比，结构更紧凑。通过测量荧光素酶的表达来确定 CS / DS-pDNA 纳米粒子对中度（AGS）和高度分化（N87）胃腺癌细胞系的转染效率，同时使用 MTT 还原评估细胞活力。包含包封的 pDNA 的 CS / DS 纳米粒子能够转染 AGS 和 N87 细胞系，对于分化程度较低的 AGS 细胞系更有效。在转染 24 小时后，获得了最高的酶活性，包封了 20％的 pDNA。CS / DS 纳米粒子无论是含有 pDNA 还是不含 pDNA，细胞毒性均较低，这表明它可能是 pDNA 粘膜传递的新潜在载体。
