Department of Pharmaceutics, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
J Pharm Pharm Sci. 2010;13(3):320-35. doi: 10.18433/j3r30t.
Leishmaniasis is a major health problem in many tropical and sub-tropical countries and development of a safe and easily-available vaccine has high priority. Although several antigens potentially capable of inducing protective immunity have been studied, in the absence of pharmaceutical industry interest they have remained as fine publications only. Amongst them, Cathepsin L-like cysteine proteinases (CPs) have received considerable attention and type I and II CPs have been used in a heterologous prime-boost vaccination regime for experimental visceral leishmaniasis in dogs. Due to the promising results of the mentioned vaccination regime, we aimed to evaluate cationic solid lipid nanoparticles (cSLNs) for in vitro delivery of cpa, cpb and cpb(CTE) intended to be used as a cocktail DNA vaccine in our forthcoming studies.
cSLNs were formulated of cetyl palmitate, cholesterol, DOTAP and Tween 80 via melt emulsification method followed by high shear homogenization. Different formulations were prepared by anchoring pDNAs on the surface of cSLNs via charge interaction. The formulations were characterized according to their size and zeta potential as well as pDNA integrity and stability against DNase I treatment. Lipoplexes' cytotoxicity was investigated on COS-7 cells by MTT test. The effect of the DOTAP:pDNA ratio on protection ability and cytotoxicity was also studied. In vitro transfection efficiency was qualified by fluorescent microscopy and quantified using flow cytometry technique.
cSLN-pDNA complexes were formulated with suitable size and zeta potential. Efficiency/cytotoxicity ratio of cSLN-pDNAs formulations was comparable to linear PEI-25KD-pDNAs polyplexes while exhibiting significantly lower cytotoxicity.
Tested formulations were able to deliver immunogenic CP genes efficiently. This data proves the ability of this system as a promising DNA vaccine carrier for leishmaniasis to cover the main drawback of naked pDNA delivery that is rapid elimination from the circulation.
利什曼病是许多热带和亚热带国家的一个主要卫生问题，因此开发安全且易于获得的疫苗成为当务之急。尽管已经研究了几种具有潜在诱导保护免疫能力的抗原，但由于制药行业缺乏兴趣，它们仍然只是优秀的出版物。在这些抗原中，组织蛋白酶 L 样半胱氨酸蛋白酶（CPs）受到了相当多的关注，I 型和 II 型 CPs 已被用于犬实验性内脏利什曼病的异源初免-加强免疫接种方案中。由于所述疫苗接种方案的良好效果，我们旨在评估阳离子固体脂质纳米粒（cSLN）在体外递送至 cpa、cpb 和 cpb（CTE）中的应用，这些物质将用于我们即将进行的研究中作为鸡尾酒 DNA 疫苗。
通过熔融乳化法随后进行高剪切匀化制备 cSLN，其中包含十六烷醇棕榈酸酯、胆固醇、DOTAP 和 Tween 80。通过电荷相互作用将不同的 pDNA 固定在 cSLN 的表面上，从而制备不同的配方。根据其大小和 zeta 电位以及 pDNA 完整性和对 DNase I 处理的稳定性来对制剂进行表征。通过 MTT 试验研究脂质体对 COS-7 细胞的细胞毒性。还研究了 DOTAP:pDNA 比对保护能力和细胞毒性的影响。通过荧光显微镜和流式细胞术技术定性和定量研究体外转染效率。
cSLN-pDNA 复合物的粒径和 zeta 电位适宜。cSLN-pDNA 制剂的效率/细胞毒性比与线性 PEI-25KD-pDNAs 多聚物相当，同时表现出明显较低的细胞毒性。
所测试的制剂能够有效地递送电免疫原性 CP 基因。该数据证明了该系统作为利什曼病的有前途的 DNA 疫苗载体的能力，能够克服裸露 pDNA 传递的主要缺点，即从循环中迅速消除。
