通过冷冻干燥和喷雾干燥制备用于非病毒基因递送的干粉分散体。

Preparation of dry powder dispersions for non-viral gene delivery by freeze-drying and spray-drying.

作者信息

Seville P C, Kellaway I W, Birchall J C

机构信息

Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, UK.

出版信息

J Gene Med. 2002 Jul-Aug;4(4):428-37. doi: 10.1002/jgm.282.

DOI:10.1002/jgm.282

PMID:12124985

Abstract

BACKGROUND

Dry powder dispersion devices offer potential for delivering therapeutic macromolecules to the pulmonary epithelia. Previously, freeze-drying (lyophilisation) has been the accepted method for preparing dried formulations of proteins and non-viral gene vectors despite the respirability of such powders being inadequate without further processing. In this study we compare the utility of freeze-drying and spray-drying, a one-step process for producing dry and respirable powders, as methods for preparing non-viral respiratory gene delivery systems.

METHODS

Lipid:polycation:pDNA (LPD) vectors comprising 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), protamine sulphate and pEGFP-N1 in 3% lactose solution were either snap-frozen and lyophilised or spray-dried. Lyophilised powder was used as recovered or following coarse grinding. Structural integrity of dehydrated pDNA was assessed by agarose gel electrophoresis and powder particle size determined by laser diffraction. The apparent structure of the systems was visualised by scanning and transmission electron microscopy with the biological functionality quantified in vitro (A549 human lung epithelial cell line) by Green Fluorescent Protein (GFP) associated fluorescence.

RESULTS

Lyophilisation produced large, irregularly shaped particles prior to (mean diameter approximately 21 microm) and following (mean diameter approximately 18 microm) coarse grinding. Spray-drying produced uniformly shaped spherical particles (mean diameter approximately 4 microm). All dehydrated formulations mediated reporter gene expression in A549 cells with the spray-dried formulation generally proving superior even when compared with freshly prepared LPD complexes. Biological functionality of the LPD dry powders was not adversely affected following 3 months storage.

CONCLUSIONS

Spray-drying has utility for producing stable, efficient and potentially respirable non-viral dry powder systems for respiratory gene delivery.

摘要

背景

干粉分散装置为将治疗性大分子递送至肺上皮细胞提供了可能。此前，尽管未经进一步处理的此类粉末的可吸入性不足，但冷冻干燥一直是制备蛋白质和非病毒基因载体干燥制剂的公认方法。在本研究中，我们比较了冷冻干燥和喷雾干燥（一种制备干燥且可吸入粉末的一步法）作为制备非病毒呼吸道基因递送系统方法的实用性。

方法

将包含1,2 - 二油酰基 - 3 - 三甲基铵丙烷（DOTAP）、硫酸鱼精蛋白和pEGFP - N1的脂质：聚阳离子：pDNA（LPD）载体溶解于3%乳糖溶液中，要么速冻后进行冷冻干燥，要么进行喷雾干燥。冷冻干燥的粉末可直接使用，也可经过粗磨后使用。通过琼脂糖凝胶电泳评估脱水pDNA的结构完整性，通过激光衍射测定粉末颗粒大小。通过扫描电子显微镜和透射电子显微镜观察系统的表观结构，并通过绿色荧光蛋白（GFP）相关荧光在体外（A549人肺上皮细胞系）对生物功能进行定量。

结果

冷冻干燥在粗磨前产生大且形状不规则的颗粒（平均直径约21微米），粗磨后颗粒平均直径约18微米。喷雾干燥产生形状均匀的球形颗粒（平均直径约4微米）。所有脱水制剂均介导A549细胞中的报告基因表达，即使与新鲜制备的LPD复合物相比，喷雾干燥制剂通常也表现更优。LPD干粉在储存3个月后其生物功能未受到不利影响。