Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan.
J Control Release. 2010 Jun 1;144(2):221-6. doi: 10.1016/j.jconrel.2010.02.018. Epub 2010 Feb 22.
Spray-freeze drying (SFD) is an attractive technique to prepare highly porous dry powders for inhalation. However, there have been few reports of its application to dry powder inhalers (DPIs). Therefore, in this study, we prepared dry plasmid DNA (pDNA) powders with different molecular ratios of chitosan to pDNA (N/P ratios) by SFD. All the pDNA powders were spherical and highly porous, with particles approximately 20-40microm in geometric diameter. The morphology changed little with the alteration of the N/P ratio. On electrophoresis, a band of linear pDNA was detected in the preparation without chitosan, suggesting the destabilization of pDNA through SFD. However, the addition of chitosan protected pDNA from destabilization. Moreover, the pDNA powders were evaluated for pulmonary gene transfection efficiency using an in vivo dual imaging technique for gene DPIs developed previously. Maximum gene expression was observed at 9-12h following pulmonary administration of the powders into mice. The powder with the N/P ratio of 10 had the highest gene transfection efficiency. A higher affinity of chitosan for pDNA and a smaller (approximately 100nm) pDNA/chitosan complex (N/Pf10) were found at pH 6.5 (in lung) than at pH 7.4 (in physiological conditions), suggesting that the effective compaction of pDNA by chitosan at the N/P ratio of 10 at pH 6.5 contributes to the gene transfection efficiency in the lung. These results suggest inhalable dry pDNA powders with chitosan prepared by SFD to be a suitable formulation for pulmonary gene therapy.
喷雾冷冻干燥（SFD）是一种有吸引力的技术，可用于制备用于吸入的高多孔干燥粉末。然而，将其应用于干粉吸入器（DPIs）的报道很少。因此，在这项研究中，我们通过 SFD 用不同的壳聚糖与 DNA（N/P 比）摩尔比制备了干燥的质粒 DNA（pDNA）粉末。所有的 pDNA 粉末都是球形的，高度多孔，几何直径约为 20-40μm。形态变化很小，随着 N/P 比的改变。在电泳中，在没有壳聚糖的制剂中检测到线性 pDNA 带，表明 SFD 使 pDNA 不稳定。然而，壳聚糖的添加保护了 pDNA 免受不稳定的影响。此外，使用先前开发的用于基因 DPIs 的体内双成像技术评估了 pDNA 粉末的肺基因转染效率。在将粉末经肺部给药后 9-12 小时观察到最大的基因表达。N/P 比为 10 的粉末具有最高的基因转染效率。在 pH 6.5（肺部）下发现壳聚糖与 pDNA 的亲和力更高，并且 pDNA/壳聚糖复合物（N/Pf10）更小（约 100nm），而在 pH 7.4（生理条件下）下发现壳聚糖与 pDNA 的亲和力更高，并且 pDNA/壳聚糖复合物（N/Pf10）更小（约 100nm），这表明在 pH 6.5 下 N/P 比为 10 时壳聚糖对 pDNA 的有效压实有助于肺部的基因转染效率。这些结果表明，通过 SFD 制备的具有壳聚糖的可吸入干燥 pDNA 粉末是一种适合肺部基因治疗的制剂。
