Department of Pharmacy; Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-Universitaet Muenchen, Munich, Germany.
Eur J Pharm Biopharm. 2013 Oct;85(2):294-305. doi: 10.1016/j.ejpb.2013.05.010. Epub 2013 Jun 5.
Polyplexes based on precise oligoaminoamides exhibited promising results in non-viral siRNA delivery. However, one serious limitation is insufficient stability of polyplexes in liquid, which raises the demand for lyophilized, long-term stable formulations. Two different siRNA/oligoaminoamide polyplexes were prepared. Freeze-thaw experiments were performed, in order to test various formulations containing sucrose, trehalose, lactosucrose, and hydroxypropyl-β-cyclodextrin for their cryoprotective potential and to investigate the influence of the oligoaminoamide structure on particle stability. Selected formulations were lyophilized and tested for storage stability up to 6 months. Moreover, reconstitution of the lyophilisates in reduced volume as a technique to prepare higher concentration formulations was studied. Samples were analyzed for particle size, gene silencing, cytotoxicity, turbidity, subvisible particles, osmolarity, residual moisture content, glass transition temperature, and morphology. Depending on the oligoaminoamide, siRNA polyplexes maintained particle size and gene silencing efficiency in the absence or presence of low amounts (7%) of stabilizers after freeze-thawing, lyophilization, and reconstitution. Particle stability was highly dependent on the oligoaminoamide used, but independent of the presence of cysteines that form intra-particular disulfide bridges. In contrast to all other excipients, hydroxypropyl-β-cyclodextrin did not provide sufficient stability. For lyophilized 5%/10% sucrose and 7% lactosucrose formulations, long-term stability was demonstrated at 40 °C with retained particle size, retained gene silencing activity, unchanged turbidity values, low numbers of subvisible particles, low residual moisture level, and sufficiently high glass transition temperature. Hence, this work is a promising approach in order to provide long-term stable siRNA polyplex formulations that are ready to use after a simple reconstitution step.
基于精确寡聚氨基酸酰胺的超分子聚合物在非病毒 siRNA 递送上显示出了良好的效果。然而，一个严重的局限性是超分子聚合物在液体中的稳定性不足，这就需要制备冻干的、长期稳定的制剂。我们制备了两种不同的 siRNA/寡聚氨基酸酰胺超分子聚合物。进行了冻融实验，以测试含有蔗糖、海藻糖、乳蔗糖和羟丙基-β-环糊精的各种配方在冷冻保护方面的潜力，并研究寡聚氨基酸酰胺结构对颗粒稳定性的影响。选择的配方进行了冻干，并测试了长达 6 个月的储存稳定性。此外，还研究了减少体积复溶的方法，以制备更高浓度的配方。对样品进行了粒径、基因沉默、细胞毒性、浊度、亚可见颗粒、渗透压、残余水分含量、玻璃化转变温度和形态分析。根据寡聚氨基酸酰胺的不同，siRNA 超分子聚合物在冻融、冻干和复溶后，在没有或存在少量（7%）稳定剂的情况下，保持了粒径和基因沉默效率。颗粒稳定性高度依赖于所用的寡聚氨基酸酰胺，但与形成颗粒内二硫键的半胱氨酸的存在无关。与所有其他赋形剂不同，羟丙基-β-环糊精不能提供足够的稳定性。对于冻干的 5%/10%蔗糖和 7%乳蔗糖配方，在 40°C 下进行了长期稳定性测试，结果表明粒径、基因沉默活性、浊度值、亚可见颗粒数量、残余水分含量和玻璃化转变温度均保持不变。因此，这项工作为提供长期稳定的 siRNA 超分子聚合物制剂提供了一种有前景的方法，这些制剂在经过简单的复溶步骤后即可使用。
