Bielski Elizabeth, Zhong Qian, Mirza Hamad, Brown Matthew, Molla Ashura, Carvajal Teresa, da Rocha Sandro R P
Department of Chemical Engineering and Materials Science, Wayne State University, Detroit, MI, 48202, USA; Department of Pharmaceutics, School of Pharmacy & Department of Chemical and Life Science Engineering, School of Engineering, Virginia Commonwealth University, Richmond, VA, 23284, USA.
Department of Chemical Engineering and Materials Science, Wayne State University, Detroit, MI, 48202, USA.
Int J Pharm. 2017 Jul 15;527(1-2):171-183. doi: 10.1016/j.ijpharm.2017.05.046. Epub 2017 May 23.
The regulation of genes utilizing the RNA interference (RNAi) mechanism via the delivery of synthetic siRNA has great potential in the treatment of a variety of lung diseases. However, the delivery of siRNA to the lungs is challenging due to the poor bioavailability of siRNA when delivered intraveneously, and difficulty in formulating and maintaining the activity of free siRNA when delivered directly to the lungs using inhalation devices. The use of non-viral vectors such as cationic dendrimers can help enhance the stability of siRNA and its delivery to the cell cytosol. Therefore, in this work, we investigate the ability of a triphenylphosphonium (TPP) modified generation 4 poly(amidoamine) (PAMAM) dendrimer (G4NH-TPP) to enhance the in vitro transfection efficiency of siRNA in a model of the pulmonary epithelium and their aerosol formulations in pressurized metered dose inhalers (pMDIs) and dry powder inhalers (DPIs). Complexes of siRNA and G4NH-TPP were prepared with varying TPP densities and increasing N/P ratios. The complexation efficiency was modulated by the presence of the TPP on the dendrimer surface, allowing for a looser complexation compared to unmodified dendrimer as determined by gel electrophoresis and polyanion competition assay. An increase in TPP density and N/P ratio led to an increase in the in vitro gene knockdown of stably green fluorescent protein (eGFP) expressing lung alveolar epithelial (A549) cells. G4NH-12TPP dendriplexes (G4NH PAMAM dendrimers containing 12 TPP molecules on the surface complexed with siRNA) at N/P ratio 30 showed the highest in vitro gene knockdown efficiency. To assess the potential of TPP-dendriplexes for pulmonary use, we also developed micron particle technologies for both pMDIs and DPIs and determined their aerosol characteristics utilizing an Andersen Cascade Impactor (ACI). Mannitol microparticles encapsulating 12TPP-dendriplexes were shown to be effective in producing aerosols suitable for deep lung deposition for both pMDI formulations (fine particle fraction of 50-53%) and DPI formulations (fine particle fraction of 39%) with no impact on the in vitro gene knockdown efficiency of the siRNA. This work demonstrates the potential benefits of utilizing TPP-conjugated dendrimers in the formation of dendriplexes for siRNA delivery to the pulmonary epithelium and their aerosol formulation for local delivery to the lungs using portable inhalers.
通过递送合成小干扰RNA（siRNA）利用RNA干扰（RNAi）机制调控基因，在多种肺部疾病的治疗中具有巨大潜力。然而，将siRNA递送至肺部具有挑战性，因为静脉内递送时siRNA的生物利用度较差，且使用吸入装置直接递送至肺部时，游离siRNA的制剂和活性维持存在困难。使用阳离子树枝状聚合物等非病毒载体有助于提高siRNA的稳定性及其向细胞溶质的递送。因此，在本研究中，我们研究了三苯基鏻（TPP）修饰的第4代聚（酰胺胺）（PAMAM）树枝状聚合物（G4NH-TPP）在肺上皮模型中增强siRNA体外转染效率的能力，以及它们在压力定量吸入器（pMDIs）和干粉吸入器（DPIs）中的气雾剂制剂。制备了具有不同TPP密度和不断增加的N/P比的siRNA与G4NH-TPP的复合物。树枝状聚合物表面TPP的存在调节了复合效率，与未修饰的树枝状聚合物相比，通过凝胶电泳和聚阴离子竞争试验确定其复合更松散。TPP密度和N/P比的增加导致稳定表达绿色荧光蛋白（eGFP）的肺泡上皮（A549）细胞的体外基因敲低增加。N/P比为30时，G4NH-12TPP树枝状复合物（表面含有12个TPP分子的G4NH PAMAM树枝状聚合物与siRNA复合）显示出最高的体外基因敲低效率。为了评估TPP-树枝状复合物用于肺部应用的潜力，我们还开发了用于pMDIs和DPIs的微粒技术，并使用Andersen级联撞击器（ACI）测定了它们的气雾剂特性。结果表明，包封12TPP-树枝状复合物的甘露醇微粒对于pMDI制剂（细颗粒分数为50-53%）和DPI制剂（细颗粒分数为39%）均能有效产生适合于肺部深部沉积的气雾剂，且对siRNA的体外基因敲低效率没有影响。这项工作证明了利用TPP共轭树枝状聚合物形成树枝状复合物用于将siRNA递送至肺上皮及其气雾剂制剂以便使用便携式吸入器局部递送至肺部的潜在益处。
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