Borgheti-Cardoso Lívia Neves, Depieri Lívia Vieira, Kooijmans Sander A A, Diniz Henrique, Calzzani Ricardo Alexandre Junqueira, Vicentini Fabiana Testa Moura de Carvalho, van der Meel Roy, Fantini Márcia Carvalho de Abreu, Iyomasa Mamie Mizusaki, Schiffelers Raymond M, Bentley Maria Vitória Lopes Badra
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café, s/n, 14040-903 Ribeirão Preto, SP, Brazil.
Laboratory of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands.
Eur J Pharm Sci. 2015 Jul 10;74:103-17. doi: 10.1016/j.ejps.2015.04.017. Epub 2015 Apr 25.
The development of delivery systems able to complex and release siRNA into the cytosol is essential for therapeutic use of siRNA. Among the delivery systems, local delivery has advantages over systemic administration. In this study, we developed and characterized non-viral carriers to deliver siRNA locally, based on polyethylenimine (PEI) as gene carrier, and a self-assembling drug delivery system that forms a gel in situ. Liquid crystalline formulations composed of monoglycerides (MO), PEI, propylene glycol (PG) and 0.1M Tris buffer pH 6.5 were developed and characterized by polarized light microscopy, Small Angle X-ray Scattering (SAXS), for their ability to form inverted type liquid crystalline phases (LC2) in contact with excess water, water absorption capacity, ability to complex with siRNA and siRNA release. In addition, gel formation in vivo was determined by subcutaneous injection of the formulations in mice. In water excess, precursor fluid formulations rapidly transformed into a viscous liquid crystalline phase. The presence of PEI influences the liquid crystalline structure of the LC2 formed and was crucial for complexing siRNA. The siRNA was released from the crystalline phase complexed with PEI. The release rate was dependent on the rate of water uptake. The formulation containing MO/PEI/PG/Tris buffer at 7.85:0.65:76.5:15 (w/w/w/w) complexed with 10 μM of siRNA, characterized as a mixture of cubic phase (diamond-type) and inverted hexagonal phase (after contact with excess water), showed sustained release for 7 days in vitro. In mice, in situ gel formation occurred after subcutaneous injection of the formulations, and the gels were degraded in 30 days. Initially a mild inflammatory process occurred in the tissue surrounding the gel; but after 14 days the tissue appeared normal. Taken together, this work demonstrates the rational development of an in situ gelling formulation for local release of siRNA.
能够将小干扰RNA（siRNA）包裹并释放到细胞质中的递送系统的开发对于siRNA的治疗应用至关重要。在各种递送系统中，局部递送比全身给药具有优势。在本研究中，我们基于聚乙烯亚胺（PEI）作为基因载体，开发并表征了用于局部递送siRNA的非病毒载体，以及一种可原位形成凝胶的自组装药物递送系统。由甘油单酯（MO）、PEI、丙二醇（PG）和pH 6.5的0.1M Tris缓冲液组成的液晶制剂被开发出来，并通过偏光显微镜、小角X射线散射（SAXS）对其在与过量水接触时形成反相液晶相（LC2）的能力、吸水能力、与siRNA复合的能力以及siRNA释放情况进行了表征。此外，通过在小鼠皮下注射制剂来确定其在体内的凝胶形成情况。在水过量的情况下，前体液制剂迅速转变为粘性液晶相。PEI的存在影响了所形成的LC2的液晶结构，并且对于siRNA的复合至关重要。siRNA从与PEI复合的晶相中释放出来。释放速率取决于吸水速率。含有7.85:0.65:76.5:15（w/w/w/w）的MO/PEI/PG/Tris缓冲液并与10 μM siRNA复合的制剂，其特征为立方相（菱形型）和反相六方相的混合物（与过量水接触后），在体外显示出7天的持续释放。在小鼠中，皮下注射制剂后发生原位凝胶形成，并且凝胶在30天内降解。最初在凝胶周围的组织中发生轻度炎症过程；但14天后组织看起来正常。综上所述，这项工作证明了一种用于局部释放siRNA的原位凝胶制剂的合理开发。
