Abstract
The local controlled release of siRNA is an attractive and rational strategy to enhance and extend the effectiveness of gene therapy. Since naked and unmodified siRNA has a limited cell uptake and knockdown efficiency, the complexation of siRNA with non-viral carriers is often necessary for the delivery of bioactive RNA. We evaluated the performance of three different non-viral siRNA carriers, including DOTAP lipoplexes (DL), chitosan polyplexes (CP), and solid lipid complexes (SLC). The physicochemical properties of the siRNA-nanocarriers were characterized by dynamic light scattering and gel electrophoresis. After in vitro characterization, the carrier with the most appropriate properties was found to be the DL suspension, which was subsequently loaded into a gellan gum hydrogel matrix and examined for its drug load, stability, and homogeneity. The hydrogels microstructure was investigated by rheology to assess the impact of the rheological properties on the release of the siRNA nanocarriers. A controlled release of complexed siRNA over 60 days in vitro was observed. By comparing the results from fluorescence imaging with data received from HPLC measurements, fluorescence imaging was found to be an appropriate tool to measure the release of siRNA complexes. Finally, the bioactivity of the siRNA released from hydrogel was tested and compared to free DL for its ability to knockdown the GFP expression in a DLD1 colon cancer cell model. The results indicate controlled release properties and activity of the released siRNA. In conclusion, the developed formulation is a promising system to provide local controlled release of siRNA over several weeks.
Highlights
After the discovery of RNA interference in 1998 by Fire et al [1], the downregulation of proteins became a valuable approach for the treatment of various diseases [2]
We focused our efforts on the development of hydrogels for controlled siRNA delivery with respect to a high translational potential
For the solid lipid complexes (SLC), 4% (m/V) cetyl palmitate was molten at 60 ◦ C, mixed with 1% (m/V) cationic lipid cetylpyridinium chloride and produced by a hot emulsion technique in 100 mL water with a high-pressure homogenizer (Emulsiflex C5, Avestin Inc., Ottawa, Canada)
Summary
After the discovery of RNA interference in 1998 by Fire et al [1], the downregulation of proteins became a valuable approach for the treatment of various diseases [2]. Besides its uses for wound dressing, bone regeneration material, and further applications in biomedicine, gellan gum is used as drug delivery vehicle [26] It is a linear negatively charged polysaccharide that forms cross-linked hydrogels in the presence of ions. Goyal et al [29] developed gellan gum PEI-DNA-siRNA nanocomposites, in which gellan gum improved bioactivity in vitro and after intravenous injection, in vivo Due to these properties, gellan gum offers a wide range of applicability as therapeutic drug depots. Besides the external administration for ocular, dermal, or vaginal diseases, the injectability would allow the application for subcutaneous, intra-articular, or intratumoral indications Due to these promising attributes, our study aimed to develop a siRNA-loaded gellan gum hydrogel for local and controlled release applications. The development can be separated into three parts in which the carrier finding and characterization is part I, the drug loading and hydrogel formulation is part II and the controlled release is part III
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