Laboratory research studies involving nucleic acid therapies, such as delivery of short, interfering RNAs (siRNAs) for RNA interference (RNAi), are typically performed on two-dimensional (2D) cell cultures. However, this is not an ideal model of the three-dimensional (3D) in vivo environment. As such, we are seeking to optimize transfection of siRNAs in 3D, specifically to cells cultured in collagen gels. Commercial transfection reagents with proven efficacy in 2D siRNA transfections have difficulty silencing cells suspended within hydrogel matrices, primarily due to interactions between the transfection complexes and the matrix.
In this study, we used multicolor fluorescence confocal microscopy and flow cytometry to analyze the cellular uptake and efficiencies of siRNAs delivered through 3D collagen matrices. Delivery was compared using Lipofectamine 2000 (LF2K) and 3-D FectIN, a transfection reagent developed specifically for hydrogel delivery. Although our results showed siRNA uptake under multiple conditions, effective silencing has only been achieved through using 3-D FectIN for delivery during gel formation. Additionally, treatments after gel formation demonstrate significant siRNA accumulation at the top of the matrix with minimal penetration throughout the gel, even at longer transfection times (e.g., 72 h). We will discuss our recent work explaining this phenomenon as well as other methods to aid in delivery throughout the collagen scaffolds.