Abstract
The disruption of the cellular pathways of protein biosynthesis through the mechanism of RNA interference has been recognized as a tool of great diagnostic and therapeutic significance. However, in order to fully exploit the potential of this phenomenon, efficient and safe carriers capable of overcoming extra- and intracellular barriers and delivering siRNA to the target cells are needed. Recently, attention has focused on the possibility of the application of multifunctional nanoparticles, dendrimers, as potential delivery devices for siRNA. The aim of the present work was to evaluate the formation of dendriplexes using novel poly(lysine) dendrimers (containing lysine and arginine or histidine residues in their structure), and to verify the hypothesis that the use of these polymers may allow an efficient method of siRNA transfer into the cells in vitro to be obtained. The fluorescence polarization studies, as well as zeta potential and hydrodynamic diameter measurements were used to characterize the dendrimer:siRNA complexes. The cytotoxicity of dendrimers and dendriplexes was evaluated with the resazurin-based assay. Using the flow cytometry technique, the efficiency of siRNA transport to the myeloid cells was determined. This approach allowed us to determine the properties and optimal molar ratios of dendrimer:siRNA complexes, as well as to demonstrate that poly(lysine) dendrimers may serve as efficient carriers of genetic material, being much more effective than the commercially available transfection agent Lipofectamine 2000. This outcome provides the basis for further research on the application of poly(lysine) dendrimers as carriers for nucleic acids in the field of gene therapy.
Highlights
RNA interference (RNAi) is a biological process in which RNA molecules induce specific inhibition of target gene expression
It has been shown that PAMAM, phosphorus, and carbosilane dendrimers form stable dendriplexes [29,30] that enable effective transfection and enhancement of the cytotoxic effect of short interfering RNA (siRNA) [31]
The main goal of this work was the initial characterization of dendrimer:siRNA complexes and evaluation of the in vitro transfection efficiency to determine the optimal conditions for further research
Summary
RNA interference (RNAi) is a biological process in which RNA molecules induce specific inhibition of target gene expression. SiRNA has become the basis for drug development due to the high level of specificity, limited side effects, and the ease of synthesis of therapeutic molecules [2]. Before siRNA molecules reach their site of action in the cell cytoplasm, they have to conquer several obstacles, the number of which depends largely on the way of administration, the target tissue, as well as the physicochemical properties of the siRNA itself. The latter involve low stability, negative charge, and high structural stiffness, hampering the transport in body fluids, across cellular membranes, and between intracellular compartments [3]. SiRNA upon entering the bloodstream undergoes enzymatic degradation by nucleases or rapid renal clearance [4]
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