Folate-Targeted Transgenic Activity of Dendrimer Functionalized Selenium Nanoparticles In Vitro

Abstract

Current chemotherapeutic drugs, although effective, lack cell-specific targeting, instigate adverse side effects in healthy tissue, exhibit unfavourable bio-circulation and can generate drug-resistant cancers. The synergistic use of nanotechnology and gene therapy, using nanoparticles (NPs) for therapeutic gene delivery to cancer cells is hereby proposed. This includes the benefit of cell-specific targeting and exploitation of receptors overexpressed in specific cancer types. The aim of this study was to formulate dendrimer-functionalized selenium nanoparticles (PAMAM-SeNPs) containing the targeting moiety, folic acid (FA), for delivery of pCMV-Luc-DNA (pDNA) in vitro. These NPs and their gene-loaded nanocomplexes were physicochemically and morphologically characterized. Nucleic acid-binding, compaction and pDNA protection were assessed, followed by cell-based in vitro cytotoxicity, transgene expression and apoptotic assays. Nanocomplexes possessed favourable sizes (<150 nm) and ζ-potentials (>25 mV), crucial for cellular interaction, and protected the pDNA from degradation in an in vivo simulation. PAMAM-SeNP nanocomplexes exhibited higher cell viability (>85%) compared to selenium-free nanocomplexes (approximately 75%), confirming the important role of selenium in these nanocomplexes. FA-conjugated PAMAM-SeNPs displayed higher overall transgene expression (HeLa cells) compared to their non-targeting counterparts, suggesting enhanced receptor-mediated cellular uptake. Overall, our results bode well for the use of these nano-delivery vehicles in future in vivo studies.

Hepatocellular-Targeted mRNA Delivery Using Functionalized Selenium Nanoparticles In Vitro

“…Although Se species were reported to induce cytotoxicity through apoptosis [ 14 , 20 , 61 ], no significant apoptosis was observed in this study as evidenced in Supplementary Figure S4 and Table S2 . Similar results with low apoptotic indices were reported for mRNA delivery using modified SeNPs [ 36 ]. Overall, favorable cell viability in normal HEK293 cells bodes well for the use of these NPs in both gene and drug delivery.…”

“…CS facilitated the binding of nucleic acids to the NP via electrostatic interactions between its cationic amine groups and the negatively charged phosphate backbone of the mRNA. The 0.1% ( w / v ) CS used was reported to be the optimal concentration for homogeneity and stability of the CS encapsulated SeNPs in solution [ 36 , 37 , 47 ]. Galactose-based ligands have commonly been used for targeting the asialoorosomucoid glycoprotein receptor (ASGPR) on the surface of hepatocytes [ 48 , 49 , 50 , 51 ].…”

“…The use of mRNA therapeutics further provides a transient high level of expression of the genetic cargo upon entry into the cell, preventing the side effects caused by persistent foreign genetic elements, which could further encourage the transformation of cells into a cancerous state [ 76 ]. Transfection capabilities of novel gene vectors are often evaluated by the use of reporter genes (e.g., bioluminescence, β-galactosidase, and fluorescent proteins), which offer a mode of measuring the transcription of foreign genetic elements introduced into the cell through recognition of the accumulation of a produced protein [ 36 , 37 , 38 ]. The luciferase assay was conducted to assess the transfection potential of these FSeNPs in vitro.…”

“…The SeNPs were synthesized ( Figure 1 A) chemically by the reduction of sodium selenite (Na 2 SeO 3 ) with ascorbic acid, as described previously with slight modifications [ 34 , 35 , 36 ]. Briefly, 0.2 mL of a 0.51 M Na 2 SeO 3 solution was added dropwise with stirring to 7.5 mL of a freshly prepared 0.23 M ascorbic acid solution and left to stir for a further 30 min.…”

“…Thus, mediation of RNA often requires the use of a vector [ 31 , 32 , 33 ]. Chitosan SeNPs (Cs-SeNPs) have been explored previously for the delivery of siRNA [ 34 ] and mRNA [ 35 ], and dendrimer-SeNPs were recently used for the delivery of a luciferase reporter gene, pCMV -luc- DNA [ 36 ]. This study explores the novel use of SeNPs as liver-targeted mRNA-based nanocarriers and evaluated their luciferase gene expression as a proof of principle study.…”