Internalization of mRNA Lipoplexes by Dendritic Cells

Abstract

Lipoplexes, composed of Lipofectamine and mRNA encoding HIV Gag protein, were shown to be internalized by dendritic cells (DCs) and promote antigen presentation to stimulate HIV-specific T cell responses. Using confocal microscopy, we showed that one-third of fluorescently labeled mRNA containing lipoplexes are colocalized with late endosomes. We further investigated the effect of inhibitors, blocking phagocytosis, macropinocytosis, and clathrin- and caveolae-mediated endocytosis, on both the internalization of the lipoplexes by DCs and the transfection efficiency. We observed that chloropromazine had no effect on the cellular uptake or transfection efficiency, excluding the involvement of clathrin-mediated endocytosis. Cytochalasin D, inhibiting macropinocytosis and phagocystosis, strongly reduced internalization (50%) of the lipoplexes as well as protein expression (70%). Amiloride, which should specifically block macropinocytosis, induced only a modest reduction of uptake and transfection. Genistein and dynasore induced a strong reduction of on the level of protein expression (>70%), but not the overall uptake. Our results indicate that transfection-effective mRNA lipoplex internalization by DCs, i.e., uptake that results in protein expression, preferentially proceeds by macropinocytosis and/or phagocytosis.

Multifunctional Hierarchically Assembled Nanostructures as Complex Stage-Wise Dual-Delivery Systems for Coincidental Yet Differential Trafficking of siRNA and Paclitaxel

“…The overall data suggest that several endocytic pathways are involved in the uptake and intracellular delivery of siRNA during Lipofectamine transfection, which is supported by available literature. 47,48 The reduction in gene silencing activity of the siRNA/cSCKs complexes was unclear for all the inhibitors used, probably due to the low transfection of the cSCKs themselves, and hence, it was difficult to observe the reduction in transfection. A slight increase in transfection of cSCKs upon pre-treatment with CP was observed (not significant, p > 0.05).…”

Self-Amplifying Replicon RNA Vaccine Delivery to Dendritic Cells by Synthetic Nanoparticles

“…Replicon RNA has been delivered by coating on to gold microparticles [ 26 ], but these are neither biodegradable nor nanoparticles, and do not offer the advantages of biodegradable nanoparticles for targeting DCs and processing via DC endocytic pathways. While mRNA delivery to DCs has proven successful [ 27 , 28 , 29 , 30 , 31 , 32 ], it has only recently been shown that such nanoparticulate delivery is feasible for the much larger RepRNA molecules [ 33 ] ( Figure 2 and Figure 3 ). This success of nanoparticle-based delivery of RepRNA to DCs requires an appreciation of the DC requirements for interaction with the delivery vehicles and the subsequent intracellular delivery of the RepRNA to the RNA translation sites ( Figure 3 ).…”

“…Such an observation cannot be repeated with an RNase-sensitive pestivirus RepRNA, which does not lead to translation or induction of immune responses in vitro or in vivo when employed as “naked” RNA [ 33 ]. These observations may relate to the application of mRNA vaccines applied as “naked” RNA; such vaccines require capping and/or poly-adenylation [ 1 , 28 , 91 , 92 , 93 ]. It has been well established for over two decades that alphavirus RNAs contain 5' hypermethylated caps, together with a 3' poly(A) tail in their genomic structure [ 94 ].…”

“…Current alphavirus replicon vaccines are capped using, for example, the Vaccinia Capping system [ 83 ]. It would appear that such capped replicon molecules can be employed without the need for a delivery vehicle [ 83 , 86 ], related to what has been reported for mRNA vaccines [ 1 , 28 , 91 , 92 , 93 ]. Nevertheless, not all RNA viruses require capping or poly(A) tails.…”

Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

“…Hence, non-viral vectors availing nanotechnological advances are in the focus of investigation to improve cellular uptake and subsequent transfection of target cells. Therefore, a variety of different biocompatible and biodegradable nanoparticles (NPs) have been developed including lipid-based [ 4 – 7 ] and polymer-based [ 8 , 9 ] systems, featuring a cationic surface charge and thereby facilitating complexation of anionic mRNA. While these approaches rendered significant progress to overcome drawbacks resulting from mRNA-based delivery in vitro as well as in vivo [ 10 – 13 ], designing of suitable systems with low cytotoxicity and high transfection rate [ 14 ] remains crucial parameters and sets an indispensable precondition for mRNA-delivery.…”