Efficient Transfection of Hepatocytes Mediated by mRNA Complexed to Galactosylated Cyclodextrins

Abstract

In this study, we aimed at specific targeting of polycationic amphiphilic cyclodextrins (paCDs) to HepG2 cells via the asialoglycoprotein receptor (ASGPr). The transfection efficiencies of paCDs modified with galactose moieties were evaluated. In preliminary experiments, attempts to transfect HepG2 cells with pDNA complexed with different modified paCDs resulted in very low transfection levels. In additional series of experiments, we found out that nucleic acid/cyclodextrin complexes (CDplexes) were efficiently taken up by the cells and that photochemical internalization, which facilitates release from endosomes, did not improve transfection. Further experiments showed that pDNA can be readily released from the CDplexes when exposed to negatively charged vesicles. These observations imply that the lack of transfection cannot be attributed to a lack of internalization, release of CDplexes from the endosomal compartment, or release of free pDNA from the CDplexes. This in turn suggests that the nuclear entry of the pDNA represents the main limiting factor in the transfection process. To verify that HepG2 cells were transfected with targeted CDplexes containing mRNA, which does not require entry into the nucleus for being translated. With mRNA encoding the green fluorescent protein, fractions of GFP-positive cells of up to 31% were obtained. The results confirmed that the galactosylated complexes are specifically internalized via the ASGPr.

Cyclodextrin-based multivalent glycodisplays: covalent and supramolecular conjugates to assess carbohydrate–protein interactions

“…Rejman, García Fernández and co-workers explored this approach for the preparation of bGal-coated pGaCDs (111) intended for hepatocyte-specific nucleic acid delivery. 168 Since replacement of cationic centres by sugar motifs in paCDs was detrimental for CDplex stability, grouping the recognition elements into multivalent glycodendrons prior to paCD conjugation was judged convenient. The coating moiety consisted of a trivalent b-Dgalactopyranosyl antenna, built on a pentaerythritol scaffold, armed with an isothiocyanate group (110) for thiourea-forming conjugation.…”

“…The fact that high transfection levels were achieved with mRNA, which is translated in the cytoplasm, much higher as compared with mRNA complexes formulated with the reference compound JetPEIt-Hepatocyte, supported this assumption. 168 …”

Diving through Membranes: Molecular Cunning to Enforce the Endosomal Escape of Antibody-Targeted Anti-Tumor Toxins

“…In another study PCI failed to enhance the transfection rate of HepG2 cells transfected with polycationic amphiphilic cyclodextrin-DNA complexes but did enhance the transfection of the cells with complexed mRNA. The authors concluded that the low transfection rate for DNA was caused by problems with the complexed DNA entering the nucleus and may not be indicative that PCI per se failed to increase the delivery of complexed DNA to the cytosol [118]. An approach with low density lipoproteins as carriers for siRNA demonstrated only a moderate increase of transfection by PCI (38% gene knock-down without PCI versus 78% gene knock-down with PCI) [119].…”

The yin of exofacial protein sulfhydryls and the yang of intracellular glutathione in in vitro transfection with SS14 bioreducible lipoplexes

“…Bioreducible lipoplexes were thus added to SYBR Green I in DMEM either supplemented or not with 10% FBS. The changes in fluorescence, indicating an increase in the amount of free DNA that is the only accessible for the probe, were measured with a fluorometer [40]. Lipoplexes should remain intact upon dilution in cell culture media.…”