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.

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The delivery of plasmid DNA remains hard to achieve, especially due to the presence of the nuclear membrane barrier. During cell division, however, the nuclear membrane is temporarily disassembled. We evaluated two different strategies to optimize plasmid DNA delivery in dividing cells: 1) phosphorylation responsive peptides that release plasmid DNA preferentially during mitosis and 2) chromatin targeting peptides to anchor plasmid DNA in newly formed nuclei upon cell division. Peptide/DNA particles alone were not efficient in penetrating cells. Upon co-delivery with lipid-based carriers, however, transfection efficiency drastically improved when compared to controls. For the phosphorylation responsive peptides, the presence of the phosphorylation sequence slightly increased transfection efficiency. For the chromatin targeting peptides, however, the chromatin targeting sequence did not seem to be the main reason for the improvement of transfection efficiency when applied in living cells. In conclusion, the pre-condensation of plasmid DNA with peptides improves lipid based delivery, but the nature of the peptides (cell responsive or not) does not seem to be the main reason for the improvement. It seems that the nuclear entry of foreign plasmid DNA is still under tight control, even during the mitotic window of opportunity.

ABSTRACT. The nuclear membrane is one of the major cellular barriers in the delivery of plasmid DNA (pDNA). Cell division has a positive influence on the expression efficiency since at the end of mitosis, pDNA or pDNA containing complexes near the chromatin are probably included by a random process in the nuclei of the daughter cells. However, very little is known about the nuclear inclusion of nanoparticles during cell division. Using the Xenopus nuclear envelope reassembly (XNER) assay, we found that the nuclear enclosure of nanoparticles was dependent on size (with 100 nm and 200 nm particles being better included than the 500 nm ones) and charge (with positively charged particles being better included than negatively charged or poly-ethyleneglycolated (PEG-ylated) ones) of the 2 beads. Also, coupling chromatin-targeting peptides to the polystyrene beads or pDNA complexes improved their inclusion by 2-to 3-fold. Upon microinjection in living HeLa cells, however, nanoparticles were never observed in the nuclei of cells post-division but accumulated in a specific perinuclear region, which was identified as the lysosomal compartment. This indicates that nanoparticles can end up in the lysosomes even when they were not delivered through endocytosis. To elucidate if the chromatin binding peptides also have potential in living cells, this additional barrier first has to be tackled, since it prevents free particles to be present near the chromatin at the moment of cell division.

In cationic carrier-mediated gene delivery, the disproportional relationship between the quantity of delivered DNA and the amount of encoded protein produced is a well-known phenomenon. The numerous intracellular barriers which need to be overcome by pDNA to reach the nucleoplasm play a major role in it. In contrast to what one would expect, a partial replacement of coding pDNA by noncoding DNA does not lead to a decrease in transfection efficiency. The mechanism underlying this observation is still unclear. Therefore, we investigated which constituents of the transfection process might contribute to this phenomenon. Our data reveal that the topology of the noncoding plasmid DNA plays a major role. Noncoding pDNA can be used only in a supercoiled form to replace coding pDNA in Lipofectamine lipoplexes, without a loss in transfection levels. When noncoding pDNA is linearized or partly digested, it diminishes the transfection potential of coding pDNA, as does noncoding salmon DNA. The difference in transfection efficiencies could not be attributed to diverse physicochemical characteristics of the Lipofectamine lipoplexes containing different types of noncoding DNA or to the extent of their internalization. At the level of endosomal release, however, nucleic acid release from the endosomal compartment proceeds faster when lipoplexes contain noncoding salmon DNA. Since the half-life of pDNA in the cytosol hardly exceeds 90 min, it is conceivable that prolonged release of coding pDNA from complexes carrying supercoiled noncoding pDNA may explain its positive effect on transfection, while this depot effect does not exist when noncoding salmon DNA is used.

Due to their great instability, phosphodiester antisense oligonucleotides (PO-ODNs) are rapidly degraded in the intracellular environment, which limits their biological activity. The release of PO-ODNs during a prolonged period of time could however greatly enhance their antisense effect by creating a pool of intact PO-ODNs at any time point. Poly-β-aminoesters are biodegradable cationic polymers which show potential for the controlled release of short DNA fragments like ODNs and small interfering RNA (siRNA). In this research we evaluated biodegradable poly-β-aminoesters as carriers for PO-ODNs and compared the antisense activity with nuclease stable phosphothioate (PS) ODNs. PBAE1 polymers were not able to generate an antisense effect with PO-or PS-ODNs, most likely due to their poor cellular uptake. When complexed to PBAE2 polymers at N/P ratio 10, both PO-and PS-ODNs downregulated the targeted protein expression with 70%. By confocal imaging we observed a high concentration of released PO-ODNs that formed nuclear bodies in the nucleoplasm. The ODNs in this nuclear bodies were still intact as could be demonstrated by Fluorescence Resonance Energy Transfer (FRET) and acceptor photobleaching. This was in clear contrast to PO-ODNs delivery by cationic liposomes where the ODNs that accumulated in the nucleus were degraded and nuclear bodies were not observed. We conclude that PBAE2 shows potential for the delivery of nuclease sensitive POODNs. This occurs however not through a time controlled release profile, but rather due to the rapid delivery of a high concentration of intact PO-ODNs that form nuclear bodies in the nuclei of the cells. These nuclear bodies can most likely act as a depot of intact PO-ODNs, resulting in efficient antisense activity.keywords : poly β aminoesters, Fluorescence Correlation Spectroscopy, Fluorescence Resonance Energy Transfer, oligonucleotides, nucleases, delivery 2 INTRODUCTIONDelivery of small DNA fragments such as antisense oligonucleotides (ODNs) and small interfering RNA (siRNA) remains an attractive research domain to achieve the therapeutic regulation of gene expression. Antisense oligonucleotides are short single stranded DNA strands which naturally consist of a phosphodiester (PO) backbone. Due to the high instability of this PO backbone, however, the routinely used ODNs consist of the more stabile phosphothioate (PS) backbone (1;2). ODNs can act both in the cytoplasm and the nucleus by a sequence-specific recognition of the complementary mRNA. Through a variety of mechanisms, the complementary mRNA is then destroyed which results in the downregulation of the corresponding protein (3;4).siRNAs are short double-stranded RNA fragments with a 2 nucleotides overhang at the 3' end. They are very potent molecules since they are recognised by intracellular RNA-induced silencing complexes (RISCs) which specifically and repeatedly destroy the targeted mRNA molecules. SiRNA can be used in much lower concentrations and generally achieves a higher knockdown efficiency when comp...

The magnetic particles offer several possibilities as efficient cellular MRI contrast agents for direct in vitro or in vivo cell labeling.

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