Exogenous chondroitin sulfate glycosaminoglycan associate with arginine-rich peptide-DNA complexes to alter their intracellular processing and gene delivery efficiency.

Arginine-rich peptides have been used extensively as efficient cellular transporters. However, gene delivery with such peptides requires development of strategies to improve their efficiency. We had earlier demonstrated that addition of small amounts of exogenous glycosaminoglycans (GAGs) like heparan sulfate or chondroitin sulfate to different arginine-rich peptide-DNA complexes (polyplexes) led to an increase in their gene delivery efficiency. This was possibly due to the formation of a 'GAG coat' on the polyplex surface through electrostatic interactions which improved their extracellular stability and subsequent cellular entry. In this report, we have attempted to elucidate the differences in intracellular processing of the chondroitin sulfate (CS)-coated polyplexes in comparison to the native polyplexes by using a combination of endocytic inhibitors and co-localization with endosomal markers in various cell lines. We observed that both the native and CS-coated polyplexes are internalized by multiple endocytic pathways although in some cell lines, the coated polyplexes are taken up primarily by caveolae mediated endocytosis. In addition, the CS-coat improves the endosomal escape of the polyplexes as compared to the native polyplexes. Interestingly, during these intracellular events, exogenous CS is retained with the polyplexes until their accumulation near the nucleus. Thus we show for the first time that exogenous GAGs in small amounts improve intracellular routing and nuclear accumulation of arginine-based polyplexes. Therefore, addition of exogenous GAGs is a promising strategy to enhance the transfection efficiency of cationic arginine-rich peptides in multiple cell types.

Different roles of cell surface and exogenous glycosaminoglycans in controlling gene delivery by arginine-rich peptides with varied distribution of arginines.

The role of cell surface and exogenous glycosaminoglycans (GAGs) in DNA delivery by cationic peptides is controlled to a large extent by the peptide chemistry and the nature of its complex with DNA. We have previously shown that complexes formed by arginine homopeptides with DNA adopt a GAG-independent cellular internalization mechanism and show enhanced gene delivery in presence of exogenous GAGs. In contrast, lysine complexes gain cellular entry primarily by a GAG-dependent pathway and are destabilized by exogenous GAGs. The aim of the current study was to elucidate the factors governing the role of cell surface and soluble glycosaminoglycans in DNA delivery by sequences of arginine-rich peptides with altered arginine distributions (compared to homopeptide). Using peptides with clustered arginines which constitute known heparin-binding motifs and a control peptide with arginines alternating with alanines, we show that complexes formed by these peptides do not require cell surface GAGs for cellular uptake and DNA delivery. However, the charge distribution and the spacing of arginine residues affects DNA delivery efficiency of these peptides in presence of soluble GAGs, since these peptides show only a marginal increase in transfection in presence of exogenous GAGs unlike that observed with arginine homopeptides. Our results indicate that presence of arginine by itself drives these peptides to a cell surface GAG-independent route of entry to efficiently deliver functional DNA into cells in vitro. However, the inherent stability of the complexes differ when the distribution of arginines in the peptides is altered, thereby modulating its interaction with exogenous GAGs.

Second generation, arginine-rich (R-X'-R)(4)-type cell-penetrating α-ω-α-peptides with constrained, chiral ω-amino acids (X') for enhanced cargo delivery into cells.

The syntheses of novel N-aminoalkyl proline-derived spacers (X') in polycationic (R-X'-R)-motif cell-penetrating α-ω-α-peptides are described as improved molecular transporters and their structural features studied by CD. FACS analysis shows enhanced cellular uptake and confocal microscopy indicates predominantly cytoplasmic localization. The oligomers are efficient at transporting pDNA into cells. The chirality together with the hydrophobicity and flexibility derived from the spacer chain are found to have marked influence on the cell-penetrating and cargo delivery properties of the cell-penetrating peptides (CPPs). The peptides containing N-(3-aminopropyl)-D-proline spacers are found to be the best at cell penetration and cargo delivery in the present study.

Exogenous and cell surface glycosaminoglycans alter DNA delivery efficiency of arginine and lysine homopeptides in distinctly different ways.

Glycosaminoglycans (GAGs) expressed ubiquitously on the cell surface are known to interact with a variety of ligands to mediate different cellular processes. However, their role in the internalization of cationic gene delivery vectors such as liposomes, polymers, and peptides is still ambiguous and seems to be controlled by multiple factors. In this report, taking peptides as model systems, we show that peptide chemistry is one of the key factors that determine the dependence on cell surface glycosaminoglycans for cellular internalization and gene delivery. Arginine peptides and their complexes with plasmid DNA show efficient uptake and functional gene transfer independent of the cell surface GAGs. On the other hand, lysine peptides and complexes primarily enter through a GAG-dependent pathway. The peptide-DNA complexes also show differential interaction with soluble GAGs. In the presence of exogenous GAGs under certain conditions, arginine peptide-DNA complexes show increased transfection efficiency that is not observed with lysine. This is attributed to a change in the complex nature that ensures better protection of the compacted DNA in the case of arginine complexes, whereas the lysine complexes get destabilized under these conditions. The presence of a GAG coating also ensures better cell association of arginine complexes, resulting in increased uptake. Our results indicate that the role of both the cell surface and exogenous glycosaminoglycans in gene delivery is controlled by the nature of the peptide and its complex with DNA.

Highly efficient (R-X-R)-type carbamates as molecular transporters for cellular delivery.

The (R-X-R) motif-containing arginine-rich peptides are among the most effective cell-penetrating peptides. The replacement of amide linkages in the (R-X-R) motif by carbamate linkages as in (r-ahx-r)(4) or (r-ahx-r-r-apr-r)(2) increases the efficacy of such oligomers several-fold. Internalization of these oligomers in mammalian cell lines occurs by an energy-independent process. These oligomers show efficient delivery of biologically active plasmid DNA into CHO-K1 cells.

IFI16 and cGAS cooperate in the activation of STING during DNA sensing in human keratinocytes.

Many human cells can sense the presence of exogenous DNA during infection though the cytosolic DNA receptor cyclic GMP-AMP synthase (cGAS), which produces the second messenger cyclic GMP-AMP (cGAMP). Other putative DNA receptors have been described, but whether their functions are redundant, tissue-specific or integrated in the cGAS-cGAMP pathway is unclear. Here we show that interferon-γ inducible protein 16 (IFI16) cooperates with cGAS during DNA sensing in human keratinocytes, as both cGAS and IFI16 are required for the full activation of an innate immune response to exogenous DNA and DNA viruses. IFI16 is also required for the cGAMP-induced activation of STING, and interacts with STING to promote STING phosphorylation and translocation. We propose that the two DNA sensors IFI16 and cGAS cooperate to prevent the spurious activation of the type I interferon response.

Structural rearrangements and chemical modifications in known cell penetrating peptide strongly enhance DNA delivery efficiency.

Amphipathic peptides with unusual cellular translocation properties have been used as carriers of different biomolecules. However, the parameters which control the delivery efficiency of a particular cargo by a peptide and the selectivity of cargo delivery are not very well understood. In this work, we have used the known cell penetrating peptide pVEC (derived from VE-cadherin) and systematically changed its amphipathicity (from primary to secondary) as well as the total charge and studied whether these changes influence the plasmid DNA condensation ability, cellular uptake of the peptide-DNA complexes and in turn the efficiency of DNA delivery of the peptide. Our results show that although the efficiency of DNA delivery of pVEC is poor, modification of the same peptide to create a combination of nine arginines along with secondary amphipathicity improves its plasmid DNA delivery efficiency, particularly in presence of an endosomotropic agent like chloroquine. In addition, presence of histidines along with 9 arginines and secondary amphipathicity shows efficient DNA delivery with low toxicity even in absence of chloroquine in multiple cell lines. We attribute these enhancements in transfection efficiency to the differences in the mechanism of complex formation by the different variants of the parent peptide which in turn are related to the chemical nature of the peptide itself. These results exhibit the importance of understanding the physicochemical parameters of the carrier and complex in modulating gene delivery efficiency. Such studies can be helpful in improving peptide design for delivery of different cargo molecules.