Ikramy A. Khalil

High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression

The mechanism of the arginine-rich peptide-mediated cellular uptake is currently a controversial issue. Several factors, including the type of peptide, the nature of the cargo, and the linker between them, appear to affect uptake. One of the less studied factors, which may affect the uptake mechanism, is the effect of peptide density on the surface of the cargo. Here, we examined the mechanism of cellular uptake and intracellular trafficking of liposomes modified with different densities of the octaarginine (R8) peptide. Liposomes modified with a low R8 density were taken up mainly through clathrin-mediated endocytosis, leading to extensive lysosomal degradation, whereas those modified with a high R8 density were taken up mainly through macropinocytosis and were less subject to lysosomal degradation. Furthermore, the high density R8-liposomes were able to stimulate the macropinocytosis-mediated uptake of other particles. When plasmid DNA was condensed and encapsulated in R8-liposomes, the levels of gene expression were three orders of magnitude higher for the high density liposomes. The enhanced gene expression by the high density R8-liposomes was highly impaired by blocking uptake through macropinocytosis. The different extents of gene expression from different densities of the R8 peptide on the liposomes could be explained principally by the existence of an intracellular trafficking route, but not by the uptake amount, of internalized liposomes. These results show that the density of the R8 peptide on liposomes determines the uptake mechanism and that this is directly linked to intracellular trafficking, resulting in different levels of gene expression.

Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation

The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors.

Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery

This study describes a multifunctional envelope-type nano device (MEND) that mimics an envelope-type virus based on a novel packaging strategy. MEND particles contain a DNA core packaged into a lipid envelope modified with an octaarginine peptide. The peptide mediates internalization via macropinocytosis, which avoids lysosomal degradation. MEND-mediated transfection of a luciferase expression plasmid achieved comparable efficiency to adenovirus-mediated transfection, with lower associated cytotoxicity. Furthermore, topical application of MEND particles containing constitutively active bone morphogenetic protein (BMP) type IA receptor (caBmpr1a) gene had a significant impact on hair growth in vivo. These data demonstrate that MEND is a promising non-viral gene delivery system that may provide superior results to existing non-viral gene delivery technologies.

Octaarginine- and octalysine-modified nanoparticles have different modes of endosomal escape.

The present study examines the role of surface modification with an octaarginine peptide (R8) in liposomal escape from endocytic vesicles, using octalysine (K8) as a control cationic peptide; the mechanism of endosomal escape of liposomes was also investigated. Gene expression of condensed plasmid DNA encapsulated in R8-modified nanoparticles was more than 1 order of magnitude higher than that of K8-modified nanoparticles, and 2 orders of magnitude higher than gene expression using unmodified nanoparticles. The difference in gene expression could not be attributed to differences in uptake, as R8- and K8-modified liposomes were taken up primarily via macropinocytosis with comparable efficiency. The extent of R8-nanoparticle escape to the cytosol was double that of K8-nanoparticles. Suppression of endosome acidification inhibited R8-nanoparticle endosomal escape, but enhanced that of K8-nanoparticles. Using spectral imaging in live cells, we showed that R8- and K8-liposomes escaped from endocytic vesicles via fusion between the liposomes and the endosomal membrane. R8-liposomes fused efficiently at both acidic and neutral pH, whereas K8-liposomes fused only at neutral pH. Similar behavior was observed during in vitro lipid mixing and calcein-release experiments. Co-incubation of cells with distinctly labeled K8- and R8-modified nanoparticles confirmed a common uptake pathway and different rates of endosomal escape particularly at longer time intervals. Therefore, it was concluded that R8 on the liposome surface stimulates efficient escape from endocytic vesicles via a fusion mechanism that works at both neutral and acidic pH; in contrast, K8 mediates escape mainly at neutral pH.

Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process.

Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.

Octaarginine-modified liposomes: Enhanced cellular uptake and controlled intracellular trafficking

Gene therapy is a promising new approach for treating a variety of genetic and acquired diseases. While viral vectors are highly efficient for gene therapy, their use is associated with high toxicity and immunogenicity. Synthetic or nonviral vectors are attractive alternatives to viral vectors because of their low immunogenicity and low acute toxicity. The main disadvantage of the nonviral vectors is the low transfection efficiency compared to viral vectors. Novel functional devices to enhance the transfection activities of nonviral vectors are needed. In this review, we discuss the modification of liposomal drug carriers with a novel functional device, the octaarginine (R8) peptide, for drug and gene delivery. Decoration of liposomes with R8 enhanced their cellular uptake. In addition, by optimizing the density of the peptide as well as its topology, the liposomes could be internalized via clathrin-independent pathways, which improved the intracellular trafficking through avoiding lysosomal degradation. A special emphasis is given to the need for optimizing the conditions of using the peptide to not only enhance the cellular uptake but also to improve the intracellular trafficking of its cargos. In addition, the use of R8-modified liposomes and nano-particles in gene delivery is discussed.

Enhanced gene expression by a novel stearylated INF7 peptide derivative through fusion independent endosomal escape

An octaarginine-modified multifunctional envelope-type nano device (R8-MEND) was previously reported to be an efficient nonviral vector for the delivery of plasmid DNA, in vitro and after topical administration. We report herein on a novel stearylated derivative of the INF7 peptide, a derivative of the N-terminal domain of the HA2 protein of the influenza virus envelope, which enhances the endosomal escape of R8-MEND through a mechanism independent of fusion between the MEND coat and the endosomal membrane. The use of the novel peptide derivative would permit the gene expression of the R8-MEND to be improved, both in vitro and in vivo. R8-MEND modified with stearylated INF7 resulted in gene expression levels that were 77-fold higher than unmodified and 20-fold higher than the free INF7 peptide-modified R8-MEND with no cellular toxicity. Spectral imaging in live cells confirmed that the stearylated INF7 modification did not mediate fusion between liposomes and the endosomal membrane. The inclusion of DOPE to the R8-MEND coat was synergistic with the peptide in improving gene transfection. The intravenous injection of an R8-MEND modified with stearylated INF7 to ICR mice resulted in luciferase expression levels 240-fold higher in liver and 115-fold higher in spleen than that of the R8-MEND.

Octaarginine- and pH sensitive fusogenic peptide-modified nanoparticles for liver gene delivery.

We previously reported that octaarginine peptide modified liposomes (R8-liposomes) largely accumulated in the liver after intravenous administration and that this is dependent on the R8-density. We report herein on the development of a Multifunctional Envelope-type Nano Device modified with R8 and GALA, as a pH-sensitive fusogenic peptide (R8-GALA-MEND) for liver gene delivery. An R8-MEND encapsulating pDNA prepared using two different cores (negatively or positively charged pDNA/polyethylene imine condensed particles) failed to produce a high gene expression in the liver. Modification with GALA dramatically increased gene expression particularly in the liver only in the case of a negative core R8-MEND. Quantification of the number of gene copies delivered to liver cells and nuclei revealed that the amount of pDNA was significantly higher in the case of positive core R8-MENDs, regardless of the absence or presence of GALA. However, gene expression efficiencies per nucleus-delivered pDNA were much higher in the case of the negative core R8-MEND, especially the R8-GALA-MEND suggesting that the substantial improvement in gene expression can be explained by an improved gene expression efficiency per pDNA in the presence of GALA. A comparative study between the developed R8-GALA-MEND and a similar system containing DOTAP, a commonly used cationic lipid, instead of R8 showed that gene expression of the R8-GALA-MEND was 29 times higher than that of the DOTAP-GALA-MEND and is more selective for the liver. Collectively, these results suggest that the combination of a negatively charged core system and GALA modification of the R8-MEND is useful system for efficiently delivering genes to the liver.

Cell penetrating peptide-mediated systemic siRNA delivery to the liver

The cell-penetrating peptide (CPP) is one of the most attractive tools for efficiently delivering biomolecules to a target organelle. Here, we describe the use of octaarginine (R8)-modified lipid nanoparticles for the efficient and targeted in vivo delivery of siRNA to the liver. In this study, SR-BI (a scavenger receptor class B, member 1) was targeted by this nanoparticle. Our results demonstrate that R8-modified lipid nanoparticles can be used for the efficient and targeted delivery of liver siRNA to induce the specific knock-down of an endogenous gene with minimum liver toxicity and immune response, and that this CPP based technology holds considerable promise for further in vivo biological applications of siRNA.

Novel lipidated sorbitol-based molecular transporters for non-viral gene delivery

In this study, we investigated the possible use of novel lipidated sorbitol-based transporters as functional devices for the improvement of non-viral gene delivery. These transporters are composed of a sorbitol scaffold bearing 8 guanidine moieties that mimic the arginine residues of well-known cell-penetrating peptides. In addition, the transporters carry different lipid groups to aid DNA condensation and facilitate lipid vesicle-binding. We found that the transporters described in this study have the potential to function as plasmid DNA/siRNA-condensers and surface ligands for the enhancement of cellular uptake of lipid vesicles. Shorter lipid chains were found to be better for condensation, whereas longer chains were superior surface ligands. The differential activity of different cores might be explained by facilitated decondensation of cores prepared with transporters comprised of shorter lipid chains. However, we suggest that there is an optimum value of decondensation to achieve higher transfection activities. The proper use of the transporters presented in this study enabled us to prepare a highly efficient non-viral gene delivery system based on a core-shell structure, in which a condensed DNA core is encapsulated by a lipid envelope. A multifunctional envelope-type nano-device prepared with an optimal surface ligand favorably competes with commonly used transfection systems.