gala-peptide and octaarginine

A paradigm shift has occurred in the field of drug delivery systems (DDS), one being intracellular targeting, and the other, active targeting. An important aspect of intracellular targeting involves delivering nucleic acids such as siRNA/pDNA rather than small molecular compounds, since the mechanism responsible for their entering a target cell is usually via endocytosis, and the efficiency of endosomal escape is a critical factor in determining the functional activities of siRNA/pDNA. A multifunctional envelope-type nano device (MEND) was developed to control the intracellular trafficking of nano carriers containing siRNA/pDNA. An octaarginine (R8) modified MEND was developed to achieve this. Considerable progress has been made in active targeting to selective tissue vasculature such as tumor, adipose tissue, and the lung where endothelial barrier is tight against nanoparticles with diameters larger than 50 nm. A dual-ligand system is proposed to enhance active targeting ability by virtue of a synergistic interaction between a selective ligand and a cell penetrating ligand. Prohibitin targeted nanoparticles (PTNP) were developed to target endothelial cells in adipose tissue, which deliver apoptotic peptides/proteins to the adipose vasculature. Lung endothelial cells can be targeted by means of the GALA peptide, which is usually used to enhance endosomal escape. These active targeting systems can induce pharmacological effects in in vivo conditions. Finally, a novel strategy for producing an original ligand has been developed, especially for the tumor vasculature. This progress in DDS promises to extend the area of nanomedicine as a breakthrough technology.

Topics: Animals; Aptamers, Nucleotide; DNA; Drug Delivery Systems; Gene Transfer Techniques; Humans; Liposomes; Nanostructures; Nanotechnology; Oligopeptides; Peptides; Pharmaceutical Preparations; Plasmids; RNA, Small Interfering

Topics: Animals; Cell Line; DNA; Female; Gene Expression; Gene Transfer Techniques; Humans; Hydrogen-Ion Concentration; Lipids; Liver; Lung; Mice; Mice, Inbred ICR; Nanoparticles; Oligopeptides; Peptides; Plasmids; Transfection

The transduction of antibodies into living cells would represent a major contribution to both basic and applied biomedical fields, as currently available methods suffer from limitations such as low-uptake efficiency and endosomal entrapment. In this study, a liposome-based carrier was designed to overcome these issues. Liposomes were modified with octaarginine (R8), a cell penetrating peptide and GALA, a pH-sensitive fusogenic peptide. The presence of R8 enhanced the cellular uptake of antibodies, whereas GALA reduced endosomal entrapment, resulting in antibodies being released into the cytosol within 30 min. Moreover, compared with commercially available reagents for delivering antibodies, our system was superior in both cellular uptake and endosomal escape. In addition, specific antibodies delivered by R8-GALA liposomes were found to be associated with their epitope, confirming the preservation of functionality. This system for the efficient and high-speed cytosolic delivery of an antibody provides a valuable tool that can be useful in basic and applied research for analyzing the expression and function of intracellular molecules.

Topics: Antibodies; Antibody Specificity; Cytosol; Drug Carriers; Drug Delivery Systems; Endosomes; HeLa Cells; Humans; Liposomes; Nanostructures; Oligopeptides; Peptides

The cellular uptake pathway for a gene vector is an important factor in transgene expression. We previously constructed an original gene vector, multifunctional envelope-type nano device (MEND). The use of octaarginine (R8), a cell-penetrating peptide dramatically enhanced the transfection activity of the MEND since efficient cellular uptake via macropinocytosis, while the R8 should overcome its poor cell selectivity. Here we prepared an R8-MEND equipped with GALA (a peptide for endosomal escape) (R8/GALA-MEND) coated with hyaluronic acid (HA) (HA-R8/GALA-MEND), a natural ligand for cancer cells overexpressing CD44. We investigated the cellular uptake pathway of the HA-R8/GALA-MEND and the R8/GALA-MEND using HCT116 cells overexpressing CD44. Both carriers were taken up by cells mainly via macropinocytosis, whereas only the HA-R8/GALA-MEND was partially internalized into cells via a CD44-mediated pathway. Investigation of transgene expression showed that the HA-R8/GALA-MEND had a high transfection activity in HCT116 cells via both macropinocytotic and CD44-mediated pathways. On the other hand, the value for the HA-R8/GALA-MEND was significantly decreased compared with the value for the R8/GALA-MEND in NIH3T3 cells (CD44-negative cells). These findings indicate that the HA-coating controls the intracellular pathway for R8-modified nanocarriers, and that a CD44-mediated pathway is an important route for transgene expression.

Topics: Animals; Cell-Penetrating Peptides; Drug Carriers; Genetic Vectors; HCT116 Cells; Humans; Hyaluronan Receptors; Hyaluronic Acid; Mice; NIH 3T3 Cells; Oligopeptides; Peptides; Transfection

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.

Topics: Animals; DNA; Gene Expression; Gene Transfer Techniques; Lipids; Liver; Male; Mice; Mice, Inbred BALB C; Nanoparticles; Oligopeptides; Peptides; Plasmids

We recently developed a multifunctional envelope-type nano device (MEND) for efficient nucleic acid delivery. Here, we report on the development of an octaarigine (R8)-modified MEND encapsulating small interfering RNA (siRNA) with a tumor-specific, cleavable, polyethylene glycol (PEG)-lipid (PPD). We first determined the optimal concentration of R8 and pH-sensitive fusogenic peptide (GALA) on the lipid envelope of MEND (R8/GALA-MEND). Then, we examined the combination of optimized R8/GALA-MEND with a PEG-lipid. When a conventional PEG-lipid was used, the R8/GALA-MEND failed to knockdown expression of the target gene. On the other hand, PPD-modified R8/GALA-MEND exhibited efficient silencing activity to the level of the PEG-unmodified R8/GALA-MEND. In addition, we compared a R8/GALA-MEND with a MEND composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) that is a conventional cationic lipid used as a lipoplex component. The knockdown ability of the R8/GALA-MEND was much higher than that of the DOTAP-based MEND at the dose that is commonly employed in in vitro siRNA transfection. These results demonstrate that the R8/GALA-MEND is a promising delivery system for the transfer of siRNA to tumor cells.

Topics: Drug Delivery Systems; Fatty Acids, Monounsaturated; Gene Silencing; Gene Transfer Techniques; HeLa Cells; Humans; Luciferases; Nanoparticles; Oligopeptides; Peptides; Polyethylene Glycols; Quaternary Ammonium Compounds; RNA, Small Interfering; Transfection