Prestin binding peptides as ligands for targeted polymersome mediated drug delivery to outer hair cells in the inner ear.

Targeted delivery of treatment agents to the inner ear using nanoparticles is an advanced therapeutic approach to cure or alleviate hearing loss. Designed to target the outer hair cells of the cochlea, two 12-mer peptides (A(665) and A(666)) with affinity to prestin were identified following 3 rounds of sequential phage display. Two-round display with immobilized prestin protein was used to enrich the library for full-length prestin. The last round was performed using Cos-7 cells transiently transfected with a cCFP-prestin plasmid to display phages expressing peptides restrictive to the extracellular loops of prestin. The binding properties of A(665) and A(666) shown by flow cytometry demonstrated selectivity to prestin-expressing Chinese hamster ovary cells. PEG6K-b-PCL19K polymersomes covalently labelled with these peptides demonstrated effective targeting to outer hair cells in a rat cochlear explant study.

Size matters: versatile use of PiggyBac transposons as a genetic manipulation tool.

Transposons have been promising elements for gene integration, and the Sleeping Beauty (SB) system has been the major one for many years, although there have been several other transposon systems available, for example, Tol2. However, recently another system known as PiggyBac (PB) has been introduced and developed for fulfilling the same purposes, for example, mutagenesis, transgenesis and gene therapy and in some cases with improved transposition efficiency and advantages over the Sleeping Beauty transposon system, although improved hyperactive transposase has highly increased the transposition efficacy for SB. The PB systems have been used in many different scientific research fields; therefore, the purpose of this review is to describe some of these versatile uses of the PiggyBac system to give readers an overview on the usage of PiggyBac system.

Effect of edge activators on the formation and transfection efficiency of ultradeformable liposomes.

Highly deformable hydrophilic lipid vesicles have been studied for transdermal delivery of therapeutically active small molecules and proteins. Here, we report the effects of edge activators on the formation of ultradeformable liposomes (UL) and transdermal gene delivery. Sodium cholate, sodium deoxycholate, and Tween 80 were tested as edge activators. Of the edge activators, sodium cholate and sodium deoxycholate resulted in the smaller sizes of UL and more positive zeta potentials than did Tween 80. Moreover, sodium deoxycholate-based UL showed the highest positive zeta potentials, which might lead to the firmer binding with negatively charged DNA. Following topical application onto mice, DNA complexed with UL containing either sodium cholate or sodium deoxycholate showed substantial transdermal absorption. In contrast, DNA complexed with Tween 80-based UL did not show in vivo transdermal absorption. These data suggest that UL might be of use as a transdermal delivery system of plasmid DNA, and that the choice of edge activators may play an important role in the transdermal delivery of plasmid DNA via UL.

The use of chitosan as a condensing agent to enhance emulsion-mediated gene transfer.

Previously we have formulated a new cationic emulsion, composed of 3beta [N-(N',N'-dimethylaminoethane) carbamoyl] cholesterol and dioleoylphosphatidyl ethanolamine, castor oil and Tween 80, and it efficiently delivered plasmid DNA into various cancer cells with low toxicity. Chitosan is a natural cationic polysaccharide and is able to form polyelectrolyte complexes with DNA, in which the DNA is condensed and protected against nuclease degradation. Based on these facts, chitosan was used as a condensing agent to enhance the transfection efficiency of cationic emulsion-mediated gene delivery vehicle. The particle size, zeta potential and transmission electron micrographs of DNA/emulsion complexes were observed before and after condensation by chitosan. In vitro transfection efficiency of naked or precondensed DNA/emulsion (pcDNA/E) complexes was investigated in human hepatoma cells (HepG2) using flow cytometer, confocal microscope and western blot. In addition, in vivo gene transfer was also evaluated as GFP mRNA expression by reverse transcriptase-polymerase chain reaction. The size of transfection complexes was reduced after the condensation of DNA by chitosan. Moreover, when the pcDNA/E complexes were administered into the mice, the GFP mRNA expression was prolonged in liver and lung until day 6. These results suggest that the use of chitosan enhance the in vitro transfection efficiency and extend in vivo gene transfer.

In vitro and in vivo transfection efficiency of a novel ultradeformable cationic liposome.

Cationic lipids have been often used as one of the major components in making most promising non-viral gene delivery systems, whereas sodium cholate, a surfactant so-called edge activator has been used in preparing ultradeformable and ultraflexible liposomes called Transfersomes. Using both a cationic lipid, DOTAP and sodium cholate, a novel formulation of ultradeformable cationic liposome (UCL) has been prepared. The average particle size of this formulation was approximately 80 nm. The physical and chemical stabilities at two different temperatures (4 degrees C and 20 degrees C) were also evaluated for 60 days. The ultradeformability of new formulation was also assessed, and it has been proved that the formulation is deformable. In vitro transfection efficiency of plasmid DNA/UCL was assessed by the expression of green fluorescent protein (GFP) in four cell lines, OVCAR-3 (human ovarian carcinoma cells), HepG2 (human hepatoma cells), H-1299 (human lung carcinoma cells) and T98G (human brain carcinoma cells). The optimal ratio of DNA to liposome for maximal transfection efficiency was 1:14 (w/w) in all the cell lines except for the human brain carcinoma cells. The same formulation was tested for in vivo transfection efficiency and its retention time within the organs by applying the DNA/UCL complexes on hair-removed dorsal skin of mice non-invasively. It was found that genes were transported into several organs for 6 days once applied on intact skin.

Effects of chlorinated aliphatic hydrocarbons on the fidelity of cell division in human CYP2E1 expressing cells.

Chlorinated organic chemicals are widely used in industry and are present in the environment. Five chlorinated aliphatic hydrocarbons, namely 1-2-dichloroethane, 1,1,2-trichloroethane, trichloroethylene, 2,3-dichlorobutane and 1-chlorohexane were investigated to determine their influence upon the fidelity of cell division in cultured mammalian cells. In order to determine the influence of these chemical compounds upon the fidelity of cell division, a technique known as differential staining of chromosomes and spindle was performed with one genetically engineered cell line and its parental cell line. The genetically engineered cell line used in this study expressed a human P450 enzyme, CYP2E1. Four chemicals, 1-2-dichloroethane, trichloroethylene, 2,3-dichlorobutane and 1-chlorohexane required metabolic bioactivations in order to induce spindle damage in cultured mammalian cells whereas 1,1,2-trichloroethane was a direct-acting spindle poison.