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BackgroundGene transfer using non-viral vectors offers a non-immunogenic and safe method of gene delivery. Cellular uptake and intracellular trafficking of the nanoparticles can impact on the transfection efficiency of these vectors. Therefore, understanding the physicochemical properties that may influence the cellular uptake and the intracellular trafficking can aid the design of more efficient non-viral gene delivery systems. Recently, we developed novel amino acid-substituted gemini surfactants that showed higher transfection efficiency than their parent compound. In this study, we evaluated the mechanism of cellular uptake of the plasmid/gemini surfactant/helper lipid nanoparticles and their effect on the transfection efficiency.ResultsNanoparticles were incubated with Sf 1 Ep cells in the presence of different endocytic inhibitors and gene expression (interferon-γ) was measured using ELISA. Clathrin-mediated and caveolae-mediated uptake were found to be equally contributing to cellular internalization of both P/12-7NH-12/L (parent gemini surfactant) and P/12-7NGK-12/L (amino acid-substituted gemini surfactant) nanoparticles. The plasmid and the helper lipid were fluorescently tagged to track the nanoparticles inside the cells, using confocal laser scanning microscopy. Transmission electron microscopy images showed that the P/12-7NGK-12/L particles were cylindrical while the P/12-7NH-12/L particles were spherical which may influence the cellular uptake behaviour of these particles. Dye exclusion assay and pH-titration of the nanoparticles suggested that high buffering capacity, pH-dependent increase in particle size and balanced DNA binding properties may be contributing to a more efficient endosomal escape of P/12-7NGK-12/L compared to the P/12-7NH-12/L nanoparticles, leading to higher gene expression.ConclusionAmino-acid substitution in the spacer of gemini surfactant did not alter the cellular uptake pathway, showing similar pattern to the unsubstituted parent gemini surfactant. Glycyl-lysine substitution in the gemini spacer improved buffering capacity and imparted a pH-dependent increase of particle size. This property conferred to the P/12-7NGK-12/L nanoparticles the ability to escape efficiently from clathrin-mediated endosomes. Balanced binding properties (protection and release) of the 12-7NGK-12 in the presence of polyanions could contribute to the facile release of the nanoparticles internalized via caveolae-mediated uptake. A more efficient endosomal escape of the P/12-7NGK-12/L nanoparticles lead to higher gene expression compared to the parent gemini surfactant.

The aim of this work was to elucidate the structure-activity relationship of new peptide-modified gemini surfactant-based carriers. Glycyl-lysine modified gemini surfactants that differ in the length and degree of unsaturation of their alkyl tail were used to engineer DNA nano-assemblies. To probe the optimal nitrogen to phosphate (N/P) ratio in the presence of helper lipid, in vitro gene expression and cell toxicity measurements were carried out. Characterization of the nano-assemblies was accomplished by measuring the particle size and surface charge. Morphological characteristics and lipid organization were studied by small angle X-ray scattering technique. Lipid monolayers were studied using a Langmuir-Blodgett trough. The highest activity of glycyl-lysine modified gemini surfactants was observed with the 16-carbon tail compound at 2.5 N/P ratio, showing a 5- to 10-fold increase in the level of reporter protein compared to the 12 and 18:1 carbon tail compounds. This ratio is significantly lower compared to the previously studied gemini surfactants with alkyl or amino- spacers. In addition, the 16-carbon tail compound exhibited the highest cell viability (85%). This high efficiency is attributed to the lowest critical micelle concentration of the 16-tail gemini surfactant and a balanced packing of the nanoparticles by mixing a saturated and unsaturated lipid together. At the optimal N/P ratio, all nanoparticles exhibited an inverted hexagonal lipid assembly. The results show that the length and nature of the tail of the gemini surfactants play an important role in determining the transgene efficiency of the delivery system. We demonstrated here that the interplay between the headgroup and the nature of tail is specific to each series, thus in the process of rational design, the contribution of the latter should be assessed in the appropriate context.

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The γ- diimine 1,2-(2,6-i Pr 2 -C 6 H 3 NC) 2 -C 6 H 4 (2) was synthesized by the reaction of phthalaldehyde with 2,6-diisopropylaniline. Depending on reaction conditions, 2 can cyclize to form the corresponding iminoisoindoline or isoindolinone. Unlike analogous Rand β-diimine complexes, reaction of the γ-diimine 2 with (MeCN) 2 PdCl 2 results in a dinuclear complex, [(γ-diimine)PdCl(µ-Cl)] 2 (5), where the ligand does not coordinate to the Pd(II) center in a chelating fashion but instead adopts a monodentate coordination mode. On the other hand, reaction of 2 with Pd(OAc) 2 results in C-H activation and formation of the trinuclear Pd 3 (OAc) 4 -based palladacycle {1,2-(2,6-i Pr 2 -C 6 H 3 NC) 2 -C 6 H 3 ]Pd(µ-OAc) 2 } 2 Pd (6). The resulting palladium complexes were tested as precatalysts in Heck and Suzuki coupling reactions.

In this work, four poly­(glycidyl methacrylate-co-ethylene glycol methacrylate) [PGMA-co-EDGMA] functionalized particles were synthesized and characterized, and tested in gas oil adsorption. Particles consisted of identical polymer support (PGMA-co-EDGMA, henceforth referred to as PGMA) and π-acceptor moiety (2,4,5,7-tetranitroflorenone, TENF), while the linkers (linear diamines) were varied from a two- (diaminoethane, DAE (2)), a three- (diaminopropane, DAP­(3)) to a four-carbon (diaminobutane, DAB­(4)) containing compounds. The particles notation takes the form: PGMA-DAE­(2)-TENF, PGMA-DAP­(3)-TENF, PGMA-DAB­(4)-TENF and PGMA-DAB­(4)­5-TENF, where 5 denotes the synthesis in 5% (vol./vol.) linker and 95% (vol./vol.) toluene solution, while for other three, 100% (vol./vol.) linker was used. Particles were characterized by, among others, Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), elemental carbon, hydrogen, nitrogen and sulfur analysis (CHNS) and scanning electron microscopy (SEM). The change in linker length did not result in a significant effect on loading as similar amounts of TENF were immobilized on the polymeric particles regardless of the linker length. From the adsorption studies of light gas oil (LGO), results showed that diaminopropane (PGMA-DAP­(3)-TENF) substituted particles gave the highest % adsorption of nitrogen compounds, followed by the diaminobutane (PGMA-DAB­(4)-TENF)-based particles, while no significant differences between the particles with diaminoethane (PGMA-DAE­(2)-TENF) and PGMA-DAB­(4)­5-TENF were observed. The results further showed that all the four types of particles selectively adsorbed nitrogen compounds, while the sulfur concentration in LGO remained unchanged under the following adsorption process conditions: ambient temperature (24 °C), time (24 h), particles to oil loading ratio (1:4 wt./wt.) and stirring speed (400 rpm). Generally, steric hindrance around the TENF molecules, on the surface of the particles, had a major role to play in the adsorption process than the length of the linker.

This provides a proof-of-concept that amino acid substituted gemini surfactants can be used as noninvasive mucosal (vaginal) gene delivery systems to treat diseases associated with mucosal epithelia.

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