RESUMO
Understanding the relationship of structural modifications on the assembly and disassembly of synthetic or non-viral gene delivery is crucial with regard to their rational development. This study describes the use of fluorescence correlation spectroscopy (FCS), as a new tool, to investigate the effect of systematic chemical modifications to dicationic N,N-bis(dimethylalkyl)-α,ω-alkanediammonium surfactants (gemini surfactants) on the self-assembly and physical properties of a series of gemini nanoparticles (gemini NPs). A systematic screening of 27 gemini-plasmid (GP) complexes and gemini NPs showed that their final morphology is governed by the pre-compaction of plasmid by the gemini surfactants. The assembly process of gemini-plasmid intermediate complex (GP) and the final gemini NP (or gemini-plasmid-lipid complex, GPL) was monitored by the tracking of the Cy5-labeled plasmid. Based on diffusion properties, GP complexes were larger than gemini NPs (300-500 nm for GP and 200-300 nm for GPLs). Stoichiometric analysis of the raw intensity histograms showed that both GPs and GPLs particles were composed of multiple plasmids. The final GPLs contain fewer plasmids (2-20 per particle) compared to the intermediate GP (5-35 per particle). The addition of phospholipids dispersed and stabilized GPs to form GPL, but the type of phospholipid (DOPE or DD 1:3) had little effect on the final size of the particles. The FCS data were both validated and complemented by the results of studies of dynamic light scattering (DLS), atomic force microscopy (AFM), X-ray scattering and dye-exclusion assays. A model for gemini NP assembly involving supramolecular aggregate intermediates is proposed.
RESUMO
The outermost layer of the skin, known as the stratum corneum (SC), is composed of dead corneocytes embedded in an intercellular lipid matrix consisting of ceramides, free fatty acids, and cholesterol. The high level of organization within this matrix protects the body by limiting the permeation of most compounds through the skin. While essential for its protective functions, the SC poses a significant barrier for the delivery of topically applied pharmaceutical agents. Chemical permeation enhancers (CPEs) can increase delivery of small drug compounds into the skin by interacting with the intercellular lipids through physical processes including extraction, fluidization, increased disorder, and phase separation. However, it is not clear whether these same mechanisms are involved in delivery of biotherapeutic macromolecules, such as proteins. Here we describe the effect of three categories of CPEs {solvents [ethanol, propylene glycol, diethylene glycol monoethyl ether (transcutol), oleic acid], terpenes [menthol, nerol, camphor, methyl salicylate], and surfactants [Tween 80, SDS, benzalkonium chloride, polyoxyl 40 hydrogenated castor oil (Cremophor RH40), didecyldimethylammonium bromide (DDAB), didecyltrimethylammonium bromide (DTAB)]} on the lipid organizational structure of human SC as determined by X-ray scattering studies. Small- and wide-angle X-ray scattering studies were conducted to correlate the degree of structural changes and hydrocarbon chain packing in SC lipids caused by these various classes of CPEs to the extent of permeation of interferon alpha-2b (IFNα), a 19 kDa protein drug, into human skin. With the exception of solvents, propylene glycol and ethanol, all classes of CPEs caused increased disordering of lamellar and lateral packing of lipids. We observed that the highest degree of SC lipid disordering was caused by surfactants (especially SDS, DDAB, and DTAB) followed by terpenes, such as nerol. Interestingly, in vitro skin permeation studies indicated that, in most cases, absorption of IFNα was low and that an increase in SC lipid disorder does not correspond to an increase in IFNα absorption.