Time-Resolved Fluorescence Spectroscopy Reveals Fine Structure and Dynamics of Poly(l-lysine) and Polyethylenimine Based DNA Polyplexes.

Structural dynamics of the polyethylenimine-DNA and poly(l-lysine)-DNA complexes (polyplexes) was studied by steady-state and time-resolved fluorescence spectroscopy using the fluorescence resonance energy transfer (FRET) technique. During the formation of the DNA polyplexes, the negative phosphate groups (P) of DNA are bound by the positive amine groups (N) of the polymer. At N/P ratio 2, nearly all of the DNA's P groups are bound by the polymer N groups: these complexes form the core of the polyplexes. The excess polymer, added to this system to increase the N/P ratio to the values giving efficient gene delivery, forms a positively charged shell around the core polyplex. We investigated whether the exchange between the core and shell regions of PEI and PLL polyplexes takes place. Our results demonstrated a clear difference between the two studied polymers. Shell PEI can replace PEIs previously attached to DNA in the polyplex core, while PLL cannot. Such a dynamic structure of PEI polyplexes compared to a more static one found for PLL polyplexes partially explains the observed difference in the DNA transfection efficiency of these polyplexes. Moreover, the time-resolved fluorescence spectroscopy revealed additional details on the structure of PLL polyplexes: in between the core and shell, there is an intermediate layer where both core and shell PLLs or their parts overlap.

Isolation of Intact and Functional Melanosomes from the Retinal Pigment Epithelium.

Melanosomes of retinal pigment epithelium (RPE) have many vision supporting functions. Melanosome research would benefit from a method to isolate pure and characterized melanosomes. Sucrose gradient centrifugation is the most commonly used method for isolation of RPE melanosomes, but the isolated products are insufficiently characterized and their quality is unclear. Here we introduce a new gentle method for fractionation of porcine RPE that produces intact functional melanosomes with minimal cross-contamination from other cell organelles. The characterization of isolated organelles was conducted with several methods confirming the purity of the isolated melanosomal fraction (transmission electron microscopy, immunoblotting) and presence of the melanosomal membrane (fluorescence staining of melanosomal membrane, zeta potential measurement). We demonstrate that our isolation method produces RPE melanosomes with the ability to generate free phosphate (Pi) from ATP thereby proving that many membrane proteins remain functional after isolation. The isolated porcine RPE melanosomes represented V-type H+ATPase activity that was demonstrated with bafilomycin A1, a specific V-ATPase inhibitor. We anticipate that the isolation method described here can easily be optimized for the isolation of stage IV melanosomes from other pigmented cell types and tissues.

Photopolymerized micelles of diacetylene amphiphile: physical characterization and cell delivery properties.

A series of polydiacetylene (PDA) - based micelles were prepared from diacetylenic surfactant bearing polyethylene glycol, by increasing UV-irradiation times. These polymeric lipid micelles were analyzed by physicochemical methods, electron microscopy and NMR analysis. Cellular delivery of fluorescent dye suggests that adjusting the polymerization state is vital to reach the full in vitro potential of PDA-based delivery systems.