Multilayered polyelectrolyte films fabricated from plasmid DNA and a hydrolytically degradable synthetic polycation can be used to direct the localized transfection of cells without the aid of a secondary transfection agent. Multilayered assemblies 100 nm thick consisting of alternating layers of synthetic polymer and plasmid DNA encoding for enhanced green fluorescent protein (EGFP) were deposited on quartz substrates using a layer-by-layer fabrication procedure. The placement of film-coated slides in contact with COS-7 cells growing in serum-containing culture medium resulted in gene expression in cells localized under the film-coated portion of the slides. The average percentage of cells expressing EGFP relative to the total number of cells ranged from 4.6% to 37.9%, with an average of 18.6%+/-8.2%, as determined by fluorescence microscopy. In addition to providing a mechanism for the immobilization of DNA at the cell/surface interface, a preliminary analysis of film topography by atomic force microscopy (AFM) demonstrated that polymer /DNA films undergo significant structural rearrangements upon incubation to present surface bound condensed plasmid DNA nanoparticles. These data suggest that the presence of the cationic polymer in these materials may also contribute to the internalization and expression of plasmid. The materials and design principles reported here present an attractive framework for the local or non-invasive delivery of DNA from the surfaces of implantable materials or biomedical devices.
"Une fois implanté à l'endroit souhaité de l'organisme hôte, le gel doit résister aux diverses contraintes mécaniques et conserver son volume pendant une durée suffisante tout en transmettant ces contraintes aux cellules encapsulées . Dans le cas classique des gels d'alginate, la valeur du module de compression peut être accrue en augmentant la concentration volumique en alginate ainsi que le rapport du nombre de groupements α-L-guluronique (G dans la figure 3b) au nombre de groupements β-D-mannuronique (M dans la A B Figure 8 -A) Schéma de principe de préparation de l'architecture multicouche consistant à déposer de manière séquentielle le polycation hydrolysable et le plasmide (ici le pEGFP) et de l'hydrolyse progressive du film lorsque celui-ci est placé à pH 7,2 et à 37 °C ; B) Schéma montrant la possibilité de réaliser une « expression génique localisée » lors de la mise en contact d'une lamelle de quartz fonctionnalisée avec l'architecture illustrée en A avec une couche de cellules (d'après ). "
"Solutions of SPS used for film fabrication were prepared by mixing unlabeled commercially available SPS with SPS FL in a ratio of 9:1 (w/w). Films having the structure (LPEI/SPS FL ) 10 (1/DNA) 8 were fabricated layer-by-layer on silicon substrates using methods described in past studies (Fredin et al., 2005; 2007; Jewell et al., 2005; Zhang et al., 2004), such that SPS FL was incorporated into every layer of the LPEI/SPS bilayers used to form the film. Figure 1C shows a LSCM image of a film having this structure imaged prior to incubation. "
[Show abstract][Hide abstract] ABSTRACT: Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) were used to characterize changes in nanoscale structure that occur when ultrathin polyelectrolyte multilayers (PEMs) are incubated in aqueous media. The PEMs investigated here were fabricated by the deposition of alternating layers of plasmid DNA and a hydrolytically degradable polyamine onto a precursor film composed of alternating layers of linear poly(ethylene imine) (LPEI) and sodium poly(styrene sulfonate) (SPS). Past studies of these materials in the context of gene delivery revealed transformations from a morphology that is smooth and uniform to one characterized by the formation of nanometer-scale particulate structures. We demonstrate that in-plane registration of LSCM and AFM images acquired from the same locations of films fabricated using fluorescently labeled polyelectrolytes allows the spatial distribution of individual polyelectrolyte species to be determined relative to the locations of topographic features that form during this transformation. Our results suggest that this physical transformation leads to a morphology consisting of a relatively less disturbed portion of film composed of polyamine and DNA juxtaposed over an array of particulate structures composed predominantly of LPEI and SPS. Characterization by scanning electron microscopy and energy-dispersive X-ray microanalysis provides additional support for this interpretation. The combination of these different microscopy techniques provides insight into the structures and dynamics of these multicomponent thin films that cannot be achieved using any one method alone, and could prove useful for the further development of these assemblies as platforms for the surface-mediated delivery of DNA.
Microscopy Research and Technique 09/2010; 73(9):834-44. DOI:10.1002/jemt.20830 · 1.15 Impact Factor
"This technique allows nanoscale control over the deposition of a large variety of functional materials, which are polymer layers assembled by taking advantage of attractive interactions such as electrostatic interactions, covalent bonding, and hydrogen bonding   . Recently, the LbL method has been used in developing a drug delivery system for the sustained and controlled release of incorporated molecules  . In most such research, multilayered coatings are used for their structural properties by dissociation of polymer layers. "
[Show abstract][Hide abstract] ABSTRACT: Here we describe the functionalization of a multilayered hydrogel layer on a Ti alloy with an antineoplastic agent, paclitaxel (PTX). The multilayered hydrogel was synthesized via layer-by-layer self-assembly (LbL) using selective intermolecular reactions between two water-soluble polymers, phospholipid polymer (PMBV) containing a phenylboronic acid unit and poly(vinyl alcohol) (PVA). Reversible covalent bonding between phenylboronic acid and the polyol provided the driving force for self-assembly. Poorly water-soluble PTX dissolves in PMBV aqueous solutions because PMBV is amphiphilic. Therefore, our multilayered hydrogel could be loaded with PTX at different locations to control the release profile and act as a drug reservoir. The amount of PTX incorporated in the hydrogel samples increased with the number of layers but was not directly proportional to the number of layers. However, as the step for making layers was repeated, the concentration of PTX in the PMBV layers increased. The different solubilities of PTX in PMBV and PVA aqueous solutions allow for the production of multilayered hydrogels loaded with PTX at different locations. In vitro experiments demonstrated that the location of PTX in the multilayered hydrogel influences the start and profile of PTX release. We expect that this rapid and facile LbL synthesis of multilayered hydrogels and technique for in situ loading with PTX, where the location of loading controls the release pattern, will find applications in biomedicine and pharmaceutics as a promising new technique.
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