The basic premise of gene therapy is that genes can be used to produce in situ therapeutic proteins. The controlled delivery of DNA complexes from biomaterials offers the potential to enhance gene transfer by maintaining an elevated concentration of DNA within the cellular microenvironment. Immobilization of the DNA to the substrate to which cells adhere maintains the DNA in the cell microenvironment for subsequent cellular internalization. Here, layer-by-layer (LBL) films made from poly(L-glutamic acid) (PLGA) and poly(L-lysine) (PLL) containing DNA were built in the presence of charged cyclodextrins. The biological activities of these polyelectrolyte films were tested by means of induced production of a specific protein in the nucleus or in the cytoplasm by cells in contact with the films. This type of coating offers the possibility for either simultaneous or sequential interfacial delivery of different DNA molecules aimed at cell transfection. These results open the route to numerous potential applications in patch vaccination, for example.gene delivery ͉ layer-by-layer films ͉ transfection T he basic premise of somatic gene therapy is that genes can be used to cause in vivo production of therapeutic proteins. Controlled and efficient gene delivery has implications in many fields ranging from basic science to clinical medicine. Current strategies to enhance gene delivery involve the complexation of DNA with cationic polymers or lipids delivery. Cationic polymers or lipids can self-assemble with DNA to form particles that are capable of being endocytosed by cells (1). These complexes reduce effective size and cellular degradation of DNA (2) and are often delivered as a bolus, added to culture wells in vitro. Bolus delivery of these complexes can be hindered by mass transport limitations or deactivation processes, such as degradation or aggregation. For example, in vitro studies have estimated that bolus addition of complexes to the culture media results in internalization of only 20% of the DNA added (3). These limitations motivated the development of alternative delivery strategies.The controlled delivery of DNA complexes from biomaterials offers the potential to enhance gene transfer by maintaining an elevated concentration of DNA within the cellular microenvironment (4). DNA delivery systems from biomaterials are designed to maintain elevated concentrations locally by supplying DNA to balance the loss by degradation. The continued presence of the DNA during cell division seems to facilitate entry into the nucleus (5). In recent years, considerable effort has been devoted to the design and the controlled fabrication of structured materials with functional properties (6). The layer-by-layer (LBL) buildup of polyelectrolyte films from oppositely charged polyelectrolytes (7) offers opportunities for the preparation of functionalized biomaterial coatings. This technique allows the preparation of supramolecular nano-architectures (8-13) exhibiting specific properties in terms of control of cell activation (8-11) ...

The alternate layer‐by‐layer (LBL) deposition of polycations and polyanions for the build up of multilayered polyelectrolyte films is an original approach that allows the preparation of tunable, biologically active surfaces. The resulting supramolecular nanoarchitectures can be functionalized with drugs, peptides, and proteins, or DNA molecules that are able to transfect cells in vitro. We monitor, for the first time, the embedding of a bioactive adenoviral (Ad) vector in multilayered polyelectrolyte films. Ad efficiently adsorbs on poly(L‐lysine)–poly(L‐glutamic acid) (PLL–PGA), PLL–HA (HA: hyaluronan), poly(allylamin hydrochloride)–poly(sodium‐4‐styrenesulfonate) (PAH–PSS), and CHI–HA (CHI: chitosan) films; it preserves its transduction capacity (which can reach 95 %) for a large number of cell types, and also allows vector uptake into receptor‐deficient cells, thus abrogating the restricted tropism of Ad.

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In this study, the binding of F components of the staphylococcal bicomponent leukotoxins Panton-Valentine leucocidin (LukF-PV) and gamma-hemolysin (HlgB) on polymorphonuclear neutrophils (PMNs), monocytes, and lymphocytes was determined using labeled mutants and flow cytometry. Leukotoxin activity was evaluated by measuring Ca 2؉ entry or pore formation using spectrofluorometry or flow cytometry. Although HlgB had no affinity for cells in the absence of an S component, LukF-PV had high affinity for PMNs (dissociation constant Staphylococcus aureus secretes water-soluble protein monomers which assemble to form well-characterized two-component pore-forming leukotoxins that define a toxin subfamily (17). Each leukotoxin is formed by a class S protein (LukS-PV, HlgA, HlgC, or LukE) that is associated with a class F protein (LukF-PV, HlgB, or LukD). The Panton-Valentine leucocidin (PVL) (16), composed of LukS-PV and LukF-PV, is secreted by strains isolated from humans suffering from abscesses, furuncles (6, 8), and necrotizing pneumonia (11,12). PVL has been shown to contribute to the severity of acute hematogenous osteomyelitis caused by community-acquired methicillinresistant S. aureus in children (2). Moreover, reports of hospital-acquired and community-acquired methicillin-resistant S. aureus possessing PVL genes are increasing (3). The LukELukD-secreting S. aureus strains have been shown to be associated with postantibiotic diarrhea (10). However, gamma-hemolysin (HlgA-HlgB and HlgC-HlgB) is produced by all human clinical S. aureus strains.[The main target cells of staphylococcal leukotoxins are human polymorphonuclear cells (PMNs), monocytes, and lymphocytes (13). However, PVL is not toxic for lymphocytes, and gamma-hemolysin is hemolytic. After binding to the membrane, leukotoxins assemble as a ring-shaped prepore (15) consisting of heterologous octamers with a molar ratio of 1:1 (14). They induce an increase in the intracellular Ca 2ϩ level by opening Ca 2ϩ channels (19, 1), and the deployment of stems forms beta-barrels that create transmembrane pores in target cells (7, 1). Previous perfusion studies of PMNs showed that the initial binding of S components was a prerequisite for the binding of F components to obtain toxic activity on PMNs (4). Conversely, Yokota and Kamio (22) showed that the binding of the F component LukF (HlgB) on human erythrocytes was a prerequisite for the subsequent binding of Hlg2 (HlgA). Later, Gauduchon et al. (9) determined a dissociation constant (K d ) of 0.07 nM for LukS-PV binding on PMNs and showed that HlgC competed with LukS-PV for binding but HlgA and LukE did not compete. However, no data are available yet for the binding of F components.The present study was performed to analyze the binding of the F components of leukotoxins, particularly the binding of LukF-PV compared with that of HlgB, two F components which exhibit 71% identity. LukF-PV* and HlgB* leukotoxins with cysteine mutations were labeled with fluorescein to follow their binding to human leukocytes by flow cy...