In this communication, anti-streptavidin M13 viruses were used to self-assemble various nanosized materials. We believe the anti-streptavidin M13 viruses provide a convenient method to organize a variety of nanosized materials into self-assembled ordered structures. Because the modification of the DNA insert allows controlled modification of the virus length, the spacing in the smectic layer can be genetically controlled.[12] By conjugating other nanosized materials (magnetic nanoparticles, II±VI semiconductor nanoparticles, functional chemicals, etc.) with streptavidin, we believe that this anti-streptavidin method can be used to align various nanosized materials at the desired length scale, which is defined by the smectic layers. ExperimentalThe anti-streptavidin virus was selected by a phage display method using a M13 bacteriophage library (New England Biolab). The virus was amplified in a large volume (400 mL scale, 7 10 7 pfu/lL). The virus suspension was precipitated into a pellet. 20 mg of the virus pellet was suspended with 1.0 mL of 10 nm gold nanoparticles (Abs: 2.5 at 520 nm), conjugated with a streptavidin colloidal suspension (Sigma Co.), and agitated using a rocker for 1 day. The viruses conjugated with gold nanoparticles (Au±virus) were centrifuged after adding 167 lL of poly(ethylene glycol) solution. The red colored pellet was suspended using~20 lL of Tris-buffered saline solution (pH 7.5) to form a Au± virus liquid-crystalline suspension (virus concentration: 83.2 mg mL ±1 ). In order to fabricate the Au±virus film, the Au±virus suspension was diluted tõ 6 mg mL ±1 (400 lL) and kept dry in a dessicator for two weeks. Fluorescein-Virus Cast Film Fabrication: 20 lL of virus suspension (1.9 10 ±7 M in Tris±HCl saline buffered solution (pH 7.5)) was mixed with 20 lL of 0.01 mg mL ±1 (1.9 10 ±7 M, MW: 53 200) fluorescent-streptavidin suspension. 1 lL of suspension was cast and dried on the glass substrate. The molarity of virus suspension was measured using a UV-vis spectrophotometer (extinction coefficient: 1.2 10 8 M ±1 cm ±1 at 268 nm) [13]. Phycoerythrin-Virus and Cast Film Fabrication: 20 lL of the virus suspension (~6 mg mL ±1 , 1.9 10 ±7 M, MW: 292 800 Tris±HCl saline buffered solution (pH 7.5)) was mixed with 20 lL of 0.05 mg mL ±1 (1.7 10 ±7 M in Tris±HCl saline-buffered solution (pH 7.5) with 5 % sucrose) of R-phycoerythrin-streptavidin. 1 lL of suspension was cast and dried on the glass substrate.Microscopy: POM images were obtained using an Olympus polarized optical microscope. Images were taken using a SPOT Digital camera (Diagnostic Inc.). Scanning laser microscopy images was obtained using a Leica TCS 4D and SEM images were obtained using LEO1530, operating at an accelerating voltage of 1 kV. TEM images were obtained using a Philips 208 at an accelerating voltage of 80 kV and a JEOL 2010F at 200 kV. The AFM images (Digital Instruments) were taken in air using tapping mode. The AFM probes were etched silicon with 125 lm cantilevers and spring constants of 20±100 N m ±1 driven near their...

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) ...

This article demonstrates the possibility of tuning the degradability of polysaccharide multilayer films in vitro and in vivo. Chitosan and hyaluronan multilayer films (CHI/HA) were either native or crosslinked using a water soluble carbodiimide, 1‐ethyl‐3‐(3‐dimethylamino‐propyl)carbodiimide (EDC) at various concentrations in combination with N‐hydroxysulfosuccinimide. The in‐vitro degradation of the films in contact with lysozyme and hyaluronidase was followed by quartz crystal microbalance measurements, fluorimetry, and confocal laser scanning microscopy after labeling of the chitosan with fluorescein isothiocyanate (CHIFITC). The native films were subjected to degradation by these enzymes, and the crosslinked films were more resistant to enzymatic degradation. Films made of chitosan of medium molecular weight were more resistant than films made of chitosan‐oligosaccharides. The films were also brought in contact with plasma, which induced a change in film structure for the native film but did not have any effect on the crosslinked film. The in‐vitro study shows that macrophages can degrade all types of films and internalize the chitosan. The in‐vivo degradation of the films implanted in mouse peritoneal cavity for a week again showed an almost complete degradation of the native films, whereas the crosslinked films were only partially degraded. Taken together, these results suggest that polysaccharide multilayer films are of potential interest for in‐vivo applications as biodegradable coatings, and that degradation can be tuned by using chitosan of different molecular weights and by controlling film crosslinking.