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...

We report the development of new bioactive coatings of biomaterials based on the alternate deposition of oppositely charged polyelectrolytes. We selected polylysine (PLL) and poly(glutamic acid) (PGA) for the polyelectrolytes and murine melanoma cells as a biological test model system. These cells respond specifically to a small peptide hormone, alpha-melanocortin, which is a potent stimulator of melanogenesis. We show that a synthetic alpha-melanocortin derivative, covalently coupled to PLL forming the outer layer of a multilayer film remains as biologically active as the free hormone. Furthermore, the long time activity of the hormone is maintained when embedded in multilayer architectures whereas its short time activity depends on integration depth. The embedding of bioactive molecules not only anchors them irreversibly on the biomaterial, but opens also the possibility to control their activity. In comparison to conventional coating methods, polyelectrolyte multilayers are easy to prepare and retain their biological activity after storage as dry material. These very flexible systems allow broad medical applications for implant and tissue engineering.

Adhesion of bacteria at the surface of implanted materials is the first step in microbial infection, leading to post-surgical complications. In order to reduce this adhesion, we show that poly(L-lysine)/poly(L-glutamic acid) (PLL/PGA) multilayers ending by several PLL/PGA-g-PEG bilayers can be used, PGA-g-PEG corresponding to PGA grafted by poly(ethylene glycol). Streaming potential and quartz crystal microbalance-dissipation measurements were used to characterize the buildup of these films. The multilayer films terminated by PGA and PGA-g-PEG were found to adsorb an extremely small amount of serum proteins as compared to a bare silica surface but the PGA ending films do not reduce bacterial adhesion. On the other hand, the adhesion of Escherichia coli bacteria is reduced by 72% on films ending by one (PLL/PGA-g-PEG) bilayer and by 92% for films ending by three (PLL/PGA-g-PEG) bilayers compared to bare substrate. Thus, our results show the ability of PGA-g-PEG to be inserted into multilayer films and to drastically reduce both protein adsorption and bacterial adhesion. This kind of anti-adhesive films represents a new and very simple method to coat any type of biomaterials for protection against bacterial adhesion and therefore limiting its pathological consequences.

Infection of implanted materials by bacteria constitutes one of the most serious complications following prosthetic surgery. In the present study, we developed a new strategy based on the insertion of an antimicrobial peptide (defensin from Anopheles gambiae mosquitoes) into polyelectrolyte multilayer films built by the alternate deposition of polyanions and polycations. Quartz crystal microbalance and streaming potential measurements were used to follow step by step the construction of the multilayer films and embedding of the defensin within the films. Antimicrobial assays were performed with two strains: Micrococcus luteus (a gram-positive bacterium) and Escherichia coli D22 (a gram-negative bacterium). The inhibition of E. coli D22 growth at the surface of defensin-functionalized films was found to be 98% when 10 antimicrobial peptide layers were inserted in the film architecture. Noticeably, the biofunctionalization could be achieved only when positively charged poly(L-lysine) was the outermost layer of the film. On the basis of the results of bacterial adhesion experiments observed by confocal or electron microscopy, these observations could result from the close interaction of the bacteria with the positively charged ends of the films, which allows defensin to interact with the bacterial membrane structure. These results open new possibilities for the use of such easily built and functionalized architectures onto any type of implantable biomaterial. The modified surfaces are active against microbial infection and represent a novel means of local host protection.