Bioactive Coatings Based on a Polyelectrolyte Multilayer Architecture Functionalized by Embedded Proteins

Abstract

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...

Active multilayered capsules for in vivo bone formation

“…We have shown that when in contact with planar multilayered films, the cells locally degrade the film (by the secreted proteases able to degrade PLL and PGA) and develop pseudopods. By these two mechanisms, local degradation and chimiotactism, the cells interact with active molecules incorporated into the multilayered film (21). We have also shown that by using the enantiomer D of PLL and PGA, the cells cannot degrade the film and are not able to interact with embedded active molecules into the multilayered film (21).…”

“…Previously, we determined the mechanism by which cells come in contact and interact with active proteins, peptides, drug, or DNA incorporated into the multilayered PLL/PGA films (21)(22)(23)(24). We have shown that when in contact with planar multilayered films, the cells locally degrade the film (by the secreted proteases able to degrade PLL and PGA) and develop pseudopods.…”

“…PEM films are prepared by the layer-by-layer (LbL) deposition of interacting materials, typically by the electrostatic interaction of oppositely charged polyelectrolytes (20). Therapeutics and biomolecules including peptides, proteins, and nucleic acid have been embedded in PEM films, which offer new opportunities for the preparation of functionalized bioactive coatings (19)(20)(21). These supramolecular nanoarchitectures can be designed to exhibit specific properties, including control of cell activation, inflammation (22,23) and localized drug, growth factor or nucleic acid delivery (24,25).…”

UV degradation of deoxyribonucleic acid

“…A simple and versatile method for producing these architectures is the sequential build up of layers of functional materials by the layer-by-layer (LBL) technique [12,13]. This technique, initially applied to the production of polyelectrolyte thin films, has also been found to be suitable for the production of functionalized biomolecular architectures [14,15,16,17,18] and is therefore a relevant methodology for producing biological mimics to address radiation damage studies. However, it is fundamental to characterize the radiation degradation of the biological macromolecules in vacuum.…”

Biocompatible polypeptide microcapsules via templating mesoporous silica spheres

“…polypeptides (e.g., PLL, PGA) [11][12][13][14]. For example, multilayer films of dextran/chitosan have the properties of pro-or anti-coagulation depending on the outmost layer of the film [11].…”