Synthesis and Characterization of Protein-Conjugated Silver Nanoparticles/Silver Salt Loaded Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) Film for Prevention of Bacterial Infections and Potential Use in Bone Tissue Engineering Applications

Failure of orthopedic implants due to bacterial infection has been a major concern in bone tissue engineering. To this end, we have formulated a potential orthopedic implant made of naturally occurring biodegradable polymer, i.e. poly (3-hydroxylbutyrate-co-3-hydroxylvalerate) (PHBV), modified with BSA conjugated silver nanoparticles and or silver chloride. Upon release of Ag NPs and or Ag+ in the implant region, can promote aseptic environment by inhibition of bacteria growth and also support/maintain bone cell adhesion, growth, and proliferation.;For formulating nanoparticles loaded PHBV scaffold, we exploit specific interaction between bovine serum albumin (BSA) of BSA capped silver nanoparticles and collagen of collagen immobilized PHBV scaffold. Therefore, the first part of this study dealt with synthesis and characterization of collagen immobilized PHBV film for loading of BSA stabilized silver (Ag/BSA) nanoparticles. Two different approaches were used to immobilize collagen on macroporous PHBV film. First approach uses thermal radical copolymerization with 2-hydroxyethylmethacrylate (HEMA), while the second approach uses aminolysis to functionalize macroporous PHBV film. Using collagen crosslinker, type I collagen was covalently grafted to formulate collagen immobilized PHEMA-g-PHBV and collagen immobilized NH2-PHBV films, respectively. Spectroscopic (FTIR, XPS), physical (SEM), and thermal (TGA) techniques were used to characterize the functionalized PHBV films. The Ag/BSA nanoparticles were then loaded on collagen immobilized PHBV films and untreated PHBV films. The concentration of nanoparticles loaded on PHBV film was determined by atomic absorption spectrometry and fluorescence spectroscopy. The amount of nanoparticles loaded on collagen immobilized PHBV film was found to be significantly greater than that on untreated PHBV film. The amount of Ag/BSA nanoparticles loaded on collagen immobilized PHBV film was found to depend on the concentration of Ag/BSA nanoparticles solution used for loading and on the molecular weight of type I collagen used in collagen immobilization on PHBV film. At physiological pH, optimum amount of nanoparticles was retained on Type I collagen immobilized PHBV film because at pH 7.4, protonated amino groups of collagen immobilized PHBV film promote strong electrostatic interaction with the carboxylate anions of BSA stabilized silver nanoparticles.;The second part of this study dealt with formulating AgCl/PHBV film that can potentially release silver ions for effective antimicrobial activity. In this study, we formulated AgCl/PHBV composite film by a salt exchange mechanism. Thermogravimetric analysis (TGA) was used to quantify the amount of NaCl present before and after salt exchange. The Na content in the pre-washed and partially washed NaCl/PHBV film was found to be 43.60% and 1.24% by mass, respectively. The AgCl/PBHV composite film was acid digested and assayed for Na+ and Ag+ content by using Atomic Absorption Spectrometry (AAS) and was found to be 2.15 and 10.25 ppm, respectively. XPS technique was used to characterize the surface elemental composition of the AgCl/PHBV composite film. The survey spectrum of AgCl/PHBV film showed emergence of Ag 3d and Cl 2s peaks compared to pure PHBV which was predominantly composed of C 1s and O 1s peaks. The release kinetics of silver ions from AgCl/PHBV composite film showed an initial burst of about 1.5 ppm of silver ions during the first day of desorption followed by a gradual release of silver ions at an average rate of 0.3 ppm per day during the span of two weeks studied.;Ag/BSA nanoparticles loaded collagen immobilized PHBV films and AgCl/PHBV composite films were tested for antibacterial efficacy against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Colony forming unit and optical density measurements of Ag/BSA nanoparticles loaded collagen immobilized PHBV films showed broad antimicrobial activity at low Ag/BSA nanoparticles concentration (0.19and 0.31 microg) compared to commercially available gentamicin and sulfamethoxazole/trimethoprim which showed sometimes selective antimicrobial activity and antimicrobial activity at high concentration (10 microg and 23.75/1.25 microg/disc). Additionally, a clear zone of inhibition around AgCl/PHBV composite film was noticed on a modified Kirby-Bauer disk diffusion assay. Optical density results and colony forming unit measurements showed that AgCl/PHBV composite film exhibit broad bactericidal activity.;Next, we evaluated the cytotoxicity of Ag/BSA nanoparticles loaded collagen immobilized PHBV films and AgCl/PHBV composite films towards MC3T3-E1 cells at the same concentration both films showed broad antimicrobial activity. By using MTT assay, we established that Ag/BSA nanoparticles loaded collagen immobilized PHBV film showed minimal, if any, cytotoxic effect towards MC3T3-E1 cells while AgCl/PHBV composite film showed significant cytotoxic effect compared to tissue culture polystyrene.;Our research findings provide several formulations for preparation of scaffold, if properly tuned; it can be used as a potential biocompatible and biodegradable scaffold for the prevention of bacterial infections and promotion of cell attachment and proliferation in bone tissue engineering applications.