Rational design of antimicrobial peptide conjugated graphene-silver nanoparticle loaded chitosan wound dressing.

Wound dressing with poor antibacterial properties, the tendency to adhere to the wound site, poor mechanical strength, and lack of porosity and flexibility are the major cause of blood loss, delayed wound repair, and sometimes causes death during the trauma or injury. In such cases, hydrogel-based antibacterial wound dressing would be a boon to the existing dressing as the moist environment will maintain the cooling temperate and proper exchange of atmosphere around the wound. In the present study, the multifunctional graphene with silver and ε-Poly-l-lysine reinforced into the chitosan matrix (CGAPL) was prepared as a nanobiocomposite wound dressing. The contact angle measurement depicted the hydrophilic property of CGAPL nanobiocomposite dressing (water contact angle 42°), while the mechanical property was 78.9 MPa. The antibacterial and cell infiltration study showed the antimicrobial property of CGAPL nanobiocomposite wound dressing. It also demonstrated no cytotoxicity to the L929 fibroblast cells. Chorioallantoic Membrane (CAM) assay showed the pro-angiogenic potential of CGAPL nanobiocomposite wound dressing. In-vitro scratch wound assay confirmed the migration of cells and increased cell adhesion and proliferation within 18 h of culture on the surface of CGAPL nanobiocomposite dressing. Later, the in-vivo study in the Wistar rat model showed that CGAPL nanobiocomposite dressing significantly enhanced the wound healing process as compared to the commercially available wound dressing Tegaderm (p-value <0.01) and Fibroheal@Ag (p-value <0.005) and obtained complete wound closure in 14 days. Histology study further confirmed the complete healing process, re-epithelization, and thick epidermis tissue formation. The proposed CGAPL nanobiocomposite wound dressing thus offers a novel wound dressing material with an efficient and faster wound healing property.

Rationally designed Shewanella oneidensis Biofilm Toilored Graphene-Magnetite Hybrid Nanobiocomposite as Reusable Living Functional Nanomaterial for Effective Removal of Trivalent Chromium.

Sustainable treatment of wastewater containing trivalent chromium (Cr3+) remains a significant challenge owing to the several limitations of the existing methodologies. Herein, combination of biosynthesis and Response Surface Methodology (RSM) for the fabrication and optimization of Shewanella oneidensis biofilm functionalized graphene-magnetite (GrM) nanobiocomposite was adopted as a 'living functional nanomaterial' (viz. S-GrM) for effective removal of Cr3+ ions from aqueous solution. In the biosynthetic process, S. oneidensis cells reduced the GO-akaganeite complex and adhered on the as-synthesized GrM nanocomposite to form S-GrM hybrid-nanobiocomposite. The process parameters for fabrication of S-GrM hybrid-nanobiocomposite was optimized by RSM based on four responses of easy magnetic separation, biofilm formation along with protein, and carbohydrate contents in extracellular polymeric substances (EPS). The morphology and chemical composition of S-GrM hybrid-nanobiocomposite were investigated using various spectroscopic and microscopic analyses and subsequently explored for removal of Cr3+ ions. The hybrid-nanobiocomposite effectively removed 304.64 ± 14.02 mg/g of Cr3+ at pH 7.0 and 30 °C, which is found to be very high compared to the previously reported values. The high surface area of graphene, biofilm biomass of S. oneidensis and plenty of functional groups provided a unique structure to the S-GrM hybrid-nanobiocomposite for efficient removal of Cr3+ through synergistic interaction. The FTIR and zeta potential studies confirmed that electrostatic and chelation/complexation reaction played key roles in the adsorption process. The fabrication of S-GrM nanobiocomposite thus creates a novel hybrid 'living functional nanomaterial' for low cost, recyclable, and sustainable removal of Cr3+ from wastewater.

Antibacterial and Antibiofouling Activities of Antimicrobial Peptide-Functionalized Graphene-Silver Nanocomposites for the Inhibition and Disruption of Staphylococcus aureus Biofilms.

Owing to the emergence of antibiotic-resistant strains, bacterial infection and biofilm formation are growing concerns in healthcare management. Herein, we report an eco-benign strategy for the synthesis and functionalization of graphene-silver (rGOAg) nanocomposites with an antimicrobial peptide (AMP) for the treatment of Staphylococcus aureus infection. The synthesis of rGOAg nanocomposites was carried out by simple microwave reduction, and the as-synthesized rGOAg was covalently functionalized with an AMP. As a natural AMP, poly-l-lysine (PLL) functionalization of rGOAg enhanced the antibacterial efficacy and target specificity against the S. aureus biofilm. The robust bactericidal efficiency and biofilm disruption by AMP-functionalized rGOAg (designated as GAAP) occurred through the "contact-kill-release" mode of action, where the electrostatic interaction with bacterial cells together with intracellular ROS generation induced physical disruption to the cell membrane. The internalization of GAAP into the cytoplasm through the damaged cell membrane caused an outburst of intracellular proteins and DNA. Crystal violet staining along with fluorescence and confocal microscopic images showed an effective inhibition and disruption of the S. aureus biofilm upon treatment with GAAP. PLL functionalization also prevented the dissolution of Ag+ ions and thereby minimized the in vitro toxicity of GAAP to the 3 T6 fibroblast and human red blood cells. The ex vivo rat skin disinfection model further demonstrated the potency of GAAP in eliminating the biofilm formation and disruption of the S. aureus biofilm. The obtained results demonstrated a general approach for designing a functional nanocomposite material to disrupt the mature biofilm and provided a promising strategy for treating bacterial infection.

Fabrication of Chitosan-Reinforced Multifunctional Graphene Nanocomposite as Antibacterial Scaffolds for Hemorrhage Control and Wound-Healing Application.

Accidents on battlefields and roads often lead to hemorrhage and uncontrolled bleeding. Hence, immediate hemorrhage control remains of great importance to reduce mortality and socioeconomic loss. Herein, nanobiocomposite scaffolds (film and sponge) have been fabricated for the first time through the incorporation of a graphene-silver-polycationic peptide (GAP) nanocomposite into chitosan (Cs). Ten different scaffolds viz. Cs, Cs-GAP25, Cs-GAP50, Cs-GAP75, and Cs-GAP100 were prepared in the form of films and sponges. Cs-GAP100 nanobiocomposite sponge exhibited excellent porosity, fluid absorption, and blood clotting capacity, whereas Cs-GAP100 nanobiocomposite film showed excellent mechanical strength and poor degradation property. The presence of graphene in GAP provided a unique mechanical property and prevented the natural degradation, whereas silver nanoparticles and polycationic peptide provided an efficient antimicrobial property to the scaffolds. The high surface area of graphene and the hydrophilic nature of the polycationic peptide also imparted high fluid and blood absorption capacity to Cs-GAP nanobiocomposite scaffolds. The in vitro whole blood clotting assay demonstrated that clotting efficacy improved with the concentration of GAP nanocomposite and Cs-GAP100 nanobiocomposite sponge significantly (p value <0.003) reduced the clotting time to 60 s, as compared to the pristine chitosan dressings. On the other side, the Cs-GAP100 nanobiocomposite film showed an excellent wound-healing property. The Cs-GAP100 nanobiocomposite demonstrated profound antibacterial activity against Escherichia coli and Staphylococcus aureus. The intracellular reactive oxygen species (ROS) assay explained the interfacial interaction of Cs-GAP100 nanobiocomposite and bacterial cells, resulting in cell damage and finally cell death. The obtained information thus provided a novel safe-by-design concept for fabrication of Cs-GAP100 nanobiocomposite scaffolds and demonstrated potential development of antibacterial hemostatic and wound dressing in traumacare management.

Fabrication of Nontoxic Reduced Graphene Oxide Protein Nanoframework as Sustained Antimicrobial Coating for Biomedical Application.

Bacterial colonization on medical devices is a major concern in the healthcare industry. In the present study, we report synthesis of environmental sustainable reduced graphene oxide (rGO) on the large scale through biosynthetic route and its potential application for antibacterial coating on medical devices. HRTEM image depicts formation of graphene nanosheet, while DLS and ζ potential studies reveal that in aqueous medium the average hydrodynamic size and surface charge of rGO are 4410 ± 116 nm and -25.2 ± 3.2 mV, respectively. The Raman, FTIR, and XPS data suggest in situ conjugation of protein with rGO. The as-synthesized rGO protein nanoframework exhibits dose-dependent antibacterial activity and potential of killing of 94% of Escherichia coli when treated with 80 µg/mL of rGO for 4 h. The hemolytic and cytotoxicity studies demonstrate that rGO protein nanoframework is highly biocompatible at the same concentration showing significant antimicrobial properties. The rGO coated on the glass surface obtained through covalent bonding exhibits potent antibacterial activity. Antibacterial mechanism further demonstrates that rGO-protein nanoframework in dispersed state (rGO solution) exerts bactericidal effect through physical disruption accompanied by ROS-mediated biochemical responses. The rGO subsequently entering into the cytoplasm through the damaged membrane causes metabolic imbalance in the cells. In sharp contrast, physical damage of the cell membrane is the dominant antibacterial mechanism of rGO in the immobilized state (rGO coated glass). The obtained results help indepth understanding of the antibacterial mechanism of the biosynthesized rGO and a novel way to develop nontoxic antibacterial coating on medical devices to prevent bacterial infection.

Antibacterial Effects of Biosynthesized Silver Nanoparticles on Surface Ultrastructure and Nanomechanical Properties of Gram-Negative Bacteria viz. Escherichia coli and Pseudomonas aeruginosa.

Understanding the interactions of silver nanoparticles (AgNPs) with the cell surface is crucial for the evaluation of bactericidal activity and for advanced biomedical and environmental applications. Biosynthesis of AgNPs was carried out through in situ reduction of silver nitrate (AgNO3) by cell free protein of Rhizopus oryzae and the synthesized AgNPs was characterized by UV-vis spectroscopy, high resolution transmission electron microscopy (HRTEM), dynamic light scattering (DLS), ζ-potential analysis, and FTIR spectroscopy. The HRTEM measurement confirmed the formation of 7.1 ± 1.2 nm AgNPs, whereas DLS study demonstrated average hydrodynamic size of AgNPs as 9.1 ± 1.6 nm. The antibacterial activity of the biosynthesized AgNPs (ζ = -17.1 ± 1.2 mV) was evaluated against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. The results showed that AgNPs exhibited concentration dependent antibacterial activity and 100% killing of E. coli and P. aeruginosa achieved when the cells were treated with 4.5 and 2.7 µg/mL AgNPs, respectively for 4 h. Furthermore, the intracellular reactive oxygen species (ROS) production suppressed the antioxidant defense and exerted mechanical damage to the membrane. AgNPs also induced surface charge neutralization and altered of the cell membrane permeability causing nonviability of the cells. Atomic force microscopy (AFM) studies depicted alteration of ultrastructural and nanomechanical properties of the cell surface following interaction with AgNPs, whereas FTIR spectroscopic analysis demonstrated that cell membrane of the treated cells underwent an order-to-disorder transition during the killing process and chemical composition of the cell membrane including fatty acids, proteins, and carbohydrates was decomposed following interaction with AgNPs.