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.

Microencapsulation of Probiotic Streptococcus salivarius LAB813.

Probiotics are living microorganisms that confer a health benefit on the host when administered in adequate amounts. Streptococcus salivarius, a commensal bacterium found in the oral cavity, has been shown to secrete antimicrobial peptides and can be used as probiotics. This study aimed to develop a delivery system for the probiotic LAB813, a novel S. salivarius strain first identified in the laboratory. Probiotics can be delivered and protected through the encapsulation of biomaterials such as polysaccharides. Their biocompatibility, biodegradability, user-friendliness, and ease of access make polysaccharides useful for encapsulating probiotics. Alginate (Alg) and chitosan (Ch) are naturally obtained polysaccharides and, hence, tested for LAB813 encapsulation. An extrusion method of encapsulation was performed to form Alg microcapsules (Alg-LAB813), some of which were coated with Ch (Alg-LAB813-Ch) to provide dual-layered protection. Inhibitory assays of the Alg-LAB813 and Alg-LAB813-Ch microcapsules were assayed against an indicator strain. Alg-LAB813-Ch microcapsules showed superior antibacterial properties compared to Alg-LAB813 microcapsules over 24 h and when subject to temperatures ranging from 4 to 68 °C. In addition, Alg-LAB813-Ch microcapsules retained antibacterial activity for up to 28 days of storage at 4 °C. The strong and sustained inhibitory activities of Ch-coated Alg encapsulated LAB813 signify the potential for their use to improve oral health.

Antimicrobial activity of probiotic Streptococcus salivarius LAB813 on in vitro cariogenic biofilms.

OBJECTIVE: To investigate the antimicrobial activity of a novel commensal strain of Streptococcus salivarius, LAB813, against Streptococcus mutans biofilms. METHODS: The inhibitory activity of LAB813 towards S. mutans was tested using mono-, dual-, and multi-species cariogenic biofilms formed on three types of orthodontic appliances (metal, ceramic, aligner). The activity of the commercially available probiotic, BLIS M18™ was used as control. RESULTS: LAB813 significantly inhibited S. mutans biofilms with cell killing approximating 99% for all materials. LAB813 showed effectiveness at inhibiting S. mutans in more complex multi-species biofilms with cell killing approximating 90% for all three materials. When comparing the killing kinetics of the probiotics, LAB813 had a faster rate of killing biofilms than M18. Experiments conducted with cell-free culture supernatant confirmed the presence of an inhibitory substance of proteinaceous nature. The addition of xylitol, a common sugar substitute used for human consumption, potentiated the inhibitory effects of LAB813 against S. mutans embedded in a more complex fungal-bacterial biofilm. CONCLUSIONS: LAB813 possesses strong antimicrobial activity, potent anti-biofilm properties, and enhanced antimicrobial activity in the presence of xylitol. The identification and characterization of strain LAB813 exhibiting antimicrobial activity towards S. mutans hold exciting promise for this novel strain to be developed as an oral probiotic for use in the prevention of dental caries.

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.

Fungal surface protein mediated one-pot synthesis of stable and hemocompatible gold nanoparticles.

Despite their large secretome and wide applications in bioprocesses, fungi remain underexplored in metal nanoparticles (MNP) biosynthesis. Previous studies have shown that cell surface proteins of Rhizopus oryzae play a crucial role in biomineralization of Au(III) to produce gold nanoparticles (AuNPs). Therefore, it is hypothesized that purified cell surface protein may produce in vitro AuNPs with narrow size distribution for biomedical and biocatalytic applications. However, different protein extraction methods might affect protein stability and the AuNP biosynthesis process. Herein, we have explored the extraction of cell surface proteins from R. oryzae using common detergents and reducing agent (sodium dodecyl sulfate (SDS) Triton X-100, and 1,4-dithiothreitol (DTT)) and their effect on the size and shape of the biosynthetic AuNPs. The surface proteins extracted with reducing agent (DTT) and non-ionic detergent (Triton X-100) produce spherical AuNPs with a mean particle size of 16±7nm, and 19±4nm, respectively, while the AuNPs produced by the surface protein extracted by ionic detergent (SDS) are flower-like AuNPs with broader size distribution of 43±19nm. This synthetic approach does not require use of any harsh chemicals, multistep preparation and separation process, favouring environmental sustainability. The biosynthetic AuNPs thus formed, are stable in different physiological buffers and hemocompatible, making them suitable for biomedical applications.