Preparation, characterization and efficacy of lysostaphin-chitosan gel against Staphylococcus aureus.

Lysostaphin (LST) is a bacteriocin that cleaves within the pentaglycine cross bridge of Staphylococcus aureus peptidoglycan. Previous studies have reported the high efficiency of LST even against multi drug resistant S. aureus including methicillin resistant S. aureus (MRSA). In this study, we have developed a new chitosan based hydrogel formulation of LST to exploit its anti-staphylococcal activity. The atomic interactions of LST with chitosan were studied by molecular docking studies. The rheology and the antibacterial properties of the developed LSTC gel were evaluated. The developed LST containing chitosan hydrogel (LSTC gel) was flexible, flows smoothly and remains stable at physiological temperature. The in vitro studies by agar well diffusion and ex vivo studies in porcine skin model exhibited a reduction in S. aureus survival by ∼3 Log10CFU/mL in the presence of LSTC gel. The cytocompatibility of the gel was tested in vitro using macrophage RAW 264.7 cell line and in vivo in Drosophila melanogaster. A gradual disruption of S. aureus biofilms with the increase of LST concentrations in the LSTC gel was observed which was confirmed by SEM analysis. We conclude that LSTC gel could be highly effectual and advantageous over antibiotics in treating staphylococcal-topical and biofilm infections.

Anti-staphylococcal Activity of Injectable Nano Tigecycline/Chitosan-PRP Composite Hydrogel Using Drosophila melanogaster Model for Infectious Wounds.

Compared to the current treatment modalities, the use of an injectable hydrogel system, loaded with antibiotic encapsulated nanoparticles for the purpose of treating Staphylococcus aureus (S. aureus) chronic wound infections have several advantages. These include adhesiveness to infection site, reduced frequency of dressings, sustained drug release, inhibition of bacterial growth, and increased healing. In the present work tigecycline nanoparticles were loaded into chitosan-platelet-rich plasma (PRP) hydrogel. The tigecycline nanoparticles (95 ± 13 nm) were synthesized through ionic cross-linking method using chitosan, tripolyphosphate, and tigecycline and characterized by dynamic light scattering (DLS), scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FT-IR). The synthesized nanoparticles and activated PRP powder were mixed with chitosan hydrogel to form a homogeneous gel. Rheology studies have confirmed the shear thinning property, thermal stability, and injectability of the prepared gel systems. The gel system was further assessed for its drug release property and found that it was released in a sustained manner. Hemolysis and blood-clotting assays demonstrated that the gel system was neither a hemolysin nor a hamper to the clotting cascade. Cell viability results showed that these nanoparticles were cyto-compatible. The bioactivity of PRP loaded chitosan gel toward fibroblast cell line was studied using cell proliferation and migration assay. In vitro antibacterial studies revealed that the gel system inhibited bacterial growth to a great extent. The antibacterial activity was further analyzed using ex vivo porcine skin assay. In vivo anti-Staphylococcal activity of the prepared hydrogels was studied using a Drosophila melanogaster infection model. The tigecycline and tigecycline nanoparticle incorporated chitosan gel showed a significant antibacterial activity against S. aureus. Thus, the gel system is an effective medium for antibiotic delivery and can be applied on the infection sites to effectively forestall various skin infections caused by S. aureus.

Skin and muscle permeating antibacterial nanoparticles for treating Staphylococcus aureus infected wounds.

Majority of the chronic wounds are infected with bacteria like Staphylococcus aureus (S. aureus). The deep tissue infections are difficult to treat using topical antibiotics, due to their poor tissue penetration. In order to treat S. aureus deep tissue infections we have developed an antibiotic delivery system using chitosan nanoparticles (CNPs). To enhance their tissue penetration these CNPs were further coated using lecithin (CLNPs). Antibiotic tigecycline was loaded into chitosan nanoparticles (tCNPs) and then coated with lecithin to generate lecithin coated tigecycline loaded chitosan nanoparticles (tCLNPs). The prepared nanoparticles were characterized using DLS, SEM, TEM and FT-IR. The prepared CNPs, tCNPs, CLNPs and tCLNPs have the size range of 85 ± 10, 90 ± 18, 188 ± 5 and 235 ± 20 nm, respectively. The tCLNPs shows more sustained release pattern of tigecycline. The antibacterial activity of the developed nanoparticles was confirmed against laboratory and clinical strains of S. aureus using in vitro and ex vivo experiments. The ex vivo skin and muscle permeation study ensures the enhanced delivery of tigecycline to the deeper tissue. The prepared nanoparticles were hemo-compatible and cyto-compatible. Our study suggests that the prepared tCLNPs can be effectively used for the treatment of S. aureus infected wounds.

Drug loaded bi-layered sponge for wound management in hyperfibrinolytic conditions.

Excessive bleeding due to premature clot lysis and secondary bacterial wound infection are two significant problems that contribute to increased morbidity in patients with hyperfibrinolytic conditions. In this study, we have developed a bi-layered sponge that promotes fibrin clot stability and prevents secondary bacterial wound infections. Using the technique of freeze-drying, a bi-layer matrix consisting of hyaluronic acid (HA) containing aminocaproic acid (amicar) and chitosan containing tetracycline loaded O-carboxymethyl chitosan nanoparticles (Tet-O-CMC NPs) were produced. We hypothesized that the top chitosan layer with Tet-O-CMC NPs will prevent wound infection and concomitantly act as a matrix for cellular migration and subsequent wound healing, while the amicar-containing layer would promote clot stability. Tet-O-CMC NPs and bi-layer sponges were characterized using Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM) and Fourier Transform Infra Red (FT-IR) spectroscopy. Physiochemical characterization such as porosity, swelling and mechanical testing was performed. The drug release study shows that the bi-layered sponge demonstrates a robust burst release of amicar and a sustained release of tetracycline. The ex vivo muscle permeation study indicated that Tet-O-CMC NPs have enhanced tissue permeation compared to free Tet. In vitro antibacterial activity of the bi-layer sponge towards laboratory and clinical strains of Staphylococcus aureus and Escherichia coli was proved. The ex vivo bacterial sensitivity study using porcine muscles confirmed the antibacterial activity, while the cell viability study using human dermal fibroblast (HDF) cells revealed its biocompatible nature. The in vitro antifibrinolytic study shows that the bi-layered sponge with amicar showed significant protection against streptokinase induced clot lysis. These studies suggest that the prepared amicar and tetracycline loaded chitosan-HA bi-layered sponge can be used effectively to promote better wound healing by simultaneously preventing bacterial infection, and enhancing clot stability.

Injectable alginate-O-carboxymethyl chitosan/nano fibrin composite hydrogels for adipose tissue engineering.

Injectable, biodegradable scaffolds are required for soft tissue reconstruction owing to its minimally invasive approach. Such a scaffold can mimic the native extracellular matrix (ECM), provide uniform distribution of cells and overcome limitations like donor site morbidity, volume loss, etc. So, here we report two classes of biocompatible and biodegradable hydrogel blend systems namely, Alginate/O-carboxymethyl chitosan (O-CMC) and Alginate/poly (vinyl alcohol) (PVA) with the inclusion of fibrin nanoparticles in each. The hydrogels were prepared by ionic cross-linking method. The developed hydrogels were compared in terms of its swelling ratio, degradation profile, compressive strength and elastic moduli. From these preliminary findings, it was concluded that Alginate/O-CMC formed a better blend for tissue engineering applications. The potential of the formed hydrogel as an injectable scaffold was revealed by the survival of adipose derived stem cells (ADSCs) on the scaffold by its adhesion, proliferation and differentiation into adipocytes. Cell differentiation studies of fibrin incorporated hydrogel scaffolds showed better differentiation was confirmed by Oil Red O staining technique. These injectable gels have potential in soft tissue regeneration.