In vitro and in vivo evaluation of poly-3-hydroxybutyric acid-sodium alginate as a core-shell nanofibrous matrix with arginine and bacitracin-nanoclay complex for dermal reconstruction of excision wound.

The protective layer of the body, the skin is often prone to damage due to several factors like trauma, accidents, stress and hazardous exposure. This requires the skin to regenerate itself which is a finely regulated process. To hasten the process and prevent further damage, the dressing material is of prime importance. Herein, we fabricated poly-3-hydroxybutyric acid (P)-sodium alginate (S)-(core-shell) nanofibrous matrix as protective scaffold for the skin tissue regeneration in excision wound model. The arginine (A) and layered double hydroxides-bacitracin (LB) were incorporated into the core and shell of the nanofibrous matrix using co-axial electrospinning. The core-shell nanofibers assist in the synergistic, controlled delivery of L-arginine, and bacitracin with major role in the protein synthesis, cell signaling and infection control at wound site respectively. In vitro biocompatibility was confirmed by testing on dermal fibroblasts. Furthermore, in vivo studies revealed the synergistic effect of both the components in active healing of wounds. The biochemical, histochemical and immunohistochemical studies reveal that the arginine loaded scaffold aided cellular migration and proliferation. These results suggest that the simultaneous existence of the drug bacitracin-nano clay complex and L-arginine in the shell and core respectively has conferred interesting dynamic properties to the scaffold towards wound healing.

Fabrication of Biohybrid Cellulose Acetate-Collagen Bilayer Matrices as Nanofibrous Spongy Dressing Material for Wound-Healing Application.

Tissue engineering is currently one the fastest growing engineering fields, requiring fabrication of advanced and multifunctional materials to be used as scaffolds or dressing for tissue regeneration. In this work, a bilayer matrix was fabricated by electrospinning of a hybrid cellulose acetate nanofibers (CA) containing bioactive latex or Ciprofloxacin over highly interconnected collagen (CSPG) 3D matrix previously obtained by a freeze-drying process. The bilayer matrix was fabricated with a nanofibrous part as the primary (top) layer and a spongy porous part as the secondary (bottom) layer by combining electrospinning and freeze-drying techniques to enhance the synergistic effect of both materials corresponding to physical and biological properties. The final material was physicochemically characterized using Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The bilayer matrix exhibited nanofibrous and 3D porous structure with properties such as high porosity, swelling, and stability required for soft-tissue-engineering applications. Furthermore, the in vitro biological and fluorescence properties of the matrix were tested against NIH 3T3 fibroblast and human keratinocyte (HaCaT) cell lines and showed good cell adhesion and proliferation over the bilayer matrix. Thus, the synergistic combination of nanofibrous material deposition onto to the collagenous porous material has proved efficient in the fabrication of a bilayer matrix for skin-tissue-engineering applications.

Accelerated wound healing and its promoting effects of biomimetic collagen matrices with siderophore loaded gelatin microspheres in tissue engineering.

The prolonged inflammation and elevation of Matrix Metalloproteniases (MMPs) at the wound site causes significant degradation of Extracellular matrix (ECM) which cause delays the process of wound healing. Hence the development of therapeutic dressing matrices to control and to positively regulate MMPs balance was considered important in achieving faster healing. The design of biomaterial matrices of collagen scaffold has the challenge to mimic the function of ECM and emulate to the attraction of fibroblast migration at wound site. Herein, we report the fabricated Collagen (COL) matrices impregnated with Siderophore loaded Gelatin Microspheres (SGM) as a delivery system to control both infection and protease levels in the wound site for accelerated healing. The fabricated collagen scaffold impregnated with siderophore loaded gelatin microspheres (COL-SGM) was characterized physiochemically using Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM) and swelling behaviour. The COL-SGM scaffold possesses good swelling ability and also exhibited better morphology for the cell adhesion and proliferation. The in vitro biocompatibility and in vitro fluorescence activity of the developed scaffold revealed to possess good cell proliferation and migration against NIH 3T3 fibroblast and Human keratinocytes (HaCaT) cell lines. Furthermore, the in vivo evaluation offered the advantage of neutralizing the excessive proteases and delivered the siderophore in controlled fashion depending on the level of wound exudates with modulated MMPs. Moreover, the COL-SGM scaffold exhibited with increase in the collagen synthesis and faster reepitheliazation of wounds. Thus the developed COL-SGM scaffold achieved improvements in biocompatibility and act as a potent MMP inhibitor to improve wound healing efficiency in tissue engineering application.

Development of antiviral inhibitor against dengue 2 targeting Ns3 protein: In vitro and in silico significant studies.

Dengue fever is a severe, widespread disease with more than 2 million diagnosed infections per year. The Dengue virus protease represents a cardinal target for prudent drug design. Among the four serotypes Dengue 2 is known for the occurrence of its frequent epidemics. The new compound inhibited the Dengue-2 in the low-micromolar range in cells. At the moment, protease inhibitors are not actively tried against dengue virus as therapeutic option. We have identified thiosemicarbazones derived phenyl-acetyl ketones as candidate for a novel class of protease inhibitors. Here, we report the selective and non-competitive inhibition of the Dengue virus serotype 2 in vitro and in silico. Molecular docking suggests binding at a specific active site. In addition to the docking assays, few techniques were developed to interpret these molecules's antiviral profile in vitro.

In vivo efficiency of the collagen coated nanofibrous scaffold and their effect on growth factors and pro-inflammatory cytokines in wound healing.

Exploring the importance of nanofibrous scaffold with traditionally important medicine as a wound dressing material prevents infection and aids in faster healing of wounds. In the present study, the Collagen (COL) from the marine fish skin was extracted and employed for coating the Poly(3-hydroxybutyric acid) (P)-Gelatin (G) nanofibrous scaffold with a bioactive Coccinia grandis extract (CPE) fabricated through electrospinning. Further, the fabricated collagen coated nanofibrous scaffold (PG-CPE-COL) applied to the experimental wound of rats and the wound healing was analyzed with by physiochemical and biological techniques. The increased level of hydroxyproline, hexosamine and uronic acid was observed in PG-CPE-COL treated than the other groups. The CPE and collagen in the nanofibrous scaffold accelerates the wound healing and thereby reduced the inflammation caused by the cyclooxygenase-2 (COX-2) and inducible nitric oxide synthases (iNOS) in wound healing. The nanofibrous scaffold has influenced the expression of various growth factors such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and transforming growth factor (TGF-ß). In addition, the PG-CPE-COL nanofibrous scaffold increases the deposition of collagen synthesis and accelerates reepithelialization. Thus, the results suggest that the collagen coated nanofibrous scaffold with bioactive traditional medicine enhanced the faster healing of wound.

Dual-layered 3D nanofibrous matrix incorporated with dual drugs and their synergetic effect on accelerating wound healing through growth factor regulation.

The by-product of the slaughter house was utilized for the development of promising regenerative wound dressing material. Currently, dual-layered nanofibrous spongy scaffold was fabricated for tissue engineering applications. Herein, Keratin (K)-Fibrin (F)-Gelatin (G) 3D sponge loaded with Mupirocin (M) was fabricated with the naturally derived materials from bovine origin using freeze drying method. Moreover, poly(3-hydroxybutyric acid) (P) and Gelatin(G) solution loaded with Curcumin (C) were electrospun to get the dual drug loaded dual-layered nanofibrous spongy 3D scaffold (KFG:M-PG:C). The fabricated biomaterial was assisted with physical, biological and mechanical property. The in vitro cell viability and fluorescence staining of NIH 3T3 and HaCaT cells assist in cell adhesion and proliferation of the dual-layered scaffold. Moreover, in vivo assessment using silicone splint animal model was employed. The nanofibrous surface aids in the migration of fibroblast for the increased the collagen deposition and granulation tissue formation. Nonetheless, the 3D spongy surface promotes the gaseous exchange and absorption of exudates. The fabricated KFG:M-PG:C scaffold has the ability to produce perusable material that can integrate with the host tissue. Overall, the three dimensional (3D) dual-layered nanofibrous spongy scaffold with synergistic effect of dual drugs prevents from infection and facilitates as highly durable substrate in tissue engineering application.

Nanofibrous matrixes with biologically active hydroxybenzophenazine pyrazolone compound for cancer theranostics.

The nanomaterial with the novel biologically active compounds has been actively investigated for application in cancer research. Substantial use of nanofibrous scaffold for cancer research with potentially bioactive compounds through electrospinning has not been fully explored. Here, we describe the series of fabrication of nanofibrous scaffold loaded with novel potential biologically active hydroxybenzo[a]phenazine pyrazol-5(4H)-one derivatives were designed, synthesized by a simple one-pot, two step four component condensation based on Michael type addition reaction of lawsone, benzene-1,2-diamine, aromatic aldehydes and 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one as the substrates. The heterogeneous solid state catalyst (Fe (III) Y-Zeolite) could effectively catalyze the reaction to obtain the product with high yield and short reaction time. The synthesized compounds (5a-5p) were analyzed by NMR, FTIR and HRMS analysis. Compound 5c was confirmed by single crystal XRD studies. All the compounds were biologically evaluated for their potential inhibitory effect on anticancer (MCF-7, Hep-2) and microbial (MRSA, MTCC 201 and FRCA) activities. Among the compounds 5i exhibited the highest levels of inhibitory activity against both MCF-7, Hep-2 cell lines. Furthermore, the compound 5i (BPP) was evaluated for DNA fragmentation, flow cytometry studies and cytotoxicity against MCF-7, Hep-2 and NIH 3T3 fibroblast cell lines. In addition, molecular docking (PDB ID: 1T46) studies were performed to predict the binding affinity of ligand with receptor. Moreover, the synthesized BPP compound was loaded in to the PHB-PCL nanofibrous scaffold to check the cytotoxicity against the MCF-7, Hep-2 and NIH 3T3 fibroblast cell lines. The in vitro apoptotic potential of the PHB-PCL-BPP nanofibrous scaffold was assessed against MCF-7, Hep-2 cancerous cells and fibroblast cells at 12, 24 and 48h respectively. The nanofibrous scaffold with BPP can induce apoptosis and also suppress the proliferation of cancerous cells. We anticipate that our results can provide better potential research in nanomaterial based cancer research.

A novel assembly pheromone trap for tick control in dog kennels.

A novel ecofriendly sticky tick trap device for the control of dog tick Rhipicephalus sanguineus using gold nanoparticle assembly pheromone complex as a bait was developed. Assembly pheromones comprising of guanine, xanthine and adenine in the ratio of 25:1:1 was encapsulated in gold nanoparticle. The response of the different stages of unfed R. sanguineus ticks was evaluated using petridish bioassay. Statistical analysis was done using chi-square test. Petridish bioassay with unfed stages of R. sanguineus revealed that 100% of the larvae, nymph and adults were attracted to assembly pheromone nanogold complex within 24h. Of the 952 ticks trapped, ticks of different stages trapped in total by the baited sticky trap device, 543 (57%) were engorged and 409 (43%) were unfed ticks. The study revealed that assembly pheromone baited traps has the potential to control tick infestations in dog kennels.

Design and characterization of 3D hybrid collagen matrixes as a dermal substitute in skin tissue engineering.

The highly interconnected porous dressing material was fabricated with the utilization of novel collagen (COL-SPG) for the efficient healing of the wound. Herein, we report the fabrication of 3D collagen impregnated with bioactive extract (COL-SPG-CPE) to get rid of infection at the wound site. The resultant 3D collagen matrix was characterized physiochemically using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and mechanical property. The dressing substrate possesses the high swelling ability, increase in the porosity, in vitro enzymatic degradability and antibacterial property. The in vitro biocompatibility and fluorescence activity of the collagen scaffold against both NIH 3T3 fibroblast and Human keratinocyte (HaCaT) cell lines assisted in excellent cell adhesion and proliferation over the collagen matrix. Furthermore, the in vivo evaluation of the COL-SPG-CPE 3D sponge exhibited with enhanced collagen synthesis and aids in faster reepithelialization. However, the rate of wound healing was influenced by the expression of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and transforming growth factor (TGF-ß) growth factors promotes the collagen synthesis, thereby increases the healing efficiency. Based on the results, COL-SPG-CPE has a potential ability in the remodeling of the wound with the 3D collagen as wound dressing material.

Antiviral activity of Thiosemicarbazones derived from α-amino acids against Dengue virus.

The endemicity and seasonal outbreaks of Dengue disease in most tropical and subtropical countries underscores an urgent need to develop effective prevention and control measures. Development of a Dengue vaccine, which is complicated by the Antibody Dependent Enhancement effect (ADE), a viral inhibitor, seems prudent as it would inhibit the spread of the virus. In vitro methods such as MTT assay and plaque formation unit reduction assays were employed for screening the viral inhibitory property of α-amino acid based Thiosemicarbazides. The results elicits that at concentrations not exceeding the maximum non cytotoxic concentration (MNCC), these compounds completely prevented Dengue virus infection in vero cells as indicated by the absence of cytopathic effects in a dose-dependent manner. The high potency of Bz-Trp-TSC against all four types of Dengue virus infection elevates Thiosemicarbazide as a lead antiviral agent for Dengue disease. Screening small molecules for antiviral activity against the most rapidly spreading mosquito-borne viral disease is being explored by several research groups. Our findings would help to augment the efforts to identify the lead compounds for antiviral therapy to combat the Dengue disease. J. Med. Virol. 89:546-552, 2017. © 2016 Wiley Periodicals, Inc.

Biomimetic interconnected porous keratin-fibrin-gelatin 3D sponge for tissue engineering application.

The medicated wound dressing material with highly interconnected pores, mimicking the function of the extracellular matrix was fabricated for the promotion of cell growth. In this study, keratin (K), fibrin (F) and gelatin (G) composite scaffold (KFG-SPG) was fabricated by freeze drying technique and the mupirocin (D) drug was successfully incorporated with KFG-SPG (KFG-SPG-D) intended for tissue engineering applications. The fabrication of scaffold was performed without the use of any strong chemical solvents, and the solid sponge scaffold was obtained with well interconnected pores. The porous morphology of the scaffold was confirmed by SEM analysis and exhibited competent mechanical properties. KFG-SPG and KFG-SPG-D possess high level of biocompatibility, cell proliferation and cell adhesion of NIH 3T3 fibroblast and human keratinocytes (HaCaT) cell lines thereby indicating the scaffolds potential as a suitable medicated dressing for wound healing.

Evaluation of Stress-Induced Microbial Siderophore from Pseudomonas aeruginosa Strain S1 as a Potential Matrix Metalloproteinase Inhibitor in Wound Healing Applications.

Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes capable of causing various inflammatory and various degenerative diseases if over-expressed. The active site of these enzymes is a zinc binding motif which binds to the specific site on the substrate and induce degradation. Hence an inhibitor is required to form a complex with zinc motif which hampers the binding ability of MMPs. To obtain novel MMPs inhibitor for wound healing, the chelating activity of siderophore from the microbial source was focused. During screening for siderophore production, strain S1 produced the highest amount of siderophore in the minimal salts medium. The isolate was confirmed as Pseudomonas aeruginosa strain S1 based on 16S rRNA gene sequencing and phylogenetic analysis. The activity of the siderophore was assayed using chrome azurol sulphonate and purified by the chromatographic techniques. The structural evidence through Fourier transform infrared and nuclear magnetic resonance spectra revealed that the isolated siderophore is a catecholate type with the distinctive characters. The positive results of calcein and fluozin-3 assays indicate that siderophore could bind to divalent metal ions, namely Fe(2+) and Zn(2+). As the siderophore compound focused on wound healing property, the in vitro studies revealed the viability of NH3T3 fibroblast cells and its efficiency in matrix modulating was confirmed through gelatin zymogram.

Durable keratin-based bilayered electrospun mats for wound closure.

A bilayered nanofibrous scaffold with rapid wound healing properties is found to be suitable for tissue regeneration applications. The objective of this study is to reveal the fabrication of a poly(3-hydroxybutyric acid) (P)-gelatin (G) nanofibrous mat through electrospinning, with a horn keratin-chitosan-based biosheet (KC) as a bilayered nanofibrous scaffold. The mupirocin (D)-loaded horn KC biosheet (KCD) acts as the primary layer over which PG nanofibers were electrospun to act as the secondary layer. It is shown that this engineered bilayered nanofibrous scaffold material (KC-PG) should fulfill the functions of the extracellular matrix (ECM) by elucidating its function in vitro and in vivo. The bilayered nanofibrous scaffold was designed to exhibit improved physiochemical, biological and mechanical properties, with better swelling and porosity for enhanced oxygen permeability, and it also exhibits an acceptable antibacterial property to prevent infection at the wound site. The bilayered nanofibrous scaffold assists in better biocompatibility towards fibroblast and keratinocyte cell lines. The morphology of the nanofibrous scaffold aids increased cell adhesion and proliferation with cell material interactions. This was elucidated with the help of in vitro fluorescence staining against both cell lines. The bilayered KCD-PG nanofibrous scaffold material gives accelerated wound healing efficiency during in vivo wound healing. The results showed the regulation of growth factors with enhanced collagen synthesis, thereby helping in faster wound healing.

Synthesis of highly interconnected 3D scaffold from Arothron stellatus skin collagen for tissue engineering application.

The substrate which is avidly used for tissue engineering applications should have good mechanical and biocompatible properties, and all these parameters are often considered as essential for dermal reformation. Highly interconnected three dimensional (3D) wound dressing material with enhanced structural integrity was synthesized from Arothron stellatus fish skin (AsFS) collagen for tissue engineering applications. The synthesized 3D collagen sponge (COL-SPG) was further characterized by different physicochemical methods. The scanning electron microscopy analysis of the material demonstrated that well interconnected pores with homogeneous microstructure on the surface aids higher swelling index and that the material also possessed good mechanical properties with a Young's modulus of 0.89±0.2 MPa. Biocompatibility of the 3D COL-SPG showed 92% growth for both NIH 3T3 fibroblasts and keratinocytes. Overall, the study revealed that synthesized 3D COL-SPG from fish skin will act as a promising wound dressing in skin tissue engineering.