A biologically functional bioink based on extracellular matrix derived collagen for 3D printing of skin.

Over the past few years, several advancements have been made to develop artificial skin that mimics human skin. Artificial skin manufactured using 3D printing technology that includes all epidermal and dermal components, such as collagen, may offer a viable solution. The skin-specific bioink was derived from digested chicken skin and incorporated into PVA (polyvinyl alcohol) and gelatin. The prepared bioink was further analyzed for its structure, stability, biocompatibility, and wound healing potential in in vitro, in ovo, and in vivo models. The 3D-printed skin showed excellent mechanical properties. In vitro scratch assays showed the proliferation and migration of cells within 24 h. In an in ovo assay, the 3D-printed skin facilitated the attachment of cells to the scaffolds. In the animal study, the quick cellular recruitment at the injury site accelerated wound healing. Further, hydroxyproline content was estimated to be 0.9-1.2 mg/ml, and collagen content was 7.5 %, which confirmed the epithelization. The relative expressions of MMP-9, COMP, TNF-α, and IL-6 genes were found to be increased compared to the control. These results demonstrate that 3D bioprinting represents a suitable technology to generate bioengineered skin for therapeutic and industrial applications in an automated manner.

Synergistic Effect of Zinc-Chitosan Nanoparticles and Hydroxychloroquine to Inhibit Buffalo Coronavirus.

Zinc ions can hinder the synthesis of proteins required for accomplishing several stages of the viral life cycle. The intracellular zinc concentration can be increased by using zinc ionophores which transport zinc ions into the cells and hinder viral replication. (Hydroxy)chloroquine is an example of a zinc ionophore, but both zinc and (hydroxy)chloroquine can be toxic to the host organism. The nanocarriers may serve as camouflage to evade the adverse effects of drugs, chemicals, and nanoparticles on the host. We formulated ZnO nanoparticles with flower-like morphology (ZnONFs). It was further decorated with chitosan along with hydroxychloroquine (as a zinc ionophore) (CHCZnO NPs). We have chosen the cationic polymer chitosan since it is biocompatible, biodegradable and binds easily with the cells, and enhances the transport of drugs across cell membranes. The formulation was investigated for size, shape, surface charge, and interaction of chemicals used. We evaluated the formulations for cytotoxicity, and biocompatibility in embryonated chicks and their efficacy against bovine coronavirus (BCoV) isolated from a buffalo calf, and pneumo-enteric coronaviruses isolated from a buffalo calf with promising results in comparison to ZnONFs/hydroxychloroquine alone. Furthermore, we elucidate the mechanism underlying the lysosomotropic effect of various formulations on Vero cells infected with the buffalo coronavirus.

Nanoformulation of a Trypanocidal Drug Isometamidium Chloride Ameliorates the Apurinic-Apyrimidinic DNA Sites/Genotoxic Effects in Horse Blood Cells.

Isometamidium chloride (ISM) is a trypanocide for the prophylactic and therapeutic use against vector-borne animal trypanosomosis (mainly Surra caused by Trypanosoma evansi) and African animal trypanosomosis caused by T. congolense/T. vivax/T. brucei). ISM was found to be an efficient trypanocide for therapeutic/prophylactic use against trypanosomosis; however, it produces some local and systemic detrimental effects in animals. We synthesized isometamidium chloride-loaded alginate gum acacia nanoformulation (ISM SANPS) to lessen the detrimental side effects of isometamidium chloride (ISM) while treating trypanosomal diseases. We intended to determine the cytocompatibility/toxicity, and DNA deterioration/chromosomal structural or number changes (genotoxicity) of ISM SANPs using mammalian cells in a concentration-dependent manner. Apurinic/apyrimidinic (AP) sites are one of the major types of DNA lesions formed during base excision and repair of oxidized, deaminated, or alkylated bases. The intensity of the cellular AP site is an excellent marker of the deterioration of DNA quality. We thought it pertinent to quantify the AP sites in ISM SANPs-treated cells. Our investigations established a dose-dependent cyto-compatibility or toxicity and DNA impairment (genotoxicity) in ISM SANPs-treated horse peripheral blood mononuclear cells. ISM SANPs were biocompatible at various concentrations tested on the mammalian cells.

Strangles in equines: An overview.

Strangles, caused by Streptococcus equi subspecies equi, is a highly infectious respiratory disease affecting horses and other equines. The disease is economically important and compromises the productivity of equine farm significantly. The disease is characterized by pyrexia, mucopurulent nasal discharge, and abscess formation in the lymph nodes of the head and neck of horses. The disease transmission occurs either directly by coming in contact with infectious exudates or indirectly via fomite transmission. Besides this, carrier animals are the primary and most problematic source of disease infection. The organism not only initiates outbreaks but also makes the control and prevention of the disease difficult. The diagnosis of strangles is best done by isolating and characterizing the bacteria from nasal discharge, pus from abscesses, and lymphoid tissues or by using PCR. ELISA can also be used to detect serum protein M (SeM) antibodies for diagnosis. The most popular treatment for strangles is with penicillin; however, the treatment is affected by the stage, feature and severity of the disease. Prevention and control of strangles can be achieved through vaccination and good hygiene practices. Basically, this review describes the global prevalence of S. equi, as well as general aspects of the disease, like pathogenesis, diagnosis, treatment, prevention, control and management of the disease.

Chloroquine chaos and COVID-19: Smart delivery perspectives through pH sensitive polymers/micelles and ZnO nanoparticles.

The global pandemic of COVID-19 had a consequential impact on our lives. (Hydroxy)chloroquine, a well-known drug for treatment or prevention against malaria and chronic inflammatory conditions, was also used for COVID patients with reported potential efficacy. Although it was well tolerated, however in some cases, it produced severe side effects, including grave cardiac issues. The variable reports on the administration of (hydroxy)chloroquine in COVID19 patients led to chaos. This drug is a well-known zinc ionophore, besides possessing antiviral effects. Zinc ionophores augment the intracellular Zn2+ concentration by facilitating the zinc ions into the cells and subsequently impair virus replication. Zinc oxide nanoparticles (ZnO NPs) have been reported to possess antiviral activity. However, the adverse effects of both components are also reported. We discussed in depth their possible mechanism as antiviral and smart delivery perspectives through pH-sensitive polymers/ micelles and ZnO NPs.

Toxicity Assessment and Control of Early Blight and Stem Rot of Solanum tuberosum L. by Mancozeb-Loaded Chitosan-Gum Acacia Nanocomposites.

Biopolymers such as chitosan and gum acacia are used for nanotechnological applications due to their biosafety and ecofriendly nature. The commercial fungicide mancozeb (M) was loaded into chitosan-gum acacia (CSGA) polymers to form nanocomposite (NC) CSGA-M (mancozeb-loaded) measuring 363.6 nm via the ionic gelation and polyelectrolyte complexation method. The physico-chemical study of nano CSGA-M was accomplished using dynamic light scattering (DLS), scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). Nano CSGA-M-1.0 (containing 1.0 mg/mL mancozeb) at 1.5 ppm demonstrated a maximum inhibition (83.8 ± 0.7%) against Alternaria solani, while Sclerotinia sclerotiorum exhibited a 100% inhibition at 1.0 and 1.5 ppm through the mycelium inhibition method. Commercial mancozeb showed an inhibition of 84.6 ± 0% and 100%, respectively, for both fungi. In pot house conditions, NCs were found to exhibit good antimicrobial activity. Disease control efficiency (DCE, in %) in pathogen-treated plants for CSGA-M-1.0 was 64.6 ± 5.0 and 60.2 ± 1.4% against early blight and stem rot diseases, respectively. NCs showed lower cytotoxicity than commercial mancozeb at the given concentration. In conclusion, both in vitro and in vivo antifungal efficacy for nano CSGA-M was found to be quite comparable but less toxic than mancozeb to Vero cell lines; thus, in the future, this formulation may be used for sustainable agriculture.

Metal/metal oxide nanoparticles: Toxicity concerns associated with their physical state and remediation for biomedical applications.

Metal/metal oxide nanoparticles show promise for various applications, including diagnosis, treatment, theranostics, sensors, cosmetics, etc. Their altered chemical, optical, magnetic, and structural properties have differential toxicity profiles. Depending upon their physical state, these NPs can also change their properties due to alteration in pH, interaction with proteins, lipids, blood cells, and genetic material. Metallic nanomaterials (comprised of a single metal element) tend to be relatively stable and do not readily undergo dissolution. Contrarily, metal oxide and metal alloy-based nanomaterials tend to exhibit a lower degree of stability and are more susceptible to dissolution and ion release when introduced to a biological milieu, leading to reactive oxygen species production and oxidative stress to cells. Since NPs have considerable mobility in various biological tissues, the investigation related to their adverse effects is a critical issue and required to be appropriately addressed before their biomedical applications. Short and long-term toxicity assessment of metal/metal oxide nanoparticles or their nano-formulations is of paramount importance to ensure the global biome's safety; otherwise, to face a fiasco. This article provides a comprehensive introspection regarding the effects of metal/metal oxides' physical state, their surface properties, the possible mechanism of actions along with the potential future strategy for remediation of their toxic effects.

Phytochemical Screening, Cytotoxicity and Anti-inflammatory Activities of the Leaf Extracts from Lawsonia inermis of Indian Origin to Explore their Potential for Medicinal Uses.

BACKGROUND: Lawsonia inermis Linn popularly known as Henna, plays an important role in ayurvedic or natural herbal medicines. The presence of phytoconstituents in henna, that may affect the animal or human health adversely, needs to be elucidated for L. inermis Linn species grown in India. INTRODUCTION: The aim of this research was to perform phytochemical screening, and study cytotoxicity and anti-inflammatory activities to understand the potential of leaves of Lawsonia inermis of Indian origin to provide a way forward for therapeutic use in medicine. METHODS: We assessed the phytochemical profile for the presence of phytoconstituents (alkaloids, carbohydrates, glycosides, steroids, flavonoids, saponins, tannins, proteins/amino acids and gums/mucilage) in various extracts of the plant leaves. The extracts were further purified by column chromatography for the isolation of plant constituents and monitored by TLC, analyzed by Fourier transform infrared FT-IR spectroscopy, H1NMR, and GC-MS analysis. Fractions were assessed for cytotoxicity and anti-inflammatory properties at various concentrations. We assessed the anti-inflammatory activity by nitric oxide production in various leaf extracts determined by Griess assay. RESULTS: All the spectral results suggest that the compounds from the extract contain an aromatic nucleus and OH group along with the methoxy group, allyl as well as vinyl group. Fractions of chloroform/methanolic (7:3) leaf extract of Lawsonia inermis confirmed the presence of the two constituents i.e. fraxetin and 1(3H)-isobenzofuranone. We observed a significant difference in cytotoxicity at higher concentrations in methanol and chloroform: methanol (8:2) leaf extracts (p>0.05), we could not find any significant differences amongst other leaf extracts at different concentrations. Some leaf extracts have potential cytotoxic activity on Vero cells. Reducing the chloroform concentration during extraction decreases the cytotoxic effect on cells. Nitric oxide levels decreased from 1000 µg/ml concentration to lower concentrations with varying degrees. Overall the highest nitric oxide production by CHCl3 (70%)/ MeOH (30%) was observed amongst various fractions at different concentrations. CONCLUSION: Phytochemical screening and the study of cytotoxicity and anti-inflammatory activities highlight the potential of leaves of the plant to provide a way further for their use in medicine. Fraxetin 1(3H) and isobenzofuranone structures were confirmed in fractions of CHCl3 (70%)/ MeOH (30%) extract as potent constituents. Some leaf extracts have potential cytotoxic activity on Vero cells. Reducing the chloroform concentration during extraction, it decreases the cytotoxic effect on cells. The cytotoxicity studies indicate the presence of cytotoxic compounds in some of these extracts, warranting research for the fabrication of suitable formulations comprising these constituents to reduce dose/toxicity for beneficial effects of the plant components.

Assessment of Antifungal Efficacy and Release Behavior of Fungicide-Loaded Chitosan-Carrageenan Nanoparticles against Phytopathogenic Fungi.

Biopolymeric Chitosan-Carrageenan nanocomposites 66.6-231.82 nm in size containing the chemical fungicide mancozeb (nano CSCRG-M) were synthesized following a green chemistry approach. The physicochemical study of nanoparticles (NPs) was accomplished using a particle size analyzer, SEM and FTIR. TEM exhibited clover leaf-shaped nanoparticles (248.23 nm) with mancozeb on the inside and entrapped outside. Differential scanning calorimetry and TGA thermogravimetry exhibited the thermal behaviour of the nanoform. Nano CSCRG-1.5 at 1.5 ppm exhibited 83.1% inhibition against Alternaria solani in an in vitro study and performed as well as mancozeb (84.6%). Complete inhibition was exhibited in Sclerotinia sclerotiorum at 1.0 and 1.5 ppm with the nanoformulation. The in vivo disease control efficacy of mancozeb-loaded nanoparticles against A. solani in pathogenized plants was found to be relatively higher (79.4 ± 1.7) than that of commercial fungicide (76 ± 1.1%) in pot conditions. Nanomancozeb showed superior efficacy for plant growth parameters, such as germination percentage, root-shoot ratio and dry biomass. The nanoformulation showed higher cell viability compared to mancozeb in Vero cell cultures at 0.25 and 0.50 mg/mL in the resazurin assay. CSCRG-0.5 showed slow-release behavior up to 10 h. Thus, these green nano-based approaches may help combat soil and water pollution caused by harmful chemical pesticides.

Microwave assisted fast fabrication of zinc/iron oxides based polymeric nanocomposites and evaluation on equine fibroblasts.

We aimed to provide a tissue repair material, which can be synthesized rapidly, using polymers mimicking the natural environment in the extra-cellular matrix and metals/minerals. The components should have the potential to be used in tissue repair and simultaneously, reducing the side-effects of the incorporated molecules. It is challenging to manage the dispersibility of ZnO NPs in common solutions like water. Here, we report a novel method for preparing highly dispersible suspensions of ZnO NPs. In contrast to those synthesized by conventional methods, microwave assisted method allowed synthesis of dispersible ZnO NPs and the incorporation of zinc/Iron oxides NPs within alginate and gum matrix (AG) in a short span of time providing high yield of the product. The nanoformulations were characterized for size, morphology, interaction of various chemicals used during their synthesis by transmissible electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and energy dispersive X ray Spectrum. It was also evaluated for cytotoxicity and their effect on equine fibroblast cells. Microwave-assisted fabrication of zinc/iron oxides nanoparticles provided flowerlike morphology with good dispersibility and high yield in a short span of time. Our results revealed that ZnO NPs were more cytotoxic than AG ZnO NPs and doped AG Fe3O4 doped ZnO NPs at higher concentrations. Further metal nanoparticles capped with alginate/acacia with size range less than 100 nm demonstrated high stability, good biocompatibility, re-epithelization and enhanced mineralization in horse fibroblast cells.

Accelerated healing of full thickness excised skin wound in rabbits using single application of alginate/acacia based nanocomposites of ZnO nanoparticles.

A perfect wound covering should prevent dryness of the wound and provide a favourable moist milieu at the wound interface allowing gas access but act as a barrier to the dirt and microorganisms. It is imperative to ensure early restoration of wound without scar formation at the site. Topical application of antiseptic preparation is the best for wound treatment because of its direct action. Zinc oxide nanoparticles (ZnO NPs) possess antimicrobial activity and enhance wound healing. Biocompatible polymers for inclusion of ZnO NPs can enhance the efficacy at lower doses while reducing the unwanted toxic effects. We synthesized ZnO NPs nanocomposites by impregnating the NPs in covalently attached gum acacia to the alginate exploiting the hydroxyl groups with aldehydes of glutaraldehyde, providing hydrated environment during wound application. Its topical application accelerated the full-thickness excision wound healing in rabbits. The polymers exerted synergistic effects due to their wound-healing potential. The wound-healing process was also investigated by transmission electron microscopy of regenerated tissues, collagen contents, alizared staining and histological observations to elucidate the healing mechanism compared to a commercially available ointment and negative controls. It has promising properties of biocompatibility, anti-inflammatory, cell adhesion and proliferation without any scar formation which are crucial for healing.

An Insight into Nanomedicinal Approaches to Combat Viral Zoonoses.

BACKGROUND: Emerging viral zoonotic diseases are one of the major obstacles to secure the "One Health" concept under the current scenario. Current prophylactic, diagnostic and therapeutic approaches often associated with certain limitations and thus proved to be insufficient for customizing rapid and efficient combating strategy against the highly transmissible pathogenic infectious agents leading to the disastrous socio-economic outcome. Moreover, most of the viral zoonoses originate from the wildlife and poor knowledge about the global virome database renders it difficult to predict future outbreaks. Thus, alternative management strategy in terms of improved prophylactic vaccines and their delivery systems; rapid and efficient diagnostics and effective targeted therapeutics are the need of the hour. METHODS: Structured literature search has been performed with specific keywords in bibliographic databases for the accumulation of information regarding current nanomedicine interventions along with standard books for basic virology inputs. RESULTS: Multi-arrayed applications of nanomedicine have proved to be an effective alternative in all the aspects regarding the prevention, diagnosis, and control of zoonotic viral diseases. The current review is focused to outline the applications of nanomaterials as anti-viral vaccines or vaccine/drug delivery systems, diagnostics and directly acting therapeutic agents in combating the important zoonotic viral diseases in the recent scenario along with their potential benefits, challenges and prospects to design successful control strategies. CONCLUSION: This review provides significant introspection towards the multi-arrayed applications of nanomedicine to combat several important zoonotic viral diseases.

Zoonotic Viral Diseases of Equines and Their Impact on Human and Animal Health.

INTRODUCTION: Zoonotic diseases are the infectious diseases that can be transmitted to human beings and vice versa from animals either directly or indirectly. These diseases can be caused by a range of organisms including bacteria, parasites, viruses and fungi. Viral diseases are highly infectious and capable of causing pandemics as evidenced by outbreaks of diseases like Ebola, Middle East Respiratory Syndrome, West Nile, SARS-Corona, Nipah, Hendra, Avian influenza and Swine influenza. EXPALANTION: Many viruses affecting equines are also important human pathogens. Diseases like Eastern equine encephalitis (EEE), Western equine encephalitis (WEE), and Venezuelan-equine encephalitis (VEE) are highly infectious and can be disseminated as aerosols. A large number of horses and human cases of VEE with fatal encephalitis have continuously occurred in Venezuela and Colombia. Vesicular stomatitis (VS) is prevalent in horses in North America and has zoonotic potential causing encephalitis in children. Hendra virus (HeV) causes respiratory and neurological disease and death in man and horses. Since its first outbreak in 1994, 53 disease incidents have been reported in Australia. West Nile fever has spread to many newer territories across continents during recent years.It has been described in Africa, Europe, South Asia, Oceania and North America. Japanese encephalitis has expanded horizons from Asia to western Pacific region including the eastern Indonesian archipelago, Papua New Guinea and Australia. Rabies is rare in horses but still a public health concern being a fatal disease. Equine influenza is historically not known to affect humans but many scientists have mixed opinions. Equine viral diseases of zoonotic importance and their impact on animal and human health have been elaborated in this article. CONCLUSION: Equine viral diseases though restricted to certain geographical areas have huge impact on equine and human health. Diseases like West Nile fever, Hendra, VS, VEE, EEE, JE, Rabies have the potential for spread and ability to cause disease in human. Equine influenza is historically not known to affect humans but some experimental and observational evidence show that H3N8 influenza virus has infected man. Despite our pursuit of understanding the complexity of the vector-host-pathogen mediating disease transmission, it is not possible to make generalized predictions concerning the degree of impact of disease emergence. A targeted, multidisciplinary effort is required to understand the risk factors for zoonosis and apply the interventions necessary to control it.

Biocompatibility and Targeting Efficiency of Encapsulated Quinapyramine Sulfate-Loaded Chitosan-Mannitol Nanoparticles in a Rabbit Model of Surra.

Quinapyramine sulfate (QS) produces trypanocidal effects against the parasite Trypanosoma evansi but is often poorly tolerated and causes serious reactions in animals. The encapsulation of QS in chitosan-mannitol to provide sustained release would improve both the therapeutic effect of QS and the quality of life of animals treated with this formulation. QS was encapsulated into a nanoformulation prepared from chitosan, tripolyphosphate, and mannitol nanomatrix (ChQS-NPs). ChQS-NPs were well ordered in shape, with nanoparticle size, as determined by transmission electron microscopy and atomic force microscopy. Our research revealed dose-dependent effects on biosafety and DNA damage in mammalian cells treated with ChQS-NPs. ChQS-NPs were absolutely risk-free at effective as well as many times higher doses against T. evansi ChQS-NPs were effective in rabbits, as they killed the parasites, relieving the animals from the clinical symptoms of the disease. The extent of this protection was similar to that observed with the conventional drug at higher dosages (5 mg QS/kg of body weight). ChQS-NPs are safe, nontoxic, and more effective than QS and offer a promising alternative to drug delivery against surra in animal models. ChQS-NPs may be useful for the treatment of surra due to reduced dosages and frequency of administration.

Cytotoxicity and genotoxicity of a trypanocidal drug quinapyramine sulfate loaded-sodium alginate nanoparticles in mammalian cells.

We synthesized quinapyramine sulfate loaded-sodium alginate nanoparticles (QS-NPs) to reduce undesirable toxic effects of QS against the parasite Trypanosoma evansi, a causative agent of trypanosomosis. To determine the safety of the formulated nanoparticles, biocompatibility of QS-NPs was determined using Vero, Hela cell lines and horse erythrocytes in a dose-dependent manner. Our experiments unveiled a concentration-dependent safety/cytotoxicity (metabolic activity), genotoxicity (DNA damage, chromosomal aberrations), production of reactive oxygen species and hemolysis in QS-NPs treated cells. Annexin-V propidium iodide (PI) staining showed no massive apoptosis or necrosis. However, at very high doses (more than 300 times than the effective doses), we observed more toxicity in QS-NPs treated cells as compared to QS treated cells. QS-NPs were safe at effective trypanocidal doses and even at doses several times higher than the effective dose.

Alginate/gum acacia bipolymeric nanohydrogels--promising carrier for zinc oxide nanoparticles.

Zinc oxide nanoparticles (ZnO nps) are known to be effective against a wide array of microorganisms. At nanoscale, they have higher toxicity and they need to be rendered less toxic and more biocompatible. To achieve this, ZnO nps were incorporated in nanohydrogel particles made out of sodium alginate/gum acacia and cross-linker glutaraldehyde in order to ensure their gradual and sustained release instead of burst release, and hence lowering their toxicity. The particles synthesized were in the nano-range, i.e., 70-100 nm size and their in vitro release studies indicated that release of upto 68% of ZnO nps was prolonged to over 2 weeks following the Higuchi model. Cytotoxicity studies on vero cell line (African green monkey kidney cell line) revealed that toxicity of ZnO nps-loaded nanohydrogels was substantially lower as compared to ZnO nps. At the same time, it demonstrated desired level of antibiotic activity against Pseudomonas aeruginosa, an antibiotic resistant microbial model. In conclusion, this work led to successful preparation of novel formulation of ZnO incorporated in nanohydrogels that are not only safer but also retain adequate antibacterial activity due to their ability for gradual and sustained release of the active constituent.

CpG-ODN class C-mediated immunostimulation and its potential against Trypanosoma evansi in equines.

Trypanosoma evansi is the causative agent of surra, which is the most common and widespread trypansomal disease. The infection is mainly restricted to animals, but it has also been documented in human. Trypanosomes possess the thick immunogenic surface coat known as variant surface glycoprotein (VSG). The parasite modifies the VSG constantly resulting in continuous antigenic variations and thus evades the host immune response. Due to antigenic variations, vaccination against trypanosomosis is not useful. Therefore, alternate strategies to augment the immune response are required. CpG-ODN class-C has combined immune effects of both A and B classes of CpG-ODN. In this study, we observed that CpG-ODN class-C stimulated horse peripheral blood mononuclear cells (PBMC) induce the expression of interferon-α (IFN-α), tumor necrosis factor-α (TNF-α), IL-12 and nitric oxide (NO) indicating enhanced innate immune response. We have for the first time demonstrated that co-culture of CpG-ODN with T. evansi antigen induces lymphocyte proliferative responses and result in a synergistic effect in eliciting the immune response.