A safe, cost-effective, and high-throughput SARS-CoV-2 antigen capture ELISA suitable for large-scale screening in low-resource settings.

Diagnostics employing multiple modalities have been essential for controlling and managing COVID-19, caused by SARS-CoV-2. However, scaling up Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR), the gold standard for SARS-CoV-2 detection, remains challenging in low and middle-income countries. Cost-effective and high-throughput alternatives like enzyme-linked immunosorbent assay (ELISA) could address this issue. We developed an in-house SARS-CoV-2 nucleocapsid capture ELISA, and validated on 271 nasopharyngeal swab samples from humans (n = 252), bovines (n = 10), and dogs (n = 9). This ELISA has a detection limit of 195 pg/100 µL of nucleocapsid protein and does not cross-react with related coronaviruses, ensuring high specificity to SARS-CoV-2. Diagnostic performance was evaluated using receiver operating characteristic curve analysis, showing a diagnostic sensitivity of 67.78 % and specificity of 100 %. Sensitivity improved to 74.32 % when excluding positive clinical samples with RT-qPCR Ct values > 25. Furthermore, inter-rater reliability analysis demonstrated substantial agreement (κ values = 0.73-0.80) with the VIRALDTECT II Multiplex RT-qPCR kit and perfect agreement with the CoVeasy™ COVID-19 rapid antigen self-test (κ values = 0.89-0.93). Our findings demonstrated that the in-house nucleocapsid capture ELISA is suitable for SARS-CoV-2 testing in humans and animals, meeting the necessary sensitivity and specificity thresholds for cost-effective, large-scale screening.

In-process quality control in foot-and-mouth disease vaccine production by detection of viral non-structural proteins using chemiluminescence dot blot assay.

Foot-and-mouth disease (FMD) is a contagious viral disease of cloven-footed animals. Immunization with inactivated virus vaccine is effective to control the disease. Six-monthly vaccination regimen in endemic regions has proven to be effective. To enable the differentiation of infected animals from those vaccinated, non-structural proteins (NSPs) are excluded during vaccine production. While the antibodies to structural proteins (SPs) could be observed both in vaccinated and infected animals, NSP antibodies are detectable only in natural infection. Quality control assays that detect NSPs in vaccine antigen preparations, are thus vital in the FMD vaccine manufacturing process. In this study, we designed a chemiluminescence dot blot assay to detect the 3A and 3B NSPs of FMDV. It is sensitive enough to detect up to 20 ng of the NSP, and exhibited specificity as it does not react with the viral SPs. This cost-effective assay holds promise in quality control assessment in FMD vaccine manufacturing.

Development, characterization and in vitro-in vivo evaluation of Farnesol loaded niosomal gel for applications in oral candidiasis treatment.

OBJECTIVES: The aim of the study was to formulate and characterize the farnesol loaded niosomes comprising gel formulation and perform their in vitro-in vivo evaluation for applications in the treatment of oral candidiasis infections. METHODS: Various gelling systems were evaluated for their rheological and stability properties. The formulation was statistically optimized using experimental design method (Box-Behnken). Transmission electron microscopy (TEM) and Atomic force microscopy (AFM) were used to observe the niosomal surface morphology. Centrifugation method and dialysis method were used to find out the % entrapment efficiency (%EE) and in-vitro release of Farnesol, respectively. In-vitro antifungal effect and cell biocompatibility of the Farnesol loaded niosomal gel was also performed using Candida albicans (C. albicans) as the model organism and epithelial cell line (SW480) by MTT cytotoxicity assay. In-vivo skin irritation test was performed on rabbit skin. KEY FINDINGS: Farnesol loaded niosomes were integrated into polymeric gel solution. The optimized formulation demonstrated acceptable % EE (>80%) and an optimum particle size (168.8 nm) along with a sustained release and a long-term storage stability for up to a period of 6 months. TEM and AFM observations displayed a spherical niosome morphology. Farnesol niosomal gel showed a higher antifungal efficacy, ex-vivo skin permeation and deposition in comparison to plain farnesol solution. The niosomal gel also showed negligible cytotoxicity to normal cells citing biocompatibility and was found to be non-toxic and non-irritant to rabbit skin. CONCLUSIONS: This novel niosome loaded gel-based formulation could make the oral candidiasis healing process more efficient while improving patient compliance. With the optimized methodology used in this work, such formulation approaches can become an efficient, industrially scalable, and cost-effective alternatives to the existing conventional formulations.

Design, development and in-vitro/in-vivo evaluation of intranasally delivered Rivastigmine and N-Acetyl Cysteine loaded bifunctional niosomes for applications in combinative treatment of Alzheimer's disease.

The present investigation explores the potential of novel dual drug-loaded niosomes for nasal delivery of Rivastigmine (RIV) and N-Acetyl Cysteine (NAC) to the brain. The dual niosomes showed a particle size of 162.4 nm and % entrapment efficiencies of 97.7% for RIV and 85.9% for NAC. The niosomes were statistically validated using Box-Behnken experimental design (BBD) with good significance. Ultrastructural and chemical characterization of the niosomes using various analytical techniques like Fourier Transform Infrared spectroscopy (FTIR), Differential scanning calorimetry (DSC), Transmission electron microscopy (TEM) showcased drug-excipient compatibility and robust stability of 6 months in a liquid state at 4-8 °C. The dual drug-loaded niosomes showed a sustained drug release pattern up to 2 days. Acetylcholinesterase (AChE) and DPPH (1, 1-diphenyl-2- picrylhydrazyl) enzyme inhibition assays showed a better combinative effect than the free drug solutions. A 2-day nasal permeation proved the effectiveness and biocompatibility of the niosomes. In-vivo pharmacokinetic and organ biodistribution studies revealed a better drug profile and greater distribution of the niosomes in the brain compared to other organs, thereby indicating a direct nose-to-brain delivery of the niosomes.

Physicochemical and biological assessment of silver nanoparticles immobilized Halloysite nanotubes-based resin composite for dental applications.

OBJECTIVE: The purpose of this study was to investigate the effect of Silver nanoparticle immobilized Halloysite Nanotubes (HNT/Ag) fillers on physicochemical, mechanical, and biological properties of novel experimental dental resin composite in order to compare with the properties of corresponding composites containing conventional glass fillers. METHODS: Dental resin (Bis-GMA/TEGDMA with ratio 70/30) composites were prepared by incorporation of varied mass fraction of HNT/Ag. Experimental composites were divided into six groups, one control group and five experimental groups containing mass fraction 1 to 10.0 wt. % of HNT/Ag. Mechanical properties of the dental composites were recorded. Degree of conversion and depth of cure of the dental resin composites were assessed. Antimicrobial properties were assessed using agar diffusion test and evaluation of cytotoxicity were performed on NIH-3T3 cell line. RESULTS: The inclusion of mass fractions (1-5 wt. %) of the HNT/Ag in dental resins composites, significantly improved mechanical properties. While, addition of larger mass fractions (7.5 and 10 wt. %) of the HNT/Ag did not show further improvement in the mechanical properties of dental resins composites. Theses composites also demonstrated satisfactory depth of cure and degree of conversion. A significant antibacterial activity was observed on S. mutans. No significant cytotoxicity was found on NIH-3T3 cell lines. CONCLUSION: The incorporation of HNT/Ag in Bis-GMA/TEGDMA dental resins composites resulted in enhancement in mechanical as well as biological properties for dental applications. CLINICAL SIGNIFICANCE: HNT/Ag containing dental composite is proposed to be highly valuable in the development of restorative dental material for patients with high risk of dental caries.

Physicochemical and biological assessment of flowable resin composites incorporated with farnesol loaded halloysite nanotubes for dental applications.

The aim of this study was to fabricate flowable resin composites, by incorporating Farnesol loaded Halloysite Nanotubes (Fa-HNT) as a filler and evaluate their physicochemical as well as biological properties. Chemical and morphological characterization of antibacterial filler, Fa-HNT were performed using Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), Transmission Electron Microscope (TEM), Scanning Electron Microscope (SEM). The antibacterial filler was mixed into composite material consisting of methacrylate monomers and dental glass fillers at concentrations of 1-20% (wt./wt.). It was observed that addition of mass fractions of Fa-HNT causes enhancement of compressive strength as well as flexural modulus of the composite. However, it significantly decreases flexural strength and degree of conversion. A significant antibacterial activity of dental composite was observed with increase in the area of zone of inhibition against the strains of Streptococcus mutans (S. mutans). There was no cytotoxicity observed by Fa-HNT resin composites on NIH-3T3 (mouse embryonic fibroblast cells) cell lines. A favourable integration of antibacterial filler with significant mechanical properties was achieved at concentrations from 7 to 13 wt% of Fa-HNT in dental composites, which is desirable in dentistry.

Formulation, Characterization and In-vitro and In-vivo Evaluation of Capecitabine Loaded Niosomes.

BACKGROUND: Nanocarriers improve the efficacy of drugs by facilitating their specific delivery and protecting them from external environment resulting in a better performance against diseases. OBJECTIVE: In this study, it was aimed to improve the efficacy of capecitabine against colorectal cancer by its entrapment in niosomes. Ether injection method was used to prepare niosomes composed of span 20 and cholesterol. METHODS: Niosomes were evaluated by evaluating the entrapment efficiency, in-vitro drug release and cytotoxicity of capecitabine loaded niosomes. Niosomes were characterized by particle size analysis, transmission electron microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry for surface morphology and drug excipient interactions. RESULTS: High encapsulation efficiency (90.55%) was observed, which is anticipated to resolve the multi-drug resistance problem. Reported particle size was 180.9 + 5 nm with a negative zeta potential - 21 + 0.5 mV and the kinetic study showed a concentration-dependent release of the drug from the niosome. DSC study proved entrapment of the entire drug and its non-covalent bonding with the excipients. Cytotoxicity study of niosomes on CaCO2 cell line showed an improved IC50 value as compared to the free drug. CONCLUSION: Enhanced cytotoxicity observed in the results further supports the suitability of niosome as a nanocarrier for pharmaceutical drug delivery.

Hyaluronic acid: A review on its biology, aspects of drug delivery, route of administrations and a special emphasis on its approved marketed products and recent clinical studies.

Hyaluronic acid (HA) is a large non-sulphated glycosaminoglycan that is an important component of extracellular matrix (ECM) and a biodegradable polymer. Due to a variation in its molecular weight, HA derivatives can be utilized to make different formulations like fillers, creams, gels and drops. HA based drug research has seen a recent surge largely due to some properties like mucoadhesion, biocompatibility and ease of chemical modification. Such properties of HA have led to applications in tissue regeneration, anti-aging and anti-inflammatory medications. HA can be conjugated, functionalized or used as a nanocarrier supplement with a definite increase in its cellular uptake and efficiency. HA when encapsulated in a nanocarrier may help to improve the ECM growth and provide a sustained release of agents. This review discusses the mechanistic behavior of HA pertaining to its biological synthesis and degradation. It also discusses the administration of some noteworthy and recent HA based formulations through different routes for application in various physiological conditions along with their ongoing clinical trial updates and approved marketed products.

Application of Box-Behnken Design in the Preparation, Optimization, and In Vitro Evaluation of Self-Assembly-Based Tamoxifen- and Doxorubicin-Loaded and Dual Drug-Loaded Niosomes for Combinatorial Breast Cancer Treatment.

This study was developed with the objective to prepare self-assembled niosomes to support sufficient entrapment and sustained drug release of the drugs having different solubility and mechanisms. In the current work, Tamoxifen- and Doxorubicin-loaded niosomes were prepared for combinatorial breast cancer treatment with statistical optimization by Box-Behnken experimental design. Atomic force microscopy revealed a spherical shape morphology of the niosomes. The entrapment efficiencies for the drugs were found to be 74.3% and 72.7% for Tamoxifen and Doxorubicin, respectively. The drug release experiments at different pH values displayed a sustained release up to 3 days. Fourier transform infrared spectroscopy and differential scanning calorimetry showed a robust drug-excipient compatibility. The niosomes were stable over a period of 6 months with no significant changes. In vitro cytotoxicity studies on MCF-7 cell line showed a 15-fold improvement (0.01 µg per mL) and a better synergistic effect of the niosomes in comparison to the free drug combination (0.15 µg per mL). Moreover, the nanocarrier uptake studies by fluorescence microscopy and flow cytometry showed a good distribution and greater uptake of the niosomes throughout the cells. These results suggest a profound therapeutic application of the niosomes for a combinatorial breast cancer treatment.