Nand Kishore

Generation of different states of lysozyme in presence of HFIP and TFE at pH 2 and pH 11.

Human serum albumin adsorption on graphitic carbon nitride nanorods.

The aggregation of proteins and the formation of amyloid fibrils as a result of protein misfolding are thought to be strongly linked to neurodegenerative disorders, including those driven by infectious prions, Alzheimer's disease, and Parkinson's disease, to name only a few. The rapid discovery of inhibitors that prevent protein aggregation has aided in the development of therapeutic approaches for these conditions. Plant-based extracts and chemical substances have become intriguing sources of potential inhibitors since they can be used as particular pharmaceuticals at greater doses or as nutraceuticals as part of a healthy diet. Succulent plants like aloe vera have long been valued for their medicinal and healing qualities. This research focuses on the inhibition of hen egg white lysozyme fibrillation using aloe vera extract. To verify fibril formation and assess the inhibitory potential of aloe vera, an integrated approach involving calorimetric, spectroscopic, and microscopic techniques was employed. Employing a variety of methods, it was found that aloe vera promotes the thermal and structural stability of proteins while inhibiting the formation of fibrils. Furthermore, aloin, a significant bioactive component of aloe vera, was also studied for its anti-fibrillogenic properties and proven to efficiently prevent the formation of fibrils. Aloin demonstrated effective inhibition of protein fibrillation, likely through hydrophobic and electrostatic interactions. With the growing preference for natural products due to their reduced side effects, this study highlights the potential of plant-derived therapeutics as promising inhibitors of protein aggregation.

This study investigates the interaction of solid lipid nanoparticles (SLNs) with the transport protein bovine serum albumin (BSA) in terms of thermodynamic signatures, employing both spectroscopic and calorimetric techniques. When nanoparticles are exposed to biological media, proteins are adsorbed on their surfaces, leading to protein corona formation. Therefore, controlling the formation of the protein corona is essential for in vivo therapeutic efficacy. Although SLNs have previously been explored solely as potential nano-carriers for drug delivery, no prior efforts have been made to study their interactions with biomolecules from a biophysical and mechanistic perspective. SLNs are colloidal dispersions of the solid lipid in an aqueous solution stabilized by surfactants. Herein, a hot emulsification methodology was employed to formulate SLNs, and their interactions with BSA were analyzed. The SLNs were characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques to obtain information on their size, zeta potential, and shape. Fluorescence data suggested the presence of weak interactions between the SLNs and BSA. Static quenching is confirmed using time-correlated single-photon counting (TCSPC) experiments. Differential scanning calorimetric (DSC) and fluorescence spectroscopic experiments suggest the thermal stabilization of BSA by the SLNs. This stabilization results from the enhancement of the secondary structure of the protein without significantly altering the tertiary structure. Isothermal calorimetry (ITC) results suggest weak interactions between the SLNs and BSA, although not in a site-specific manner. Overall, mechanistic insights into lipid nanoparticle–protein interactions obtained from such studies efficiently overcome the hurdles associated with targeted drug delivery.

It is crucial to comprehend protein misfolding and aggregation in the domains of biomedicine, pharmaceuticals, and proteins. Amyloid fibrils are formed when proteins misfold and assemble, resulting in the debilitating illness known as "amyloidosis". This work investigates lysozyme fibrillation with pluronics (F68 and F127). The effect of pluronics on protein aggregation and fibrillation has been studied mechanistically using a combination of calorimetric and spectroscopic techniques. TEM images and the ThT binding experiment were used to analyze the conformation of protein fibrils, and the results showed that pluronics accelerated the fibrillation process. When pluronics interact with protein at different stages of fibrillation, their pre- and postmicellar concentrations show a decrease in ΔH_m° value as the time of incubation increases. This indicates the formation of amorphous aggregates due to which endothermic enthalpy is observed. As a consequence, it was investigated if these generated aggregates can also act as drug delivery vehicle; therefore, the work was carried out with 5-fluorouracil and cytarabine. The endothermic enthalpy of interaction suggests that hydrophobic interaction is more prevalent when cytarabine is employed with protein fibrils, whereas the electrostatic interaction is more prevalent when 5-fluorouracil is combined with it. The former drug, however, showed a greater adsorption than the latter on the surface of protein fibrils. It is therefore determined that 5-fluorouracil has relatively significant adsorption on fibril surfaces, whereas cytarabine has weak adsorption and is easily desorbed in cells. Consequently, the combination of LFF127 and 5-FU is lethal to malignant cells. The drug encapsulation and delivery aspect of protein fibrils/aggregates needs further exploration.

Protein–ligand interactions are crucial for developing and identifying novel therapeutic targets. In this study, we investigate the interaction of the alkali induced state of human serum albumin (pH 11.2) with three hydroxycinnamic acid derivatives (HCDs), ferulic acid (FA), sinapic acid (SA) and trans-o-coumaric acid, which are biologically important antioxidants, and compare the outcomes with the results obtained at physiological pH (7.4). This study aims to explore the interaction of altered protein conformation with small molecules. Spectroscopic characterization methods show that the conformation of HSA and the ionic properties of HCDs are pH-dependent. Fluorescence, FRET and lifetime measurements reveal that the binding of HCDs with HSA is different at both pH 7.4 and 11.2. Despite the moderate binding of HCDs to HSA, circular dichroism and thermal denaturation studies report no conformational changes in HSA in the presence of HCDs. Isothermal titration calorimetry is employed to assess their binding based on structure and energetics using thermodynamic parameters. Standard molar enthalpy change (ΔH⁰ _m) and standard molar entropy change (ΔS⁰ _m) values vary with the change of pH from 7.4 to 11.2 with the contributions from the exothermicity and hydrophobicity of functional and aromatic groups of HCDs. Ferulic acid (FA) and sinapic acid (SA) binding to HSA is entropically driven, whereas trans-o-coumaric acid (CA) acid binding is enthalpically favourable. Our ITC studies also reveal that the involvement of –OH functional groups present in CA in binding with HSA is greater than that present in FA and SA at pH 11.2. Overall, this experimental study shows the comparable binding strength of HCDs to both the alkali-induced state of HSA and native HSA (pH 7.4). However, the mechanism of their binding is different.