Unraveling the Role of Deep Eutectic Solvents with Varying Hydrogen-Bond Acceptors on the Thermoresponsive Polymer Poly(N-isopropylacrylamide).

Deep eutectic solvents (DESs) have emerged as promising tools for crafting polymeric materials across diverse domains. This study delves into the impact of a series of DESs on the phase behavior of poly(N-isopropylacrylamide) (PNIPAM) in aqueous environments, presenting compelling insights into their performance. Specifically, we explore the conformational phase behavior of PNIPAM in the presence of four distinct lactic acid (LA)-based DESs: LA-betaine (LA-BET), LA-proline (LA-PRO), LA-choline chloride (LA-CC), and LA-urea (LA-U). By maintaining a consistent hydrogen-bond donor (HBD) while varying the hydrogen-bond acceptor (HBA), we unravel how different DES compositions modulate the phase transition behavior of PNIPAM. Our findings underscore the profound influence of DESs comprising LA as the HBD and diverse HBAs-BET, PRO, CC, and U on the thermoresponsive behavior of PNIPAM. Employing spectroscopic techniques such as ultraviolet-visible (UV-vis) spectroscopy, steady-state fluorescence, Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), ζ-potential, and transmission electron microscopy (TEM), we elucidate the preferential interactions between the HBA groups within DESs and the hydration layer of PNIPAM. Notably, temperature-dependent DLS analyses reveal a discernible decrease in the lower critical solution temperature (LCST) of PNIPAM with increasing DES concentration, ultimately disrupting the hydrogen-bond interactions and resulting in early hydrophobic collapse of the polymer, which can be clearly seen in the TEM micrographs. Furthermore, the formation of polymer composites within the mixed system leads to notable alterations in the physiochemical properties of PNIPAM, as evidenced by shifts in its LCST value in the presence of DESs. This perturbation disrupts hydrogen-bond interactions, inducing hydrophobic collapse of the polymers, a phenomenon vividly captured in TEM micrographs. In essence, our study sheds new light on the pivotal role of varying HBA groups within DESs in modulating the conformational transitions of PNIPAM. These insights not only enrich our fundamental understanding but also hold immense promise for the development of smart polymeric systems with multifaceted applications spanning bioimaging, biomedical science, polymer science, and beyond.

Green Synthesis of Silver Nanoparticles Using Drymaria cordata and Their Biocompatibility with Hemoglobin: A Therapeutic Potential Approach.

In this study, we present the synthesis and characterization of AgNPs using Drymaria cordata along with an assessment of their antioxidant, antibacterial, and antidiabetic activities. Antibacterial activities using four bacterial strains, free radical scavenging assays (DPPH and ABTS), and carbohydrate hydrolyzing enzyme inhibition assays were done to examine the therapeutic efficacy of AgNPs. Additionally, herein, we also evaluated the biocompatibility of the AgNPs using hemoglobin (Hb) as a model protein. A comprehensive analysis of Hb and AgNP interactions was carried out by using various spectroscopic, imaging, and size determination studies. Spectroscopic results showed that the secondary structure of Hb was not altered after its interaction with AgNPs. Furthermore, the thermal stability was also well maintained at different concentrations of nanoparticles. This study demonstrated a low-cost, quick, and eco-friendly method for developing AgNPs using D. cordata, and the biocompatible nature of AgNPs was also established. D. cordata-mediated AgNPs have potential applications against bacteria and diabetes and may be utilized for targeted drug delivery.

A novel amalgamation of deep eutectic solvents and crowders as biocompatible solvent media for enhanced structural and thermal stability of bovine serum albumin.

Proteins have immense untapped potential in numerous industries as a green catalyst. Thus, there is an emergent need to find a suitable co-solvent which is biocompatible with proteins and environmentally safe. In this context, the present study investigates the effect of a novel solvent medium designed by an amalgamation of macromolecular crowders and deep eutectic solvents (DESs) on bovine serum albumin (BSA). It was discovered that the presence of crowding agents such as polyethylene glycol (PEG) 12 kDa (P12), 20 kDa (P20), and Ficoll 70 kDa (F70) marginally destabilizes the conformational stability of BSA. While the thermal stability of BSA was dramatically enhanced in the presence of choline chloride (ChCl)-based deep eutectic solvents (DESs) namely ChCl-urea (DES1) and ChCl-glycerol (DES2), the order of stability was (DES1) > (DES2). It was interesting to note that DES1 possessing urea as a hydrogen bond donor leads to the exceptional thermal stability of the BSA structure. Taking a cue from this, an innovative crowded DES medium was prepared by mixing a synthetic crowder and DES1 in optimum concentration. Fascinatingly, the combination of DES1 with PEG delivers promising results, as they elevate the thermal stability of BSA by approximately 16 °C. Thus, crowded DES medium confers structural compactness to the BSA. Altogether, this work reports for the first time the potential of DESs to attenuate the adverse effects of macromolecular crowding on protein stability.

Sustainable Solvothermal Conversion of Waste Biomass to Functional Carbon Material: Extending Its Utility as a Biocompatible Cosolvent for Lysozyme.

Functional carbon material synthesis from waste biomass by a sustainable method is of prime importance and has wide variety of applications. Herein, functional carbon materials with structural variability are synthesized using a well-known solvothermal method. The leftover pulp waste biomass (PB) of citrus limetta is converted to functional carbon by treatment with a mixture of choline bitartrate (ChBt) and FeCl3 (1:2 mol ratio) as a solvent. The biomass to solvent ratio is varied as 1:1, 0.8:1, and 0.4:1 during solvothermal treatment to obtain PB-1, PB-2, and PB-3 as functional carbon materials, respectively. On characterization, PB carbon materials were found to be rich in oxygen-containing functional groups possessing different morphologies. Furthermore, results suggested the role of solvent as a soft template and catalyst during the synthesis of carbon materials. The feasibility of synthesized carbon materials as a biocompatible cosolvent for lysozyme was evaluated. In the case of PB-2 material (synthesized using 0.8:1 biomass to solvent ratio), results show an enhancement of lysozyme activity by 150%. Besides, spectroscopic and calorimetric data confirm the preservation of thermal and structural stability of lysozyme in the PB-2 solution. Thus, this study stipulates PB-2 as an excellent cosolvent for protein studies. With this work, we aim to delve into an entirely new arena of applications of biomass in the field of biotechnology.

Strategic planning of proteins in ionic liquids: future solvents for the enhanced stability of proteins against multiple stresses.

Ionic liquids (ILs) present a vast number of solvents capable of replacing toxic organic solvents in chemical, biotechnology and biomedical applications. ILs are inexpensive and environmentally friendly as the materials can be recycled conveniently. Chemists use a variety of cation and anion combinations to produce an IL that fits the requirements of the sustainable future through the pursuit of greener chemical processes. As such, the development of various types of ILs has been recognized as the emergence of environmentally friendly solvents to attain enhanced protein stability in vitro. The literature survey reveals that there exist a large number of scholarly articles as well as elegant reviews on protein stability in ILs. Biomolecules have adapted to antagonistic environmental stresses that normally denature proteins, and the mechanism of adaptation that protects the cellular components against denaturation involves the intracellular concentration of co-solvents. In this regard, recent experimental results distinctly demonstrated that ILs are stabilizing proteins against denaturing stresses, and their presence in the cells does not alter protein functional activities. However, a review focusing particularly on the refolding and counteracting effects of the ILs against denatured proteins by multiple stresses is still missing. This perspective unveils the studies that have been conducted to improve protein stabilities with ILs as well as the refolding and counteracting abilities of these ILs against the denatured proteins under the influence of multiple stresses. We believe that ILs can provide significant environmental and economic advantages for biochemical processes in the near future. Essentially, numerous investigations are required to allow us to further explore the stabilizing properties of ILs over proteins.

Designing biological fluid inspired molecularly crowded ionic liquid media as a sustainable packaging platform for cytochrome c.

Biological fluids are highly crowded due to the presence of various biomolecules. Therefore, it is essential to study the effects of molecular crowding agents to probe the behavior of a protein in a cell-like environment. Inspired by biofluids, herein, a molecularly crowded environment is created in the presence of an ionic liquid (IL), envisaging sustainable protein packaging. Interestingly, the molecularly crowded IL media enhanced the stability and catalytic activity (1.5-fold higher) of cytochrome c (Cyt c) as compared to the IL alone and the crowding agents without the IL. A similar trend was observed when the activity was recorded at 100 °C and when stored at room temperature for 30 days. Moreover, Cyt c dissolved in the molecularly crowded IL media was regenerated successfully without affecting the melting temperature of the protein, confirming the suitability of the molecularly crowded IL media as a potential and ecofriendly packaging system for Cyt c.

Does macromolecular crowding compatible with enzyme stem bromelain structure and stability?

It is essential to explore the impact of macromolecular crowding on protein in order to understand the behavior of the protein in the biological system. In this context, the consequences of macromolecular crowding on stem bromelain (BM) are explored, which is important for its industrial application. Herein, the effect of dextran (D70) and polyethylene glycol (P12 and P20) on native BM structure are investigated. The effect of crowder molecules on BM are deduced by combining the results of absorption, circular dichroism, fluorescence and activity studies. Additionally, molecular level interactions are investigated with molecular docking studies. Our results display that BM acts differently in higher and lower concentrations of crowding agents. Furthermore, crowding leads to alteration of protein structure and activity as compared to the dilute solutions. We observed that D70, is a very effective thermal stabilizing agent, while P12 is moderately stabilizing, on the other hand, P20 is destabilizing the BM structure. In the present study, the overall effect of crowders was destabilizing and deactivating on enzyme. Therefore, this study provides yet another example where soft interaction is dominating over the excluded volume effect.

Crowded milieu tuning the stability and activity of stem bromelain.

Proteins in vivo are under an extremely crowded environment because of the presence of bulky and large biological macromolecules (known as crowders). These crowders affect the proper functioning and structure of proteins in a cell. During in vitro studies, we often ignore the effect of macromolecular crowding on protein stability. However, if a large concentration of crowder is used to examine protein stability, its effects on the functioning of protein inside the cell in a confined environment, as stated, can be understood. Keeping this in context, we investigated the effects of macromolecular crowding on stem bromelain (BM) with the help of different crowding agents of varying molecular weights such as dextran (40 kDa and 6 kDa) and ficoll (70 kDa). Activity and stability of BM was examined using UV-vis, fluorescence and circular dichroism (CD) spectroscopy. Furthermore, docking methods are used to complement the crowding effects on the stability of BM. We found that stability and activity of BM are dependent on the surrounding crowder molecules. Thermal flouresence results showed that, thermal stability of BM decreses with incresing concentration of crowder except dextran40. It was observed that the decrese in stability and activity can be related to the presence of soft interactions between crowder and BM. Thus,crowding does not always stabilize the native structure, instead, it depends on degree of disorder of protein structure and on two competing effects: the excluded volume, which favors compact states, and soft interactions, which favor extended conformers.