Insulin fibrillation under physicochemical parameters of bioprocessing and intervention by peptides and surface-active agents.

Even after the centenary celebration of insulin discovery, there prevail challenges concerning insulin aggregation, not only after repeated administration but also during industrial production, storage, transport, and delivery, significantly impacting protein quality, efficacy, and effectiveness. The aggregation reduces insulin bioavailability, increasing the risk of heightened immunogenicity, posing a threat to patient health, and creating a dent in the golden success story of insulin therapy. Insulin experiences various physicochemical and mechanical stresses due to modulations in pH, temperature, ionic strength, agitation, shear, and surface chemistry, during the upstream and downstream bioprocessing, resulting in insulin unfolding and subsequent fibrillation. This has fueled research in the pharmaceutical industry and academia to unveil the mechanistic insights of insulin aggregation in an attempt to devise rational strategies to regulate this unwanted phenomenon. The present review briefly describes the impacts of environmental factors of bioprocessing on the stability of insulin and correlates with various intermolecular interactions, particularly hydrophobic and electrostatic forces. The aggregation-prone regions of insulin are identified and interrelated with biophysical changes during stress conditions. The quest for novel additives, surface-active agents, and bioderived peptides in decelerating insulin aggregation, which results in overall structural stability, is described. We hope this review will help tackle the real-world challenges of insulin aggregation encountered during bioprocessing, ensuring safer, stable, and globally accessible insulin for efficient management of diabetes.

Biogenesis, Isolation, and Detection of Exosomes and Their Potential in Therapeutics and Diagnostics.

The increasing research and rapid developments in the field of exosomes provide insights into their role and significance in human health. Exosomes derived from various sources, such as mesenchymal stem cells, cardiac cells, and tumor cells, to name a few, can be potential therapeutic agents for the treatment of diseases and could also serve as biomarkers for the early detection of diseases. Cellular components of exosomes, several proteins, lipids, and miRNAs hold promise as novel biomarkers for the detection of various diseases. The structure of exosomes enables them as drug delivery vehicles. Since exosomes exhibit potential therapeutic applications, their efficient isolation from complex biological/clinical samples and precise real-time analysis becomes significant. With the advent of microfluidics, nano-biosensors are being designed to capture exosomes efficiently and rapidly. Herein, we have summarized the history, biogenesis, characteristics, functions, and applications of exosomes, along with the isolation, detection, and quantification techniques. The implications of surface modifications to enhance specificity have been outlined. The review also sheds light on the engineered nanoplatforms being developed for exosome detection and capture.

Synthesis of iron oxide nanoparticles mediated by Camellia sinensis var. Assamica for Cr(VI) adsorption and detoxification.

Environment-benign synthesis of nanoparticles (NPs) are of great importance. Plant-based polyphenols (PPs) are electron donor analytes for the synthesis of metal and metal oxide NPs. This work produced and investigated iron oxide nanoparticles (IONPs) from PPs of tea leaves of Camellia sinensis var. assamica for Cr(VI) removal. The conditions for IONPs synthesis were using RSM CCD and found to be optimum at a time of 48 min, temperature of 26 °C, and iron precursors/leaves extract ratio (v/v) of 0.36. Further, these synthesized IONPs at a dosage of 0.75 g/L, temperature of 25 °C, and pH 2 achieved a maximum of 96% Cr(VI) removal from 40 mg/L of Cr(VI) concentration. The exothermic adsorption process followed the pseudo-second-order model, and Langmuir isotherm estimated a remarkable maximum adsorption capacity (Qm) of 1272 mg g-1 of IONPs. The proposed mechanistic for Cr(VI) removal and detoxification involved adsorption and its reduction to Cr(III), followed by Cr(III)/Fe(III) co-precipitation.

Surface modified iron-oxide based engineered nanomaterials for hyperthermia therapy of cancer cells.

Magnetic hyperthermia is emerging as a promising alternative to the currently available cancer treatment modalities. Superparamagnetic iron-oxide nanoparticles (SPIONs) are extensively studied functional nanomaterials for biomedical applications, owing to their tunable physio-chemical properties and magnetic properties. Out of various ferrite classes, spinel and inverse-spinel ferrites are widely used but are affected by particle size distribution, particle shape, particle-particle interaction, geometry, and crystallinity. Notably, their heating ability makes them suitable candidates for heat-mediated cancer cell ablation or hyperthermia therapy. Exposing SPIONs to an externally applied magnetic field of appropriate frequency and intensity causes them to release heat to ablate cancer cells. Majorly, three heating mechanisms are exhibited by magnetic nanomaterials: Nèel relaxation, Brownian relaxation, and hysteresis losses. In SPIONs, Nèel and Brownian relaxations dominate, whereas hysteric losses are negligible. These nanomaterials possess high magnetization values capable of generating heat to ablate cancer cells. Furthermore, surface functionalization of these materials imparts the ability to selectively target cancer cells and deliver cargo to the affected area sparing the normal body cells. The surface of nanoparticles can be functionalized with various physical, chemical, and biological coatings. Moreover, hyperthermia can be applied in combination with other cancer treatment modalities in order to enhance the efficiency of treatment.

siRNA Functionalized Lipid Nanoparticles (LNPs) in Management of Diseases.

RNAi (RNA interference)-based technology is emerging as a versatile tool which has been widely utilized in the treatment of various diseases. siRNA can alter gene expression by binding to the target mRNA and thereby inhibiting its translation. This remarkable potential of siRNA makes it a useful candidate, and it has been successively used in the treatment of diseases, including cancer. However, certain properties of siRNA such as its large size and susceptibility to degradation by RNases are major drawbacks of using this technology at the broader scale. To overcome these challenges, there is a requirement for versatile tools for safe and efficient delivery of siRNA to its target site. Lipid nanoparticles (LNPs) have been extensively explored to this end, and this paper reviews different types of LNPs, namely liposomes, solid lipid NPs, nanostructured lipid carriers, and nanoemulsions, to highlight this delivery mode. The materials and methods of preparation of the LNPs have been described here, and pertinent physicochemical properties such as particle size, surface charge, surface modifications, and PEGylation in enhancing the delivery performance (stability and specificity) have been summarized. We have discussed in detail various challenges facing LNPs and various strategies to overcome biological barriers to undertake the safe delivery of siRNA to a target site. We additionally highlighted representative therapeutic applications of LNP formulations with siRNA that may offer unique therapeutic benefits in such wide areas as acute myeloid leukaemia, breast cancer, liver disease, hepatitis B and COVID-19 as recent examples.

Unfolding of the SARS-CoV-2 spike protein through infrared and ultraviolet-C radiation based disinfection.

The spreading of coronavirus from contacting surfaces and aerosols created a pandemic around the world. To prevent the transmission of SARS-CoV-2 virus and other contagious microbes, disinfection of contacting surfaces is necessary. In this study, a disinfection box equipped with infrared (IR) radiation heating and ultraviolet-C (UV-C) radiation is designed and tested for its disinfection ability against pathogenic bacteria and SARS-CoV-2 spike protein. The killing of a Gram-positive, namely, S. aureus and a Gram-negative namely, S. typhi bacteria was studied followed by the inactivation of the spike protein. The experimental parameters were optimized using a statistical tool. For the broad-spectrum antibacterial activity, the optimum condition was holding at 65.61 °C for 13.54 min. The killing of the bacterial pathogen occurred via rupturing the cell walls as depicted by electron microscopy. Further, the unfolding of SARS-CoV-2 spike protein and RNase A was studied under IR and UV-C irradiations at the aforesaid optimized condition. The unfolding of both the proteins was confirmed by changes in the secondary structure, particularly an increase in ß-sheets and a decrease in α-helixes. Remarkably, the higher penetration depth of IR waves up to subcutaneous tissue resulted in lower optimum disinfection temperature, <70 °C in vogue. Thus, the combined UV-C and IR radiation is effective in killing the pathogenic bacteria and denaturing the glycoproteins.

Physicochemical factors of bioprocessing impact the stability of therapeutic proteins.

The aggregation of therapeutic proteins is potentially encountered during various steps such as bioprocessing, formulation, storage, transportation and administration. The aggregation results in irreversible drug loss and also leads to an increase in the risk of immunogenicity. The aggregated proteins have also been associated with various protein deposition diseases like amyloidosis. Various physicochemical factors like pH, temperature, salt concentrations, ionic strength, shear and surface affect the stability of proteins. Interestingly, therapeutic proteins simultaneously experience these physical, chemical and mechanical stresses during upstream, downstream and storage processes. The above physicochemical factors are reported to induce the unfolding and aggregation of proteins. The mechanistic insights of this complex aggregation behavior may allow devising strategies to limit/restrict this unwanted phenomenon. This review intends to undertake systematic descriptions of the key physicochemical factors in upstream and downstream bioprocesses and correlating their implications with the unfolding and aggregation of therapeutic proteins. The present review highlights the impacts of environmental, chemical and mechanical factors of the bioprocessing on the stability/aggregation of therapeutic proteins. The present review offers insight into this important phenomenon, which will be helpful for real-world challenges in the bioprocess and bio-therapeutic industries.

Biodegradation of waste cooking oil and simultaneous production of rhamnolipid biosurfactant by Pseudomonas aeruginosa P7815 in batch and fed-batch bioreactor.

Biosurfactants are non-toxic, surface-active biomolecules capable of reducing surface tension (ST) and emulsifying interface at a comparably lower concentration than commercial surfactants. Yet, poor yield, costlier substrates, and complex cultivation processes limit their commercial applications. This study focuses on producing biosurfactants by Pseudomonas aeruginosa P7815 in batch and fed-batch bioreactor systems using waste cooking oil (WCO) as the sole carbon source. The batch study showed a 92% of WCO biodegradation ability of P. aeruginosa producing 11 g L-1 of biosurfactant. To enhance this biosurfactant production, a fed-batch oil feeding strategy was opted to extend the stationary phase of the bacterium and minimize the effects of substrate deprivation. An enhanced biosurfactant production of 16 g L-1 (i.e. 1.5 times of batch study) was achieved at a feed rate of 5.7 g L-1d-1 with almost 94% of WCO biodegradation activity. The biosurfactant was characterized as rhamnolipid using Fourier transform infrared spectroscopy (FTIR), and its interfacial characterization showed ST reduction to 29 ± 1 mN m-1 and effective emulsification stability at pH value of 4, temperature up to 40 °C and salinity up to 40 g L-1. The biosurfactant exhibited antibacterial activity with minimum inhibitory concentration (MIC) values of 100 µg mL-1 and 150 µg mL-1 for pathogenic E. hirae and E. coli, respectively. These findings suggest that biodegradation of WCO by P. aeruginosa in a fed-batch cultivation strategy is a potential alternative for the economical production of biosurfactants, which can be further explored for biomedical, cosmetics, and oil washing/recovery applications.

Yttrium iron garnet for hyperthermia applications: Synthesis, characterization and in-vitro analysis.

Exclusive magnetocaloric properties of orthoferrites offer advantages for their application in the magnetic hyperthermia as well as imaging applications. In the present study, the effect of yttrium concentration on the magnetic characteristics of the iron oxide based nanomaterials was analyzed to assess their potential for the hyperthermia applications. The Sol-gel method was used to synthesize the Yttrium Iron Garnet (YIG) based nanoparticles, using different molar ratios of Fe and Y precursors, followed by the calcination at 900, 1000 and 1100 °C. XRD analysis determined the formation of the pure phase of yttrium iron garnet Y3Fe5O12 (YIG) at 0.5 molar ratio of yttrium at all the calcination temperatures and pure phase of yttrium iron perovskite YFeO3 (YIP) for 1 molar ratio of yttrium at 1000 and 1100 °C. The mean particle size was observed in the range of 100 to 400 nm. The magnetic characterization studies showed the highest saturation magnetization for the sample containing 0.5 molar ratio of the yttrium calcinated at 1000 °C. The magnetization values were linearly related to the contents of YIG phases in the synthesized samples. Induction heating of YIG resulted in the hyperthermia temperature (42 to 44 °C) in 13 min with the SAR values 114.65 W/g at 1 mg/ml. The prepared samples showed no in-vitro toxic effects on the MG63 cells (>90% cell viability). In addition, in-vitro treatment at hyperthermia temperature for 15 min reduced cell viability of cancer cells (A549) to 55%, while no toxic effect was observed on MG 63 cells. The present study postulates Yttrium Iron Garnet as an effective therapeutic agent for hyperthermia cancer treatment.

Production of biosurfactant by Bacillus subtilis RSL-2 isolated from sludge and biosurfactant mediated degradation of oil.

This study aims to unveil the effect of biosurfactant as stimulant in crude oil bioremediation. Isolated oil-degrading strain, B. subtilis RSL 2 was optimized for the maximum oil degradation and biosurfactant production using Response surface methodology. The produced biosurfactant was characterized and investigated for its effect on microbial oil degradation in two modes (a) sequential and (b) simultaneous. The strain produced 3.5 g/L of biosurfactant at pH 4.0, 25 °C, using 1 g/L crude oil as the only C-source in 7 days, which was characterized as lipopeptide with a critical micelle concentration (CMC) of 0.5 g/L. The biosurfactant improved surface wettability of a hydrophobic substrate i.e. increased surface energy from 30 ± 1 to 35 ± 1 mJ/m2. Further, the simultaneous feed of biosurfactant at 0.5 CMC enhanced oil biodegradation (72%) and biosurfactant production (5.2 g/L) by about 1.6 times than the sequential mode due to improvement in mobilization of oil thus making it more bioavailable.

Production of novel rhamnolipids via biodegradation of waste cooking oil using Pseudomonas aeruginosa MTCC7815.

In this paper, Pseudomonas aeruginosa MTCC7815, a biosurfactant producing strain was studied for its ability to utilize waste cooking oil (WCO) as a sole carbon source for the production of biosurfactant. Culture conditions were optimized based on surface tension reduction and biomass concentration. The obtained biosurfactant was characterized using 1H NMR, FTIR, LC-MS, and MALDI-TOF techniques. The chemical properties of the produced biosurfactant were estimated by assessing the critical micelle concentration (CMC), emulsification index (E24) and oil displacement test. The optimal culture conditions were found to be similar to the natural domestic sewage such as basic pH value of 10, temperature of 25 °C and a very high WCO concentration of 40 gL-1 (C/N ratio of 40/1). The biosurfactant yield was found to be significant as 11 ± 0.2 gL-1 upon utilizing about 90% of WCO within 5 days of incubation. The biosurfactant produced was found to be a mixture of mono- and di-rhamnolipid in nature and comprised excellent surface active properties i.e. an extremely low CMC of 8.8 ± 0.3 mgL-1, E24 of 62.5 ± 0.3% and surface tension reduction up to 26.2 ± 0.5 mNm-1. These results suggest the suitability of Pseudomonas aeruginosa for the biosurfactant production at commercial scale along with waste remediation in an economic way.

Isolation and characterization of biosurfactant producing and oil degrading Bacillus subtilis MG495086 from formation water of Assam oil reservoir and its suitability for enhanced oil recovery.

The strains isolated from the formation water were characterized and screened considering their crude oil degradation capability and biosurfactant production ability. The growth kinetics study of isolated Bacillus subtilis MG495086 was carried out by varying growth parameters i.e. carbon source, temperature, pH and salinity. The biosurfactant production was optimized adopting RSM-CCD considering carbon source (1-5%), pH (3-11) and temperature (25-65 °C) as matrix parameters. The optimum biosurfactant production (6.3 ±â€¯0.1 g/L) and the minimum surface tension 29.85 mN/m were obtained after 96 h of incubation under optimal conditions i.e. 3.8% (v/v) of light-paraffin oil as sole carbon source at 62.4 °C and pH 7.7 with the maximum oil degradation capability of 91.3 ±â€¯5%. Critical micelle concentration value of crude biosurfactant was found to be 40 mg/L with high emulsification activity of 72.45 ±â€¯0.85%. The produced biosurfactant was identified as lipopeptide (Surfactin) and characterized using various analytical techniques to establish its suitability for microbial enhanced oil recovery.

Synthesis, characterization and in vitro analysis of α-Fe2O3-GdFeO3 biphasic materials as therapeutic agent for magnetic hyperthermia applications.

The use of gadolinium orthoferrite for biomedical application like as contrast agents for magnetic resonance imaging (MRI) has been found to be very promising due to its fascinating properties. The present study focuses on the determination of the effect of the gadolinium concentration in the formation biphasic α-Fe2O3-GdFeO3 for hyperthermia applications. An in-situ sol-gel technique was adopted for the synthesis of biphasic orthoferrites with four different gadolinium concentrations. The XRD analysis confirmed the formation of gadolinium orthoferrites after heat treatment at 1000 °C, 1100 °C, and 1200 °C. The presence of α-Fe2O3 in trace amounts was observed in the materials with low gadolinium concentrations. VSM (Vibrating-sample magnetometer) analysis was performed to ensure the magnetic properties of the materials, which were found to be weakly ferromagnetic. The biocompatibility of the materials was investigated through MTT assay and no cytotoxic effect was observed. The assessment of heating ability of the materials was performed under an alternating magnetic field using an induction heating instrument and all the samples showed temperature rise in the range of hyperthermia temperature with a maximum temperature of 55.71 °C in 6 min. The heating experiments at 44 °C in the absence of samples established the vulnerability of cancer cells as compared to normal cells.

Removal of methylene blue dye from aqueous solution using immobilized Agrobacterium fabrum biomass along with iron oxide nanoparticles as biosorbent.

A nano-biosorbent for the removal of methylene blue (MB) was prepared by encapsulating iron oxide nanoparticles (NPs) and Agrobacterium fabrum strain SLAJ731, in calcium alginate. The prepared biosorbent was optimized for the maximum adsorption capacity at pH 11, 160 rpm, and 25 °C. Adsorption kinetics was examined using pseudo-first-order, pseudo-second-order, and intra-particle diffusion (IPD) models. The kinetic data agreed to pseudo-second-order model indicating chemisorption of MB, which was also explained by FTIR analysis. The adsorption rate constant (k2) decreased and initial adsorption rate (h, mg g-1 min-1) increased, with an increase in initial dye concentration. The dye adsorption process included both IPD and surface adsorption, where IPD was found to be a rate-limiting step after 60 min of adsorption. The adsorption capacity was found to be 91 mg g-1 at 200 mg L-1 dye concentration. Adsorption data fitted well to Freundlich isotherm; however, it did not fit to Langmuir isotherm, indicating adsorbent surfaces were not completely saturated (monolayer formed) up to the concentration of 200 mg L-1 of MB. Thermodynamic studies proposed that the adsorption process was spontaneous and exothermic in nature. Biosorbent showed no significant decrease in adsorption capacity even after four consecutive cycles. The present study demonstrated dead biomass along with NPs as a potential biosorbent for the treatment of toxic industrial effluents.

Fabrication and characterization of chitosan, polyvinylpyrrolidone, and cellulose nanowhiskers nanocomposite films for wound healing drug delivery application.

This study describes the preparation of composite film using chitosan (CS) and polyvinylpyrrolidone (PVP) with incorporated cellulose nanowhiskers (CNWs) for drug delivery application. CNWs were prepared by acid hydrolysis of cellulose with sulfuric acid. Field emission scanning electron microscopy studies revealed nanofibrous morphology of CNWs with 20-30 nm diameter and 200-250 nm in length. X-ray powder diffraction analysis confirmed highly crystalline nature of CNWs with 92.81% crystallinity. Incorporation of CNWs enhanced the thermal and mechanical properties of films. Fourier transform infrared spectroscopy data showed physical interactions between polymer-polymer and polymer-drug. Films prepared with CNWs showed improved swelling behavior which resulted in sustained drug release from polymeric matrix. In vitro curcumin release data were fitted with two-step release model; Step 1 as desorption from the outer surface of the film, and Step 2 as diffusion from within the film and subsequent desorption. The release kinetics confirmed biphasic release profile with different release rates along with diffusion controlled curcumin release. Prepared films showed high biocompatibility with excellent antibacterial activities. Overall, the performed studies confirmed CS-PVP-CNWs based release system can as a potential candidate for wound dressing applications with sustained drug release. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2391-2404, 2017.

Deciphering the mechanistic insight into the stoichiometric ratio dependent behavior of Cu(II) on BSA fibrillation.

Various metal ions are recently implicated in protein aggregates and are associated with numerous neurodegenerative diseases. In the present work, we have scrutinized the effect of stoichiometric variation of Cu(II) on BSA fibrillation at physiological pH 7.4 through Thioflavin-T dye binding study, residual protein concentration, Fourier Transform Infrared spectroscopy (FTIR), Dynamic Light Scattering (DLS), Atomic Force Microscopy (AFM) and Isothermal Titration Calorimetry (ITC). Fibrillation kinetics was studied through ThT fluorescence, which illustrated dependency on stoichiometric ratio of Cu(II). The ThT intensity data were fitted to a single exponential expression to determine the aggregation rate, k, which revealed a slight lower k value for 1:1 ratio of BSA-Cu(II) reaction as compared to BSA alone whereas k value for 1:2 ratio of BSA-Cu(II) reaction was higher. Higher equilibrium residual concentration in case of 1:1 ratio of BSA-Cu(II) agreed to the lower aggregation rate. IR spectroscopy revealed the presence of increased ß-sheet proportion to the detriment of α-helix conformation with increasing concentration of Cu(II) and illustrated maximum ß-sheet proportion in 1:2 ratio of BSA-Cu(II). These results were combined with scattering results that showed the higher average hydrodynamic radius (Z-average value) of aggregates in 1:2 ratio of BSA-Cu(II) with respect to BSA and 1:1 ratio of BSA-Cu(II). AFM analysis confirmed the fibril formation. ITC analysis has shown the presence of two binding sites with many fold difference in binding affinity at prescribed in vitro conditions. An N-terminal binding site with many fold higher binding affinity was found as the first Cu(II) binding site. The free thiol group of the cysteine residue positioned at 34 (cys-34) in BSA was covalently capped and this modified BSA also showed the presence of two binding sites, which declined the cys-34 site as the second Cu(II) bind site. Therefore Zn(II) binding site was predicted as the second Cu(II) binding site. The binding of Cu(II) at N-terminal binding site marks a square planner geometry; while that at zinc binding site marks a rigid coordination geometry. Conclusively, analyzed data has concluded that the variation in Cu(II) efficacy in the different stoichiometric ratio results because of two different coordination types in different stoichiometric ratios.