Structural and mechanical behavior of type-I collagen fibrils in presence of induced electrostatic interactions through ionic liquids.

Tuning the self-assembly of collagen has broad applications in the biomedical field owing to their desired biological performance as collagenous materials with tunable functionalities can further determine cellular responses. In this work, an attempt has been made to tune the self-assembly of collagen using ionic liquids, viz., imidazolium chloride (IC) and choline dihydrogen phosphate (CDHP) at its physiological pH, followed by probing assembled systems using various characterization methods. Turbidity measurements of fibrillar networks were performed to ascertain the rate of fibril formation in addition of imidazolium chloride and choline dihydrogen phosphate to collagen at physiological pH. Morphological changes were examined using Scanning Electron Microscope (SEM), binding affinities were measured by Microscale Thermophoresis (MST), in addition to, changes in the shear viscosity, mechanical strength of collagen fibrils when interacted with imidazolium and choline based ILs were carried out using rotational rheometer and Quartz Crystal Microbalance (QCM) measurements. Experimental result depicts that CDHP imparts better crosslinking as well as mechanical strength compare to IC, which is already known for destabilizing the triple helix structure is inhibiting the fibril formation. This self-assembled, ionic-liquid treated collagen-fibrillar system would accelerate various force modulated fibrillar network study, for mimicking the ECM and tissue engineering application.

Insights into protein-ionic liquid interaction: A comprehensive overview on theoretical and experimental approaches.

Owing to highly tunable nature, ionic liquids are nesting stance in the scientific community for a wide variety of applications ranging from electrochemistry to product purification, from chemical and biomedical applications to biotechnological interventions and proteomics. Proteins are unstable in its native form and several attempts have been made to stabilize them by addition of various additives. This review focusses on the studies conducted to improve protein stability with ionic liquids along with an emphasis on the mechanism of interaction. This review also specifies and discusses about the brief introduction to ionic liquids, evolution of first-, second-, and third generation of liquids over the years and their selection criterion and applications. Though, there are several elegant reviews available on proteins-ionic liquids interaction, this review systematically highlights the effect of ionic liquids viz., imidazolium, ammonium, phosphonium and choline-based ionic liquids (amino acid-based anions & classical anions) on fibrous proteins viz., collagen and keratin and globular proteins viz., bovine serum albumin and cytochrome c. Thus, this review elaborates the thorough investigations conducted to explore the stabilizing properties of ionic liquids over fibrous and globular proteins.

Self-association of type I collagen directed by thymoquinone through alteration of molecular forces.

Type I collagen is a vital structural component of the extracellular matrix providing the connective tissues with biomechanical support. One of the interesting properties of collagen is to self-associate into fibrils. The present work aims to direct the self-assembly of collagen through different molecular forces, which are tuned on the addition of thymoquinone a well-known phytochemical. A change in relative viscosity and stress of collagen-thymoquinone blends influenced the interfibrillar aggregates around its hydration shell. Further, secondary structural integrity was studied via cotton curve effect, and vibrational frequency shifts showed a characteristic interaction of thymoquinone at the N-terminal residues of the triple helix. Finally, the spontaneous self-association of fibrils was tracked by calculating the rate of fibril growth kinetics, which potentially decreased with increase in thymoquinone concentration. The fibrils were eventually visualized under the high resolution-scanning microscope showing morphological variations. Therefore, such a protein-phytochemical interaction may tend to play with the hydration network of collagen and covalently interact with its imino acid residues. It may be speculated that such an inhibitory process portrayed by thymoquinone may have a fortune in the targeted and sustainable delivery to the site of action for certain diseases, which includes collagen accumulation. Moreover, its directed assembly could be utilized for designing templates as in manipulating the collagen as a nanoporous membrane to make nanofibers and further tuned by small molecules for nanoparticle synthesis application.

Trigonelline hydrochloride: A promising inhibitor for type I collagen fibrillation.

Collagen is a major structural protein, with properties such as low toxicity, low immune response and promoting cellular growth. If there is an excess of collagen accumulation, especially in the myocardium it leads to fibrosis, which in turn impairs the normal functioning of the myocardial tissues. In order to overcome collagen fibrillation, trigonelline hydrochloride has been used in this study as a potential agent for inhibiting the spontaneous self-assembly of type I collagen. Trigonelline hydrochloride, a nicotinic acid derivative is largely found in Trigonella foenum-graecum L. (fenugreek). Experimental work on turbidity assay shows that there is an increase in the lag phase compared to the native collagen indicating a delay in fibrillation. According to rheological aspects, viscosity of the collagen composite decreases due to the increase in shear rate, which disentangles the aggregated particles of collagen fibrils and allows it to align along the direction of rate. Morphological assessments through AFM and HR-SEM suggest that there is an effective reduction in fibrillation with an increase in the concentration of trigonelline hydrochloride. FT-IR (ATR) studies indicate that compactness of secondary structure helicity occurs on treatment with trigonelline hydrochloride. Such promising effect of trigonelline on collagen fibrillogenesis can be judiciously used for targeting specific sites of collagen accumulation via innovative drug delivery systems.

Choline-Based Amino Acid ILs-Collagen Interaction: Enunciating Its Role in Stabilization/Destabilization Phenomena.

Given the potential of productive interaction between choline-based amino acid ionic liquids (CAAILs) and collagen, we investigated the role of four CAAILs, viz., choline serinate, threoninate, lysinate, and phenylalaninate, and the changes mediated by them in the structure of collagen at different hierarchical orderings, that is, at molecular and fibrillar levels. The rheological, dielectric behavior and the secondary structural changes signify the alteration in the triple helical structure of collagen at higher concentrations of CAAILs. A marginal swelling and slight decrease in the thermal stability of rat tail tendon collagen fibers were observed for choline serinate and threoninate, albeit distortions in banding patterns were noticed for choline lysinate and phenylalaninate, suggesting chaotropicity of the ions at the fibrillar level. This signifies the changes in the hydrogen-bonding environment of collagen with increasing concentrations of CAAILs, which could be due to competitive hydrogen bonding between the carbonyl group of amino acid ionic liquids and the hydroxyl groups of collagen.

Exploiting oleuropein for inhibiting collagen fibril formation.

Collagen fibrils accumulate in excessive amounts and impair the normal functioning of the organ; therefore it stimulates the interest for identifying the compounds that could prevent the formation of fibrils. Herein, inhibition of self-assembly of collagen using oleuropein has been studied. The changes in the physico-chemical characteristics of collagen on interaction with increasing concentration of oleuropein has been studied using techniques like viscosity, UV-vis, CD and FT-IR. The inhibitory effect of oleuropein on fibril formation of collagen was proved using SEM. Circular dichroism and FT-IR spectra elucidates the alterations in the secondary structure of collagen suggesting non-covalent interactions between oleuropein and collagen. The decreased rate of collagen fibril formation also confirms the inhibition in the self-assembly of collagen. Hence, our study suggests that inhibition of the self-assembly process using oleuropein may unfold new avenues to treat fibrotic diseases.

Role of Preferential Ions of Ammonium Ionic Liquid in Destabilization of Collagen.

Ions play a key role in the destabilization of collagen. This study explores the effect of diethyl methyl ammonium methane sulfonate (AMS), an ionic liquid (IL), on different hierarchical orderings of collagen, namely, at the molecular and fibrillar levels. The rheological behavior and secondary structural changes reveal changes in the hydrogen-bonding environment of collagen, leading to alterations in the triple helical structure of collagen. An increase in the concentration of AMS resulted in swelling of rat-tail tendon fibers, and also, decreased thermal stability signifies that ions are obliged to destabilize collagen at the fibrillar level. Molecular modeling studies confirm that anions are judiciously held responsible for structural deformities in collagen, whereas cations have a tenuous effect. Thus, the preferential role of ions present in an ammonium IL has been elucidated in this study.

Electrostatic Forces Mediated by Choline Dihydrogen Phosphate Stabilize Collagen.

Cross-linkers aid in improving biostability of collagen via different mechanisms. Choline dihydrogen phosphate (cDHP), a biocompatible ionic liquid, has been reported as a potential cross-linker for collagen. However, its mechanism is yet unclear. This study explores the effect of cDHP on the physicochemical stability of collagen and nature of its interaction. Dielectric behavior of collagen-cDHP composites signifies that cDHP enhances intermolecular forces. This was demonstrated by an increase in cross-linked groups and high denaturation temperature of collagen-cDHP composites. XRD measurements reveal minor conformational change in helices. Molecular modeling studies illustrate that the force existing between collagen and cDHP is electrostatic in nature. Herein, it is postulated that dihydrogen phosphate anion attaches to cationic functional groups of collagen, resulting in closer vicinity of various side chains of collagen, forming physical and chemical cross-links within collagen, contributing to its structural stability. Our study suggests that dihydrogen phosphate anions can be employed for developing a new class of biocompatible cross-linkers.

Calorimetric analysis of gelatine-glycosaminoglycans blend system.

Gelatine is one of the most valuable natural polymers used for drug delivery applications. Gelatine-GAGs based composite system has been shown to act as good scaffolds for tissue engineering. The objective of the present study is to investigate the calorimetric properties of microporous gelatine-GAGs based blend, which were modified by co-crosslinking with a naturally occurring crosslinking agent genipin. The melting temperature (T(m)), enthalpy change (ΔH(m)) and heat capacity change (ΔC(p)) were systematically calculated over the experimentally observed systems using differential scanning calorimeter (DSC). The thermoporometry results suggest that the concentration of the glycosaminoglycans plays an important role in the pore size distribution of the blend matrices. The circular dichroism (CD) spectroscopy study, scanning electron microscopy (SEM) studies provide the valuable information about the structural features of the biodegradable blend that can be utilized for various biomedical applications. The results provide new insights into the thermal stability of blend and suggest potential strategies for its manipulation.

Degree of crosslinking of collagen at interfaces: adhesion and shear rheological indicators.

Work of adhesion (ΔW) and surface rheology at solid/air and solution/air interface have been used as indicators to study the stabilization of collagen by different crosslinking agents like basic chromium sulfate (BCS), tannic acid, catechin and formaldehyde. The results show that an increase in rate of ΔW would promote adsorption while a decrease leads to hindered adsorption. Shear rheological studies on collagen demonstrate an increase in both shear viscosity and elasticity with time while for collagen with polyphenols like catechin and tannic acid there is an unusual breakdown of these values. A correlation between the rheological properties and the work of adhesion suggests that the time frame in which the viscoelastic behavior is initiated for collagen with different crosslinking agents determines the final macroscopic property of the protein. The study attempts to quantify the degree of crosslinking of collagen through the dynamics and strength of the water molecules in the assembly of hydrated protein and the crosslinking agents.

Structure and dynamics of water in native and tanned collagen fibers: Effect of crosslinking.

The influence of crosslinking on the hydration structure of collagen has been investigated. Nuclear magnetic resonance, dielectric relaxation and thermoporometry were used to investigate water structure in native and crosslinked collagen fibers on both wet and dried specimen. Measurements reveal the influence of different chemical treatments on the transverse relaxation time and polarization of the collagen fibers. The frequency dependence of dielectric constant of collagen fibers displays an induction behavior on low frequencies. Bound water constrained in collagen fibers seems to provide signatures for changes induced by crosslinking agents on the pore diameter and distribution in collagen fibers. A correlation of transverse relaxation time of water in dry and wet states presented in this study presents an experimental tool for examining the differences in efficacy of crosslinking agents. Changes in the dielectric relaxation, dynamics of water structure and hydroporometric structure of collagen are dependent on the nature of crosslinking material.

Effect of surfactants on the thermal, conformational and rheological properties of collagen.

Collagen is an important biomaterial and its interaction with surfactant is important in light of its use in various cosmetics and dermatological applications. Presently, the effect of surfactants on the physico-chemical properties of collagen has been studied. The thermal stability of collagen is reduced by sodium dodecyl sulfate and hexadecyltrimethylammmonium bromide, whereas Triton X-100 does not. The viscosity of collagen is influenced greatly depending on the surfactant concentrations. The secondary structure of collagen shows changes only in the molar ellipticity. The role of charge and concentration of surfactants in influencing the various physico-chemical properties of collagen has been elucidated.

Influence of crosslinking agents on the pore structure of skin.

Analysis of pore structure of skin is important to understand process of diffusion and adsorption involved during any application of the skin matrix. In this study, the effect of thermal shrinkage on the pore structure of chromium and vegetable treated skin has been analyzed as these tanning agents are known to bring about thermal stability to the matrix. The changes brought about in the pore structure have been studied using mercury intrusion porosimetry and scanning electron microscopy. Response of the chromium treated and vegetable tanning treated skin structure to heat has been found to be quite different from each other. About 41% decrease in porosity is observed for chromium treated skin as against 97% decrease for the skin treated with vegetable tannins. This is primarily attributed to the basic nature of these materials and the nature of interaction of them towards skin.

Biodegradability of leathers through anaerobic pathway.

Leather processing generates huge amounts of both solid and liquid wastes. The management of solid wastes, especially tanned leather waste, is a challenging problem faced by tanners. Hence, studies on biodegradability of leather become imperative. In this present work, biodegradability of untanned, chrome tanned and vegetable tanned leather under anaerobic conditions has been addressed. Two different sources of anaerobes have been used for this purpose. The effect of detanning as a pretreatment method before subjecting the leather to biodegradation has also been studied. It has been found that vegetable tanned leather leads to more gas production than chrome tanned leather. Mixed anaerobic isolates when employed as an inoculum are able to degrade the soluble organics of vegetable tanned material and thus exhibit an increased level of gas production during the initial days, compared to the results of the treatments that received the anaerobic sludge. With chrome tanned materials, there was not much change in the volume of the gas produced from the two different sources. It has been found that detanning tends to improve the biodegradability of both types of leathers.

Mercury intrusion porosimetry, nitrogen adsorption, and scanning electron microscopy analysis of pores in skin.

Stability of collagenous matrixes such as skin and leather with respect to changes in their dimensions on heating has long been correlated with degree and type of cross links formed and short-range ordering in angstrom unit scales. Macroscopic dimensional changes may be expected to involve alterations in the long-range order as well as supramolecular assemblies in skin and leather. This study relates thermal shrinkage of skin matrixes with alterations observed in micro-, meso-, and macroporic structures. Changes in the pore structure of skin associated with thermal shrinkage have been studied using nitrogen adsorption and mercury intrusion porosimetry measurements. A comparison of results obtained using both techniques has been made. These results indicate that although the percentage porosity of the matrix decreases, the BET specific surface area increases on shrinkage. An insight into the changes in the pore systems of skin induced by thermal shrinkage has been gained.