Extraction of dermatan sulfate using ionic liquid-assisted enzymatic digestion: An efficient approach.

Dermatan sulfate is one of the major glycosaminoglycan (GAG) present in the animal hides, which is a waste/byproduct from meat industry. Efficient utilization of these meat industry wastes is garnering attention because these wastes render a possibility for their conversion into useful products. With the increased concerns over health, various initiatives have been developed to permit more efficient utilization of these by-products and thereby directly impacting environmental sustainability. Herein, we demonstrate for the first time an efficient and environmentally safe ionic liquid-assisted enzymatic process for the extraction of dermatan sulfate from buffalo hides. Dermatan sulfate has been extracted, separated, and purified from the GAG mixture using IL-assisted enzymatic digestions and chromatographic separations. NMR, FT-IR, and ESI-MS measurements showed typical characteristic peaks for dermatan sulfate. The advantages of this eco-friendly process adopted include i) use of fewer chemicals, ii) elimination of harsh chemicals, iii) elimination of various steps and sub-steps, iv) reduction in process time (12 h), and v) increase in extraction yield by 75% when compared to conventional enzymatic process (57%). Thus, the use of ionic liquids alongside enzymes will serve as an efficient methodology for the futuristic development of these derived GAGs for their potential applications.

Improved filtration for dye removal using keratin-polyamide blend nanofibrous membranes.

Dyes from industrial wastewaters represent one of the most hazardous pollutants as they are not effectively biodegradable. The present work is focused to study the novel properties of keratin-polyamide blend nanofibrous filtration membranes for treating wastewaters containing dye. Keratin protein was extracted from goat hair, a tannery waste through sulphitolysis process. The extracted keratin was blended with polyamide to prepare a nanofibrous membrane through the electrospinning process. The fabricated pristine polyamide and keratin-altered polyamide membranes were characterized and compared for their properties. Effects of solution pH, dye concentration, membrane flux, and membrane capacity have been examined. Very fine nanofibers and enhanced porosity drive the membrane to enhanced flux and higher filtration efficiencies. At pH 2, the dye removal efficiency of the blend membranes was 100, 99, 98, 90, and 83% for 100, 200, 250, 300, and 400 ppm concentrations of dye, respectively. The keratin-polyamide blend membrane exhibited better properties in all aspects. The results of this present investigation indicate that the presence of keratin in filtration membranes is promising for dye removal from the effluents.

Collagen stabilization using ionic liquid functionalised cerium oxide nanoparticle.

Nanoparticles owing to their size have a substantial influence on the biological behavior of collagen, thereby opening new channels to unfold the propensity of nanoparticles in terms of collagen stabilization. The present study aims to synthesize and characterize cerium oxide nanoparticles and to investigate their crosslinking efficiency on collagen. Cerium oxide nanoparticles, known biocatalysts, form an effective oxidation system due to their variable oxidation state. Ionic liquid functionalizes cerium oxide nanoparticles (IL-CONP) have been synthesis by the sonication method and characterizes using techniques such as Dynamic Light Scattering, and X-ray Diffraction. The hydrodynamic diameter, Zeta potential and polydispersity index of nanoparticles is 192.3 ± 2.14 nm, -13.76 ± 1.5 mV and 0.387, respectively. Changes in the secondary structure of collagen upon treatment with increasing concentration of IL-CONP indicate conformational modifications at the molecular level. Differential scanning calorimetry studies on rat tail tendon collagen fibers with IL-CONP indicate an increase in thermal stability of collagen from 61 to 87 °C. Thus, cerium oxide-based nanoparticle crosslinking invokes a considerable array of interest as a potential crosslinking agent for collagen.

Governing the Inhibition of Reconstituted Collagen Type I Assemblies Mediated Through Noncovalent Forces of (±)-α Lipoic Acid.

Type I collagen is a fibrous protein, which is highly biocompatible and biodegradable and exhibits low immunogenicity with its unique feature of undergoing a spontaneous self-assembly process. However, the excessive accumulation of collagen may lead to a condition known as fibrosis in vertebrates. Recently, saturated fatty acids have gained much attention as biomedical and therapeutic agents. Therefore, drawing inspiration from the biological and structural tunability of these fatty acids, this work aims to inhibit the self-assembly of type I collagen using (±)-α-lipoic acid (ALA). Reconstituted collagen and its blends with (±)-ALA under physiological conditions were subjected to fibril growth kinetics measurements, which exhibited the decrease in the rate of fibrillogenesis ( t1/2) with an increase in the concentration of ALA. Variations in the viscoelasticity of collagen and ALA blend with respect to rate and frequency showed significant changes. Further, the frequency shifts of different functional groups via FT-IR (ATR) and the morphological changes associated with fibril inhibition were visualized using a cryoscanning electron microscope. Molecular dynamics simulation of the collagen-like peptide with the (±)-ALA molecule at different molar ratios proved that (±)-ALA had a strong potential to bind at various sites of collagen mediated by conventional secondary or noncovalent forces. Thus, the protein-small molecule interaction dominates the forces prevailing between protein-protein binding, leading to the inhibition of the self-assembly process. Such inhibitory effects by a fatty acid may unfold newer avenues for development of targeted and sustainable drug delivery systems for fibrotic diseases.

Electrospinning of casein nanofibers with silver nanoparticles for potential biomedical applications.

Casein, a major protein content in the milk has been extensively used in drug delivery due to its unique structural features. Fabrication of nanofibers from casein along with nanoparticles for tissue engineering applications has been explored in this study. Nanofibers fabrication is achieved by co-electrospinning of casein with poly (ethylene oxide) in sodium dodecyl sulphate (SDS) aqueous solution. Stabilization of silver nanoparticles has been achieved by the presence of SDS in the nanofiber matrix. The influence of conductivity on the nanofiber fabrication has also been studied. The nanofibrous mats have been characterized using techniques such as scanning electron microscope (SEM) and high resolution-transmission electron microscope (HR-TEM). Antimicrobial properties of the nanofibers have been assessed and the cellular biocompatibility of the material has been evaluated using cultured fibroblast (NIH-3T3) cells. Silver nanoparticles incorporated nanofibers showed good antimicrobial property against both gram negative and gram positive bacteria. In addition, the nanofiber matrix exhibited good biocompatibility for the fibroblast cell proliferation. These results pave the way for extending the use of casein based nanofibers in the skin care applications.

Anti-oxidant enriched hybrid nanofibers: Effect on mechanical stability and biocompatibility.

Despite being a favorable candidate in wound dressing, collagen based biomaterials possess inferior mechanical properties which limit their usage. Collagen based hybrid nanofibers with other polymers can enhance their mechanical strength as well as their biological properties. Herein, we report collagen-silk fibroin hybrid nanofibers incorporated with fenugreek, an antioxidant, as a bioactive wound dressing material. The nanofiber mats were characterized using various experimental techniques. From the results, it was found that an increase in silk fibroin content in nanofibers improves the fiber diameter and tensile strength. The nanofibers also showed good antioxidant properties estimated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay. Presence of collagen in the nanofibers enhanced the biocompatibility of the nanofibers. Fenugreek released from the matrix enhanced the migration of fibroblasts in vitro. In vivo studies showed that collagen-silk fibroin-fenugreek nanofibers enhanced the wound closure via minimal inflammation and early epithelialization than the untreated and silk fibroin-fenugreek nanofibers treated wounds. Our study suggests that the fenugreek incorporated collagen-silk fibroin nanofibers is a potential candidate for wound dressings in clinical applications.

Polymeric Micelle of a Gelatin-Oleylamine Conjugate: A Prominent Drug Delivery Carrier for Treating Triple Negative Breast Cancer Cells.

Protein-based polymeric micelles are proven as effective colloidal drug carriers due to a high drug loading efficiency, sustained release, biocompatibility, and ease of permeation into the cell. Gelatin-based polymeric micelles find applications in treating rare cancerous cells like triple negative breast cancer cells (TNBC), which do not overexpress receptors on its surface. In the present work, we have modified the hydrophilic nature of gelatin into amphiphilic by conjugating with oleylamine using genipin as a cross-linking agent. Owing to amphiphilicity, gelatin-oleylamine conjugate (GOC) self-assembles to form micelles in the aqueous medium. NMR, FTIR, and UV-vis characterizations were used to identify cross-linkage between gelatin and oleylamine, while the results of DLS, confocal, and TEM confirmed aggregation of GOC monomers into micelles. Fluorescence measurement has revealed that the critical micellar concentration of GOC was 0.04 ± 0.01 mg/mL. According to DLS measurements, hydrodynamic size, ζ potential, and polydispersity index of GOC micelles were 230.6 ± 0.4 d. nm, -23.4 ± 0.2 mV, and 0.175 ± 0.008, respectively, proving its colloidal stability in solution at pH 7.4. Catechin was taken as a model antioxidant drug, and drug encapsulation efficiency of GOC micelle was determined to be 62 ± 3%. The cytotoxicity, fluorescent cell imaging, and flow cytometry analyses revealed that TNBC-type cells (MDA-MB-231) internalized drug-bound GOC nanocarriers (CT-GOC) and were involved in cell cycle arrest through G2/M phase-inducing cellular apoptosis. Further, CT-GOC exhibited a higher cellular toxicity to MDA-MB-231 cancerous cells but not in normal cells (NIH-3T3). The overall outcomes of physicochemical and biological measurements suggest that the prepared GOC micelles might be a promising drug carrier for novel anticancer agents in TNBC chemotherapy.

Gelatin-Cerium Oxide Nanocomposite for Enhanced Excisional Wound Healing.

Researchers are keen on formulating composites blending biomacromolecules with functional nanoparticles to achieve greater efficacy to expedite the wound healing process. In the present work, we have engineered a genipin cross-linked gelatin hydrogel composite containing optimized concentration of cerium oxide nanoparticles (G-ONp) for the purpose of wound healing. The concentration of cerium oxide nanoparticles in G-ONp has been optimized to be 250 µg/mL, which shows more than 80% cell viability in cytotoxicity study. X-ray diffractogram of ONp displays characteristic lattice planes of cubic fluorite structure, and transmission electron micrograph reveals that the particles are sized between 2.5-6.5 nm. The genipin dimeric cross-linkage in G-ONp has been confirmed by UV-vis peak at 603 nm. Swelling ratio of G-ONp (25.3 ± 1.2) has been found to be three-fold to that of native gelatin (9.2 ± 1.4). As far as pore size distribution is concerned, lyophilized sponges of gelatin and G-ONp had microsized pores in the range of 1-140 µm and 1-19 µm, respectively, and hydrogels of the same determined by thermoporometry had nanosized pores in the range of 7-48 nm and 7-24 nm, respectively. The in vivo wound healing and histological examination have revealed that G-ONp treated rat group has shown more infiltration of leukocytes and larger deposition of collagen when compared to gelatin and control groups and has healed the wound in 12 days. These findings suggest that the composite of G-ONp is superior to gelatin in increasing wound healing and can be envisaged as a wound dressing material in future.

Impact of Ionic Liquids on the Structure and Dynamics of Collagen.

The changes in the structure and dynamics of collagen treated with two different classes of ionic liquids, bis-choline sulfate (CS) and 1-butyl-3-methyl imidazolium dimethyl phosphate (IDP), have been studied at the molecular and fibrillar levels. At the molecular level, circular dichroic studies revealed an increase in molar ellipticity values for CS when compared with native collagen, indicating cross-linking, albeit pronounced conformational changes for IDP were witnessed indicating denaturation. The impedance was analyzed to correlate the conformational changes with the hydration dynamics of protein. Changes in the dielectric properties of collagen observed upon treatment with CS and IDP reported molecular reorientation in the surrounding water milieu, suggesting compactness or destabilization of the collagen. This was further confirmed by proton transverse NMR relaxation time measurements, which demonstrated that the water mobility changes in the presence of the ILs. At the fibrillar level, differential scanning calorimetry thermograms for rat tail tendon collagen fibers treated with CS show a 5 °C increase in denaturation temperature, suggesting imparted stability. On the contrary, a significant temperature decrease was noticed for IDP, indicating the destabilization of collagen fibers. The obtained results clearly indicate that the changes in the secondary structure of protein are due to the changes in the hydration dynamics of collagen upon interaction with ILs. Thus, this study on the interaction of collagen with ionic liquids unfolds the propensity of ILs to stabilize or destabilize collagen depending on the changes invoked at the molecular level in terms of structure and dynamics of protein, which also got manifested at the fibrillar level.

Fenugreek Incorporated Silk Fibroin Nanofibers-A Potential Antioxidant Scaffold for Enhanced Wound Healing.

Free radicals are generated by various biochemical pathways in the living system, causing severe oxidative damage to the biomolecules leading to adverse disease conditions. Hence, there is an increasing interest in antioxidant studies for preventing the effects of these free radicals. Herein, we propose a novel electrospun scaffold with antioxidant properties that can be used as wound healing material. Fenugreek, a natural antioxidant incorporated silk fibroin nanofiber, was prepared in four different ratios by the co-electrospinning method. The biocompatibility of the nanofibers and its antioxidant activity were evaluated through 3-(4, 5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay, respectively. The experimental observations indicate that the incorporation of fenugreek increases the thermal and mechanical properties of silk fibroin nanofibers. DPPH assay proves that the antioxidant property is enhanced with increasing concentration of fenugreek in nanofiber mats, and the Swiss albino 3T6 fibroblasts show better proliferation on the nanofibrous scaffolds. Further, the wound healing efficiency of fenugreek incorporated silk fibroin nanofibrous scaffolds was evaluated using full thickness excisional wounds in rat model. Wound healing was accelerated in silk fibroin-fenugreek nanofibers treated wounds with complete re-epithelialization and enhanced collagen deposition. The present study validates the use of fenugreek incorporated silk fibroin nanofiber mats as antioxidant scaffolds in wound healing applications.

Self-assembly of keratin peptides: Its implication on the performance of electrospun PVA nanofibers.

Drawing inspiration from the field of designer self-assembling materials, this work is aimed to focus on the self-assembling nature of extracted peptides. Hair keratin, a proteinacious reject in tanning industry has been chosen since they have been extracted and used for wide range of applications. Keratin source was subjected to five hydrolysis treatments (viz., sulphitolysis, ß-mercaptoethanol, ionic liquid, thioglycolic acid and alkali) and assayed for functional groups. This was followed by the prediction of secondary structure using circular dichroism, determining the microstructural level to which the extracted peptide has self-assembled. Sulphitolysis and thioglycolic acid based hydrolysates exist in monomeric conformation, whereas ß-mercaptoethanol based hydrolysate exhibited dimeric conformation. The subsequent part of the study is to incorporate these peptides into the nanofibers to study the structural implication of keratin peptides on its characteristics. Accordingly, the peptides were electrospun with PVA and subjected to morphological, mechanical, thermal and biological characterizations. Monomeric nanofiber mat has high tensile strength of around 5.5 MPa and offered lower mass transport resistance, whereas dimeric mat has high Tm of around 290 °C and was more biocompatible. These results help in understanding the extraction-structure-function aspect of the hydrolysates stressing the role of extraction methods on the choice of application.

A gelatin based antioxidant enriched biomaterial by grafting and saturation: towards sustained drug delivery from antioxidant matrix.

Proteins grafted with antioxidant molecules have drawn much attention due to their increased life time and biocompatibility. When protein macromolecules are cross linked chemically and physically with antioxidant molecules, they can act as antioxidant biomaterials as well as scaffolds to release the antioxidant molecules by diffusion. In our work, we have attempted to release catechin molecules from the matrix of glutathione grafted gelatin. Conjugation of glutathione and cross linkage was done by carbodiimide method to achieve smaller pores in the gelatin matrix and the characterization was performed by means of FTIR-ATR and calorimetric analyses. The glutathione grafted gelatin (GGSH) has been shown to have more thermal stability and pores with lesser radii than blank gelatin (bGEL). Free radical scavenging activity of GGSH was also found to be more than that of bGEL. Catechin was added to GGSH and bGEL by physical blending in order to achieve short term release of antioxidant molecules. CD spectra revealed that significant conformational changes occurred in secondary structure of gelatin upon interaction with catechin. Slower rate of catechin release from GGSH reflected the influence of cross linkage and physical interactive forces on the drug release properties. We conclude that the mixture of catechin with GGSH can be a potent antioxidant biomaterial releasing catechin at slower rate than the mixture of catechin with bGEL.

Microfabrication of gelatin-polycaprolactone composites for customized drug delivery.

Dynamic properties of water molecules present in the vicinity of protein are sensitive to its local conformational motions. Water mobility at the protein surface/interfaces is affected by its polar and charged groups, which are capable of anchoring water molecules through H-bonds. Differential scanning calorimetry, ATR-FTIR spectroscopy and circular dichroic analysis have been employed to substantiate the changes in hydration of gelatin, interacting with polycaprolactone. Enthalpy of denaturation and decrease in melting temperature indicate alteration in water-bridges around gelatin. In vitro drug release studies substantiate the influence of hydration on its release kinetics. These studies would aid in exploration of potential drug carrier.

Go natural and smarter: fenugreek as a hydration designer of collagen based biomaterials.

Collagen-based biomaterials have received considerable attention for smarter biomedical applications due to their inherent superior mechano-biological properties. However, accumulating evidence suggests that water, as a probe liquid bound in collagen, might be investigated to explore the influence of additives on the static and dynamic solvation behavior of collagen. The structure and dynamics of water near the surface/interface of collagen-fenugreek composites were demonstrated via circular dichroic spectroscopy, thermoporometry and impedimetric measurements to enlighten about the configuration-function relationship of collagen. Thermodynamic parameters of the composites signify the fenugreek concentration dependent structural robustness of collagen. Thermodynamic parameters such as free energies for unfolding, enthalpies, entropies and activation energies indicate that the residual structure modulates the stability of the denatured state up to 22 kcal mol(-1) and the parameters correlate with structural data for collagen complexed with fenugreek. The association constant of fenugreek is found to be 0.5807 M(-1). The binding of fenugreek influences rearrangement of the collagen-water network, resulting in the transition from a disordered (high entropy) unbound state to a structured (lower entropy) bound state. Fenugreek concentration plays a crucial role in shaping up the free energy that governs the folding, structure and stability of collagen. Dielectric data emphasize the effect of hydrophobic and hydrophilic clusters on the side chain motion constraints. The thermoporometry technique probes the pore size distributions of the composites. These methods provide insights into the role of excluded volume, chain stiffness and stability of a new collagen-galactomannan based composite, expanding its utility in "smart biomaterial applications".

Porosity and dielectric properties as tools to predict drug release trends from hydrogels.

Conventional studies on hydrogel properties such as viscosity, pH and swelling provide information without treating the components of hydrogel, viz., water and polymer individually. Water and hydrophilic polymers need to be studied individually to understand their relationship with each other to relate their influence on drug release. In this context, we have assigned the combination of porosity and dielectric properties as tools to explore the hydrogels. Porosity and dielectric properties have been analyzed using thermoporometry and alternative current impedance measurements, respectively. A well-known hydrogel genipin cross linked gelatin-chitosan (GC) composite, with catechin as model drug has been studied. The increasing concentration of chitosan in the hydrogel composites led to increase in bound water content and incorporation of charge entrapping moieties. Controlled and medium drug release are observed for GC1 whereas the native hydrogels and composites with lower ratio of chitosan yield immediate release and composites with higher ratio effects in slow release for limited duration (9 hours) of drug delivery process. This trend of drug release is in accordance with the results obtained from porosity and dielectric properties where reduction in pore radii to lower range and increase in relaxation time of polymeric components were observed at higher concentration of chitosan. Thus, these properties can be judiciously used for predicting drug release and designing biomaterials according to it.

Can green solvents be alternatives for thermal stabilization of collagen?

"Go Green" campaign is gaining light for various industrial applications where water consumption needs to be reduced. To resolve this, industries have adopted usage of green, organic solvents, as an alternative to water. For leather making, tanning industry consumes gallons of water. Therefore, for adopting green solvents in leather making, it is necessary to evaluate its influence on type I collagen, the major protein present in the skin matrix. The thermal stability of collagen from rat tail tendon fiber (RTT) treated with seven green solvents namely, ethanol, ethyl lactate, ethyl acetate, propylene carbonate, propylene glycol, polyethylene glycol-200 and heptane was determined using differential scanning calorimetry (DSC). Crosslinking efficiency of basic chromium sulfate and wattle on RTT in green solvents was determined. DSC thermograms show increase in thermal stability of RTT collagen against heat with green solvents (>78°C) compared to water (63°C). In the presence of crosslinkers, RTT demonstrated thermal stability >100°C in some green solvents, resulting in increased intermolecular forces between collagen, solvent and crosslinkers. The significant improvement in thermal stability of collagen potentiates the capability of green solvents as an alternative for water.

Effect of ionic liquids on the different hierarchical order of type I collagen.

The effect of ionic liquids (ILs) on proteins has been gaining huge interest due to easy tunability of cation and anion for generating the desired effect. This study explores the effect of alkyl imidazolium chloride ILs on collagen at molecular, inter-fibrillar and skin matrix level. Circular dichroic studies reveal that at the molecular level, the secondary structure of collagen was not affected by imidazolium ILs and there was no change in thermal stability as well. However, collagen at the inter-fibrillar level behaved differently. With increase in concentration of ILs, remarkable decrease in thermal stability of rat tail tendon (RTT) collagen fibers with marginal swelling effect was seen. SEM micrographs of skin matrix treated with IL show opening up of pores. This kind of exquisite behavior of ILs at different hierarchal order of collagen indicates that ILs are endowed with potential lyotropic action, which can be judiciously employed for biomedical applications.

Elucidation of hydration dynamics of locust bean gum-collagen composites by impedance and thermoporometry.

The intricacy of the different parameters involved in the hydration dynamics of collagen influences its performance as biomaterials. This work presents the molecular motions of collagen originating from the solvents and locust bean gum (LBG), which reveal the changes in solvation dynamics of the biopolymers affecting the surface as well as interfacial properties. Water, as a probe liquid bound in collagen has been investigated using a combination of thermoporometry, ATR-FTIR, circular dichroic spectroscopy, dielectric spectroscopy and SEM to explore the influence of LBG on collagen with respect to static and dynamic behaviour. The relaxation process of collagen in the frequency range of 0.01 Hz to 10(5)Hz and thermoporometry results indicate that the interfacial hydration dynamics are dependent on the applied concentration of LBG. This investigation explicitly reflects the rearrangements of the structural water clusters around the charged amino acids of collagen. These results can be employed to redesign the approach towards the development of collagen based biomaterials.

Gelatin-carrageenan hydrogels: role of pore size distribution on drug delivery process.

Naturally occurring biomaterials, such as gelatin and carrageenan are known to act as good drug delivering agents. The physical properties of these hydrogels are derived from their pore network. The effect of pore size distribution of hydrogel on the drug delivery process has been studied in this work. Gelatin-carrageenan hydrogel has been characterized using DSC, TGA and SEM. Thermoporometry technique has been used since it offers the measurement to be carried out in native state without drying the sample. Release of quercetin (Q,3,5,7,3',4'-pentahydroxyflavone), a member of the flavonoids family, which exerts many beneficial health effects has been studied using gelatin-carrageenan hydrogel. The addition of gelatin to carrageenan is found to improve the thermal stability of the gelatin-carrageenan fibers in the composite hydrogels. The in vitro drug release studies have shown that an increase in porosity results in the improved drug release. The tuning of pore size distribution for drug delivery applications using thermoporometry is feasible.

Influence of PCL on the material properties of collagen based biocomposites and in vitro evaluation of drug release.

Formulation of biodegradable collagen-poly-ε-caprolactone (PCL) based biomaterials for the sustained release of insulin is the main objective of the present work. PCL has been employed to modulate the physico-chemical behavior of collagen to control the drug release. Designed formulations were employed to statistically optimize insulin release parameter profile at different collagen to PCL molar ratios. Circular dichroism, thermoporometry, FTIR, impedance and scanning electron microscopy techniques have been employed to investigate the effect of PCL on hydration dynamics of the collagen molecule, which in turn changes the dissolution parameters of the drug from the systems. Drug entrapment efficiency has been found to be maximum for collagen to PCL molar ratio of 1:2 (>90%). In vitro dissolution test reveals that 99% of the drug was released from composite at collagen to PCL molar ratio of 1:3 and 1:4 within 2h, which indicates that hydrophobicity of the matrix results in weak interaction between lipophilic drug and carrier materials. The least burst release was observed for collagen to PCL molar ratio at 1:2 as synergistic interactions between collagen and PCL was maximum at that particular polymer-polymer ratios. The drug release data indicates super case-II transport of drug (n>1.0).