Development of bio-composites with novel characteristics through enzymatic grafting

Enzymatic grafting of biopolymers has recently been the focus of green chemistry technologies due to the growing environmental concerns, and subsequent legal restrictions. Over the last decade, research covering various applications of enzymes like lipases and laccases has been increased rapidly, particularly in the field of polymer science, to graft multi-functional polymers. In this context, a series of bio-composites e.g. poly3-hydroxybutyrate [P(3HB)]grafted ethyl cellulose (EC) and bacterial cellulose (BC) [i.e., P(3HB)-g-EC/BC] and keratin-g-EC bio-composites were successfully synthesised by introducing enzymebased grafting where lipase and laccase were used as model bio-catalysts. Furthermore, various natural phenols e.g., caffeic acid (CA), gallic acid (GA), p-4-hydroxybenzoic acid (HBA), and thymol (T) were grafted onto the newly developed P(3HB)-EC and keratin-EC-based composites under laccase-assisted environment. Subsequently, the resulting bio-composites were removed from their respective casting surfaces under ambient environment and characterised using different analytical and imaging techniques. This project shows improvement in the thermo-mechanical properties of the biocomposites as compared to the individual components. The tensile strength, elongation at break point, and Young’s modulus values of the bio-composites reached very high levels in comparison to the films prepared with untreated P(3HB) and keratin that were too fragile to be measured for any of the above mentioned characteristics. Morphological analysis of the newly developed bio-composites surfaces through SEM showed a uniform distribution of the P(3HB) and keratin within the backbone polymer(EC). Interestingly, untreated P(3HB) was hydrophobic in nature and after lipase treatment P(3HB) and P(3HB)-EC-based graft composites attained a higher level of hydrophilicity. The phenol grafted bio-composites were critically evaluated for their antibacterial and biocompatibility features, as well as their degradability in a soil. In particular, the results of the antibacterial evaluation indicated that 20CA-g-P(3HB)-EC, 15GA-g-P(3HB)-EC, 15HBA-g-P(3HB)-EC, 15T-g-P(3HB)-EC, 15CA-g-keratin-EC, 15GAg-keratin-EC, 10HBA-g-keratin-EC and 20T-g-keratin-EC exerted strong bactericidal and bacteriostatic activity against Gram+ bacteria Bacillus subtilis NCTC 3610 and Staphylococcus aureus NCTC 6571 and Gram- bacteria Escherichia coli NCTC 10418 and Pseudomonas aeruginosa NCTC 10662 strains, respectively. This study shows further that at various phenolic concentrations the newly synthesised bio-composites remained cytocompatible with human keratinocyte-like HaCaT skin cells, as 100% cell viability was recorded after 5 days of incubation. From the degradation point of view,an increase in the degradation rate was recorded during the soil burial analyses. This study provides novel and additional knowledge that encourage greater utilisation of biopolymers in the development of bio-composites with novel and sophisticated characteristics for potential applications

Poly(3-hydroxybutyrate)-ethyl cellulose based bio-composites with novel characteristics for infection free wound healing application

A series of bio-composites including poly3-hydroxybutyrate [P(3HB)] grafted ethyl cellulose (EC) stated as P(3HB)-EC were successfully synthesised. Furthermore, natural phenols e.g., p-4-hydroxybenzoic acid (HBA) and ferulic acid (FA) were grafted onto the newly developed P(3HB)-EC-based bio-composites under laccase-assisted environment without the use of additional initiators or crosslinking agents. The phenol grafted bio-composites were critically evaluated for their antibacterial and biocompatibility features as well as their degradability in soil. In particular, the results of the antibacterial evaluation for the newly developed bio-composites indicated that 20HBA-g-P(3HB)-EC and 15FA-g-P(3HB)-EC bio-composites exerted strong bactericidal and bacteriostatic activity against Gram- E. coli NTCT 10418 as compared to the Gram+ B. subtilis NCTC 3610. This study shows further that at various phenolic concentrations the newly synthesised bio-composites remained cytocompatible with human keratinocyte-like HaCaT skin cells, as 100% cell viability was recorded, in vitro. As for the degradation, an increase in the degradation rate was recorded during the soil burial analyses over a period of 42 days. These findings suggest that the reported bio-composites have great potential for use in wound healing; covering the affected skin area which may favour tissue repair over shorter periods

Development of bio-composites with novel characteristics: Evaluation of phenol-induced antibacterial, biocompatible and biodegradable behaviours

This paper describes a laccase-assisted grafting of gallic acid (GA) and thymol (T) as functional entities onto the previously developed P(3HB)-g-EC composite. GA-g-P(3HB)-g-EC and T-g-P(3HB)-g-EC bio-composites were prepared by laccase-assisted free radical-induced graft polymerisation of GA and T onto the P(3HB)-g-EC based composite using surface dipping and incorporation technique. The results of the antibacterial evaluation for the prepared composites indicated that 15GA-g-P(3HB)-g-EC, 15T-g-P(3HB)-g-EC and 20T-g-P(3HB)-g-EC composites possessed the strongest bacteriostatic and bactericidal activities against Gram-positive B. subtilis NCTC 3610 and S. aureus NCTC 6571 and Gram-negative E. coli NTCT 10418 and P. aeruginosa NCTC 10662 strains. In this study, we have also tested GA-g-P(3HB)-g-EC and T-g-P(3HB)-g-EC bio-composites for their ability to support and maintain multilineage differentiation of human keratinocyte-like (HaCaT) skin cells in-vitro. From the cytotoxicity results, the tested composites showed 100% viability and did not induce any adverse effect on a HaCaT’s morphology. Finally, in soil burial evaluation, a progressive increase in the degradation rate of GA-g-P(3HB)-g-EC and T-g-P(3HB)-g-EC bio-composites was recorded with the passage of time up to 6 weeks. In summary, our current findings suggest that GA-g-P(3HB)-g-EC and T-g-P(3HB)-g-EC bio-composites are promising candidates for biomedical type applications such as skin regeneration, multiphasic tissue engineering and/or medical implants

Bacterial Cellulose: A sustainable source to develop value-added products - A review

In recent decades, worldwide economic and environmental issues have prompted research scientists to re-direct their interests to bio-based resources, which are sustainable in nature. In this context, microbial polysaccharides, such as bacterial cellulose (BC), also known as microbial cellulose (MC), are some of the upcoming and emergent resources and have potential application in various bio- and non-bio-based sectors of the modern world. Many researchers have already established novel BC/MC production methods, and many new studies have been published on lab-scale and large-scale production aspects of BC/MC to date. To further expand the novel use of this sustainable source, significant progress toward the development of BC/MC has appeared in recent years. Specifically, there have been many publications and/or research reports on the valorization of BC/MC in the food, paper, materials, biomedical, pharmaceutical, and cosmeceutical industries, among others. This review will address the novel application aspects of BC/MC today, with the aim of demonstrating the importance of this sustainable and novel source in the development of value-added products

Development of chitosan, pullulan, and alginate based drug-loaded nano-emulsions as a potential malignant melanoma delivery platform

Melanoma is the most aggressive form of skin cancer and various treatments have been investigated to treat this disease, but drug resistance remains an important factor in the failure of conventional therapeutics. Here we describe the development, optimisation and characterisation of alginate, chitosan, pullulan, and their combined nano-emulsions as drug delivery platforms for potential application for melanoma. A novel nano-emulsion delivery system was designed and assessed by determining in vitro drug release, cell viability (MTT), cellular apoptosis (ELISA) and confocal microscopy. A comparative analysis of the effect of the nano-emulsions on BRAF-mutant melanoma (A375) and keratinocyte (HaCaT) cells was conducted, with the “pullulan-chitosan” nano-emulsion chosen as an approach for melanoma drug delivery. Increased apoptosis induction of melanoma cells was recorded as 90% after 72 h of treatment with doxorubicin-loaded optimal nano-emulsion. Similarly, in the same treatment, the viability of melanoma cells was decreased by 70%. More importantly, A375 cells treated with naïve doxorubicin were 100% viable compared to cells treated with doxorubicin-loaded nano-emulsion which were only 30%viable. Achieved results are indicating the importance of the drug carrier’s polymeric combination and the impact of the drug release pattern on the efficiency of the treatment. This offers potential for the abrogation of drug- efflux-related chemo-resistance

Laccase bio-grafting a versatile modification tool from green chemistry technologies

Today more than 99% of plastics are petroleum-based because of availability and cost of the raw material. The durability of these disposed plastics contributes to the environmental problems as waste and their persistence in the environment causes deleterious effects on the ecosystem. Environmental pollution awareness and the demand for green technology have drawn considerable attention of both academia and industry into biodegradable polymers. In this regard green chemistry technology has the potential to provide solution to this problematic issue. Laccase bio-grafting has recently been the focus of green chemistry technologies due to the growing environmental concerns, legal restrictions and increasing availability of scientific knowledge. In the last several years, research covering various applications of laccases has been increased rapidly particularly in the field of grafting. In principle, laccase-assisted graft co-polymerization may impart a variety of new functionalities to a polymer. The modified polymers through grafting have a bright future and their development is practically boundless. In present work, novel biodegradable graft copolymers combining the advantages of bacterial cellulose backbone and PHB side chains will be prepared by introducing enzymatic grafting technique. The present research will be a first step in the biopolymer modification. To date no report has been found in literature explaining the enzymatic grafting of PHAs. The technique would also provide an efficient modulation approach to improve the biodegradability and biocompatibility of the graft copolymer. The newly grafted copolymers will exhibit unique functionalities with wider range of potential applications mainly in tissue engineering, biosensors, pharmaceutical industry (drug delivery systems) and bio-plastics

Laccase-assisted grafting of poly(3-hydroxybutyrate) onto the bacterial cellulose as backbone polymer: development and characterisation

Bacterial cellulose (BC) exhibits high purity, mechanical strength and an ultra-fine fibrous 3-D network structure with bio-compatible and bio-degradable characteristics, while P(3HB) are a bio-degradable matrix material derived from natural resources. Herein, we report a mild and eco-friendly fabrication of indigenously isolated P(3HB) based novel composites consisting of BC (a straight-chain polysaccharide) as a backbone polymer and laccase was used as a grafting tool. The resulting composites were characterised by Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC), dynamic mechanical analyser (DMA) and water contact angle analyser (WCA). The FTIR spectra of the pure P(3HB) and P(3HB) containing graft composites [P(3HB)-g-BC] showed their strong characteristic bands at 3358 cm−1, 1721 cm−1 and 1651 cm−1, respectively. A homogenous dispersion of P(3HB) in the backbone polymer of BC was achieved as evident by the SEM micrographs. XRD pattern for P(3HB) showed distinct peaks at 2θ values that represent the crystalline nature of P(3HB). While, in comparison with those of neat P(3HB), the degree of crystallinity for P(3HB)-g-BC decreased and this reduction is mainly because of the new cross-linking of P(3HB) within the backbone polymer that changes the morphology and destroys the crystallites. Laccase-assisted graft composite prepared from P(3HB) and BC was fairly flexible and strong, judged by the tensile strength (64.5 MPa), elongations at break (15.7%), and Young's modulus (0.98 GPa) because inherently high strength of BC allowed the mechanical properties of P(3HB) to improve in the P(3HB)-g-BC composite. The hydrophilic property of the P(3HB)-g-BC was much better than that of the individual counterparts which is also a desired characteristic to enhance the biocompatibility of the materials for proper cell adhesion and proliferation

Advances in the valorization of lignocellulosic materials by biotechnology: an overview

In view of the worldwide economic and environmental issues associated with the extensive use of petro-chemicals, there has been increasing research interest during the past decade in the value of residual biomass. Because of its renewable nature and abundant availability, residual biomass has attracted considerable attention as an alternate feedstock and potential energy source. To expand the range of natural bio-resources, significant progress related to the lignocellulose bio-technology has been achieved, and researchers have been re-directing their interests to biomass-based fuels, ligninolytic enzymes, chemicals, and biocompatible materials, which can be obtained from a variety of lignocellulosic waste materials. This review article focuses on the potential applications of lignocellulosic materials in biotechnology, including the production of bio-fuels, enzymes, chemicals, the pulp and paper, animal feed, and composites

Laccase-assisted bio-grafting of polyhydroxy-alkanoates (PHAs) onto the ethyl cellulose (EC) backbone

In the present study, a novel enzyme-based methodology for grafting Polyhydroxyalkanoates (PHAs) onto the ethyl cellulose (EC) as a backbone polymer was developed. Laccase assisted copolymerization was carried out under mild and eco-friendly reaction conditions. The resulting homogeneous composite membranes were characterized by Fourier-transformed infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC), and Atomic Force Microscopy (AFM). The FTIR spectra of pure PHAs and PHAs containing graft composites (PHAs-g-EC) showed their strong characteristic bands at 1721 cm1, 1651 cm-1 and 1603 cm-1 respectively. Other accompanying bands in the range of 900-1300 cm-1 correspond to C=O vibration and C-O-C bond stretching, which could be contributed from PHAs and EC, respectively. The high intensity of the 3358 cm-1 band in the graft composite may have corresponded to the degradation of the carboxylic group from PHAs and also showed an increase of hydrogen-bonded groups at that distinct band region. The morphology was examined by SEM, which showed the well dispersed PHAs crystals in the backbone polymer of EC. XRD pattern for PHAs showed distinct peaks at 2-Theta values of 28o, 32o, 34o, 39o, 46o, 57o, 64o, 78o and 84o that represent the crystalline nature of PHAs. In comparison with those of neat PHAs, the degree of crystallinity for PHAs-g-EC decreased and this reduction is mainly because of the new cross-linking of PHAs within the EC backbone that changes the morphology and destroys the crystallites. Improved mechanical properties were observed for the PHAs-g-EC as compared to the individual components due to the impregnation of EC as reinforcement into the PHAs matrix. Improved mechanical strength enhanced thermal properties, along with low crystallinity of the present PHAs-g-EC suggesting its potential for various industrial and bio-medical applications