Chemical characterization of silk proteins

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CHAPTER THREE ELECTROSPINNING AND SILK FIBROIN NANOFIBRES

Introduction

Fibres serve different purposes in nature and have also found use in numerous technological applications. When these fibres are reduced to nano-scale dimensions, new and/or improved physicochemical properties are realized such as high surface area to volume ratio, superior thermal and electrical properties and mechanical strength [18, 151]. Nanofibres have thus found application in various industries including reinforcement of composites [152], protective textiles [153], filtration [154], catalysis [155], tissue engineering [156, 157] energy storage [158], sensors [159] and enzyme immobilization [160].
Fabrication of nanofibres can be achieved using several techniques such as the template method [162], self assembly [163], phase separation [164], drawing [165], melt blowing [166], ultrasonic techniques [167], nanolithography [168] and electrospinning [157-159].
From a practical view point, most of the above mentioned techniques have limitations with respect to cost, production rate and the variety of polymers that can be processed (Table 3.1). Electrospinning is the only process that can easily produce large volumes of nanofibres from a wide range of synthetic and natural polymers at a reasonably low cost. It has thus become the most studied and developed nanostructuring technique by many tertiary and research institutes worldwide. Several companies worldwide, such as eSpin Technologies (United States) and Elmarco (Czech Republic), have harnessed this technology for materials production and have been successful in commercializing electrospun products [169-171].

Background of electrospinning

Electrospinning, originally termed electrostatic spinning, involves the generation of nanofibres from a polymer solution or melt using electrostatic force. Cooley and Moore filed patents that depicted apparatus for spraying of liquids using electrical charges over a 100 years ago [172]. The more famous patent was filed by Formhals in which he described the electrospinning of plastics [173]. A few decades later, there was a renewed interest in the process with a discovery made by Taylor named after himself, the “Taylor cone”. This phenomenon is the deformation of the droplet at the end of the tip into a conical shape after application of the electrical field [174]. Reneker and co-workers then made significant contributions in understanding the process as they explored electrospinning of many different polymers [175, 176]. Over the years, knowledge and application of the process has evolved with continued research and development. Several review articles have been published describing the numerous advancements that have been made in various aspects of the technique [177-179].

Electrospinning setup and process

The most basic electrospinning setup is shown in Figure 3.1. It consists of a high voltage power supply, syringe pump, collector, and a syringe fitted with a needle. The polymer solution or melt is pumped through the needle and charged with a large electric potential. When voltage is applied and the electric potential reaches a critical level, the electrostatic repulsion of the polymer solution overcomes the surface tension at the tip of the spinneret, and a fine jet of entangled polymer chains is drawn out [178]. This jet whips through the air towards the grounded target, creating a dry fibre that collects on the target as the solvent evaporates.
Characteristics such as nanofibre diameter and morphology can be controlled by varying parameters that affect the electrospinning process. These parameters include processing, solution and ambient conditions [161, 181]. These parameters interact and influence each other during the electrospinning process and have to be optimized for nanofibres to form. Table 3.2 highlights how these parameters have affected nanofibre formation using different polymers.
Apart from the different parameters affecting electrospinning, some applications require tailoring of nanofibres to suit specific functions. Electrospinning offers multiple possibilities in fabricating customized nanofibrous structures by modification of components such as the needle system [193] and the collecting manifold [194].

Electrospinning of silk fibroin

In addition to its other attractive material properties, regenerated silk fibroin (RSF) is extrudable, making it a good candidate for electrospinning. It can also be easily chemically modified or blended with other polymers to improve electrospinnability.
Early recorded reports on electrospinning of silk were by Zarkoob and co-workers where they prepared nanofibres from Bombyx mori silk and spider dragline silk from Nephila clavipes
[195]. From then, Sukigara and co-workers successfully employed electrospinning to prepare silk nanofibres whilst also studying how the various electrospinning parameters impact on the properties of the nanofibres [184, 189, 196]. The studies determined that silk concentration was the most important parameter for successful fabrication of silk nanofibres [184].
Though SF from a number of different silk species has been successfully electrospun, the electrospinnability has been greatly influenced by the type of silk as the molecular weight varies for different silk species. Ohgo et al. highlighted this when they found different spinning concentrations for B. mori, Samia Cynthia ricini and genetically engineered recombinant silk fibroins using hexafluoroacetone hydrate as an electrospinning solvent [197]. In other work, indigenous Thai silkworms (Nang-Lai) and Chinese/Japanese hybrid silkworms showed different electrospinnability [198]. In addition to differences in electrospinnability, B. mori silk fibroin, Tussah silk fibroin (TSF) and (SF/TSF) hybrid nanofibres showed differences in properties such as moisture absorption as well as cell attachment and spreading [199]. Other studies have shown that electrospinning of SF is also dependent on the processing methods used for regeneration of the silk. Wadbua and co-workers reported different electrospinning concentrations when they electrospun nanofibres from separated light-chain and heavy-chain fibroins [200]. The nanofibres produced from light-chain and heavy chain fibroins also exhibited differences in properties such as hydrophilic nature, water uptake ability, degradation rate and cell adhesion. It is therefore important to study electrospinning of SF from different species individually as they may require different conditions to produce nanofibres and/or produce nanofibres exhibiting varied properties.
Table 3.3 highlights extensive work done on electrospinning of SF, alone or blended with various polymers or active agents, for different applications and studies. The most utilized solvents for electrospinning silk fibroin shown in Table 3.3 include formic acid (FA), hexafluoro-2-propanol (HFIP), trifluoroacetic acid (TFA), hexafluoroacetone (HFA) and trifluoroethanol (TFE). The use of these harsh organic solvents was put under the spotlight for potentially compromising the biocompatibility and mechanical properties of the silk nanofibres.
Although it has since been deduced that proper removal these solvents do not compromise the use of silk fibroin nanofibres in biological environments, the concern over them still resulted in studies being conducted to determine more ‘bio-friendly’ electrospinning solvents. Water was the solvent of choice and a number of researchers successfully electrospun fibroin aqueous solutions [205-207]. A general observation is that in aqueous media, higher SF concentrations are required for producing good nanofibres. Ionic liquids are powerful solvents that are considered to be environmentally friendly and some researchers have successfully utilized them for electrospinning silk fibroin [209].

Post treatment of silk nanofibres

The crystalline structure of fibroin is degraded during processing into regenerated silk fibroin (RSF) used to produce biomaterial formats such as nanofibres. RSF structure is mainly composed of α-helix and random coil conformations resulting in poor mechanical property and water resistance. Mechanical integrity is significant in many applications such as tissue and vessel engineering. RSF matrices therefore require post treatment mostly to restore and/or enhance their mechanical properties and water resistance. The common way of improving mechanical properties of silk fibroin matrices is by inducing crystallinity using polar protonic organic solvents such as methanol, ethanol, propanol and isopropanol. The solvents bring about aggregation of the fibroin molecule chains resulting in structural transition from random coil and α-helix to mainly β-sheet conformation [231]. Improved mechanical properties have been observed for different SF matrices such as films [232] and nanofibres [143]. Methanol is widely reported as the most used solvent for treating SF matrices. It has however been found to induce crystallization of SF in an uncontrollable manner, forming extensive β-sheet structures which result in very brittle matrices [104]. Ethanol has been reported to have a similar but milder effect to methanol as the transition is to a smaller extent [143, 233]. Similar to immersion methods, solvent vapour treatment is also a fast and effective way to alter the secondary structure of SF nanofibres [104]. Water vapour also successfully induced structural transition comparable to the organic solvents. Time and temperature of the treatment were the influential parameters determining the extent of structural change [213].
Recently, cross-linking agents such as glutaraldehyde and genipin have been used to achieve structural transition in SF matrices [234-236]. The advantage these compounds have is that they can be incorporated into the electrospinning thereby enabling conformational transition in one step and eliminating the post spinning treatment. The secondary structure of SF scaffolds is an important factor to consider as it has an impact on properties such as degradation [237], thermal behaviour [232], water absorption [134] as well as oxygen and water vapour permeability [134, 238]. Silk II is the most important and exploited structure in biomaterial applications as it can be tailored to suit various uses.
Alternative to structural modification, blending of SF with other polymers can help improve mechanical properties. SF blend nanofibres with polyethylene oxide (PEO) [239], carbon nanotubes [209], chitosan [211] and cellulose acetate (CA) [212] all exhibited better mechanical properties as compared to pure SF nanofibres. Besides improving and/or modifying structural and physical properties, post treatment for SF matrices can serve as a method for removing residual solvents used in the electrospinning process [30].

Application of electrospun SF in wound healing

Electrospun SF nanofibres find use in many applications such as those highlighted in Table 3.3. Regenerative medicine is one of the major areas where there has been significant utilization of SF nanofibres. From a structural perspective, the porous three-dimensional nature of electrospun nanofibres resembles the nanoscale fibrous composition of the extracellular matrix (ECM) of living systems (Figure 3.2). The high surface area provided by nanofibres enables functions such as cell attachment and migration, which are key for regeneration of tissues and organs [240- 242]. The ECM is also composed of many proteins such as collagen, fibronectins, laminin and vitronectin thus making SF a prime material to be used in various facets of regenerative medicine.
Several articles have highlighted the use of SF in many areas of regenerative medicine; however, this review will focus on its applications in wound healing and regeneration. Wound healing involves the restoration or regeneration of dermal and epidermal tissues after an individual has been wounded [244]. The healing process itself is complex and several aspects need to be carefully considered when trying to create an ideal wound dressing. However, the basic desirable properties for a wound dressing are to create an optimum environment to allow reepithelialisation, allow effective gaseous exchange and create a barrier against harmful microorganisms [245, 246].
As wound dressings are in contact with biological environments, biopolymers are mostly more effective as wound dressings and accelerate the wound healing process compared to synthetic polymers [247]. One of the most important steps in wound healing is the closure of the wounded area by epithelial cells, a process that restores the epidermal barrier and protects the underlying tissue. Fibroin has shown potential in this regard by successfully supporting adhesion and growth of human keratinocytes which are the skin cells responsible for reepitheliazation of wounds [203, 213, 248, 249]. Fibroin has also been reported to promote growth and proliferation of human skin fibroblasts, which are also important in the wound healing process [249-253]. Min et al. further examined the effect of the nature of the SF matrices on the cell adhesion and spreading. They determined that the nanofibres are better suited for applications such as wound dressings and tissue engineering scaffolds than films and microfibre matrices [248].

Bioactive agents in wound dressings

For several decades, researchers have been searching for ways of improving the wound healing process. Advances in cellular and molecular biology have brought about knowledge that has made it possible for improvement and development of new therapies for treatment of wounds. A major advancement are the new generation ‘smart’ wound dressings that incorporate single or a combination of therapeutic agents that can target different aspects of wound healing. The bioactive agents may be antibiotics [254], antibacterials [255], analgesics [256], growth factors [215], vitamins [216], antifungals [257], or a combination [258].
There are, however, some current drawbacks in wound healing regarding some commonly used additives such as toxicity and increased antibiotic resistance. For example, silver is the active agent in several commercial antimicrobial wound dressings. However, it has been reported that prolonged use of silver particles and nanoparticles may be toxic to DNA and mitochondria [259, 260]. In addition to this, silver in wound dressings is the cause of some irritation and discoloration of wounds and surrounding tissue [261]. This therefore raises concerns regarding safety in long term use of silver. There are also major concerns regarding the increase of antibiotic resistant bacterial strains. Bacterial resistance has been observed with various groups of drugs such as tetracyclines, sulphonamides, macrolides and aminoglycosides [262, 263]. There is also evidence of some microbes also having developed resistance to silver [264].
These illustrations highlight the great need for new or alternative topical bioactive agents to address the shortcomings of the current ones. In our opinion, biological active components derived from plants are significant and important as new drugs that are likely to lead to better treatments for various ailments and diseases.

Plant phenolics in wound dressings

Phenolics are secondary metabolites found commonly in many foods that are integral in the human diet such as vegetables, fruits, grains, teas, herbs and fruits [265]. The compounds are classified under several groups though their commonality lies in each compound possessing a phenol group in its structure. One of the groups of phenolics, the polyphenols, exhibit an array of valuable biological activities, including antiviral, antibacterial, antioxidant, anti-inflammatory, and anti-carcinogenic activity [266] and have great prospects as medicinal products. Major emphasis has been placed on polyphenols being taken orally as dietary supplements. The same multiple benefits can be afforded to the skin when polyphenols are applied topically [267, 268] as they possess a number of properties beneficial for wound healing and combating a wide range of skin disorders. It is often necessary for these compounds to be carried in formulations or in materials to be delivered to their targets. Fibroin is attractive and has success as a carrier for bioactive molecules due to its chemical and structural properties. A wide range of molecules can thus be encapsulated and stabilized within the fibroin through hydrophilic and/or hydrophobic interactions [356, 357].
SF matrices functionalized with polyphenols have provided scaffolds that are excellent candidates for wound healing and tissue engineering materials. Polyphenolic extracts from the plants Pistacia terebinthus, Pistacia lentiscus, and Hypericum empetrifolium were adsorbed on silk fibroin/chitosan blend films resulting in a scaffold with both antibacterial and antioxidant properties [269]. Kasoju & Bora prepared a curcumin releasing porous SF scaffold that can be used as a wound dressing or for therapy against cancerous tumours [270]. In addition to the inherent properties of SF, the high surface area and porosity of SF nanofibres make them great candidates for carrying compounds such as polyphenols. However, there are few reports on tissue engineering applications of SF nanofibres with polyphenolic compounds. Elakkiya et al. described fabrication of curcumin loaded SF nanofibres by coelectrospinning for drug delivery applications. The scaffold was reported to have high porosity (85%) and water uptake (150%) [204]. In vitro release studies showed at least 80% release after 10 days for formulations with up to 1.5 wt % curcumin. This report is based on B. mori SF and SF from African silkworm species have not been studied in this capacity.
Resveratrol (3,4′,5-Trihydroxystilbene) is one of the more potent phenolics that can be found in grapes, berries and peanuts and red wine [271]. Resveratrol exists as two isomers; cis- and trans-resveratrol. Trans-resveratrol (Figure 3.3) is the natural form of the compound and is more active and stable than the cis- isomer [272]. Resveratrol has been found to be active against several species of bacteria and dermatophytes that cause infections and a variety of skin conditions [273-275]. However, its applications have mostly been centred on its anti-inflammatory and anti-cancer properties [276, 277]. For topical applications, fibroin promotes cell growth and proliferation but lacks other properties required for wound healing specific to mitigation of bacterial and pathogen growth. SF blended with polyphenolic compounds such as resveratrol provides a great opportunity to exploit the combined benefits of these natural products. As far as we could ascertain, there have been no recorded studies on the incorporation of resveratrol into silk nanofibres for any applications.

Techniques for incorporating bioactive compounds onto nanofibres

Therapeutic compounds can be integrated into a biomaterial matrix via adsorption, covalent bonding to the matrix polymer, or physical entrapment or encapsulation. Encapsulation can be effected by co-electrospinning the compounds with the carrier polymer [278]. This procedure is very easy and convenient though it may result in diminished activity of the sensitive compounds when harsh electrospinning conditions are used. It can also be challenging to homogeneously mix some compounds in the polymer solution. Addition of some compounds can actually alter polymer solution properties, thus influencing the electrospinning process. For instance, the incorporation of the antibiotic cefazolin into poly(lactic-co-glycolic acid) (PLGA) resulted in larger nanofibre diameters as compared to as-spun PLGA nanofibres [279].
It is also possible to load compounds on prefabricated nanofibres via physical adsorption or chemical immobilization methods. Physical adsorption is a straightforward and easy technique for loading bioactive molecules on nanofibrous scaffolds due to the high surface area to volume ratio of nanofibres. The nanofibre mat is simply placed in a solution with the bioactive agents or the solution poured onto the nanofibres for adsorption to occur [280]. The compounds bind onto the nanofibre scaffold by non-covalent interactions such as van der Waals forces, hydrogen bonding, or hydrophobic interactions. Introduction of bioactive materials in this manner often prevents degradation or loss of activity of the bioactive molecules [280]. The chemical immobilization method involves covalent attachment of the bioactive molecules onto the surface of electrospun nanofibres. The disadvantage, however, is that the technique may result is some partial inactivation of the immobilized molecules as some active sites can become chemically modified during the process [281].
With co-electrospinning, solubility of the bioactive compound in the polymer will influence its distribution in the nanofibre scaffold as well as the drug release [282]. A rapid drug release profile has been observed with drugs loaded using the adsorption technique and these scaffolds are particular useful in early treatment [280]. In the case of chemical immobilization, the drugs are not as easily released from the surface of the nanofibres when incubated over an extended period. The drugs covalently bonded to the polymers are released over an extended period of time as opposed to the burst release offered by simple adsorption. Other factors that affect the release of drugs from nanofibre are the morphology of the fibres and additional interactions with the drug [283]. The concentration of the drug in the fibres also has an effect on the drug release kinetics. A pronounced burst has been observed with higher concentration, which is attributed to enrichment of the drug on the surface [283]. Using the above techniques, scaffolds for both local and sustained drug delivery can be produced depending on the compounds and polymers being used.

TABLE OF CONTENTS
Declaration
Abstract
Dedication
Acknowledgements
List of Figures
List of Tables.
Scientific contributions
CHAPTER 1: Introduction
1.1 General Introduction
1.2 Background
1.3 Motivation of the study
1.4 Objectives
1.5 Thesis Layout
CHAPTER 2: Silk and its properties
2.1 Introduction
2.2 Composition of the silk thread
2.3 Chemical characterization of silk proteins
2.4 Primary and secondary structure of silk proteins
2.5 Physical properties of silk fibroin
2.6 Silk fibroin as a biomaterial
2.7 Preparation of silk fibroin matrices for biomaterial applications
CHAPTER 3: Electrospinning and silk fibroin nanofibres
3.1 Introduction
3.2 Background of electrospinning
3.3 Electrospinning setup and process
3.4 Electrospinning of silk fibroin
3.5 Post treatment of silk nanofibres
3.6 Application of silk nanofibres in wound healing
3.7 Bioactive agents in wound dressings
3.8 Techniques for incorporating bioactive compounds onto nanofibres
CHAPTER 4: Experimental procedures
4.1 Introduction
4.2 Materials
4.3 Processing of silk fibroin
4.4 Preparation of silk fibroin nanofibres
4.5 Adsorption and release of resveratrol on silk nanofibres
4.6 Characterization studies
CHAPTER 5: Chemical and physical properties of silk fibroin
5.1 Introduction
5.2 Development of MEKC separation for PTC-modified amino acids
5.2.1 MEKC with borate buffer
5.2.2 MEKC with phosphate buffer
5.3 Method Validation
5.4 Quantification of amino acids in fibroin samples
5.5 Cocoon and fibre properties
5.6 FTIR and Raman spectroscopy
5.7 XRD analysis
5.8 Thermal analysis
5.9 Mechanical properties
CHAPTER 6: Fabrication and properties characterization of electrospun silk nanofibres
6.1 Introduction
6.2 Dissolution of degummed silk fibroin
6.3 Electrospinning of RSF
6.4 FTIR spectroscopy
6.5 Post treatment of RSF nanofibres
6.6 Morphology of the treated nanofibres
6.7 Water solubility of SF nanofibres pre and post- solvent treatment
6.8 Thermal properties of silk nanofibres
6.9 Loading of resveratrol onto Gonometa SF nanofibres
6.10 Release of resveratrol from silk nanofibres
6.11 Cytotoxicity of silk nanofibres
6.12 Antibacterial, antioxidant and anticancer properties of resveratrol loaded silk nanofibres
CHAPTER 7: Conclusions and future perspectives
6.1 Introduction
6.2 Conclusions
6.3 Future perspectives
References
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