INTRODUCTION:

Silks belong to a group of high molecular weight organic polymers characterized by repetitive hydrophobic and hydrophilic peptide sequences¹. Due to the highly repetitive primary domains, fibrous proteins and especially silk fibroin (SF) assemble into regular structures during materials formation and can be considered as Nature's equivalents to synthetic block copolymers^{1, 2, 3, 4, 5}. Silks are naturally produced by spiders or insects, such as Nephilaclavipes and Bombyxmori, respectively^{6, 7}. The primary sequence of SFs have achieved wide evolutionary adaptation to such diverse needs as spinning underwater nets to trap air for underwater breathing, lifelines, and prey capture, common features associated with the formation of robust and stable material structures.

Bombyxmori race nistari is a resistant variety of multivoltine mulberry silkworm which contributes up to a great extent in the commercial production of cocoon in varying ecological conditions in our country. The larval stages of silkworm are monophagous, since a diet of which the chemical content is completely known, both quantitatively and qualitatively⁸.

The unique properties of silk fibroin (SF) such as slow biodegradation, superior mechanical properties, favourable process ability in combination with biocompatibility, have fuelled wide interest in this material for a variety of applications, ranging from textiles to biomedical use⁹. In fact, SF is currently emerging as an important protein biomaterial of broad biomedical applicability. This review is particularly directed towards the application of native and modified SF in pharmaceuticals and biomedicals. A variety of polymers have been investigated for drug delivery purposes. Synthetic macromolecules including polyesters, polyorthoesters, polyanhydrides, polyphosphazenes, and polyphosphoesters have found broad application¹⁰. Natural polymers including alginates, chitosan, cellulose, collagen, gelatin and elastin remain attractive due to their biocompatibility and biodegradability, their similarity to biological macromolecules and the potential for chemical or physical modification^{11, 12}. However, there remains a need for biomaterials that can be highly controlled in terms of composition and sequence, structure and architecture, mechanical properties and function. To address these requirements, the exploration of SF as a biomaterial for controlled drug delivery has widely expanded over the last few years. This article will review recent developments in this area of research.

SF is a natural polymer produced by a variety of insects and spiders. It is, therefore, subject to a wide diversity in sequence, structure and properties. The best characterized silks are the cocoon silk from the domesticated silkworm Bombyxmori and the dragline silk from the spider Nephilaclavipes¹³. Silkworm silk has been studied more widely in the field of drug delivery than other silks. Silkworms are easier to domesticate and the silk can be obtained more simply in comparison to spider silk. Therefore, spider silks have been looked at only sparingly in the context of drug delivery and the focus of this review will mainly be on the use of SF obtained from silkworms.

Types of Silk fibroins:

1. Silkworm Silk fibroins:

Silkworm fibroin is used as a biomedical suture from many years, it also used in textile production for clothing, because of the fact that silkworms are easier to domesticate and obtain silk in comparison to spider silks. The silk fibroin from the cocoon of silkworm Bombyxmori, the most studied silkworm silk proteins, contain two major components, light (~25 kDa) and heavy chain (~325 kDa) fibroins. The core sequence in the heavy chain includes alanine-glycine repeats. In silkworm cocoons, these two fibroins are encased in a sericin coat, glue-like proteins, to form the composite fibers of the cocoon. Various methods are used now-a-days to extract and regenerate silk fibroin^{14, 15}, and several silk-based biomaterials, such as silk porous scaffolds, silk films, hydrogels, and electrospun nanofibers, can be processed from silk solutions.

2. Spider Silk Fibroins:

Nephilaclavipes is the most common and widely studied spider silk in terms of structure and function is dragline silk which is secreted as a mixture of two proteins from specialized columnar epithelial cells of the major ampullate gland of orb-weaver spinning spiders^{7, 16}. The molecular weight of these proteins ranges from 70 to 700 kDa depending on source. Partial cDNA clones encoding the two types of dragline silks have been isolated and analyzed from two species of orb-web weaving spiders, Araneusdiadematus (ADF-3 and ADF4) and N. clavipes (MaSpI and MaSpII)^{17, 18}. These silk proteins are characterized as block copolymers, composed of large hydrophobic blocks with highly conserved repetitive sequences consisting of short side-chain amino acids, such as glycine and alanine, with intervening small hydrophilic blocks with more complex sequences that consist amino acids with bulkier side chain and charged amino acids¹⁹. The hydrophobic blocks from beta-sheets, or physically cross-linked crystalline domains in silk fibers. The impressive tensile strength of silk fibers is due to the ordered hydrophobic and less ordered hydrophilic regions, in combination with chain orientation achieved during spinning²⁰.

Extraction of Silk fibroins from Silk:

Bombyxmori silk cocoons are cut into small pieces and the silkworms are then disposed. The dried silk cocoons are then boiled for 30 minutes in 0.02M sodium carbonate (Na₂CO₃₎.The fibers are then rinsed thrice for20 minutes. The excess water is then squeezed out and allowed to dry overnight. After overnight drying, the extracted silk fibroin solution was prepared by dissolving degummed silk in 9.30M Lithium bromide solution at 60°C for 4 hours²¹ or 70°C for 2.5 hours. The fibroin solution was dialyzed in a cellulose membrane based dialysis cassette against deionized water for 3 days changing water every 6 hours in order to remove lithium bromide. After dialysis, concentrated solution was obtained by centrifuging silk fibroin solution at 5-10˚C at 9000 rpm for 20 minutes²². The concentrated solution was stored at 4°C for further studies.

Silk Properties:

Drug loading, release kinetics and physico-chemical stability of a drug delivery system are features, which can be tailored by changing the properties of its matrix. Here we review SF properties that we expect to affect the controlled release of a drug from a SF based delivery system and its application.

Molecular properties of silk:

One of the best characterized silk is the cocoon silk from the domesticated silkworm B. mori¹³. Silk from B. mori is made of two structural proteins, the fibroin heavy chain (~325 kDa) and light chain (~25 kDa). The heavy chain of SF contains alternating hydrophobic and hydrophilic blocks similar to those seen in amphiphilic block copolymers. The hydrophobic blocks consist of highly conserved sequence repeats of GAGAGS and less conserved repeats of GAGAGX (where X is either V or Y) that make up the crystalline regions of SF by folding into intermolecular β-sheets. The hydrophilic part of the core is non-repetitive and very short compared to the size of the hydrophobic repeats^23,
24.

Physical properties of silk:

Molecular weight:

The molecular weight of a polymer strongly influences its mechanical properties and biodegradability, and, therefore, its field of application in drug delivery. For instance, because of the variety of available molecular weights affecting its biodegradation and, therefore, its drug release kinetics, customized polyesters such as poly (lactide-co-glycolide) (PLGA) of various lactide: glycolide ratios have been widely used for drug delivery applications^{25, 26, 27}.

Natural silks exhibit large molecular weights. Lower molecular weights of SF can be obtained by processing silk. For instance, prolonged boiling of silk cocoons in Na₂CO₃ solutions, a common method to remove sericin from fibroin, was shown to cause extensive hydrolytic degradation of the SF protein²⁸. However, this method is not well documented and controlled, and might produce a wide molecular weight distribution. SF with diverse molecular weights may be also obtained by adding sodium hydroxide to aqueous SF solutions at different processing temperatures²⁹ or by enzymatic degradation. A better controlled approach to produce SF analogues with distinct molecular weights is genetic engineering³⁰.

Crystallinity and water insolubility:

Crystallinity is the basis for the stability of SF. Nevertheless, an excessive increase in crystallinity reduces its flexibility and leads to more brittle materials. Annealing in water or water vapour treatments were shown to induce less β-sheet structure and retain better elasticity as compared to methanol treatments^{31, 32}. Conformational analysis of SF films and nanofibers with ¹³C-MAS NMR demonstrated the percentage of silk II after water vapour treatment (30 to 47%) to be lower as compared to a treatment with methanol (74%)^{31, 33}.

Recently, a time dependent increase in β-sheet structure was observed when treating SF microparticles with saturated sodium chloride (NaCl) solution, obtaining β-sheet contents of about 34%, 51% and 67% when treating for 1 h, 4 h and 15 h, respectively. The β-sheet content of SF microparticles treated with methanol for 30minwas analysed to be 58%, comparable to longer treatments with NaCl³⁴. Additionally, inclusion of elastin-like domains in SF could reduce crystallinity^{30, 33}. Alterations in both crystallinity and hydrophobicity offer options to affect the interaction between drug molecules and SF. Moreover, a change in crystallinity influences the degradation rate of SF, which could be an attractive approach towards drug delivery systems with distinct release kinetics.

Solubility:

Crystalline SF is insoluble in most solvents that are widely used to dissolve polymers typical for drug delivery applications, as well as in water. Commonly applied to dissolve SF are highly concentrated salt solutions of lithium bromide, lithium thiocyanate, calcium thiocyanate or calcium chloride²³.

Stability:

The stability of a polymer is an important feature for the storage of a drug delivery device. Untreated SF matrices are low in β-sheet content; remain hygroscopic and thus highly sensitive to humidity. Incubation at high humidity has been shown to change the conformational state of SF, leading to increased β-sheet contents^{35, 36}. SF displays an exceptional thermal stability, being virtually unaffected by temperatures up to 140°C. The glass transition temperature, Tg of proteins is considered to be a major determinant of protein self-assembly. Dry SF films prepared from silk taken from the posterior part of the middle division of the silk gland of the silkworm B. mori demonstrated a Tg of approximately 175°C, above which there is free molecular movement to transform into the stable β-sheet conformation, showing stability up to around 250°C³⁷. The Tg of frozen SF solution was reported to be in the range of about −34 to −20°C³⁸. The higher the pre-freezing temperature above the glass transition, the longer the time needed for ice crystals to form and grow in size. This affects the pore size within SF matrices, leading to bigger pores³⁹, as well as an increase in crystallinity^{38, 40}.

Swelling Properties:

The release of drugs from matrices such as hydrogels depends partially on the degree of swelling, which in turn depends on the ionization of the network, its degree of crosslinking and its hydrophilic/hydrophobic balance⁴¹. The hydrophilicity of the network is due to the presence of chemical residues such as hydroxylic (-OH), carboxylic (-COOH), amidic (-CONH-), primary amidic (-CONH₂), sulphonic (-SO₃H), and others that can be found within the polymer backbone or as lateral chains⁴². Changes in polymer compositions can influence the degree of swelling. The swelling ratio of SF scaffolds has also been shown to decrease with an increase in SF concentration. Blending of SF with other materials such as chitosan⁴³, hyaluronic acid⁴⁴ and chitosan⁴⁵ led to increased swelling when compared with plain SF.

Mechanical Properties:

Many scaffolds prepared from polymers including PLGA and collagen lack sufficient mechanical strength for load-bearing purposes^{46, 47}. Enhanced mechanical strength, however, is an important issue when a drug delivery device is also used as a scaffold with load-bearing function, as frequently needed in bone repair. Since it offers great advantages of biocompatibility and biodegrability with adjustable properties and capable of being processed to form a variety of objects, PLGA finds extensive biomedical applications, such as sutures, implantable devices, and drug delivery systems⁴⁸. The compressive strength and modulus of those SF scaffolds was reported to increase with increasing SF concentration, decreasing pore size and more uniform pore distribution⁴⁹.

Degradation and Biodegradation:

The hydrolytic degradation of water vapour treated SF scaffolds in vitro without the presence of proteolytic enzymes is minor, with only about 4% mass loss within 7 weeks²⁷. Being a protein, biodegradation of SF predominantly occurs through proteolytic enzymes, with non-toxic degradation products and unproblematic metabolisationin vivo. The implantation site, the mechanical environment and the size and morphology of the drug delivery device are likely to affect the degradation rate in vivo.

Applications of Silk Fibroin as a Drug Delivery:

Fig. 1: Overview on applications of silk fibroin (SF).

Implants, tubes and scaffolds:

An implant is a kind of medical device made to replace and act as a missing biological structure. The surface of implants that contact the body might be made of a biomedical material such as titanium, silicone or apatite depending on what is the most functional⁵⁰. Silk-based 3D scaffolds are attractive biomaterials for bone tissue regeneration because of their biocompatibility and mechanical properties. The 3D silk fibroin scaffolds loaded with bone morphogenetic protein-2 (BMP-2) were successfully developed for sustained release of BMP-2 in order to induce human bone marrow stromal cells to undergo osteogenic differentiation when the seeded scaffolds were cultured in vitro and in vivo with osteogenic stimulants for 4 weeks. Horseradish peroxidase (HRP) enzyme gradients were also immobilized on silk 3D scaffolds to prepare new functional scaffolds including regional patterning of the gradients to control cell and tissue outcomes. Recently, adenosine release via silk-based implants to the brain has been studied for refractory epilepsy treatments. Silk-based implants to release adenosine demonstrated therapeutic ability, including the sustained release of adenosine over a period of two weeks via slow degradation of silk, biocompatibility, and the delivery of predetermined dose of adenosine. Ongoing studies have shown that this new multiparticulate drug delivery system is suitable for achieving new implant delivery system with low risk of dose-dumping, capable of being modulated to exhibit varying release patterns, reproducible, easily applicable and well-tolerable⁵¹. Nerve growth factor (NGF)-loaded silk fibroin nerve conduits have been studied to guide the sprouting of axons and to physically protect the axonal cone for peripheral nerve repair. NGF release from the differently prepared silk fibroin-nerve conduits was prolonged over 3 weeks, while the total amount of NGF released depended on the drying procedures used in the preparation of the nerve conduits, such as air drying or freeze drying^{52, 53, 54}.

Silk fibroin scaffolds containing insulin-like growth factor I (IGF-I) were prepared for controlled IGF-I release in the context of cartilage repair. Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) was observed, starting after 2 weeks and more strongly after 3 weeks. Tropical tasar silkworm Antheraeamylitta silk-based 3D matrices were also evaluated for in vitro drug release and for the study of cell surface interactions. The silk-based matrices contained two different model compounds, bovine serum albumin (66 kDa) and FITC-inulin (3.9 kDa), to characterize release profiles. Silk fibroin protein blended calcium alginate beads resulted in prolonged drug release without initial burst for 35 days as compared to calcium alginate beads without silk fibroin. Additionally, silk-based micro molded matrices supported a significant enhancement in cell attachment, spreading, mitochondrial activity and proliferation with feline fibroblasts in comparison to polystyrene plates as controls. The introduction and delivery of DNA, proteins, or drug molecules into living cells is important to therapeutics. Nanotubes exhibit advanced physical properties promising for various biological applications, including new molecular transporters⁵⁵.

These studies indicate the potential use of slow degrading silk fibroin 3D scaffolds and tubes loaded with bioactive molecules such as BMP, HRP, and adenosine for drug-releasing biomaterials⁵⁶.

Films:

Silk films have been used with covalent decoration of functional peptides as implants for bone formation and drug delivery. For bone regeneration, BMP-2, RGD, and parathyroid hormone (PTH) can be directly immobilized on silk films using carbodiimide chemistry. Differentiation of human bone marrow-derived stem cells cultured with the decorated silk films was induced by immobilized BMP-2. Also, the utility of silk films to promote long-term adenosine release from adenosine kinase deficient embryonic stem cells has been investigated. These studies demonstrated that silk fibroin constitutes a suitable material for the directed differentiation of embryonic stem cells and for cell-mediated therapeutic release of adenosine. Silk films decorated with bioactive molecules could therefore be used for local drug delivery via direct implantation^{57, 58}. Silk films have also been used to promote stabilization of entrained molecules such as enzymes or therapeutic proteins. Glucose oxidase, lipase, and HRP were entrapped in silk films over 10 months and significant activity if the enzymes were retained, even when stored at 37°C. Silk films result in stabilization of enzymes without the need for cryoprotectants, emulsifiers, covalent immobilization or other techniques. Further, the stabilization of enzymes in silk films is amenable to environmental distribution without refrigeration, and offers potential use in vivo such as the delivery of bioactive molecules⁵⁹.

Nanofibers:

Scaffolds for tissue engineering may mimic the structure and biological function of the extracellular matrix. The natural extracellular matrix is a composite material with fibrous collagens embedded in proteoglycans. The collagen fibers are organized in a 3D porous network that form hierarchical structures from nanometer length scale multi-fibrils to macroscopic tissue architectures. The structures generated by electrospinning contain nanoscale fibers with microscale interconnected pores, resembling the topographic features of the extracellular matrix. Therefore, silk fibroin fiber scaffolds formed by electrospinning have potential as scaffolds. Silk fibroin fiber scaffolds containing BMP-2 and/or nanoparticles of hydroxyapatite (HAP) prepared via electrospinning have been studied for in vitro bone formation from hMSCs. The bioactivity of BMP-2 was retained after the aqueous-based electrospinning process, and the nanofibrous electrospun scaffolds with co-processed BMP-2 supported high calcium deposition and enhanced transcript levels of bone-specific markers, indicating that the electrospun scaffolds were an efficient delivery system for HAP nanoparticles and BMP- 2⁶⁰.

Nanoparticles:

Drug delivery systems via silkworm silk-based nanoparticles have been investigated. Biologically derived silk fibroin-based nanoparticles (100 nm) for local and sustained therapeutic curcumin delivery to cancer cells were fabricated by blending with noncovalent interactions to encapsulate curcumin in various proportions with pure silk fibroin or silk fibroin with chitosan. The concept of enhanced permeability and retention (EPR) in the solid tumor and the micro environment of the tumor (physiological drug resistance) play a vital role to the enhancement of nanoparticles uptake⁶¹. Silk nanoparticles from silk fibroin solutions of domesticated Bombyxmori and tropical tasar silkworm Antheraeamylitta were stable, spherical, negatively charged, 150-170 nm in average diameter and showed no toxicity. The silk nanoparticles were observed in the cytosol of murine squamous cell carcinoma cells, and the growth factor release from the nanoparticles showed significantly sustained release over 3 weeks, implying potential application as a growth factor delivery system. The silk-based nanoparticles containing curcumin showed a higher efficiency against breast cancer cells and have potential to treat in vivo breast tumors by local, sustained, and long-term therapeutic delivery. Silk sericin-poloxamer nanoparticles loaded with both hydrophilic and hydrophobic drugs were reported to be stable in aqueous solution, small size (100-110 nm) and rapidly taken up by cells^62,
63.

Microspheres:

Silk fibroin microspheres were processed using spray-drying, however, the sizes of the microspheres were above 100 μm, which is suboptimal for drug delivery. Other methods to prepare silk microspheres include lipid vesicles as a template to efficiently load bioactive molecules for local controlled releases was reported recently. The lipid is subsequently removed by methanol or NaCl, resulting in silk microspheres consisting beta-sheet structure and approximately 2 μm in diameter³⁴. The silk microspheres loaded with HRP, used as a model drug, demonstrated controlled and sustained release of active enzyme over 10-15 days. Growth factor delivery via the silk microspheres in alginate gels was also reported to be more efficient in delivering BMP-2 than insulin-like growth factors, probably due to the sustained release of the growth factor. Additionally, growth factors successfully formed linear concentration gradients in scaffolds to control osteogenic and chondrogenic differentiation of hMSCs during culture. This silk microsphere/polymeric scaffold system is an option for the delivery of multiple growth factors with spatial control for in vitro and in vivo 3D cultures. In more recent studies, a new mode to generate micro- and nanoparticles from silk, based on blending with polyvinyl alcohol was reported. This method simplifies the overall process compared with lipid templating and provides high yield and good control over the feature sizes, from 300 nm to 20 μm, depending on the ratio of polyvinyl alcohol/silk used⁶⁴.

Coatings:

There is a critical need in medicine to develop simple and versatile methods to assemble robust, biocompatible, and functional biomaterial coatings that direct cell outcomes. Coatings of silk fibroin have been studied to provide interfaces for biomaterials^{65, 66}. The driving force of self-assembly to form coatings is hydrophobic and some electrostatic interactions. The flexibility of silk-based coatings has been investigated using an aqueous stepwise deposition process with B. mori silk solution, which can control the structure and stability of the silk fibroin in layer-by-layer films. The thickness of one layer was reported to be around 10 nm when deposited from a 1 mg/mL silk aqueous solution. The secondary structure of silk fibroin in the coatings was regulated to control the biodegradation rate, which indicates that release of drugs from these coatings can be controlled via layer thickness, numbers of layers and secondary structure of the layers. The silk coatings have also been formed on poly (lactide-co-glycolic acid) (PLGA) and alginate microspheres for protein delivery. The silk coatings on PLGA microspheres was reported to be ~1 μm and discontinuous, while those on alginate microspheres was ~10 μm thick and continuous. These coatings provide mechanically stable shells as well as a diffusion barrier to the encapsulated protein drugs. Nanolayer coatings of silk fibroin to contain model compounds of small molecule drugs and therapeutically relevant proteins, such as rhodamine B and azoalbumin, have been prepared using the stepwise deposition method. Multilayered silk-based coatings have been developed and used as drug carriers and delivery systems to evaluate vascular cell responses to heparin, paclitaxel, and clopidogrel. Cell attachment and viability with human aortic endothelial cells and human coronary artery smooth muscle cells on the drug incorporated silk coatings demonstrated that paclitaxel and clopidogrel inhibited smooth muscle cell proliferation and retarded endothelial cell proliferation^{67, 68}. The silk multilayers with heparin promoted human aortic endothelial cell proliferation while inhibiting human coronary artery smooth muscle cell proliferation, which was a desired outcome for the prevention of restenosis. Solid adenosine powder coated with silk fibroin was investigated for local and sustained delivery of the anticonvulsant adenosine from encapsulated reservoirs. Reservoir coating thickness was varied through manipulation of the silk coating solution concentration and the number of coatings applied. An increase in either coating thickness or crystallinity delayed adenosine burst, decreased average daily release rate, and increased the duration of release⁶⁹.

Miscellaneous Applications of Silk fibroin:

There are various applications of silk fibroin as mentioned below which are not limited to:

· Silk fibers as smart materials towards medical textiles: The recent study reveals that Tetracycline hydrochloride drug can be used to dye Silk fabric by the exhaust process⁷⁰.

· Silk protein based hydrogels: The multidisciplinary advances in hydrogel technologies have spurred development in management of wound healing and tissue engineering⁷¹.

· Silk fibroin for flexible electronic devices: Advances in silk-based electronic devices would open new avenues for employing biomaterials in the design and integration of high-performance biointegrated electronics for future applications in consumer electronics, computing technologies, and biomedical diagnosis, as well as human–machine interfaces⁷².

· Silk fibroin for regenerative dentistry/medicine: It regenerates the tissues by stimulating irreparable organs to heal themselves. Regenerative medicine has the potential to solve the problem of shortage of organs by enabling the scientists to grow tissues and organs in laboratory⁷³.

CONCLUSION:

Owing to its biocompatibility, SF is experiencing increased popularity for applications as implants, films, nanofibers, nanoparticles, microspheres, coatings as well as various controlled release applications and its aqueous process ability allows the fabrication of highly tuneable morphologies under mild conditions. The simplicity of adjusting release profiles by tools such as varying its concentration, applying multiple coatings or changing the β-sheet content, makes SF especially attractive. Moreover, simple surface modifications may affect drug binding and release, and even allow targeted drug delivery.

Despite the excellent properties of SF that make it an attractive biomaterial for controlled delivery, a number of challenges remain. As a natural product the properties of silk may vary between both species and individuals of the same species. Moreover, inconsistencies in the degumming process may render the quality control of SF delivery systems and predictions for their release kinetics difficult. Genetically engineered SF proteins may overcome such deficiencies.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 14.02.2017 Modified on 19.03.2017

Res. J. Pharm. Dosage Form. & Tech. 2017; 9(3): 85-92.

DOI: 10.5958/0975-4377.2017.00015.5