Silk fibroin nanoparticle as a novel drug delivery system

    Silk fibroin nanoparticle as a novel drug delivery system Fatemeh Mottaghitalab, Mehdi Farokhi, Mohammad Ali Shokrgozar, Fatemeh Atyabi, Hossein Hosseinkhani PII: DOI: Reference:

S0168-3659(15)00186-8 doi: 10.1016/j.jconrel.2015.03.020 COREL 7605

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

11 January 2015 17 March 2015 18 March 2015

Please cite this article as: Fatemeh Mottaghitalab, Mehdi Farokhi, Mohammad Ali Shokrgozar, Fatemeh Atyabi, Hossein Hosseinkhani, Silk fibroin nanoparticle as a novel drug delivery system, Journal of Controlled Release (2015), doi: 10.1016/j.jconrel.2015.03.020

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ACCEPTED MANUSCRIPT Silk fibroin nanoparticle as a novel drug delivery system

Nanotechnology Research Center, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14174, Iran 4

Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology, Taipei 10607, Taiwan

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Center of Excellence in Nanomedicine, National Taiwan University of Science and Technology, Taipei 10607, Taiwan

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National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran

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Fatemeh Mottaghitalab1, Mehdi Farokhi2,*, Mohammad Ali Shokrgozar2, Fatemeh Atyabi3, Hossein Hosseinkhani4,5

*Corresponding author: Dr. [email protected]

Mehdi

Farokhi,

1

Tel/Fax:

+982166492595,

E-mail:

ACCEPTED MANUSCRIPT Abstract

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The design and synthesis of efficient drug delivery systems are of vital importance for medicine and healthcare. Nanocarrier-based drug delivery systems, in particular nanoparticles, have generated great excitement in the field of drug delivery since they provide new opportunities to overcome the limitations of conventional delivery methods with regards to the drugs. Silk fibroin (SF) is a naturally occurring protein polymer with several unique properties that make it a suitable material for incorporation into a variety of drug delivery vehicles capable of delivering a range of therapeutic agents. SF matrices have been shown to successfully deliver anticancer drugs, small molecules, and biomolecules. This review will provide an in-depth discussion of the development of SF nanoparticle-based drug delivery systems.

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Keywords: Drug delivery, Silk fibroin, Nanoparticles.

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ACCEPTED MANUSCRIPT Contents 1. Introduction

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2. Drug delivery systems 2.1 Drug delivery systems based on nanotechnology vs. conventional carriers

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2.2 Important parameters for nanoparticle-based drug delivery

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2.3 Polymeric nanosystems for drug delivery 3. Characteristics of silk protein

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3.1 Silkworms 3.2 Molecular properties of silk protein

3.4 Biodegradation rate of silk protein

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3.3 Crystallinity of silk protein

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3.5 Particulate silk protein preparation techniques

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3.6 Potential characteristics of silk fibroin for drug delivery applications 3.7 Silk fibroin nanoparticles vs. other silk fibroin based carriers

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4. Silk protein as a drug carrier

4.1 Silk fibroin nanoparticulate for protein delivery 4.2 Silk fibroin nanoparticulate for small molecules delivery 4.3 Silk fibroin nanoparticulate for anticancer delivery 5. Conclusions References

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ACCEPTED MANUSCRIPT 1. Introduction

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In recent years, in order to optimize the efficacy of therapeutics, many drug delivery systems have been designed to administer multiple drugs and release them in a controlled manner [1-4]. The use of these systems offers many advantages such as enhancing the bioavailability of drugs by reducing their degradation rate, improving cellular uptake, allowing targeting and control of drug release, and reducing side effects [5]. To date, both synthetic and natural polymers have been used for drug delivery applications. Among wide range of applied synthetic polymers, polyesters, polyorthoesters, polyanhydrides, polyphosphazenes, and polyphosphoesters have found extensive application [6-8]. However, despite the wide range of available materials, the majority of licensed drug delivery systems are based on the FDA (U.S. Food and Drug Administration)- approved polymer, poly(lactic-co-glycolic acid) (PLGA), because of properties such as suitable pharmacokinetics and controllable degradation rate [9, 10]. However, the usefulness of PLGA is limited in applications such as protein therapeutics due to some of its intrinsic properties and processing requirements [11-14]. Therefore, natural polymers (e.g., alginates, chitosan, collagen, dextran, pullulan and gelatin) represent an attractive alternative with higher biocompatibility and biodegradability than PLGA [15-24]. In addition to their composition, the structure of drug delivery systems also needs serious consideration. To date, many systems have been designed with different morphologies and structures, including films, gels, foams, microparticles, and nanoparticles [25]. In the 1960s, liposomal carriers were the first nano-systems to be approved for the delivery of proteins and drugs [26]. Additionally, many studies have reported the high capacity of nanoparticles for therapeutic molecules [27-29]. In most cases, the use of particulate carriers reduces the rate of delivery of solubilized drugs by introducing a second limiting step [30, 31]. Furthermore, nanoparticles have many features, which are useful for drug delivery such as a high surface to volume ratio [32], an ability to act as modifiable platforms [33], and a tunable size [34]. Therefore, applying the principals of nanotechnology to the design of drug delivery systems will not only improve their therapeutic efficacy but could also preserve the properties of bioactive molecules [35]. It is necessary to consider the properties of biomaterials in terms of composition, structure, mechanical properties, and function to fabricate particulate drug delivery carriers. Silk proteins are FDA-approved polymers that have been used successfully as both sutures and drug delivery systems. These proteins have excellent mechanical properties, a flexible preparation process, and high biocompatibility [25]. So far, many review papers have been published concerning the use of silk proteins in the field of tissue engineering. While there are many original articles about the application of silk nanoparticles as drug delivery vehicles, to the best of our knowledge, no comprehensive review on their use for drug delivery has yet been published. Therefore, in this review, we provide an extensive overview on recent efforts in constructing silk protein nanostructures for drug delivery. Firstly, we introduce the different applications of nanotechnology-based systems. Secondly, the properties of silk proteins are discussed clearly, and finally, nanoparticulate silk proteins are considered in detail. 2. Drug delivery systems 2.1 Drug delivery systems based on nanotechnology vs. conventional carriers Historically, drug delivery systems were usually based on orally administered or injectable drugs. However, these have been found to be inappropriate for novel therapeutics such as 4

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proteins and nucleic acids. Novel technologies are required for delivery of new drug molecules in order to reduce their side effects, optimize their efficacy, and enhance patient compliance. Recently, the use of nanotechnology has led to the development of many novel carriers capable of controlled release and targeted delivery of a wide range of small molecules, proteins, peptides, and genes [36-41]. These devices can have many different structures, including liposomes, micelles, quantum dots, dendrimers, fullerenes, ferritin, and nanoparticles [42-45]. Among them, nanoparticles based on biodegradable and biocompatible polymers have potential applications in cancer therapy and as sustained drug delivery vehicles. These carriers can also be designed as low toxicity systems with suitable physical and chemical structures and specific targeting properties [46]. It was reported that particle size is the most important factor when designing drug delivery systems. Therefore, it is crucial to use nanoparticles for the delivery and targeting therapeutic molecules [47, 48]. These systems also have other advantages, including prolonged drug half-life, improved solubility of hydrophobic drugs, reduced immunogenicity, and reduced administration frequency [49]. As mentioned earlier, the possibility of targeted drug delivery is one of the main advantages of nanoparticles. It is strongly believed that the conjugation of different ligands to nanoparticles could improve the targeting efficacy of them as compared to conventional therapeutics [50]. The small size of nanoparticles also affects the targeting efficacy. Generally, nanosized particles experience efficient uptake, selective drug accumulation in the targeted site, and are able to penetrate into the endothelium at inflammatory sites, epithelium (e.g., intestinal tract and liver), tumors, or microcapillaries [51, 52]. The ability to co-deliver multiple drugs is another advantage of nano-based drug delivery systems in comparison to conventional systems [53]. Co-delivery of drugs offers several benefits such as the possibility of synergistic effects [54], suppressed drug resistance [55] and the ability to adjust the dosage of drugs to the level of a single nanoparticle carrier.

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2.2 Important parameters for nanoparticle-based drug delivery Understanding the interactions between nanomaterials and cells/lipid bilayers is important in the fields of phototherapy, imaging, and drug/gene delivery. The reason for this is the small size of nanoparticles as compared to microparticles. In order to address this issue, Desai et al. have shown that the cellular uptake of 100-nm nanoparticles was 2.5 and six times higher than 1-µm and 10-µm microparticles, respectively [51]. It was also reported in a similar study that the cellular uptake of 100-nm nanoparticles was 15–250 times higher than 1- and 10-µm microparticles [56]. Chithrani et al. have claimed that efficient uptake of nanoparticles depends on their size. To this end, they have shown that 50-nm gold particles undergo the most effective uptake [57]. They have also stated that spherical nanoparticles experience five times greater uptake than rod-shaped particles. Therefore, it appears that size and shape are the two important factors that affect the cellular uptake of particles [57]. It is also known that, in addition to influencing the cellular uptake, particle size can also influence drug loading, drug release, and the stability of nanoparticles [58]. Along with size and shape, the nanoparticle’s surface properties are also important. Surface characteristics such as hydrophobicity and hydrophilicity determine the level of absorbance of blood components such as opsonins [59, 60]. However, it has been shown in in vitro studies that there is a connection between the extent of opsonization and the surface charge of nanoparticles and that less opsonization occurs in neutrally charged particles in comparison to charged particles [61]. For this reason, the use of shielding groups that is capable of blocking the electrostatic and hydrophobic interactions result in the binding of

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opsonin to the surface of nanoparticles. A brief description of the effects of particle size and the surface properties of nanoparticles is summarized in diagram 1.

Diagram 1. Properties that define the behavior of silk nanoparticles (SFNPs).

2.3 Polymeric nanosystems for drug delivery Polymeric nanoparticles have been of great interest to many researchers for decades. Since the discovery of dendrimeric ―starburst‖ polymers in the 1980s, numerous other drug delivery strategies such as self-assembled micelles and encapsulated drug molecules have been developed [62, 63]. Biodegradable polymeric nanocarriers are predominantly used for their ability to control the release rate of drugs, and for this purpose, many synthetic and natural polymers have been used [36]. Synthetic polymers have greater structural integrity and higher purity, which makes the preparation of nanoparticles more reproducible, than natural polymers [36]. However, only synthetic polymers with an appropriate biodegradability and low cytotoxicity are suitable for drug delivery applications [64]. Additionally, the ability of synthetic polymers to control the release rate of drugs is much higher than natural polymers. Synthetic polymers are capable of controlling the release rate of drugs over a longer duration than natural polymers, which have relatively short durations of release. However, the harsher reaction conditions and use of organic solvents, which are necessary to prepare synthetic polymers, have limited their application. Poly(lactic acid) (PLA), poly(glycolic acid) (PGA), PLGA, and poly(alkylcyano acrylates) (PCA) are the most promising synthetic polymers for the development of drug delivery platforms for clinical applications [65] which are listed in Table 1. 6

ACCEPTED MANUSCRIPT Table 1. Potential synthetic nanoparticles for drug delivery applications. Processing method

Loaded Molecule

size (nm)

Key findings

Ref.

PLA1

Emulsificationsolvent evaporation

Aureusidine

231-376

Improving aureusidin's water solubility and light sensitivity

[66]

Effective cytotoxic T-lymphocyte responses and tumor antigen-specific cytotoxicity

[67]

Significant uptake of nanoparticles in the estrogen receptor positive MCF-7 cell line

[68]

[69]

150- 200

Highly negative zeta potential and good biocompatibility of nanoparticles

675

Prolonged BSA release to 30 h

[70]

Enhanced encapsulation efficacy, high uptake and retention by macrophages, increased drug efficacy against M. tuberculosis residing in macrophages

[71]

Involvement of different pathways depending on the surface properties of nanoparticle in the interaction between nanoparticles and the blood–brain barrier

[72]

Long-circulating properties of nanoparticles, reduced liver accumulation

[73]

86

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Double emulsion

PBCA7

PBCA8

Poly (hexadecylcy anoacrylate)

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250 – 300

Precipitation and dialysis method

Ovalbumin

Water-in-oil emulsion technology plus cyclic freezingthawing process

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PVA5

Tamoxifen

BSA6

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g-PGA4

Solvent displacement

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PCL3

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PLGA2

Anti-OX40 monoclonal antibody

Anionic polymerisation

Moxifloxacin

Anionic polymerisation

Doxorubicin

Nanoprecipitation/

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418

202-246

150

solvent diffusion

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Material

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Poly(lactic acid), 2Poly(DL-lactide-co-glycolide), 3Poly(ε-caprolactone), 4Poly(g-glutamicacid), 5Poly(vinyl alcohol),6Bovine serum albumin, 7Poly(butyl cyanoacrylate), 8Poly(butyl cyanoacrylate)

In contrast, natural polymers have variable purity, lower stability, and require further modification and crosslinking prior to use [74]. Many studies have been fabricated nanoparticles based on natural polymer which are listed in Table 2.

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ACCEPTED MANUSCRIPT Table 2. Potential natural nanoparticles for drug delivery applications. Processing method

Loaded molecule

Size (nm)

HSA1

Desolvation

Cetuximab

200-250

BSA2

Emulsificationdispersion

Tacrolimus

189

Collagen

Alkaline hydrolysis

17β-estradiolhemihydrate

Gelatin

Desolvation

Paclitaxel

Protamine

Self-assembly

CpGoligodeoxynucle otide

Gliadin

Desolvation

Targeting colon carcinoma cells

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Proteins

Key findings

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Material

Ref.

[75]

[76]

Prolonged estradiol release and absorption

[77]

600-1000

Rapid drug release, targeting human bladder cancer cells

[78]

215

Suppress T-helper 2 immune response, suitable for immunotherapy of allergy

[79]

Al-trans-retinoic acid

500

Good drug loading efficiency, biphasic releasing pattern

[80]

Coacervation

Folic acid

150

Folic acid encapsulation, release prevention in an acid environment, enhanced oral bioavailability

[81]

pH-coacervation

Methylene blue

250

Increased release with decrease in glutaraldehyde/lysine ratio

[82]

Thermoresponsive self-assembly

BMP3-2 and BMP-14

237

High encapsulation efficacy of BMPs with sustained release pattern, maintenance of growth factors activity

[83]

Chitosan

Ionic gelation

Cyclosporin A

293

High corneal-conjunctival intact, enhanced external ocular delivery compared to inner ocular

[84]

Alginate

Water-in-oil microemulsion

GFP-encoding plasmids

55-100

Suitable for gene therapy

[85]

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Enhanced accumulation in blood rather than other organs e.g. kidney

Casein

Legumin

Elastin

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Polysaccharides

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Methotrexate

390

Sustained drug release, enhanced drug delivery

[86]

Starch

Reactive extrusion

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160-300

160 nm starch particles preparation by appropriate crosslinkers addition

[87]

Dextran

Nanoprecipitation

lidocaine

86–256

High drug encapsulation efficacy

[88]

Pullulan

Dialysis

Adriamycin

156

High drug loading, pHsensitive in vitro drug release, enhanced cellular uptake

[89]

Hyaluronic acid

Self-assembly

-

Active targeting by strong receptor-binding affinity of nanoparticles to CD44

[90]

237–424

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Pectin

Human serum albumin, 2 Bovine serum albumin, 3Bone morphogenetic protein

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3. Characteristics of silk protein

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Successful polymer-based delivery systems need to be biocompatible, biodegradable, have low toxicity, appropriate mechanical properties, require ambient processing conditions, and provide sustained release. Silk is a natural polymeric biomaterial that can address these requirements because of its unique structural properties, self-assembling ability, mechanical strength, processing flexibility, biodegradability, and biocompatibility [91]. 3.1 Silkworms

Wide varieties of silkworms produce natural silk worldwide. In general, the silk proteins produced by different silkworms differ in their structure and properties, and not all of the species are commercially viable. For instance, the Bombycoidea family contains eight subspecies, and only two members of this family – Bombycidae (Mulberry) and Saturniidae (non-mulberry) – are commercially important [92]. Bombyx mori is the commercial source of mulberry silk that is produced by the Bombycidae family. Mulberry silkworms are entirely domesticated and need human care for their growth and reproduction, which does not occur naturally [92]. The nonmulberry/mulberry silkworm classification originates from the feeding habits of silk producing insects, which belong to the Saturniidae and Lasiocampidae families [93]. Non-mulberry silkworms are comprised of the following species: the tropical (Antheraea mylitta) and temperate (A. pernyi, A. roylei, A. proylei, and A. frithi) tasar silkworms, eri silkworms (Philosamia ricini/Samia ricini), muga silkworms (A. assamensis), fagaria silkworms (Attacus atlas), and shashe silkworms (Gonometa postica) [94]. These species live principally in the wild and have been found in polymorphic forms in a variety of host plants in different geographical regions. For this reason, the silk produced by the species listed above varies in luster, color, and tensile strength [95]. 3.2 Molecular properties of silk protein

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The silk fibroin (SF) fibers from B mori fibers have a diameter of about 10-25 mm with three structural protein subunits, including a light chain (~26 kDa), a heavy chain (~390 kDa), and a small glycoprotein named P25 (of ca. 30 kDa). A disulfide bond links the light and heavy chains together, and P25 is attached to the fiber via non-covalent hydrophobic bond [95-97]. The heavy chain of SF has an amphiphilic nature and contains both hydrophobic and hydrophilic blocks. The hydrophobic blocks have a repeating sequence of Gly-Ala-Gly-Ala-Gly-Ser, which is responsible for generating the crystalline structure of SF by folding into β-sheets. However, the hydrophilic region is a short and non-repetitive segment in comparison to the hydrophobic region [96, 98]. Fibroin could be therefore be considered a hydrophobic glycoprotein; hence, it is insoluble in water [99]. Another component of B. mori SF fibers is sericin (20 kDa to 310 kDa). Sericin contains two subunits α-sericin and β-sericin, which are found in the external and inner layers of the cocoon, respectively. Sericin [98] can be isolated as a hydrophilic protein from fibroin via a thermochemical procedure called degumming [100]. Sericin has an amorphous and glue-like structure that is responsible for binding two fibroin fibers together, and thus it maintains the structural integrity of the cocoons [101].

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As mentioned previously, SF is a semi-crystalline biopolymer and consists of crystalline and amorphous regions. The crystalline part has two prominent structures, i.e., silk I and silk II [102]. Unstable and water-soluble state silk I is obtained from spinning dope [103, 104]. Silk I can be made more stable after transforming it to silk II during the spinning process. The crystalline structure of B. mori fibers consists principally of silk II structure as a result of its β-sheet conformation, while the amorphous part of fibroin is in random coil conformation [105-107]. It should be mentioned that the β-sheet has an asymmetrical structure featuring hydrogen side chains from glycine on one side and methyl side chains from alanine on the other, creating hydrophobic domains. Strong hydrogen bonds and van der Waals forces between the methyl and hydrogen groups on opposing sides allow inter-sheet stacking in the crystals and thus, generates a thermodynamically stable structure [96]. Most of the characteristic physical and chemical properties of SF such as high strength and resistance to chemicals and micro-organisms and low elasticity and extensibility are derived from its crystalline structure [108]. There are many methods to enrich SF with β-sheet structure, and they are listed in Table 3.

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ACCEPTED MANUSCRIPT Table 3. Methods capable of enriching SF in β-sheet structure.

50% (v/v) methanol solution

Silk concentration

Treatment time

20–25% (w/v)

β-sheet induction

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Casting

Treatment

2 days

Yes

Laminar jet break-up

Water vapor

3% and 9%

Film

Casting

High temperatures

1–2%

24 hour

Yes

Not reported

Yes

Capillary shape solidified sample

Membrane

Freeze drying

Freezing temperature

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Sphere

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Film

Method

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Structure

Solidification

Casting

Shear force

Ion concentration

2% (v/v)

24 hour

39 wt.%

Not reported

3% (w/v)

Not reported

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To somewhat

Yes

Yes

FTIR or Raman finding Amide I shift to 1665 cm1, appearance of new maxima at 1262, 1236 cm1 (amide III), and 1084 cm1 Shifts of 1658 cm-1 (amide I) and at 1540 cm-1 (amide II) bands to 1629 cm-1 (amide I) and 1517 cm-1 (amide II) Not reported

Ref.

[109]

[110]

[111]

Appearance of absorption band at 1265 cm-1 (amide III) by increasing the freezing temperature -60 to -10°C

[112]

Appearance of three major characteristic bands of silk II at 1085, 1232 and 1667 cm−1

[113]

Shift of amide I band from 1657 to 1667 cm-1 after increasing the ion concentration

[114]

ACCEPTED MANUSCRIPT 3.4 Biodegradation of silk protein

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In order to develop a successful formulation of nanoparticle based drug carriers, it is necessary to consider their biodegradation rate. Many factors affect the release rate of nanoparticles, including desorption of surface-bond/adsorbed drug molecules, diffusion through the nanoparticle matrix, diffusion through the polymer wall (in the case of nanocapsules), erosion of nanoparticle matrix, and a combined erosion/diffusion process [115]. Generally, in contrast to natural polymers, synthetic polymers such as PLA, PGA, and PLGA produce acidic compounds as by-products of hydrolytic degradation, and this is potentially undesirable at the targeted sites [116]. The sensitivity of natural polymers such as collagen, fibrinogen, hyaluronic acid to enzymatic degradation is sometimes higher than the synthetic polymers [117]. As a natural polymer, SF usually undergoes proteolytic degradation resulting in non-toxic by-products [26]. However, US Pharmacopeia has classified SF as non-degradable material because it maintains 50% of its tensile integrity 60 days post-implantation [95]. However, we have recently reported that immersing freeze-dried SF scaffold in phosphate buffered saline (PBS) solution can induce hydrolysis degradation. In our study, the structural, physical, and mechanical properties of the SF scaffold were different 12 weeks after incubation in PBS (Figure 1). Surprisingly, hydrolysisinduced changes to the surface of the scaffold affected the behavior of osteoblast cells in terms of biocompatibility, alkaline phosphatase production, and even the expression of some bone gene markers [118].

Figure 1. Scanning electron micrographs of: (a) Fresh SF scaffold, (b) Treated SF scaffold in PBS after 12 weeks. The interconnection between pores is treated and larger pores with unusual structure are formed as a function of exposure time. Wenk et al. have also reported that in the absence of proteolytic degradation, only 4% of SF scaffold weight is degraded within 7 weeks by hydrolysis [119]. In contrast to synthetic polymers like PLGA, SF usually undergoes surface erosion and degradation by-products diffuse into the active site. This phenomenon results in a reduction of drug stability, drug activity, and safety [120]. Moreover, Wang et al. have reported an SF porous scaffold that is not only biodegradable but also bioresorbable due to degradation by macrophages [121]. In general, the possibility of enzymatic, surface-mediated biodegradation and controlled biodegradation of SF could be considered an important factor in its role as a drug delivery agent. An overview of various enzymatic methods used for SF degradation is shown in Table 4. Generally, Biodegradable nanoparticles as well as silk polymer have shown great interest for imaging, cancer treatment, medical tools, bone treatment, drug delivery, diagnostic tests, and drug

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ACCEPTED MANUSCRIPT development [122-124]. These particles have the potential to enable early detection, prevention, and to essentially improve diagnosis, treatment and follow-up of diseases [125-129]. Table 4. An overview of various enzymatic methods used for SF degradation

Recombinant honeybee silk protein (AmelF3)

Nanofiber

In vitro

Microtube

Intervals (1, 3, 8, 24, 48 or 96 h)

Bright field microscopy, SDS-PAGE spectra

Protease XIV (2 U/ml)

56 days

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In vitro

Technical characterization

In vitro

Protease XIV (5.3 units/mg)

Mass loss evaluation, SEM, histology, cell metabolism study

10 days

Mass loss evaluation

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Bombyx mori

3D porous scaffold

Incubation time

MA

Bombyx mori

Enzyme model Alphachymotrypsin (54 U mg–1) and trypsin (313 U mg–1)

PT

In vivo/ In vitro

RI

Silk structure

SC

Type of silk

Key findings

Ref.

Cleavage of protein by both proteases

[130]

High degradation rate of scaffold affected osteogenesis and cell metabolism Production of more brittle silk microtubes, after incubation and loss of mechanical integrity Higher degradation rate during 9 days, slower degradation until 18 days, morphology changes after in vivo degradation

[131]

[132]

[133]

Conduit

In vitro and in vivo

Protease XIV (1.0 U/ml)

10 days for in vitro and 24 weeks for in vivo

UV spectroscopy, gross observation, SEM analysis, mass loss evaluation, SDSPAGE spectra, histology

Bombyx mori

Porous SF sheets

In vitro

Alphachymotrypsin collagenase IA and protease XIV (1.0 U/ml)

1, 3, 6, 9, 12, and 15 days

Mass loss evaluation, XRD, SEM, Molecularweight distribution

Degradation of sheets by collagenase IA and protease XIV in contrast to Alphachymotrypsin

[134]

Bombyx mori

SF mats

In vitro and in vivo

Protease XIV (1 U/mL)

Within 24 days for in vitro and 8-weeks

Mass loss evaluation, SEM, FTIR, XRD, histology

Degradation of 65% of the electrospun SF scaffolds within 24 day

[135]

Bombyx mori

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In vivo

-

Within one-year

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Histology, Histochemistry

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Porous SF scaffold

SC

Bombyx mori

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for in vivo

in protease XIV, complete degradation after 8 weeks in vivo Complete degradation of prepared scaffolds in aqueous medium between 2 and 6 months, beyond 1 year degradation rate for scaffolds prepared in organic solvents

[121]

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3.5 Particulate silk protein preparation techniques

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In recent years, many micro and nanoparticulate systems have been used for biomedical and pharmaceutical applications. These systems are suitable as drug carriers as they have the ability to control the release rate of drugs and increase the likelihood of positive therapeutic outcomes [136]. When designing particles for drug delivery applications, it is important to consider their biocompatibility, biodegradability, size, drug loading and release [137]. However, selecting the appropriate types of biomaterials and finding the correct processing methods needed to prepare particles is challenging. For instance, it is necessary to avoid organic solvents, surfactants, initiators, or cross-linking agents during particle preparation [138-140]. Silk as a flexible polymer is commonly prepared in a powder form by using a chemical process; this is known as the bottom-up approach [141]. During preparation via this method, the intermolecular forces between β-sheets within the silk proteins are broken down by dissolving in different solvents (e.g. chaotropic salts, ionic liquids, and fluorinated solvents) leading to the generation of silk particles [142-149]. Consequently, in order to make the silk particles water insoluble, the particles are usually treated with kosmotropic salts like potassium phosphate or alcohols (usually methanol) to induce the formation of β-sheets [150]. Another method to prepare silk particles is the top-down approach. For this method, silk fibers are cut with different milling machines to mechanically prepare fine silk particles in the powder form [151]. The key drawback of using chemical procedures is the denaturation of the silk protein structure. In addition, the removal of chemical agents from silk particles is a lengthy process [151]. This drawback makes the use of mechanical methods more appealing as it avoids the limitations inherent in the chemical approaches and allows the direct preparation of silk particles. However, this method is limited by the viscoelastic nature of silk proteins, which need a milling time of up to 40 h to produce fine particles [152]. A brief description of some important research activities concerning silk powder preparation are summarized in Table 5.

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ACCEPTED MANUSCRIPT Table 5. A brief description of important research activities concerning silk powder preparation. Loaded molecule

Key findings

Ref.

[146]

Formic acid

< 80 nm

Spherical nanopowder

FTIR1, XRD2, SEM3

200 nm

Spherical powder

Laser particle analyzer and SEM

500 nm2 µm

Spherical powder

Potassium phosphate

Rotary and planetary ball milling

Water-in-oil emulsificationdiffusion

Self-assembly

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Ethanol and PVA

980 nm

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Self-assembly

Decreasing the average size of SF particle

-

Narrow size distribution of SF particles at pH 10

Alcian blue, rhodamine B, and crystal violet

Negative surface charge, higher loading capacity of charged small molecules with controlled release

[153]

UV-Vis spectrometry, FTIR, SEM, light microscopy, DLS4

Rhodamine B, dextran and BSA

Appropriate drug loading capacity, controlled release of hydrophobic drugs, higher uptake efficiency

[154]

UV–Vis spectrometry, SEM, light microscopy, particle size analysis

-

RI

Potassium hydrogen phthalate and hydrochloric acid

PT

Characteristic method

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Phase separation

Particle type

SC

Bead milling

Particle size

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Electro-spraying

Chemical solution

MA

Method

Spherical powder

[152]

-

200 nm

Fibrous particle

Laser particle analyzer and SEM

-

Ethyl acetate, Diethyl ether, DCM5, Chloroform

48- 148 µm

Bowl-like and spherical

FTIR and SEM

-

Ethanol

0.2 to 1.5 µm

[151]

Spherical

Photoncorrelation spectroscopy, particle size analysis, SEM, AFM6, FTIR

Water-in-oil 15

-

Fine particles production

Relation of shape and size of SF microparticles to the type of organic phase

[155]

Particle size and size distribution dependence on SF microspheres to the amount of ethanol, SF concentration, freezing, and temperature

[156]

High BSA absorption

[157]

ACCEPTED MANUSCRIPT <80-150 >

Spherical

µm

BSA8

FTIR, SEM, Particle size analysis

-

101-440 μm

Spherical

FTIR, SEM, Particle size analysis

Phase separation

PVA9

300 nm20 μm

Spherical

FTIR, SEM, DSC10, DLS

BSA, rhodamine B

efficiency, lower size of SF microsphere Bioactivity preservation of growth factor, sustained release over 7 weeks Controlling the size and distribution of SF sphere by varying the concentrations of SF and PVA

[110]

[158]

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Laminar jet break-up

Salicylic acid, propranolol hydrochloride , IGF-I7

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Paraffin

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emulsion solvent evaporation

Fourier transform infrared spectroscopy, 2X-ray diffraction, 3Scanning electron microscopy, 4Dynamic light scattering, 5Dichloromethane, 6Atomic force microscopy, 7Insulin-like growth factor, 8Bovine serum albumin, 9 Polyvinyl alcohol, 10Differential scanning calorimetry.

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3.6 Potential characteristics of silk fibroin for drug delivery applications

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SF has many unique properties that established its reputation among other synthetic and natural polymers for controlled drug delivery. For this, the investigation of SF as a drug carrier has widely expanded over the last few years due to highly controllable composition and sequence, structure and architecture, mechanical properties and function. One of the main advantages of using SF as a carrier is performing mild all-aqueous processes for loading sensitive drugs such as protein and nucleic acid therapeutics in order to provide good resistance to dissolution, thermal and enzymatic degradation [159, 160]. This can be achieved by conformational transition of αhelix and random coil to highly crystalline β-sheets through water vapor annealing, mechanical stretching and ultrasonic treatments. This avoids the use of any harsh processing conditions which make silk as a potential system for drug delivery applications [161]. Additionally, SF protein consists of a diverse range of amino acids with functional groups including amines, alcohols, phenols, carboxyl groups, and thiols that simplify the attachment of different biomolecules or antibodies for specific cell types. It is suggested that different drugs with different kinetics could be introduced to SF biomaterial by varying the degree of functionalization per SF molecule that could provide a variety of drug release systems [25]. This is the significant advantage of using SF compared with many other relatively inert polymeric systems [25, 162]. Moreover, it is possible to modify the properties of SF by genetic manipulation. These novel structures are consisting of silk sequence that self-assemble into the desired morphological structures and the sequence of a polypeptide that endow novel functionalities. The functional domains can provide binding sites for receptors, enzymes, drugs, metals or sugars, among others [163]. Another main feature of silk among other polymers is its potential as a lysosomotropic drug delivery platform. A wide range of synthetic and natural polymers have been used as lysosomotropic delivery systems. However, silk as a natural polymer has an intrinsic capability to response to pH changes in order to initiate drug release. Therefore, drug release is achieved in response to pH without any chemical modifications [164]. When using biomaterials for drug delivery applications, it is necessary to consider their clearance mechanism from the body. Absorption is the main mechanism for clearing of some biomaterials. Additionally, by-products of biomaterials could be cleared by liver, kidneys or lungs [165]. As 16

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3.7 Silk fibroin nanoparticles vs. other SF based carriers

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silk can be degraded by such proteolytic enzymes, absorption is the most likely mechanism of elimination from the body without any side effects. The binding mechanism of biomolecules to silk is another important feature for controlling drug release kinetics. It is assumed that electrostatic interaction is the main possible mechanism for release and loading drug on SF biomaterial [153]. The negatively charged silk particles (−24 to −26 mV) provide opportunities for strong electrostatic force with hydrophobic and positively charged drugs compared with hydrophilic or negatively charged drugs. These strong interactions electrostatic could avoid the significant burst release as is normally seen with many polymeric carrier particles [158, 166].

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To date, different structures of SF including micro and nanospheres, hydrogels, micro- and nanoparticles have been explored for drug delivery applications. Generally, SF microspheres with mucoadhesive properties are more relevant for long-acting delivery of depot drug. They are able to adhere to the mucous membrane of oral and nasal routes and release encapsulated drug [167]. The common drug release mechanism of microspheres is diffusion of drug molecules from degraded polymeric matrix. Silk microspheres can be processed using different methods but some of these approaches (e.g. spray-drying method) apply harsh processing conditions and high temperature. In addition, the microspheres generated by this mode of processing are large, above 100 μm, which is suboptimal for drug delivery [148]. Another useful structure of SF for drug delivery applications is SF nanosphere that is usually used as short-acting delivery carriers. The other advantage of using SF nanospheres as a drug carrier is that they could be used either by fluidizing with a liquid carrier or as a solid powder [168, 169]. Drug release mechanism from nanosphers is identical to microspheres as described above. In some cases, the small size of nanospheres is not suitable for targeted drug delivery such as pulmonary drug delivery. Therefore, these spheres are incorporated to larger spheres using flocculation, spray drying and etc. to have a desired size in order target a specific disease site [170]. Rather than size limitation of nanospheres for drug delivery applications, the shape of this structure has also a significant impact on degradation of polymeric matrix and drug release [171]. Therefore, it seems that fabricating SF nanopsheres is more challenging for drug delivery. The main challenges of fabricating SF nanospheres are high molecular weight and protein nature of SF. Moreover, when expose to heat, salt, pH change and high shear, the SF tends to self-assemble into fibers or gels [158, 172]. Moreover, in both nanosphere and microsphere structures, uncontrollable size and shape and using harsh preparation conditions such as organic solvent are challenging for using these structures in drug delivery applications. Therefore, researchers have developed a new SF based structure applicable as drug carriers. For example, hydrogels are attracted more attention in the field of drug delivery. Generally, many factors including hydrogel hydration, crosslinking, pore size, degradability, hydrophobicity, charge and polymer concentration affect the release rate of different drug molecules from hydrogel. It is confirmed that the β-sheet content is increased after SF gelation. So, there is no need to post-treat the SF hydrogels with solvents in order to induce water insolubility. SF hydrogels are simply prepared by lowering the pH of a SF solution (with pI of about 4.2) in the presence of the drug with acidic solution to pH 4 in order to induce gelation. Although, this pH may be suitable for some drugs, but this may have harmful effects on others. Therefore, it is considered that despite of favorable characteristics of SF hydrogel for drug delivery, applying this structure is related to the type of incorporated drug. Moreover, the low mechanical properties are another limitation of using hydrogels in biomedical applications [173-176]. Microparticulate SF carriers have also considerable potential to be used as 17

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therapeutics delivery platform [25, 177]. Using SF microparticles have many advantages in comparison to other mentioned structures including preservation of drug from degradation and denaturation, controlling the release rate of drugs and the high potential for targeted drug delivery [153]. Despite of many potential properties of using microparticles as drug carriers, the harsh preparation processes including non-aqueous solvents, water/solvent interfaces, crosslinking reagents for hardening, or high temperatures have limited their applications [178, 179]. In this regards, it is necessary to optimize formulation protocols in order to preserve the stability and potency of drugs. Despite of potential advantages of above SF based delivery systems, their limitation tend to investigate SF nanoparticles based delivery systems. As mentioned above, the application of SF nanoparticles has expanded due to advantages such as biocompatibility, controlled degradation, size and shape as well as drug loading and release. The small size of SF nanoparticles allows them to penetrate through small capillaries which enhance the cellular uptake of an encapsulated drug or therapeutic molecule [165]. Moreover, SF nanoparticles have high potential for targeted drug delivery as they could get into cells and deliver anticancer to the tumor site. Highly efficient clearance systems are also applied for unused nanoparticles or their degraded products from the body. Therefore, SF nanoparticles are suggested as potential delivery platform in comparison to other conventional systems [165, 166]. 4. Silk protein as a drug carrier

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4.1 Silk fibroin nanoparticulate for protein delivery

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In the context of protein-based drug delivery systems, silk is a potentially useful natural polymer, which has been used to deliver peptide and protein molecules (Table 6). Unfortunately, few studies have discussed the role of silk nanostructure in protein delivery systems. Hofer et al. investigated the use of recombinant spider silk protein eADF4 (C16) particles for carrying the high molecular weight protein, lysozyme. With an efficiency of almost 100% lysozyme (positively charged) was loaded onto negatively charged eADF4 (C16) particles (size: 521 ± 8.3 nm) taking advantage of the electrostatic interaction between the negatively charged and positively charged groups. They also reported that substantial quantities of lysozyme diffused into the matrix of these particles rather than simply absorbing onto the surface. The release of lysozyme from eADF4 (C16) particles was related to the pH and ionic strength of the medium used. The maximum release of lysozyme was observed after 24 h in medium with acidic pH, and no significant release was observed in medium with a neutral pH even after 28 days [180]. It was reported that SF and biological molecules could interact via electrostatic interaction, hydrophobic attraction and Coulomb forces [25, 181, 182]. However, there is still less knowledge about the exact mechanism of these interactions and needs additional investigations. In this regards, Germershaus et al. have investigated the mechanism of SF interaction with polylysine and protamine in cosmotropic or chaotropic environment. The positive net charge of these biomolecules was responsible for inducing electrostatic interaction with negatively charged SF protein. The ionic strength and the type of deployed affected the interaction between SF nanoparticle and two basic model proteins. Increasing ionic strength using sodium chloride has decreased the zeta potential of SF and its electrostatic interactions but the impact on micelle stability was minimal. In contrast, chaotropic environments were lead to micelle destabilization due to hydrophobic collapse of SF and efficient coacervates formation. The opposite was observed for cosmotropic conditions (micelle stability and abolished coacervate formation)

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(Illustration 1). As a result, the interaction of SF with basic model proteins can be optimized by rationale salt selection [183].

Illustration 1. The effect of chaotropic and cosmotropic salts on SF micelle stability.

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The loading efficiency and release patterns of hydrophobic and protein drugs from SF particles were evaluated by Shi et al [154]. Self-assembled SF particles with average size of 980 nm were prepared in order to evaluate the drug loading capacity. The actual loading of fluorescein isothiocyanate-labeled bovine serum albumin (FITC–BSA) was 0.27% with 80.06% encapsulation efficiency, while these for rhodamine B (RhB) were 0.153% and 45.87%, respectively. The high encapsulation efficiency of drug was related to protein structure and high molecular weight of FITC–BSA than RhB. The low molecular weight of RhB (479 Da) in comparison to FITC–BSA (66,000) was lead to easy diffuse out from SF particles and thus less encapsulation efficiency. Additionally, this study showed that about 23% FITC-BSA and 34% RhB were released from the silk particles in 50 days [154]. It was well known that the half-life of an enzyme could be increased by immobilizing it on a polymeric substrate by using covalent conjugation or physical attachment [184-186]. There are many reports of the immobilization of enzymes on woven silk and diazotized silk fiber [187]. Besides these substrates, there are only a few investigations concerning the use of silk powder for enzyme immobilization. For instance, it was reported in 1981 that both neutral and alkaline proteases could be immobilized in silk powder at their isoelectric point in pH 5.0–5.5 [143, 188]. In addition, some researchers have described that covalently binding of insulin, L-asparaginase (ASNase), and β-glucosidase to SF nanoparticles enhances their biological stability and activity in vitro [189-191]. Zhang et al. have shown that rapid introduction of SF solution containing ASNase into excess acetone could not inactivate the enzyme and was able to embed and immobilize the enzyme in simultaneously formed SF nanoparticles. The crystalline globular SF nanoparticle–ASNase bioconjugates had 50–120 nm diameter with 90% enzyme activity recovery. The enzyme-entrapped SF nanoparticles produce high temperature resistance in dry conditions, trypsin digestion resistance, higher stability in serum and greater storage stability in solution. Although, the exact formation mechanism, and the reason of using acetone as a preferable agent to prepare SF nanoparticles or SF nanoparticles–ASNases with good bioactivity, is unknown, but the authors stated that this 19

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phenomena might be attributed to the cleaved micelles aggregated by these degraded polypeptide chains of the regenerated SF and the moderate polarity and hydrophilicity of acetone [192]. Another important application of protein delivery is in tissue engineering. Many growth factors have been incorporated in different scaffolds to enhance tissue regeneration. In addition, there has been extensive research on improving vascularization during the development and repair of tissue [193, 194]. Many strategies can be used for this purpose such as enhancing the pore size and interconnectivity of scaffolds [195], promoting in vitro prevascularization by using cocultures of endothelial cells or mesenchymal stem cells [196, 197], and incorporating angiogenic factors, including vascular endothelial growth factor (VEGF), transforming growth factor (TGF), platelet derived growth factor (PDGF), and fibroblast growth factor (FGF) [198]. Recently, SF nanoparticles with an average size of 150–170 nm have been used to control the release rate of VEGF in a sustained manner. In this study, it was shown that the SF nanoparticles that had accumulated in the cytoplasm of the cells were nontoxic and that normal cell cycle distribution was maintained [166]. We have recently fabricated a bio-hybrid SF/calcium phosphate/PLGA nanocomposite scaffold for controlling the release profile of VEGF in order to enhance bone regeneration. In this study, we first prepared a porous scaffold based on SF and calcium phosphate by using freeze-drying method. After that, VEGF/SF was electrospun onto the surface of the freeze-dried scaffold. PLGA nanofibers were electrospun onto the VEGF loaded SF/calcium phosphate scaffold in order to control the release profile of VEGF (Figure 2). The release profile of VEGF that we obtained over 28 days established the efficacy of this scaffold as a sustained delivery system. The bioactivity of the released VEGF was measured to be about 83% [199].

Figure 2. Schematic illustration of scaffold fabrication processes. (a) SF solution containing CaP powder, (b) freeze-drying of SF/CaP solution, (c) loading VEGF/SF nanofibers on porous SF/CaP substrate using electrospinning, (d) electrospinning of PLGA on VEGF loaded SF/CaP scaffold. Despite the potential of VEGF as an angiogenic factor, it produces immature blood vessels with high permeability [3, 200]. Thus, the incorporation of mature angiogenic factor such as PDGF is required [200]. It seems that the delivery of VEGF and PDGF within scaffolds could mimic the process of natural tissue regeneration. Therefore, in another study, we designed a nanocomposite scaffold based on silk/calcium phosphate/PLGA for the sustained release of PDGF and VEGF, concomitantly [201]. PDGF showed a slower release rate than VEGF because it was loaded deeper in the scaffold and was embedded within two layers of PLGA. In a rabbit model, neovascularization occurred 10 weeks after the implantation of the scaffold (Figure 3). These studies have shown that the electrospinning of SF with different types of angiogenic factors such as PDGF and VEGF could be an effective nanosystem for drug delivery and tissue regeneration applications [201]. 20

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Figure 3. Histological examination of rabbit bones without scaffolds (a and b), SF/CaP scaffold (c and d) and angiogenic loaded scaffold (e and f). Preexisted bone (*), newly formed bone (B), fibrous tissue (F), newly formed capillaries (arrows) and the materials (----). Bars: 50µm (a, c and e) and 30 µm (b, d and f). Magnification: 400× (a, c and e), 500× (b, d and f). Table 6. SF protein as matrix for protein delivery in different tissue constructs.

NGF1

IGF-I2

FGF-23 BMP5

IgG16 monoclonal antibody

Application

Type of delivery system

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Types of peptide or protein

Preparation method

Release time

Axon

Nerve conduit

Freeze drying

More than 3 weeks

Cartilage

Porous scaffold

Freeze drying

For 25 days

Not reported

Film

Bone

Disk shaped scaffold

-

Hydrogels and lyogel

Key findings

Ref.

Maintenance of fully bioactive NGF after release, induction of PC12 cell differentiation and neurite outgrowth

[202]

[203]

Diazonium coupling reaction

-

Sonication

For 6 days

Not reported

For 80 days

Chondrogenesis induction Reduced metabolic activities of hMSC4, increased levels of pERK1/2 Promotion of neoosteogenesis after 8 week implantation Antibody release dependence on hydrophobic interactions, hydration resistance, and ionic repulsions

[204]

[205]

[181]

[206]

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EGF7

Brain

Film

Adenosine-releasing scaffold with neuroprotective, anti-ictogenic and anti-epileptogenic properties

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Adenosine

For 2 Weeks

[207]

Silk films, Casting and Not lamellar electrospinning reported porous silk films and electrospun silk nanofibers 1 Nerve growth factor, 2Insulin-like growth factor I, 3Fibroblast growth factor 2, 4Human mesenchymal stem cell, 5Bone morphogenetic protein, 6Immunoglobulin G, 7Epidermal growth factor.

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Skin

Increasing wound healing rate, re-epithelialization, dermis proliferation

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4.2 Silk fibroin nanoparticulate for small molecules delivery

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Nowadays, the use of nanoparticles to deliver small molecules is rapidly growing in many fields [208-211]. Although small molecules have been used as chemotherapeutic agent for many years, there are several disadvantages to their use, including hydrophobicity, nonspecific targeting and biodistribution, and drug resistance shortly after initial treatment. The unique properties of nanoparticles could overcome the limitations of using small molecules as therapeutic agents in biomedical applications [212]. The first polymeric carrier used to deliver small molecules was PGA [213]. To date, there are two PGA based carriers approved for clinical trials: Xyotax (PGA–paclitaxel) [214] and CT-2106 (PGA–camptothecin) [215, 216]. As a natural polymer, silk attracts extensive attention as a possible method of delivering bioactive small molecules [110, 217-219]. Silk is used as a vehicle to deliver small molecules in various forms, including films, 3D porous scaffolds, microspheres, microcapsules, transdermal microneedles, nanospheres, and hydrogels [10]. Different small molecules such as alcian blue, rhodamine B, and crystal violet could be incorporated to silk particles as reported by Lammel et al.[153]. Silk particles produced with 1.25 M potassium phosphate has shown dominate silk II (crystalline) and silk I (less crystalline) in pH 6 and pH 9, respectively. Additionally, they showed that small molecules had 95% loading efficiency because of a charge-charge interaction with silk particles (The negative surface charge of silk particles with positively charged small molecules). They also have found that the release of these three molecules is highly related to their charge and the structure of silk. For instance, the structure of silk II and the pH of solution could induce the burst release of molecules at all-time intervals [153]. It is necessary in many diseases to administer two or more drugs concomitantly. For this purpose, dual drug delivery systems have been introduced in recent years to control the release rate of all the incorporated drugs and achieve optimal therapeutic efficacy [2, 3, 220]. However, no significant successes in clinical trials have been reported regarding the design of suitable carriers with the ability to control the release rate of each molecule independently with all the drugs embedded in the same polymeric matrix [221]. Hydrogels are a versatile polymeric network that could be used for the delivery of cells, drugs, antibodies, proteins, peptides, and genes because their structure changes depending on salt concentration, pH, and temperature [222-224]. Kaplan et al. showed that silk hydrogels could be prepared by initiating the gelation of silk solutions by changing pH or applying ultrasonication and vortexing [158, 225, 226]. Similarly, Naumata et al. have fabricated a dual 22

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delivery system based on silk nanoparticles incorporated into silk hydrogels consisting of silkmicrofibril networks. Silk nanoparticles had approximately 175 ± 3 nm diameter. The atomic force microscopy (AFM) has shown that silk nanoparticles were aggregated prior to ethanol treatment while no aggregation was observed after ethanol treatment and homogeneous size distribution. It was reported that silk nanoparticles had encapsulation efficiency of approximately 35% for Texas Red (TR) as well as that of around 55% for RhB and FITC. They have also claimed that the release rate of dyes from silk nanoparticles was related to the presence of protease XIV, which is known to degrade the β-sheet structures of silk proteins, because no significant release of RhB was observed from silk nanoparticles without using protease. None of the dyes exhibited burst release, indicating that they were not attached at the surface but rather had been incorporated into the silk particles. However, after enzymatic treatment, significant amounts of RhB and TR were released from the silk nanoparticles through enzymatic degradation especially after 9 h. RhB and TR have shown similar release pattern as a result of similar hydrophobicity and size. On the contrary, more hydrophobic nature of FITC was responsible for slower release behavior in 24 h due to stronger hydrophobic interactions between the silk molecules and FITC. The authors claimed that the release rate of molecules from their system was highly dependent on physical properties such as β-sheet content, the size of the silk nanoparticles and the network size of the silk hydrogels [225]. In addition to hydrogels, micro and nanoparticulate systems also have unique properties, which make them a great candidate for biomedical applications [136, 227]. Generally, the fabrication of silk nanospheres is a more challenging area of research than the fabrication of silk microspheres because of the high molecular weight and protein nature of silk [110, 148, 228]. However, it was reported that by using 70% (v/v) water-miscible protonic and polar aprotonic organic solvents, it is possible to fabricate silk nanospheres in a size range of 300–400 nm [172]. Silk micro and nanospheres could also be prepared with controlled size and shape by using poly(vinyl alcohol) (PVA) as the continuous phase. Silk and PVA were phase separated at a weight ratio of 1:1 and 1:4, respectively. It was observed that using 1/4 ratio sample could generate more homogenous spheres with size distribution ranging from 300 nm to 20 µm. The concentration of silk and PVA played an important role in generating spheres of different sizes and shapes (Figure 4). The loading efficiency of RhB, tetramethylrhodamine conjugated bovine serum albumin (TMR-BSA) and tetramethylrhodamine conjugated dextran (TMR-Dextran) were 95%, 51% and 1.2% respectively. It is suggested that strong binding of RhB to silk is due to hydrophobic and electrostatic attraction that lead to high loading efficiency and a slow release rate. The release rate of TMR-BSA and RhB were less than 5% of total loading during 2 weeks. However, total loading for dextran was 60% with faster release profile within 2 weeks. Since the molecular weight of RhB is much lower than that of TMR-BSA and TMR-dextran, the results confirmed that the interaction between silk and encapsulated drug is controlled by drug release rather than diffusion [158].

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Figure 4. SEM images of silk spheres with size controlled by varying silk and PVA concentration. Silk/PVA weight ratio was 1/4. The samples of 5 and 1 wt% silk and PVA concentration were dominated by silk microspheres with a size range of 1–30 µm (A,B,D,E) whereas the samples of 0.2 wt% was dominated by nanospheres with a size lower than 400 nm (C,F). Upper panel shows low magnification images, scale bar; 10 µm in A, B to show multiple microspheres in general; 1 µm in C to show multiple nanospheres. Lower panel show high magnification images, scale bar; 2 µm in D,E; 200 nm in F to show detailed structure of nanospheres 4.3 Silk fibroin nanoparticulate for anticancer delivery Despite the investment of billions of dollars into the investigation of the mechanisms of tumorigenesis, more than 10 million people are exposed to different cancers, annually. Cancer is the leading cause of death worldwide, and its rate of incidence is increasing every year. It is estimated that 15 million people will suffer from cancer until 2020 [229-232]. Many strategies have been developed for cancer treatment: immunotherapy, thermal therapy, phototherapy [233, 234], surgery, gene therapy, chemotherapy, and radiotherapy. Each of these methods has its own advantages and disadvantages [235]. Many researchers are keen to develop new strategies for improved cancer therapy with fewer side effects and improved tumor-targeting ability [236]. Recently, nanotechnology has offered a new avenue for cancer treatment by providing unique nanoparticles based on synthetic and natural polymers for drug delivery applications. Some of these vehicles are in the pre-clinical or clinical phases of development [237-239]. Silk has many unique properties that make it suitable for preparing drug delivery vehicles, including fibers, films, 3D scaffold, and gels [25]. For example, it was reported that by using a capillary-microdot technique, SF nanoparticles could be produced with an average size (<100 nm) appropriate for delivering curcumin as a therapeutic agent for cancer. The greatest entrapment, intracellular uptake, and controlled release of curcumin was observed by encapsulating them in SF nanoparticles and administering them to breast cancer cells [240]. Genetically engineered silk24

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elastin-like protein polymers (SELPs) were also used in order to prepare nanoparticles for hydrophobic anticancer delivery such as doxorubicin. Three different ratios of silk to elastin block (1:8, 1:4, and 1:2) were considered as SE8Y, S2E8Y, S4E8Y, respectively. The drug loading efficiency of SE8Y, S2E8Y and S4E8Y were 6.5%, 6% and 4%, with the average hydrodynamic radii (Rh) of doxorubicin-loaded nanoparticles about 50 ± 10, 72 ± 11, and 142 ± 10 nm, respectively. While, the average Rh of SE8Y before adding doxorubicin was only 5.2 ± 1.8 nm that might be related to free chains. This observation exhibited the role of doxorubicin in promoting the self-assembly of SE8Y into nanoparticles. In addition, the average Rh of drugloaded SE8Y nanoparticles under physiological conditions was increased from 142 to 181 nm with considerable increase of polydispersity as a result of the possible interaction between nanoparticles and serum proteins. The authors have also reported 1.8-fold higher cytotoxicity of doxorubicin-loaded SE8Y nanoparticles compared with free drug [241]. Recently, Seib and colleagues have described that the release of doxorubicin from silk nanoparticles is pHdependent. They have prepared spherical SF nanoparticles with 98 nm size with desolvation method using acetone. They exhibited that at concentrations of up to 0.04 μg doxorubicin/μg of SF, more than 95% of the drug could be absorbed by SF. SF nanoparticles have also showed pHdependent release (pH 4.5 >> 6.0 > 7.4) (Illustration 2). Two important features of SF nanoparticles including pH-dependent drug release and lysosomal accumulation of SF nanoparticles have confirmed the ability of drug-loaded SF nanoparticles to serve as a lysosomotropic anticancer nanomedicine [164].

Illustration 2. Schematic representation of silk pH dependent release of doxorubicin from a silk nanoparticle. Consistent with this study, another publication revealed that a greater release of doxorubicin was observed at pH 4.5 in comparison to pH 7.4 from folate conjugated SF nanoparticles. It was 25

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suggested that the greater release of doxorubicin at pH 4.5 was due to many factors, including weak binding between drug and the carboxylic group of silk, reprotonation of the amino groups of doxorubicin, and higher solubility of the drug at this pH [242]. SF nanoparticles are also useful for other anticancers delivery. For example, paclitaxel-encapsulated SF nanoparticles were used for locoregional gastric cancer therapy. The drug loading and encapsulation efficiency were 10 ± 2% and 52 ± 2%, respectively. Paclitaxel had a burst release of 40.9 ± 2.7% during the first 8 h followed by steady and sustained release about 47.2 ± 1.5% for 100 h. The authors have claimed that this release behavior could be related to locating paclitaxel at the hydrophobic/hydrophilic interface inside of the nanoparticles that resulted in initial paclitaxel diffusion from the nanoparticles. Another reason was that the release speed was depended on the drug payload and drug density in the nanoparticles, which was higher paclitaxel loading leading to faster release. Additionally, SF nanoparticles could be taken up by human gastric cancer cells and paclitaxel released from paclitaxel-SF-nanoparticles kept its pharmacological activity [243]. SF nanospheres have also been used for paclitaxel delivery. The maximum loading of paclitaxel in 270- to 520-nm nanospheres was about 6.9%, which was released over 9 days as reported by Chen and colleagues. The authors described that the drug loading, encapsulation efficiency, and release rate of paclitaxel-loaded SF nanospheres was highly dependent on SF concentration and the initial paclitaxel-loading capacity [244]. It is beyond doubt that combining SF with other natural and synthetic polymers could improve its structural, mechanical, and degradation properties along with its drug release profile, drug retention, and encapsulation efficiency [245, 246]. For example, the synthesis of self-aggregating SF-albumin nanoparticles for delivering methotrexate as an antitumor drug was evaluated by Subia and co-workers. Drug loading and encapsulation efficiency were 15–24% and 83–87%., respectively. It was observed that bulk SF and SF-albumin blends (1:1, 2:1) have higher drug loading and encapsulation efficiency than any other blended particles (1:2 and bulk albumin). These observations may be related to hydrophobic nature of SF protein and the greater electrostatic interaction between SF and albumin. Moreover, 35% of drug was released from nanoparticles during the first 48 h and this was increased with time. The drug release from SF-albumin (2:1) was 82%, while 70% of the total drug was released from SF-albumin (1:1). This suggested the higher drug loading ability and controlled release rate of the particles mainly due to surface localization. Increasing the SF content in nanoparticles causes faster diffusion of entrapped drug into the polymer matrix, which may be due to the hydrophobicity of SF protein [247]. Finally, more intensive examinations of the potential role of SF in drug delivery, and its ability to mediate sustained release may lead to further applications of SF-based drug delivery vehicles. 5. Conclusions Many efforts have been made to improve the therapeutic efficacy of biomolecules for various biomedical applications. Nanotechnology has led to the development of nanoparticle-based drug delivery vehicles in the ―nanometer‖ size range in order to overcome the side effects associated with chemotherapeutics. Silk-based nanoparticles have been developed to deliver proteins, small molecules, and anticancer drugs as described in this review. SF has many unique characteristics, including appropriate mechanical properties, versatile processability in an aqueous environment, biocompatibility, and a controlled degradation rate that make it an excellent candidate for drug delivery applications. For this reason, SF nanoparticles have been successfully designed and are able to control the release rate of biomolecules in a sustained manner with high stability. Overall, 26

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Acknowledgments

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This study was funded by the National Science Council of Taiwan through research grant NSC 1032221-E-011-032.

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