Silk fibroin nanoparticle as a novel drug delivery system

Fatemeh Mottaghitalab, Mehdi Farokhi, Mohammad Ali Shokrgozar,

Fatemeh Atyabi, Hossein Hosseinkhani

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

To appear in: Journal of Controlled Release

Received date: 11 January 2015

Revised date: 17 March 2015
Accepted date: 18 March 2015

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

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
 ACCEPTED MANUSCRIPT

Silk fibroin nanoparticle as a novel drug delivery system

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

PT
1
Nanotechnology Research Center, School of Pharmacy, Tehran University of Medical Sciences,
Tehran, Iran

RI
2
National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran

SC
3
Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of
Medical Sciences, Tehran 14174, Iran

NU
4
Graduate Institute of Biomedical Engineering, National Taiwan University of Science and
Technology, Taipei 10607, Taiwan
5
Center of Excellence in Nanomedicine, National Taiwan University of Science and Technology,
MA
Taipei 10607, Taiwan
D
P TE
CE
AC

*Corresponding author: Dr. Mehdi Farokhi, Tel/Fax: +982166492595, E-mail:

mehdifarkhi83@gmail.com

1
 ACCEPTED MANUSCRIPT

Abstract
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

PT
(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

RI
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.

SC
Keywords: Drug delivery, Silk fibroin, Nanoparticles.

NU
MA
D
P TE
CE
AC

2
 ACCEPTED MANUSCRIPT

Contents
1. Introduction
2. Drug delivery systems

PT
2.1 Drug delivery systems based on nanotechnology vs. conventional carriers
2.2 Important parameters for nanoparticle-based drug delivery

RI
2.3 Polymeric nanosystems for drug delivery

SC
3. Characteristics of silk protein
3.1 Silkworms

NU
3.2 Molecular properties of silk protein
3.3 Crystallinity of silk protein MA
3.4 Biodegradation rate of silk protein
3.5 Particulate silk protein preparation techniques
D

3.6 Potential characteristics of silk fibroin for drug delivery applications

TE

3.7 Silk fibroin nanoparticles vs. other silk fibroin based carriers

4. Silk protein as a drug carrier

P

4.1 Silk fibroin nanoparticulate for protein delivery

CE

4.2 Silk fibroin nanoparticulate for small molecules delivery

4.3 Silk fibroin nanoparticulate for anticancer delivery
AC

5. Conclusions
References

3
 ACCEPTED MANUSCRIPT

1. Introduction
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

PT
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,

RI
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

SC
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

NU
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
MA
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
D

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
TE

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
P

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
CE

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
AC

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
 ACCEPTED MANUSCRIPT

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,

PT
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

RI
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

SC
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

NU
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.
MA
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
D

conventional systems [53]. Co-delivery of drugs offers several benefits such as the possibility of
TE

synergistic effects [54], suppressed drug resistance [55] and the ability to adjust the dosage of
drugs to the level of a single nanoparticle carrier.

2.2 Important parameters for nanoparticle-based drug delivery

P
CE

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
AC

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

5
 ACCEPTED MANUSCRIPT

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.

PT
RI
SC
NU
MA
D
P TE

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

CE

2.3 Polymeric nanosystems for drug delivery

AC

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.

Material Processing method Loaded Molecule size (nm) Key findings Ref.

PLA1 Emulsification- Aureusidine 231-376 Improving aureusidin's water [66]

PT
solvent evaporation solubility and light sensitivity

Anti-OX40 Effective cytotoxic [67]

RI
monoclonal T-lymphocyte responses and
PLGA2 Double emulsion antibody 86 tumor antigen-specific
cytotoxicity

SC
Significant uptake of [68]
nanoparticles in the estrogen

NU
PCL3 Solvent Tamoxifen 250 – 300 receptor positive MCF-7 cell
displacement line

Highly negative zeta potential [69]

MA
and good biocompatibility of
g-PGA4 Precipitation and Ovalbumin 150- 200 nanoparticles
dialysis method

Water-in-oil
D

emulsion technology
PVA5 plus cyclic freezing- BSA6 675 Prolonged BSA release to 30 h [70]
TE

thawing process

Enhanced encapsulation [71]

P

efficacy, high uptake and

PBCA7 Anionic Moxifloxacin 418 retention by macrophages,
CE

polymerisation increased drug efficacy against

M. tuberculosis residing in
macrophages
AC

Involvement of different [72]

pathways depending on the
PBCA8 Anionic Doxorubicin 202-246 surface properties of
polymerisation nanoparticle in the interaction
between nanoparticles and the
blood–brain barrier

Poly Nanoprecipitation/ - 150 Long-circulating properties of [73]

(hexadecylcy nanoparticles, reduced liver
anoacrylate) solvent diffusion accumulation
1
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.

7
 ACCEPTED MANUSCRIPT

Table 2. Potential natural nanoparticles for drug delivery applications.

Material Processing method Loaded Size (nm) Key findings Ref.
molecule

Proteins

PT
HSA1 Desolvation Cetuximab 200-250 Targeting colon carcinoma [75]
cells

RI
BSA2 Emulsification- Tacrolimus 189 Enhanced accumulation in [76]
dispersion blood rather than other

SC
organs e.g. kidney

Collagen Alkaline hydrolysis 17β-estradiol- 123 Prolonged estradiol release [77]

NU
hemihydrate and absorption

Gelatin Desolvation Paclitaxel 600-1000 Rapid drug release, targeting [78]

human bladder cancer cells
MA
Protamine Self-assembly CpG- 215 Suppress T-helper 2 immune [79]
oligodeoxynucle response, suitable for
otide immunotherapy of allergy
D

Gliadin Desolvation Al-trans-retinoic 500 Good drug loading [80]

TE

acid efficiency, biphasic releasing

pattern
P

Casein Coacervation Folic acid 150 Folic acid encapsulation, [81]

release prevention in an acid
CE

environment, enhanced oral

bioavailability
AC

Legumin pH-coacervation Methylene blue 250 Increased release with [82]

decrease in
glutaraldehyde/lysine ratio

Elastin Thermoresponsive BMP3-2 and 237 High encapsulation efficacy [83]

self-assembly BMP-14 of BMPs with sustained
release pattern, maintenance
of growth factors activity

Polysaccharides

Chitosan Ionic gelation Cyclosporin A 293 High corneal-conjunctival [84]

intact, enhanced external
ocular delivery compared to
inner ocular

Alginate Water-in-oil GFP-encoding 55-100 Suitable for gene therapy [85]

microemulsion plasmids

8
 ACCEPTED MANUSCRIPT

Pectin Ionotropic gelation Methotrexate 390 Sustained drug release, [86]

enhanced drug delivery

Starch Reactive extrusion - 160-300 160 nm starch particles [87]

preparation by appropriate
crosslinkers addition

PT
Dextran Nanoprecipitation lidocaine 86–256 High drug encapsulation [88]
efficacy

RI
Pullulan Dialysis Adriamycin 156 High drug loading, pH- [89]
sensitive in vitro drug

SC
release, enhanced cellular
uptake

NU
Hyaluronic acid Self-assembly - 237–424 Active targeting by strong [90]
receptor-binding affinity of
MA nanoparticles to CD44
1
Human serum albumin, 2 Bovine serum albumin, 3Bone morphogenetic protein

3. Characteristics of silk protein

D

Successful polymer-based delivery systems need to be biocompatible, biodegradable, have low

toxicity, appropriate mechanical properties, require ambient processing conditions, and provide
TE

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].
P
CE

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
AC

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 non-
mulberry/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

9
 ACCEPTED MANUSCRIPT

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.

PT
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

RI
[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

SC
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

NU
maintains the structural integrity of the cocoons [101].
3.3 Crystallinity of silk protein
MA
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
D

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
TE

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
P

hydrophobic domains. Strong hydrogen bonds and van der Waals forces between the methyl and
CE

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
AC

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.

10
 ACCEPTED MANUSCRIPT

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

Structure Method Treatment Silk Treatment β-sheet FTIR or Ref.

concentration time induction Raman
finding
Amide I shift [109]

PT
to 1665 cm1,
appearance of
Film Casting 50% (v/v) 20–25% (w/v) 2 days Yes new maxima

RI
methanol at 1262, 1236
solution cm1 (amide
III), and 1084

SC
cm1
Shifts of 1658 [110]
cm-1 (amide I)
and at 1540

NU
Sphere Laminar jet Water vapor 3% and 9% 24 hour Yes cm-1
break-up (amide II)
bands to 1629
cm-1 (amide I)
MA
and 1517 cm-1
(amide II)
Film Casting High 1–2% Not reported Yes Not reported [111]
temperatures
D

Appearance of [112]
absorption
TE

band at 1265
cm-1 (amide
III) by
Particle Freeze drying Freezing 2% (v/v) 24 hour To somewhat increasing the
P

temperature freezing
temperature
CE

-60 to -10°C

Appearance of [113]
AC

Capillary three major

shape characteristic
solidified Solidification Shear force 39 wt.% Not reported Yes bands of silk
sample II at 1085,
1232 and
1667 cm−1

Shift of amide [114]

I band
Ion from 1657 to
Membrane Casting concentration 3% (w/v) Not reported Yes 1667 cm-1
after
increasing the
ion
concentration

11
 ACCEPTED MANUSCRIPT

3.4 Biodegradation of silk protein

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,

PT
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

RI
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

SC
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

NU
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, hydrolysis-
MA
induced 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].
D
P TE
CE
AC

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

12
 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
Type of silk Silk In vivo/ Enzyme Incubation Technical Key findings Ref.

PT
structure In vitro model time characterization
Alpha- Cleavage of [130]
Recombinant Nanofiber In vitro chymotrypsin Intervals Bright field protein by
(54 U mg–1)

RI
honeybee silk (1, 3, 8, 24, microscopy, both
protein and trypsin 48 or 96 h) SDS-PAGE proteases
(AmelF3) (313 U mg–1) spectra

SC
High [131]
Mass loss degradation
Bombyx mori 3D porous In vitro Protease XIV 56 days evaluation, SEM, rate of
scaffold (2 U/ml) histology, cell scaffold

NU
metabolism study affected
osteogenesis
and cell
metabolism
MA
Production of [132]
more brittle
Bombyx mori Microtube In vitro Protease XIV 10 days Mass loss silk
(5.3 units/mg) evaluation microtubes,
D

after
incubation
TE

and loss of
mechanical
integrity
Higher [133]
P

Bombyx mori Conduit In vitro Protease XIV 10 days for UV spectroscopy, degradation
and (1.0 U/ml) in vitro gross observation, rate
CE

in vivo and SEM analysis, during 9

24 weeks mass loss days, slower
for in vivo evaluation, SDS- degradation
AC

PAGE spectra, until 18 days,

histology morphology
changes after
in vivo
degradation

Bombyx mori Porous SF In vitro Alpha- 1, 3, 6, 9, Mass loss Degradation [134]

sheets chymotrypsin 12, and 15 evaluation, XRD, of sheets by
collagenase days SEM, Molecular- collagenase
IA and weight IA and
protease XIV distribution protease XIV
(1.0 U/ml) in contrast to
Alpha-
chymotrypsin

Bombyx mori SF mats In vitro Protease XIV Within 24 Mass loss Degradation [135]
and (1 U/mL) days for evaluation, SEM, of 65% of the
in vivo in vitro FTIR, XRD, electrospun
and histology SF scaffolds
8-weeks within 24 day

13
 ACCEPTED MANUSCRIPT

for in vivo in protease

XIV,
complete
degradation
after 8 weeks
in vivo

PT
Complete [121]
degradation
of prepared
Bombyx mori Porous SF In vivo - Within Histology, scaffolds in

RI
scaffold one-year Histochemistry aqueous
medium
between 2

SC
and 6 months,
beyond 1 year
degradation

NU
rate for
scaffolds
prepared in
organic
MA solvents

3.5 Particulate silk protein preparation techniques

D

In recent years, many micro and nanoparticulate systems have been used for biomedical and
TE

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
P

biocompatibility, biodegradability, size, drug loading and release [137]. However, selecting the
appropriate types of biomaterials and finding the correct processing methods needed to prepare
CE

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
AC

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.

14
 ACCEPTED MANUSCRIPT

Table 5. A brief description of important research activities concerning silk powder preparation.
Method Chemical Particle Particle Characteristic Loaded Key findings Ref.
solution size type method molecule

PT
Decreasing the [146]
Electro-spraying Formic acid < 80 nm Spherical FTIR1, XRD2, - average size of SF
nanopowder SEM3 particle

RI
[151]
Potassium Laser particle Narrow size

SC
Bead milling hydrogen 200 nm Spherical analyzer and - distribution of SF
phthalate powder SEM particles at pH 10
and
hydrochloric

NU
acid

Negative surface [153]

Phase separation Potassium 500 nm- Spherical UV-Vis Alcian blue, charge, higher
MA
phosphate 2 µm powder spectrometry, rhodamine B, loading capacity
FTIR, SEM, and crystal of charged small
light violet molecules with
microscopy, controlled release
DLS4
D

Appropriate drug [154]

UV–Vis Rhodamine loading capacity,
TE

Self-assembly Ethanol and 980 nm Spherical spectrometry, B, dextran controlled release

PVA powder SEM, light and BSA of hydrophobic
microscopy, drugs, higher
P

particle size uptake efficiency

analysis
CE

[152]
Rotary and
planetary ball - 200 nm Fibrous Laser particle - Fine particles
milling particle analyzer and production
AC

SEM

Ethyl acetate, Relation of shape [155]

Water-in-oil Diethyl ether, 48- 148 Bowl-like FTIR and SEM - and size of SF
emulsification- DCM5, µm and microparticles to
diffusion Chloroform spherical the type of
organic phase

Photon- Particle size and [156]

correlation size distribution
Self-assembly Ethanol 0.2 to 1.5 Spherical spectroscopy, - dependence on SF
µm particle size microspheres to
analysis, SEM, the amount of
AFM6, FTIR ethanol, SF
concentration,
freezing, and
temperature

High BSA [157]

Water-in-oil absorption

15
 ACCEPTED MANUSCRIPT

emulsion solvent Paraffin <80-150 > Spherical FTIR, SEM, BSA8 efficiency,
evaporation µm Particle size lower size of SF
analysis microsphere

Bioactivity [110]
Salicylic acid, preservation of

PT
Laminar jet - 101-440 Spherical FTIR, SEM, propranolol growth factor,
break-up μm Particle size hydrochloride sustained release
analysis , IGF-I7 over 7 weeks
Controlling the

RI
size and
Phase separation PVA9 300 nm- Spherical FTIR, SEM, BSA, distribution of SF [158]
20 μm DSC10, DLS rhodamine B sphere by varying

SC
the concentrations
of SF and PVA

NU
1
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.
MA
3.6 Potential characteristics of silk fibroin for drug delivery applications
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
D

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
TE

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 α-
P

helix and random coil to highly crystalline β-sheets through water vapor annealing, mechanical
CE

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,
AC

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
 ACCEPTED MANUSCRIPT

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

PT
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].

RI
3.7 Silk fibroin nanoparticles vs. other SF based carriers

SC
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

NU
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
MA
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
D

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
TE

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
P

etc. to have a desired size in order target a specific disease site [170]. Rather than size limitation
CE

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
AC

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
 ACCEPTED MANUSCRIPT

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, cross-

PT
linking 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

RI
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

SC
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

NU
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].
MA
4. Silk protein as a drug carrier
4.1 Silk fibroin nanoparticulate for protein delivery
D

In the context of protein-based drug delivery systems, silk is a potentially useful natural polymer,
TE

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
P

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
CE

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
AC

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)

18
 ACCEPTED MANUSCRIPT

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

PT
RI
SC
NU
MA
Illustration 1. The effect of chaotropic and cosmotropic salts on SF micelle stability.

The loading efficiency and release patterns of hydrophobic and protein drugs from SF particles
D

were evaluated by Shi et al [154]. Self-assembled SF particles with average size of 980 nm were
TE

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%,
P

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
CE

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
AC

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
 ACCEPTED MANUSCRIPT

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

PT
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 co-
cultures of endothelial cells or mesenchymal stem cells [196, 197], and incorporating angiogenic

RI
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

SC
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

NU
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
MA
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
D

83% [199].
P TE
CE
AC

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
 ACCEPTED MANUSCRIPT

PT
RI
SC
NU
MA
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).
D
TE

Table 6. SF protein as matrix for protein delivery in different tissue constructs.

Types of Application Type of Preparation Release Key findings Ref.
peptide or delivery method time
P

protein system
Maintenance of fully [202]
CE

bioactive NGF after

NGF1 Axon Nerve Freeze drying More than release, induction of
conduit 3 weeks PC12 cell
differentiation and
AC

neurite outgrowth
[203]
IGF-I2 Cartilage Porous Freeze drying For 25 days Chondrogenesis
scaffold induction
Reduced metabolic [204]
Diazonium activities of hMSC4,
FGF-23 Not reported Film coupling reaction For 6 days increased levels of
pERK1/2
Promotion of neo- [205]
BMP5 Bone Disk shaped - Not osteogenesis after 8
scaffold reported week implantation

Antibody release [181]

dependence on
IgG16 hydrophobic
monoclonal - Hydrogels Sonication For 80 days interactions,
antibody and lyogel hydration resistance,
and
ionic repulsions

[206]

21
 ACCEPTED MANUSCRIPT

Adenosine-releasing
- For 2 scaffold with
Adenosine Brain Film Weeks neuroprotective,
anti-ictogenic and
anti-epileptogenic
properties

PT
Increasing wound [207]
healing rate,
EGF7 Skin Silk films, Casting and Not re-epithelialization,

RI
lamellar electrospinning reported dermis proliferation
porous silk
films and

SC
electrospun
silk
nanofibers
1
Nerve growth factor, 2Insulin-like growth factor I, 3Fibroblast growth factor 2, 4Human mesenchymal stem cell, 5Bone

NU
morphogenetic protein, 6Immunoglobulin G, 7Epidermal growth factor.

4.2 Silk fibroin nanoparticulate for small molecules delivery

MA
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
D

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
TE

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,
P

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
CE

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
AC

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
 ACCEPTED MANUSCRIPT

delivery system based on silk nanoparticles incorporated into silk hydrogels consisting of silk-
microfibril 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

PT
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

RI
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

SC
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

NU
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
MA
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
D

molecular weight and protein nature of silk [110, 148, 228]. However, it was reported that by
TE

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
P

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
CE

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)
AC

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].

23
 ACCEPTED MANUSCRIPT

PT
RI
SC
NU
MA
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
D

concentration were dominated by silk microspheres with a size range of 1–30 µm (A,B,D,E)
TE

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
P

magnification images, scale bar; 2 µm in D,E; 200 nm in F to show detailed structure of

nanospheres
CE

4.3 Silk fibroin nanoparticulate for anticancer delivery

AC

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 silk-

24
 ACCEPTED MANUSCRIPT

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 ±

PT
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 drug-

RI
loaded 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

SC
doxorubicin-loaded SE8Y nanoparticles compared with free drug [241]. Recently, Seib and
colleagues have described that the release of doxorubicin from silk nanoparticles is pH-
dependent. They have prepared spherical SF nanoparticles with 98 nm size with desolvation

NU
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 pH-
dependent release (pH 4.5 >> 6.0 > 7.4) (Illustration 2). Two important features of SF
MA
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].
D
P TE
CE
AC

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
 ACCEPTED MANUSCRIPT

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

PT
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

RI
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

SC
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

NU
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
MA
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
D

methotrexate as an antitumor drug was evaluated by Subia and co-workers. Drug loading and
TE

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
P

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
CE

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
AC

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
 ACCEPTED MANUSCRIPT

applications of SF in drug delivery promise further opportunities for the future use of both
natural and engineered silk proteins.

Acknowledgments

PT
This study was funded by the National Science Council of Taiwan through research grant NSC 103-
2221-E-011-032.

RI
SC
NU
MA
D
P TE
CE
AC

27
 ACCEPTED MANUSCRIPT

References
[1] J. Lehár, A.S. Krueger, W. Avery, A.M. Heilbut, L.M. Johansen, E.R. Price, R.J. Rickles, G.F.
Short Iii, J.E. Staunton, X. Jin, Synergistic drug combinations tend to improve therapeutically
relevant selectivity, Nature biotechnology, 27 (2009) 659-666.
[2] J. Wei, F. Chen, J.-W. Shin, H. Hong, C. Dai, J. Su, C. Liu, Preparation and characterization

PT
of bioactive mesoporous wollastonite–polycaprolactone composite scaffold, Biomaterials, 30
(2009) 1080-1088.
[3] T.P. Richardson, M.C. Peters, A.B. Ennett, D.J. Mooney, Polymeric system for dual growth

RI
factor delivery, Nature biotechnology, 19 (2001) 1029-1034.
[4] Z. Ghasemi, R. Dinarvand, F. Mottaghitalab, M. Esfandyari-Manesh, E. Sayari, F. Atyabi,

SC
Aptamer decorated hyaluronan/chitosan nanoparticles for targeted delivery of 5-fluorouracil to
MUC1 overexpressing adenocarcinomas, Carbohydrate Polymers, 121 (2015) 190-198.
[5] Y. Zhang, H.F. Chan, K.W. Leong, Advanced materials and processing for drug delivery: the

NU
past and the future, Advanced drug delivery reviews, 65 (2013) 104-120.
[6] L.S. Nair, C.T. Laurencin, Polymers as biomaterials for tissue engineering and controlled
drug delivery, in: Tissue engineering I, Springer, 2006, pp. 47-90.
MA
[7] N. Aboudzadeh, M. Imani, M.A. Shokrgozar, A. Khavandi, J. Javadpour, Y. Shafieyan, M.
Farokhi, Fabrication and characterization of poly (D, L‐lactide‐co‐glycolide)/hydroxyapatite
nanocomposite scaffolds for bone tissue regeneration, Journal of Biomedical Materials Research
Part A, 94 (2010) 137-145.
D

[8] M. Farokhi, S. Sharifi, Y. Shafieyan, Z. Bagher, F. Mottaghitalab, A. Hatampoor, M. Imani,

M. Shokrgozar, Porous crosslinked poly (ε‐caprolactone fumarate)/nanohydroxyapatite
TE

composites for bone tissue engineering, Journal of Biomedical Materials Research Part A, 100
(2012) 1051-1060.
[9] S. Mao, C. Guo, Y. Shi, L.C. Li, Recent advances in polymeric microspheres for parenteral
P

drug delivery-part 1, Expert opinion on drug delivery, 9 (2012) 1161-1176.

CE

[10] T. Yucel, M.L. Lovett, D.L. Kaplan, Silk-Based Biomaterials for Sustained Drug Delivery,
Journal of Controlled Release, (2014).
[11] T. Estey, J. Kang, S.P. Schwendeman, J.F. Carpenter, BSA degradation under acidic
AC

conditions: a model for protein instability during release from PLGA delivery systems, Journal of
pharmaceutical sciences, 95 (2006) 1626-1639.
[12] A. Giteau, M.-C. Venier-Julienne, A. Aubert-Pouëssel, J.-P. Benoit, How to achieve
sustained and complete protein release from PLGA-based microparticles?, International journal
of pharmaceutics, 350 (2008) 14-26.
[13] H. Tamber, P. Johansen, H.P. Merkle, B. Gander, Formulation aspects of biodegradable
polymeric microspheres for antigen delivery, Advanced drug delivery reviews, 57 (2005) 357-
376.
[14] C.F. van der Walle, G. Sharma, M. Ravi Kumar, Current approaches to stabilising and
analysing proteins during microencapsulation in PLGA, (2009).
[15] A.O. Elzoghby, W.M. Samy, N.A. Elgindy, Protein-based nanocarriers as promising drug
and gene delivery systems, Journal of Controlled Release, 161 (2012) 38-49.
[16] P.B. Malafaya, G.A. Silva, R.L. Reis, Natural–origin polymers as carriers and scaffolds for
biomolecules and cell delivery in tissue engineering applications, Advanced drug delivery
reviews, 59 (2007) 207-233.
[17] J. Mano, G. Silva, H.S. Azevedo, P. Malafaya, R. Sousa, S. Silva, L. Boesel, J.M. Oliveira,
T. Santos, A. Marques, Natural origin biodegradable systems in tissue engineering and

28
 ACCEPTED MANUSCRIPT

regenerative medicine: present status and some moving trends, Journal of the Royal Society
Interface, 4 (2007) 999-1030.
[18] H. Hosseinkhani, M. Hosseinkhani, H. Kobayashi, Design of tissue-engineered nanoscaffold
through self-assembly of peptide amphiphile, Journal of bioactive and compatible polymers, 21
(2006) 277-296.

PT
[19] H. Hosseinkhani, T. Kushibiki, K. Matsumoto, T. Nakamura, Y. Tabata, Enhanced
suppression of tumor growth using a combination of NK4 plasmid DNA-PEG engrafted
cationized dextran complex and ultrasound irradiation, Cancer Gene Therapy, 13 (2006) 479-

RI
489.
[20] H. Hosseinkhani, T. Aoyama, O. Ogawa, Y. Tabata, Ultrasound enhances the transfection of
plasmid DNA by non-viral vectors, Current pharmaceutical biotechnology, 4 (2003) 109-122.

SC
[21] M. Konishi, Y. Tabata, M. Kariya, H. Hosseinkhani, A. Suzuki, K. Fukuhara, M. Mandai, K.
Takakura, S. Fujii, In vivo anti-tumor effect of dual release of cisplatin and adriamycin from
biodegradable gelatin hydrogel, Journal of controlled release, 103 (2005) 7-19.

NU
[22] Y. Tabata, Tissue regeneration based on growth factor release, Tissue Engineering, 9 (2003)
5-15.
[23] F. Mottaghitalab, M. Farokhi, V. Mottaghitalab, M. Ziabari, A. Divsalar, M.A. Shokrgozar,
MA
Enhancement of neural cell lines proliferation using nano-structured chitosan/poly (vinyl
alcohol) scaffolds conjugated with nerve growth factor, Carbohydrate Polymers, 86 (2011) 526-
535.
[24] M.A. Shokrgozar, F. Mottaghitalab, V. Mottaghitalab, M. Farokhi, Fabrication of porous
D

chitosan/poly (vinyl alcohol) reinforced single-walled carbon nanotube nanocomposites for

TE

neural tissue engineering, Journal of biomedical nanotechnology, 7 (2011) 276-284.

[25] E. Wenk, H.P. Merkle, L. Meinel, Silk fibroin as a vehicle for drug delivery applications,
Journal of Controlled Release, 150 (2011) 128-141.
P

[26] A.D. Bangham, Membrane models with phospholipids, Progress in biophysics and
molecular biology, 18 (1968) 29-95.
CE

[27] J. Kreuter, S. Mills, S. Davis, C. Wilson, Polybutylcyanoacrylate nanoparticles for the

delivery of [< sup> 75</sup> Se] norcholestenol, International Journal of Pharmaceutics, 16
(1983) 105-113.
AC

[28] D. Scherer, Einfluß von Polybutylcyanoacrylat-Nanopartikeln auf die orale Absorption von
Arzneistoffen, Johann Wolfgang Goethe Universitèat, 1992.
[29] C.S. Maia, W. Mehnert, M. Schäfer-Korting, Solid lipid nanoparticles as drug carriers for
topical glucocorticoids, International journal of pharmaceutics, 196 (2000) 165-167.
[30] B. Müller, J. Kreuter, Enhanced transport of nanoparticle associated drugs through natural
and artificial membranes—a general phenomenon?, International journal of pharmaceutics, 178
(1999) 23-32.
[31] M. Ricci, C. Puglia, F. Bonina, C.D. Giovanni, S. Giovagnoli, C. Rossi, Evaluation of
indomethacin percutaneous absorption from nanostructured lipid carriers (NLC): in vitro and in
vivo studies, Journal of pharmaceutical sciences, 94 (2005) 1149-1159.
[32] C. Burda, X. Chen, R. Narayanan, M.A. El-Sayed, Chemistry and properties of nanocrystals
of different shapes, Chemical reviews, 105 (2005) 1025-1102.
[33] A.K. Boal, V.M. Rotello, Fabrication and self-optimization of multivalent receptors on
nanoparticle scaffolds, Journal of the American Chemical Society, 122 (2000) 734-735.
[34] C. Murray, D.J. Norris, M.G. Bawendi, Synthesis and characterization of nearly
monodisperse CdE (E= sulfur, selenium, tellurium) semiconductor nanocrystallites, Journal of

29
 ACCEPTED MANUSCRIPT

the American Chemical Society, 115 (1993) 8706-8715.

[35] J. McKinnie, Nanobiotechnology offers a promising solution overcoming common drug
delivery failures, Drug Delivery Technology, 5 (2006) 11-15.
[36] S.M. Moghimi, A.C. Hunter, J.C. Murray, Long-circulating and target-specific
nanoparticles: theory to practice, Pharmacological reviews, 53 (2001) 283-318.

PT
[37] M.R. Avadi, A.M.M. Sadeghi, N. Mohammadpour, S. Abedin, F. Atyabi, R. Dinarvand, M.
Rafiee-Tehrani, Preparation and characterization of insulin nanoparticles using chitosan and
Arabic gum with ionic gelation method, Nanomedicine: Nanotechnology, Biology and Medicine,

RI
6 (2010) 58-63.
[38] H. Hosseinzadeh, F. Atyabi, R. Dinarvand, S.N. Ostad, Chitosan–Pluronic nanoparticles as
oral delivery of anticancer gemcitabine: preparation and in vitro study, International journal of

SC
nanomedicine, 7 (2012) 1851.
[39] A. Tahamtan, A. Tabarraei, A. Moradi, M. Dinarvand, M. Kelishadi, A. Ghaemi, F. Atyabi,
Chitosan nanoparticles as a potential nonviral gene delivery for HPV-16 E7 into mammalian

NU
cells, Artificial cells, nanomedicine, and biotechnology, (2014) 1-7.
[40] H. Hosseinkhani, Y. Tabata, Self assembly of DNA nanoparticles with polycations for the
delivery of genetic materials into cells, Journal of nanoscience and nanotechnology, 6 (2006)
MA
2320-2328.
[41] S. Abdullah, W.Y. Wendy-Yeo, H. Hosseinkhani, M. Hosseinkhani, E. Masrawa, R.
Ramasamy, R. Rosli, S.A. Rahman, A.J. Domb, Gene transfer into the lung by nanoparticle
dextran-spermine/plasmid DNA complexes, BioMed Research International, 2010 (2010).
D

[42] T. Lammers, F. Kiessling, W.E. Hennink, G. Storm, Drug targeting to tumors: principles,
TE

pitfalls and (pre-) clinical progress, Journal of controlled release, 161 (2012) 175-187.
[43] Y. Ding, S. Li, G. Nie, Nanotechnological strategies for therapeutic targeting of tumor
vasculature, Nanomedicine, 8 (2013) 1209-1222.
P

[44] S. Nie, Y. Xing, G.J. Kim, J.W. Simons, Nanotechnology applications in cancer, Annu. Rev.
Biomed. Eng., 9 (2007) 257-288.
CE

[45] F. Alexis, E.M. Pridgen, R. Langer, O.C. Farokhzad, Nanoparticle technologies for cancer
therapy, in: Drug Delivery, Springer, 2010, pp. 55-86.
[46] M. Kanapathipillai, A. Brock, D.E. Ingber, Nanoparticle targeting of anti-cancer drugs that
AC

alter intracellular signaling or influence the tumor microenvironment, Advanced drug delivery
reviews, (2014).
[47] S.A. Galindo-Rodriguez, E. Allemann, H. Fessi, E. Doelker, Polymeric nanoparticles for
oral delivery of drugs and vaccines: a critical evaluation of in vivo studies, Critical Reviews™ in
Therapeutic Drug Carrier Systems, 22 (2005).
[48] D.F. Emerich, C.G. Thanos, The pinpoint promise of nanoparticle-based drug delivery and
molecular diagnosis, Biomolecular engineering, 23 (2006) 171-184.
[49] J. Shi, A.R. Votruba, O.C. Farokhzad, R. Langer, Nanotechnology in drug delivery and
tissue engineering: from discovery to applications, Nano letters, 10 (2010) 3223-3230.
[50] D.W. Bartlett, H. Su, I.J. Hildebrandt, W.A. Weber, M.E. Davis, Impact of tumor-specific
targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality
in vivo imaging, Proceedings of the National Academy of Sciences, 104 (2007) 15549-15554.
[51] M.P. Desai, V. Labhasetwar, E. Walter, R.J. Levy, G.L. Amidon, The mechanism of uptake
of biodegradable microparticles in Caco-2 cells is size dependent, Pharmaceutical research, 14
(1997) 1568-1573.
[52] J. Panyam, S.K. Sahoo, S. Prabha, T. Bargar, V. Labhasetwar, Fluorescence and electron

30
 ACCEPTED MANUSCRIPT

microscopy probes for cellular and tissue uptake of poly (d, l-lactide-< i> co</i>-glycolide)
nanoparticles, International journal of pharmaceutics, 262 (2003) 1-11.
[53] F. Greco, M.J. Vicent, Combination therapy: opportunities and challenges for polymer–drug
conjugates as anticancer nanomedicines, Advanced drug delivery reviews, 61 (2009) 1203-1213.
[54] S. Mitragotri, Synergistic effect of enhancers for transdermal drug delivery, Pharmaceutical

PT
Research, 17 (2000) 1354-1359.
[55] C. Walsh, Molecular mechanisms that confer antibacterial drug resistance, Nature, 406
(2000) 775-781.

RI
[56] M.P. Desai, V. Labhasetwar, G.L. Amidon, R.J. Levy, Gastrointestinal uptake of
biodegradable microparticles: effect of particle size, Pharmaceutical research, 13 (1996) 1838-
1845.

SC
[57] B.D. Chithrani, W.C. Chan, Elucidating the mechanism of cellular uptake and removal of
protein-coated gold nanoparticles of different sizes and shapes, Nano letters, 7 (2007) 1542-
1550.

NU
[58] R. Singh, J.W. Lillard Jr, Nanoparticle-based targeted drug delivery, Experimental and
molecular pathology, 86 (2009) 215-223.
[59] I. Brigger, C. Dubernet, P. Couvreur, Nanoparticles in cancer therapy and diagnosis,
MA
Advanced drug delivery reviews, 54 (2002) 631-651.
[60] R. Müller, S. Maaben, H. Weyhers, W. Mehnert, Phagocytic uptake and cytotoxicity of solid
lipid nanoparticles (SLN) sterically stabilized with poloxamine 908 and poloxamer 407, Journal
of drug targeting, 4 (1996) 161-170.
D

[61] M. Roser, D. Fischer, T. Kissel, Surface-modified biodegradable albumin nano-and

TE

microspheres. II: effect of surface charges on in vitro phagocytosis and biodistribution in rats,
European journal of pharmaceutics and biopharmaceutics, 46 (1998) 255-263.
[62] D.A. Tomalia, H. Baker, J. Dewald, M. Hall, G. Kallos, S. Martin, J. Roeck, J. Ryder, P.
P

Smith, A new class of polymers: starburst-dendritic macromolecules, Polymer Journal, 17 (1985)

117-132.
CE

[63] G.S. Kwon, T. Okano, Polymeric micelles as new drug carriers, Advanced Drug Delivery
Reviews, 21 (1996) 107-116.
[64] E. Locatelli, M.C. Franchini, Biodegradable PLGA-b-PEG polymeric nanoparticles:
AC

synthesis, properties, and nanomedical applications as drug delivery system, Journal of

Nanoparticle Research, 14 (2012) 1-17.
[65] S.J. Holland, B.J. Tighe, P.L. Gould, Polymers for biodegradable medical devices. 1. The
potential of polyesters as controlled macromolecular release systems, Journal of Controlled
Release, 4 (1986) 155-180.
[66] M. Roussaki, A. Gaitanarou, P.C. Diamanti, S. Vouyiouka, C. Papaspyrides, P. Kefalas, A.
Detsi, Encapsulation of the natural antioxidant aureusidin in biodegradable PLA nanoparticles,
Polymer Degradation and Stability, 108 (2014) 182-187.
[67] M. Chen, H. Ouyang, S. Zhou, J. Li, Y. Ye, PLGA-nanoparticle mediated delivery of anti-
OX40 monoclonal antibody enhances anti-tumor cytotoxic T cell responses, Cellular
immunology, 287 (2014) 91-99.
[68] J.S. Chawla, M.M. Amiji, Biodegradable poly (ε-caprolactone) nanoparticles for tumor-
targeted delivery of tamoxifen, International journal of pharmaceutics, 249 (2002) 127-138.
[69] T. Akagi, T. Kaneko, T. Kida, M. Akashi, Preparation and characterization of biodegradable
nanoparticles based on poly (γ-glutamic acid) with L-phenylalanine as a protein carrier, Journal
of Controlled Release, 108 (2005) 226-236.

31
 ACCEPTED MANUSCRIPT

[70] J.K. Li, N. Wang, X.S. Wu, Poly (vinyl alcohol) nanoparticles prepared by freezing–thawing
process for protein/peptide drug delivery, Journal of controlled release, 56 (1998) 117-126.
[71] K. Kisich, S. Gelperina, M. Higgins, S. Wilson, E. Shipulo, E. Oganesyan, L. Heifets,
Encapsulation of moxifloxacin within poly (butyl cyanoacrylate) nanoparticles enhances efficacy
against intracellular Mycobacterium tuberculosis, International journal of pharmaceutics, 345

PT
(2007) 154-162.
[72] B. Petri, A. Bootz, A. Khalansky, T. Hekmatara, R. Müller, R. Uhl, J. Kreuter, S. Gelperina,
Chemotherapy of brain tumour using doxorubicin bound to surfactant-coated poly (butyl

RI
cyanoacrylate) nanoparticles: revisiting the role of surfactants, Journal of Controlled Release,
117 (2007) 51-58.
[73] M. Peracchia, E. Fattal, D. Desmaele, M. Besnard, J. Noel, J. Gomis, M. Appel, J. d’Angelo,

SC
P. Couvreur, Stealth® PEGylated polycyanoacrylate nanoparticles for intravenous administration
and splenic targeting, Journal of Controlled Release, 60 (1999) 121-128.
[74] M. Hans, A. Lowman, Biodegradable nanoparticles for drug delivery and targeting, Current

NU
Opinion in Solid State and Materials Science, 6 (2002) 319-327.
[75] K. Löw, M. Wacker, S. Wagner, K. Langer, H. von Briesen, Targeted human serum albumin
nanoparticles for specific uptake in EGFR-Expressing colon carcinoma cells, Nanomedicine:
MA
Nanotechnology, Biology and Medicine, 7 (2011) 454-463.
[76] L. Zhao, Y. Zhou, Y. Gao, S. Ma, C. Zhang, J. Li, D. Wang, X. Li, C. Li, Y. Liu, Bovine
serum albumin nanoparticles for delivery of tacrolimus to reduce its kidney uptake and
functional nephrotoxicity, International journal of pharmaceutics, 483 (2015) 180-187.
D

[77] M. Nicklas, W. Schatton, S. Heinemann, T. Hanke, J. Kreuter, Preparation and

TE

characterization of marine sponge collagen nanoparticles and employment for the transdermal
delivery of 17β-estradiol-hemihydrate, Drug development and industrial pharmacy, 35 (2009)
1035-1042.
P

[78] Z. Lu, T.-K. Yeh, M. Tsai, J.L.-S. Au, M.G. Wientjes, Paclitaxel-loaded gelatin nanoparticles
for intravesical bladder cancer therapy, Clinical cancer research, 10 (2004) 7677-7684.
CE

[79] I. Pali-Schöll, H. Szöllösi, P. Starkl, B. Scheicher, C. Stremnitzer, A. Hofmeister, F. Roth-

Walter, A. Lukschal, S.C. Diesner, A. Zimmer, Protamine nanoparticles with CpG-
oligodeoxynucleotide prevent an allergen-induced Th2-response in BALB/c mice, European
AC

Journal of Pharmaceutics and Biopharmaceutics, 85 (2013) 656-664.

[80] I. Ezpeleta, J.M. Irache, S. Stainmesse, C. Chabenat, J. Gueguen, Y. Popineau, A.-M.
Orecchioni, Gliadin nanoparticles for the controlled release of all-trans-retinoic acid,
International Journal of Pharmaceutics, 131 (1996) 191-200.
[81] R. Penalva, I. Esparza, M. Agüeros, C.J. Gonzalez-Navarro, C. Gonzalez-Ferrero, J.M.
Irache, Casein nanoparticles as carriers for the oral delivery of folic acid, Food Hydrocolloids, 44
(2015) 399-406.
[82] T. Mirshahi, J. Irache, J. Gueguen, A. Orecchioni, Development of drug delivery systems
from vegetal proteins: legumin nanoparticles, Drug development and industrial pharmacy, 22
(1996) 841-846.
[83] P.C. Bessa, R. Machado, S. Nürnberger, D. Dopler, A. Banerjee, A.M. Cunha, J.C.
Rodríguez-Cabello, H. Redl, M. van Griensven, R.L. Reis, Thermoresponsive self-assembled
elastin-based nanoparticles for delivery of BMPs, Journal of Controlled Release, 142 (2010) 312-
318.
[84] A.M. De Campos, A. Sánchez, M.a.J. Alonso, Chitosan nanoparticles: a new vehicle for the
improvement of the delivery of drugs to the ocular surface. Application to cyclosporin A,

32
 ACCEPTED MANUSCRIPT

International Journal of Pharmaceutics, 224 (2001) 159-168.

[85] J.O. You, C.A. Peng, Calcium‐Alginate Nanoparticles Formed by Reverse Microemulsion as
Gene Carriers, in: Macromolecular Symposia, Wiley Online Library, 2005, pp. 147-153.
[86] C. Chittasupho, M. Jaturanpinyo, S. Mangmool, Pectin nanoparticle enhances cytotoxicity
of methotrexate against hepG2 cells, Drug delivery, 20 (2013) 1-9.

PT
[87] D. Song, Y.S. Thio, Y. Deng, Starch nanoparticle formation via reactive extrusion and
related mechanism study, Carbohydrate Polymers, 85 (2011) 208-214.
[88] K. Kaewprapan, P. Inprakhon, E. Marie, A. Durand, Enzymatically degradable nanoparticles

RI
of dextran esters as potential drug delivery systems, Carbohydrate Polymers, 88 (2012) 875-881.
[89] H. Guo, Y. Liu, Y. Wang, J. Wu, X. Yang, R. Li, Y. Wang, N. Zhang, pH-sensitive pullulan-
based nanoparticle carrier for adriamycin to overcome drug-resistance of cancer cells,

SC
Carbohydrate polymers, 111 (2014) 908-917.
[90] K.Y. Choi, H. Chung, K.H. Min, H.Y. Yoon, K. Kim, J.H. Park, I.C. Kwon, S.Y. Jeong, Self-
assembled hyaluronic acid nanoparticles for active tumor targeting, Biomaterials, 31 (2010) 106-

NU
114.
[91] K. Numata, D.L. Kaplan, Silk-based delivery systems of bioactive molecules, Advanced
drug delivery reviews, 62 (2010) 1497-1508.
MA
[92] S. Kundu, Silk Biomaterials for Tissue Engineering and Regenerative Medicine, Elsevier,
2014.
[93] B. Mahendran, S.K. Ghosh, S.C. Kundu, Molecular phylogeny of silk producing insects
based on internal transcribed spacer DNA1, Journal of biochemistry and molecular biology, 39
D

(2006) 522.
TE

[94] M.S. Jolly, S. Sen, M.M. Ahsan, Tasar culture, Ambika publishers Bombay, 1974.
[95] G.H. Altman, F. Diaz, C. Jakuba, T. Calabro, R.L. Horan, J. Chen, H. Lu, J. Richmond, D.L.
Kaplan, Silk-based biomaterials, Biomaterials, 24 (2003) 401-416.
P

[96] K. McGrath, D. Kaplan, Protein-based materials, Springer, 1997.

[97] S. Inoue, K. Tanaka, F. Arisaka, S. Kimura, K. Ohtomo, S. Mizuno, Silk fibroin of Bombyx
CE

mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-
chain, and P25, with a 6: 6: 1 molar ratio, Journal of Biological Chemistry, 275 (2000) 40517-
40528.
AC

[98] E. Bini, D.P. Knight, D.L. Kaplan, Mapping domain structures in silks from insects and
spiders related to protein assembly, Journal of molecular biology, 335 (2004) 27-40.
[99] T. Gamo, T. Inokuchi, H. Laufer, Polypeptides of fibroin and sericin secreted from the
different sections of the silk gland in< i> Bombyx mori</i>, Insect Biochemistry, 7 (1977) 285-
295.
[100] M. urovec, C. ang, D. Kodr k, F. Sehnal, Identification of a novel type of silk protein
and regulation of its expression, Journal of Biological Chemistry, 273 (1998) 15423-15428.
[101] M.-p. Ho, H. Wang, K.-t. Lau, Effect of degumming time on silkworm silk fibre for
biodegradable polymer composites, Applied Surface Science, 258 (2012) 3948-3955.
[102] S. Hofmann, C. Wong Po Foo, F. Rossetti, M. Textor, G. Vunjak-Novakovic, D. Kaplan, H.
Merkle, L. Meinel, Silk fibroin as an organic polymer for controlled drug delivery, Journal of
Controlled Release, 111 (2006) 219-227.
[103] O. Kratky, E. Schauenstein, A. Sekora, An unstable lattice in silk fibroin, Nature, 165
(1950) 319-320.
[104] L.F. Drummy, D.M. Phillips, M.O. Stone, B. Farmer, R.R. Naik, Thermally induced α-
helix to β-sheet transition in regenerated silk fibers and films, Biomacromolecules, 6 (2005)

33
 ACCEPTED MANUSCRIPT

3328-3333.
[105] T. Asakura, J. Ashida, T. Yamane, T. Kameda, Y. Nakazawa, K. Ohgo, K. Komatsu, A
repeated β-turn structure in Poly (Ala-Gly) as a model for silk I of< i> Bombyx mori</i> silk
fibroin studied with two-dimensional spin-diffusion NMR under off magic angle spinning and
rotational echo double resonance, Journal of molecular biology, 306 (2001) 291-305.

PT
[106] J.P. Anderson, Morphology and crystal structure of a recombinant silk-like molecule,
SLP4, (1998).
[107] C. Zhao, J. Yao, H. Masuda, R. Kishore, T. Asakura, Structural characterization and

RI
artificial fiber formation of Bombyx mori silk fibroin in hexafluoro‐iso‐propanol solvent system,
Biopolymers, 69 (2003) 253-259.
[108] P. Garside, P. Wyeth, Crystallinity and degradation of silk: correlations between analytical

SC
signatures and physical condition on ageing, Applied Physics A, 89 (2007) 871-876.
[109] P. Monti, G. Freddi, A. Bertoluzza, N. Kasai, M. Tsukada, Raman spectroscopic studies of
silk fibroin from Bombyx mori, Journal of Raman spectroscopy, 29 (1998) 297-304.

NU
[110] E. Wenk, A.J. Wandrey, H.P. Merkle, L. Meinel, Silk fibroin spheres as a platform for
controlled drug delivery, Journal of Controlled Release, 132 (2008) 26-34.
[111] A. Motta, L. Fambri, C. Migliaresi, Regenerated silk fibroin films: thermal and dynamic
MA
mechanical analysis, Macromolecular Chemistry and Physics, 203 (2002) 1658-1665.
[112] J. Nam, Y.H. Park, Morphology of regenerated silk fibroin: Effects of freezing temperature,
alcohol addition, and molecular weight, Journal of Applied Polymer Science, 81 (2001) 3008-
3021.
D

[113] F. Xie, H. Zhang, H. Shao, X. Hu, Effect of shearing on formation of silk fibers from
TE

regenerated< i> Bombyx mori</i> silk fibroin aqueous solution, International journal of
biological macromolecules, 38 (2006) 284-288.
[114] X.-H. Zong, P. Zhou, Z.-Z. Shao, S.-M. Chen, X. Chen, B.-W. Hu, F. Deng, W.-H. Yao,
P

Effect of pH and copper (II) on the conformation transitions of silk fibroin based on EPR, NMR,
and Raman spectroscopy, Biochemistry, 43 (2004) 11932-11941.
CE

[115] K.S. Soppimath, T.M. Aminabhavi, A.R. Kulkarni, W.E. Rudzinski, Biodegradable
polymeric nanoparticles as drug delivery devices, Journal of controlled release, 70 (2001) 1-20.
[116] K.A. Athanasiou, A.R. Shah, R.J. Hernandez, R.G. LeBaron, Basic science of articular
AC

cartilage repair, Clinics in sports medicine, 20 (2001) 223-247.

[117] E. Dawson, G. Mapili, K. Erickson, S. Taqvi, K. Roy, Biomaterials for stem cell
differentiation, Advanced drug delivery reviews, 60 (2008) 215-228.
[118] M. Farokhi, F. Mottaghitalab, J. Hadjati, R. Omidvar, M. Majidi, A. Amanzadeh, M.
Azami, S.M. Tavangar, M.A. Shokrgozar, J. Ai, Structural and functional changes of silk fibroin
scaffold due to hydrolytic degradation, Journal of Applied Polymer Science, 131 (2014).
[119] E. Wenk, A.J. Meinel, S. Wildy, H.P. Merkle, L. Meinel, Microporous silk fibroin scaffolds
embedding PLGA microparticles for controlled growth factor delivery in tissue engineering,
Biomaterials, 30 (2009) 2571-2581.
[120] R.L. Horan, K. Antle, A.L. Collette, Y. Wang, J. Huang, J.E. Moreau, V. Volloch, D.L.
Kaplan, G.H. Altman, In vitro degradation of silk fibroin, Biomaterials, 26 (2005) 3385-3393.
[121] Y. Wang, D.D. Rudym, A. Walsh, L. Abrahamsen, H.-J. Kim, H.S. Kim, C. Kirker-Head,
D.L. Kaplan, < i> In vivo</i> degradation of three-dimensional silk fibroin scaffolds,
Biomaterials, 29 (2008) 3415-3428.
[122] H. Hosseinkhani, M. Hosseinkhani, Biodegradable polymer-metal complexes for gene and
drug delivery, Current Drug Safety, 4 (2009) 79-83.

34
 ACCEPTED MANUSCRIPT

[123] K. Subramani, H. Hosseinkhani, A. Khraisat, M. Hosseinkhani, Y. Pathak, Targeting

nanoparticles as drug delivery systems for cancer treatment, Current Nanoscience, 5 (2009) 135-
140.
[124] H. Hosseinkhani, M. Hosseinkhani, E.V. Farahani, M.N. Haghighi, In vitro sustained
release and degradation study of biodegradable poly (d, l-lactic acid) microspheres loading

PT
theophylline, Advanced Science Letters, 2 (2009) 70-77.
[125] M. Mahmoudi, H. Hosseinkhani, M. Hosseinkhani, S. Boutry, A. Simchi, W.S. Journeay,
K. Subramani, S. Laurent, Magnetic resonance imaging tracking of stem cells in vivo using iron

RI
oxide nanoparticles as a tool for the advancement of clinical regenerative medicine, Chemical
reviews, 111 (2010) 253-280.
[126] R. Amini, H. Hosseinkhani, A. Jalilian, S. Abdullah, R. Rosli, Engineered smart

SC
biomaterials for gene delivery, Gene Ther Mol Biol, 14 (2012) 72-86.
[127] H. Hosseinkhani, 3D in vitro technology for drug discovery, Current drug safety, 7 (2012)
37-43.

NU
[128] K. Subramani, S. Pathak, H. Hosseinkhani, Recent trends in diabetes treatment using
nanotechnology, Dig J Nanomat Biostructures, 7 (2012) 85-95.
[129] H. Hosseinkhani, P.-D. Hong, D.-S. Yu, Self-assembled proteins and peptides for
MA
regenerative medicine, Chemical reviews, 113 (2013) 4837-4861.
[130] C.R. Wittmer, X. Hu, P.-C. Gauthier, S. Weisman, D.L. Kaplan, T.D. Sutherland,
Production, structure and in vitro degradation of electrospun honeybee silk nanofibers, Acta
biomaterialia, 7 (2011) 3789-3795.
D

[131] S.-H. Park, E.S. Gil, H. Shi, H.J. Kim, K. Lee, D.L. Kaplan, Relationships between
TE

degradability of silk scaffolds and osteogenesis, Biomaterials, 31 (2010) 6162-6172.

[132] M. Lovett, C. Cannizzaro, L. Daheron, B. Messmer, G. Vunjak-Novakovic, D.L. Kaplan,
Silk fibroin microtubes for blood vessel engineering, Biomaterials, 28 (2007) 5271-5279.
P

[133] Y. Yang, Y. Zhao, Y. Gu, X. Yan, J. Liu, F. Ding, X. Gu, Degradation behaviors of nerve
guidance conduits made up of silk fibroin in vitro and in vivo, Polymer Degradation and
CE

Stability, 94 (2009) 2213-2220.

[134] M. Li, M. Ogiso, N. Minoura, Enzymatic degradation behavior of porous silk fibroin
sheets, Biomaterials, 24 (2003) 357-365.
AC

[135] J. Zhou, C. Cao, X. Ma, L. Hu, L. Chen, C. Wang, In vitro and in vivo degradation
behavior of aqueous-derived electrospun silk fibroin scaffolds, Polymer Degradation and
Stability, 95 (2010) 1679-1685.
[136] F. Chiellini, A.M. Piras, C. Errico, E. Chiellini, Micro/nanostructured polymeric systems
for biomedical and pharmaceutical applications, (2008).
[137] D.S. Kohane, Microparticles and nanoparticles for drug delivery, Biotechnology and
bioengineering, 96 (2007) 203-209.
[138] M. Vandelli, F. Rivasi, P. Guerra, F. Forni, R. Arletti, Gelatin microspheres crosslinked
with D, L-glyceraldehyde as a potential drug delivery system: preparation, characterisation, in
vitro and in vivo studies, International journal of pharmaceutics, 215 (2001) 175-184.
[139] R. Arshady, Review: Biodegradable microcapsular drug delivery systems: manufacturing
methodology, release control and targeting prospects, Journal of Bioactive and Compatible
Polymers, 5 (1990) 315-342.
[140] G.A. Digenis, T.B. Gold, V.P. Shah, Cross‐linking of gelatin capsules and its relevance to
their in vitro‐in vivo performance, Journal of pharmaceutical sciences, 83 (1994) 915-921.
[141] J. Gosline, P. Guerette, C. Ortlepp, K. Savage, The mechanical design of spider silks: from

35
 ACCEPTED MANUSCRIPT

fibroin sequence to mechanical function, Journal of Experimental Biology, 202 (1999) 3295-
3303.
[142] Y. Horikawa, K. Ohtomo, Finely powdered fibroin and process for producing same, in,
Google Patents, 1980.
[143] D. Akiyama, K. Hirabayashi, Effect of preparation method of powdered silk on the

PT
mechanical properties of moulded silk, Polymer, 35 (1994) 2355-2358.
[144] T. Hino, M. Tanimoto, S. Shimabayashi, Change in secondary structure of silk fibroin
during preparation of its microspheres by spray-drying and exposure to humid atmosphere,

RI
Journal of Colloid and interface science, 266 (2003) 68-73.
[145] J.-H. Yeo, K.-G. Lee, Y.-W. Lee, S.Y. Kim, Simple preparation and characteristics of silk
fibroin microsphere, European Polymer Journal, 39 (2003) 1195-1199.

SC
[146] A. Gholami, H. Tavanai, A. Moradi, Production of fibroin nanopowder through
electrospraying, Journal of Nanoparticle Research, 13 (2011) 2089-2098.
[147] P. Shi, J.C. Goh, Self-assembled silk fibroin particles: Tunable size and appearance,

NU
Powder Technology, 215 (2012) 85-90.
[148] X. Wang, E. Wenk, A. Matsumoto, L. Meinel, C. Li, D.L. Kaplan, Silk microspheres for
encapsulation and controlled release, Journal of Controlled Release, 117 (2007) 360-370.
MA
[149] J.G. Hardy, T.R. Scheibel, Composite materials based on silk proteins, Progress in Polymer
Science, 35 (2010) 1093-1115.
[150] C. Fu, Z. Shao, V. Fritz, Animal silks: their structures, properties and artificial production,
Chemical Communications, (2009) 6515-6529.
D

[151] M. Kazemimostaghim, R. Rajkhowa, T. Tsuzuki, X. Wang, Production of submicron silk

TE

particles by milling, Powder Technology, 241 (2013) 230-235.

[152] R. Rajkhowa, L. Wang, X. Wang, Ultra-fine silk powder preparation through rotary and
ball milling, Powder technology, 185 (2008) 87-95.
P

[153] A.S. Lammel, X. Hu, S.-H. Park, D.L. Kaplan, T.R. Scheibel, Controlling silk fibroin
particle features for drug delivery, Biomaterials, 31 (2010) 4583-4591.
CE

[154] P. Shi, J.C. Goh, Release and cellular acceptance of multiple drugs loaded silk fibroin
particles, International journal of pharmaceutics, 420 (2011) 282-289.
[155] Y. Baimark, P. Srihanam, Y. Srisuwan, P. Phinyocheep, Preparation of porous silk fibroin
AC

microparticles by a water‐in‐oil emulsification‐diffusion method, Journal of Applied Polymer

Science, 118 (2010) 1127-1133.
[156] Z. Cao, X. Chen, J. Yao, L. Huang, Z. Shao, The preparation of regenerated silk fibroin
microspheres, Soft Matter, 3 (2007) 910-915.
[157] Y. Srisuwan, P. Srihanam, Y. Baimark, Preparation of silk fibroin microspheres and its
application to protein adsorption, Journal of Macromolecular Science®, Part A: Pure and
Applied Chemistry, 46 (2009) 521-525.
[158] X. Wang, T. Yucel, Q. Lu, X. Hu, D.L. Kaplan, Silk nanospheres and microspheres from
silk/pva blend films for drug delivery, Biomaterials, 31 (2010) 1025-1035.
[159] V. Karageorgiou, L. Meinel, S. Hofmann, A. Malhotra, V. Volloch, D. Kaplan, Bone
morphogenetic protein‐2 decorated silk fibroin films induce osteogenic differentiation of human
bone marrow stromal cells, Journal of Biomedical Materials Research Part A, 71 (2004) 528-537.
[160] V. Karageorgiou, M. Tomkins, R. Fajardo, L. Meinel, B. Snyder, K. Wade, J. Chen, G.
Vunjak‐Novakovic, D.L. Kaplan, Porous silk fibroin 3‐D scaffolds for delivery of bone
morphogenetic protein‐2 in vitro and in vivo, Journal of Biomedical Materials Research Part A,
78 (2006) 324-334.

36
 ACCEPTED MANUSCRIPT

[161] B. Kundu, R. Rajkhowa, S.C. Kundu, X. Wang, Silk fibroin biomaterials for tissue
regenerations, Advanced drug delivery reviews, 65 (2013) 457-470.
[162] R. Rajkhowa, X. Wang, S. Kundu, Silk powder for regenerative medicine, Silk
Biomaterials for Tissue Engineering and Regenerative Medicine, (2014) 191.
[163] K. Jastrzebska, K. Kucharczyk, A. Florczak, E. Dondajewska, A. Mackiewicz, H. Dams-

PT
Kozlowska, Silk as an innovative biomaterial for cancer therapy, Reports of Practical Oncology
& Radiotherapy, (2014).
[164] F.P. Seib, G.T. Jones, J. Rnjak‐Kovacina, Y. Lin, D.L. Kaplan, pH‐Dependent Anticancer

RI
Drug Release from Silk Nanoparticles, Advanced healthcare materials, 2 (2013) 1606-1611.
[165] A.B. Mathur, V. Gupta, Silk fibroin-derived nanoparticles for biomedical applications,
Nanomedicine, 5 (2010) 807-820.

SC
[166] J. Kundu, Y.-I. Chung, Y.H. Kim, G. Tae, S. Kundu, Silk fibroin nanoparticles for cellular
uptake and control release, International journal of pharmaceutics, 388 (2010) 242-250.
[167] K.P.R. Chowdary, Y. Srinivasa Rao, Mucoadhesive microspheres for controlled drug

NU
delivery, Biological and pharmaceutical Bulletin, 27 (2004) 1717-1724.
[168] P.H. Hoet, I. Brüske-Hohlfeld, O.V. Salata, Nanoparticles–known and unknown health
risks, Journal of nanobiotechnology, 2 (2004) 12.
MA
[169] R.C. Mundargi, V.R. Babu, V. Rangaswamy, P. Patel, T.M. Aminabhavi, Nano/micro
technologies for delivering macromolecular therapeutics using poly (D, L-lactide-co-glycolide)
and its derivatives, Journal of Controlled Release, 125 (2008) 193-209.
[170] E. Rytting, J. Nguyen, X. Wang, T. Kissel, Biodegradable polymeric nanocarriers for
D

pulmonary drug delivery, (2008).

TE

[171] J.A. Champion, Y.K. Katare, S. Mitragotri, Particle shape: a new design parameter for
micro-and nanoscale drug delivery carriers, Journal of Controlled Release, 121 (2007) 3-9.
[172] Y.-Q. Zhang, W.-D. Shen, R.-L. Xiang, L.-J. Zhuge, W.-J. Gao, W.-B. Wang, Formation of
P

silk fibroin nanoparticles in water-miscible organic solvent and their characterization, Journal of
Nanoparticle Research, 9 (2007) 885-900.
CE

[173] K. Numata, 19 - Silk hydrogels for tissue engineering and dual-drug delivery, in: S.C.
Kundu (Ed.) Silk Biomaterials for Tissue Engineering and Regenerative Medicine, Woodhead
Publishing, 2014, pp. 503-518.
AC

[174] X. Hu, Q. Lu, L. Sun, P. Cebe, X. Wang, X. Zhang, D.L. Kaplan, Biomaterials from
ultrasonication-induced silk fibroin− hyaluronic acid hydrogels, Biomacromolecules, 11 (2010)
3178-3188.
[175] X. Wang, J.A. Kluge, G.G. Leisk, D.L. Kaplan, Sonication-induced gelation of silk fibroin
for cell encapsulation, Biomaterials, 29 (2008) 1054-1064.
[176] U.-J. Kim, J. Park, C. Li, H.-J. Jin, R. Valluzzi, D.L. Kaplan, Structure and properties of
silk hydrogels, Biomacromolecules, 5 (2004) 786-792.
[177] P.C. Bessa, E.R. Balmayor, H.S. Azevedo, S. Nürnberger, M. Casal, M. Van Griensven, R.
Reis, H. Redl, Silk fibroin microparticles as carriers for delivery of human recombinant BMPs.
Physical characterization and drug release, Journal of tissue engineering and regenerative
medicine, 4 (2010) 349-355.
[178] L.-Y. Wang, Y.-H. Gu, Z.-G. Su, G.-H. Ma, Preparation and improvement of release
behavior of chitosan microspheres containing insulin, International journal of pharmaceutics, 311
(2006) 187-195.
[179] U. Bilati, E. Allémann, E. Doelker, Strategic approaches for overcoming peptide and
protein instability within biodegradable nano-and microparticles, European Journal of

37
 ACCEPTED MANUSCRIPT

Pharmaceutics and Biopharmaceutics, 59 (2005) 375-388.

[180] M. Hofer, G. Winter, J. Myschik, Recombinant spider silk particles for controlled delivery
of protein drugs, Biomaterials, 33 (2012) 1554-1562.
[181] N.A. Guziewicz, A.J. Massetti, B.J. Perez-Ramirez, D.L. Kaplan, Mechanisms of
monoclonal antibody stabilization and release from silk biomaterials, Biomaterials, 34 (2013)

PT
7766-7775.
[182] A. Matsumoto, J. Chen, A.L. Collette, U.-J. Kim, G.H. Altman, P. Cebe, D.L. Kaplan,
Mechanisms of silk fibroin sol-gel transitions, The Journal of Physical Chemistry B, 110 (2006)

RI
21630-21638.
[183] O. Germershaus, V. Werner, M. Kutscher, L. Meinel, Deciphering the mechanism of
protein interaction with silk fibroin for drug delivery systems, Biomaterials, 35 (2014) 3427-

SC
3434.
[184] L.M. Holle, Pegaspargase: an alternative?, Annals of Pharmacotherapy, 31 (1997) 616-624.
[185] S.L. Berg, F.M. Balis, C.L. McCully, K.S. Godwin, D.G. Poplack, Pharmacokinetics of

NU
PEG-l-asparaginase and plasma and cerebrospinal fluidl-asparagine concentrations in the rhesus
monkey, Cancer chemotherapy and pharmacology, 32 (1993) 310-314.
[186] M.J. Keating, R. Holmes, S. Lerner, D.H. Ho, L-asparaginase and PEG asparaginase-past,
MA
present, and future, Leukemia & lymphoma, 10 (1993) 153-157.
[187] L. Grasset, D. Cordier, A. Ville, Woven silk as a carrier for the immobilization of enzymes,
Biotechnology and bioengineering, 19 (1977) 611-618.
[188] S. Inoue, Y. Matsunaga, H. Iwane, M. Sotomura, T. Nose, Entrapment of phenylalanine
D

ammonia-lyase in silk fibroin for protection from proteolytic attack, Biochemical and
TE

biophysical research communications, 141 (1986) 165-170.

[189] H.-B. Yan, Y.-Q. Zhang, Y.-L. Ma, L.-X. Zhou, Biosynthesis of insulin-silk fibroin
nanoparticles conjugates and in vitro evaluation of a drug delivery system, Journal of
P

Nanoparticle Research, 11 (2009) 1937-1946.

[190] Y.-Q. Zhang, R.-L. Xiang, H.-B. Yan, X.-X. Chen, Preparation of silk fibroin nanoparticles
CE

and their application to immobilization of L-asparaginase, Chemical Journal of Chinese

Universities, 29 (2008) 628-633.
[191] . . hou, .Q. hang, Biosynthesis of β-glucosidase-silk fibroin nanoparticles conjugates
AC

and enzymatic characteristics, Advanced Materials Research, 175 (2011) 186-191.

[192] Y.-Q. Zhang, Y.-J. Wang, H.-Y. Wang, L. Zhu, Z.-Z. Zhou, Highly efficient processing of
silk fibroin nanoparticle-l-asparaginase bioconjugates and their characterization as a drug
delivery system, Soft Matter, 7 (2011) 9728-9736.
[193] M. Grellier, L. Bordenave, J. Amedee, Cell-to-cell communication between osteogenic and
endothelial lineages: implications for tissue engineering, Trends in biotechnology, 27 (2009)
562-571.
[194] J.J. Moon, J.L. West, Vascularization of engineered tissues: approaches to promote angio-
genesis in biomaterials, Current topics in medicinal chemistry, 8 (2008) 300.
[195] S. Yang, K.-F. Leong, Z. Du, C.-K. Chua, The design of scaffolds for use in tissue
engineering. Part I. Traditional factors, Tissue engineering, 7 (2001) 679-689.
[196] R.E. Unger, A. Sartoris, K. Peters, A. Motta, C. Migliaresi, M. Kunkel, U. Bulnheim, J.
Rychly, C. James Kirkpatrick, Tissue-like self-assembly in cocultures of endothelial cells and
osteoblasts and the formation of microcapillary-like structures on three-dimensional porous
biomaterials, Biomaterials, 28 (2007) 3965-3976.
[197] J. Rouwkema, J.D. Boer, C.A.V. Blitterswijk, Endothelial cells assemble into a 3-

38
 ACCEPTED MANUSCRIPT

dimensional prevascular network in a bone tissue engineering construct, Tissue engineering, 12

(2006) 2685-2693.
[198] D. Kaigler, Z. Wang, K. Horger, D.J. Mooney, P.H. Krebsbach, VEGF scaffolds enhance
angiogenesis and bone regeneration in irradiated osseous defects, Journal of Bone and Mineral
Research, 21 (2006) 735-744.

PT
[199] M. Farokhi, F. Mottaghitalab, M.A. Shokrgozar, J. Ai, J. Hadjati, M. Azami, Bio-hybrid
silk fibroin/calcium phosphate/PLGA nanocomposite scaffold to control the delivery of vascular
endothelial growth factor, Materials Science and Engineering: C, 35 (2014) 401-410.

RI
[200] N. Ferrara, H.-P. Gerber, J. LeCouter, The biology of VEGF and its receptors, Nature
medicine, 9 (2003) 669-676.
[201] M. Farokhi, F. Mottaghitalab, J. Ai, M.A. Shokrgozar, Sustained release of platelet-derived

SC
growth factor and vascular endothelial growth factor from silk/calcium phosphate/PLGA based
nanocomposite scaffold, International journal of pharmaceutics, 454 (2013) 216-225.
[202] L. Uebersax, M. Mattotti, M. Papaloïzos, H.P. Merkle, B. Gander, L. Meinel, Silk fibroin

NU
matrices for the controlled release of nerve growth factor (NGF), Biomaterials, 28 (2007) 4449-
4460.
[203] L. Uebersax, H.P. Merkle, L. Meinel, Insulin-like growth factor I releasing silk fibroin
MA
scaffolds induce chondrogenic differentiation of human mesenchymal stem cells, Journal of
controlled release, 127 (2008) 12-21.
[204] E. Wenk, A.R. Murphy, D.L. Kaplan, L. Meinel, H.P. Merkle, L. Uebersax, The use of
sulfonated silk fibroin derivatives to control binding, delivery and potency of FGF-2 in tissue
D

regeneration, Biomaterials, 31 (2010) 1403-1413.

TE

[205] C. Kirker-Head, V. Karageorgiou, S. Hofmann, R. Fajardo, O. Betz, H. Merkle, M. Hilbe,

B. Von Rechenberg, J. McCool, L. Abrahamsen, BMP-silk composite matrices heal critically
sized femoral defects, Bone, 41 (2007) 247-255.
P

[206] C. Szybala, E.M. Pritchard, T.A. Lusardi, T. Li, A. Wilz, D.L. Kaplan, D. Boison,
Antiepileptic effects of silk-polymer based adenosine release in kindled rats, Experimental
CE

neurology, 219 (2009) 126-135.

[207] E.S. Gil, B. Panilaitis, E. Bellas, D.L. Kaplan, Functionalized silk biomaterials for wound
healing, Advanced healthcare materials, 2 (2013) 206-217.
AC

[208] R.-D. Hofheinz, S.U. Gnad-Vogt, U. Beyer, A. Hochhaus, Liposomal encapsulated anti-
cancer drugs, Anti-Cancer Drugs, 16 (2005) 691-707.
[209] R. Duncan, The dawning era of polymer therapeutics, Nature Reviews Drug Discovery, 2
(2003) 347-360.
[210] R. Duncan, Polymer conjugates as anticancer nanomedicines, Nature Reviews Cancer, 6
(2006) 688-701.
[211] D.J. Bharali, M. Khalil, M. Gurbuz, T.M. Simone, S.A. Mousa, Nanoparticles and cancer
therapy: a concise review with emphasis on dendrimers, International journal of nanomedicine, 4
(2009) 1.
[212] Z.G. Chen, Small-molecule delivery by nanoparticles for anticancer therapy, Trends in
molecular medicine, 16 (2010) 594-602.
[213] C. Li, Poly (L-glutamic acid)–anticancer drug conjugates, Advanced drug delivery
reviews, 54 (2002) 695-713.
[214] P. Sabbatini, C. Aghajanian, D. Dizon, S. Anderson, J. Dupont, J.V. Brown, W.A. Peters, A.
Jacobs, A. Mehdi, S. Rivkin, Phase II study of CT-2103 in patients with recurrent epithelial
ovarian, fallopian tube, or primary peritoneal carcinoma, Journal of clinical oncology, 22 (2004)

39
 ACCEPTED MANUSCRIPT

4523-4531.
[215] K. Cho, X. Wang, S. Nie, D.M. Shin, Therapeutic nanoparticles for drug delivery in cancer,
Clinical cancer research, 14 (2008) 1310-1316.
[216] J. Homsi, G.R. Simon, C.R. Garrett, G. Springett, R. De Conti, A.A. Chiappori, P.N.
Munster, M.K. Burton, S. Stromatt, C. Allievi, Phase I trial of poly-L-glutamate camptothecin

PT
(CT-2106) administered weekly in patients with advanced solid malignancies, Clinical Cancer
Research, 13 (2007) 5855-5861.
[217] A.S. Gobin, R. Rhea, R.A. Newman, A.B. Mathur, Silk-fibroin-coated liposomes for long-

RI
term and targeted drug delivery, International journal of nanomedicine, 1 (2006) 81.
[218] X. Wang, X. Hu, A. Daley, O. Rabotyagova, P. Cebe, D.L. Kaplan, Nanolayer biomaterial
coatings of silk fibroin for controlled release, Journal of Controlled release, 121 (2007) 190-199.

SC
[219] E.M. Pritchard, T. Valentin, B. Panilaitis, F. Omenetto, D.L. Kaplan, Antibiotic‐Releasing
Silk Biomaterials for Infection Prevention and Treatment, Advanced functional materials, 23
(2013) 854-861.

NU
[220] C.M. Owens, C. Mawhinney, J.M. Grenier, R. Altmeyer, M.S. Lee, A.A. Borisy, J. Lehár,
L.M. Johansen, Chemical combinations elucidate pathway interactions and regulation relevant to
Hepatitis C replication, Molecular systems biology, 6 (2010).
MA
[221] K. Karthikeyan, S. Guhathakarta, R. Rajaram, P.S. Korrapati, Electrospun zein/eudragit
nanofibers based dual drug delivery system for the simultaneous delivery of aceclofenac and
pantoprazole, International journal of pharmaceutics, 438 (2012) 117-122.
[222] J.K. Oh, D.J. Siegwart, K. Matyjaszewski, Synthesis and biodegradation of nanogels as
D

delivery carriers for carbohydrate drugs, Biomacromolecules, 8 (2007) 3326-3331.

TE

[223] M.S. Muthu, M.K. Rawat, A. Mishra, S. Singh, PLGA nanoparticle formulations of
risperidone: preparation and neuropharmacological evaluation, Nanomedicine: Nanotechnology,
Biology and Medicine, 5 (2009) 323-333.
P

[224] J.T. Zhang, S.W. Huang, Y.N. Xue, R.X. Zhuo, Poly (N‐isopropylacrylamide)
Nanoparticle‐Incorporated PNIPAAm Hydrogels with Fast Shrinking Kinetics, Macromolecular
CE

rapid communications, 26 (2005) 1346-1350.

[225] K. Numata, S. Yamazaki, N. Naga, Biocompatible and biodegradable dual-drug release
system based on silk hydrogel containing silk nanoparticles, Biomacromolecules, 13 (2012)
AC

1383-1389.
[226] D.N. Rockwood, R.C. Preda, T. Yücel, X. Wang, M.L. Lovett, D.L. Kaplan, Materials
fabrication from Bombyx mori silk fibroin, Nature protocols, 6 (2011) 1612-1631.
[227] M.E. Davis, Nanoparticle therapeutics: an emerging treatment modality for cancer, Nature
reviews Drug discovery, 7 (2008) 771-782.
[228] X. Wang, E. Wenk, X. Hu, G.R. Castro, L. Meinel, X. Wang, C. Li, H. Merkle, D.L.
Kaplan, Silk coatings on PLGA and alginate microspheres for protein delivery, Biomaterials, 28
(2007) 4161-4169.
[229] J. Ferlay, H.R. Shin, F. Bray, D. Forman, C. Mathers, D.M. Parkin, Estimates of worldwide
burden of cancer in 2008: GLOBOCAN 2008, International journal of cancer, 127 (2010) 2893-
2917.
[230] A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, T. Murray, M.J. Thun, Cancer statistics, 2008,
CA: a cancer journal for clinicians, 58 (2008) 71-96.
[231] L. Brannon-Peppas, J.O. Blanchette, Nanoparticle and targeted systems for cancer therapy,
Advanced drug delivery reviews, 64 (2012) 206-212.
[232] S.-S. Feng, Nanoparticles of biodegradable polymers for new-concept chemotherapy,

40
 ACCEPTED MANUSCRIPT

(2004).
[233] X. Jia, L. Jia, Nanoparticles improve biological functions of phthalocyanine
photosensitizers used for photodynamic therapy, Current drug metabolism, 13 (2012) 1119-1122.
[234] J. Shao, Y. Dai, W. Zhao, J. Xie, J. Xue, J. Ye, L. Jia, Intracellular distribution and
mechanisms of actions of photosensitizer Zinc (II)-phthalocyanine solubilized in Cremophor EL

PT
against human hepatocellular carcinoma HepG2 cells, Cancer letters, 330 (2013) 49-56.
[235] Y. Gao, J. Xie, H. Chen, S. Gu, R. Zhao, J. Shao, L. Jia, Nanotechnology-based intelligent
drug design for cancer metastasis treatment, Biotechnology advances, (2013).

RI
[236] X. Wang, L. Yang, Z.G. Chen, D.M. Shin, Application of nanotechnology in cancer therapy
and imaging, CA: a cancer journal for clinicians, 58 (2008) 97-110.
[237] O.C. Farokhzad, R. Langer, Impact of nanotechnology on drug delivery, ACS nano, 3

SC
(2009) 16-20.
[238] S. Biswas, V.P. Torchilin, Nanopreparations for organelle-specific delivery in cancer,
Advanced drug delivery reviews, 66 (2014) 26-41.

NU
[239] J.K. Vasir, V. Labhasetwar, Targeted drug delivery in cancer therapy, Technology in cancer
research & treatment, 4 (2005) 363-374.
[240] V. Gupta, A. Aseh, C.N. Ríos, B.B. Aggarwal, A.B. Mathur, Fabrication and
MA
characterization of silk fibroin-derived curcumin nanoparticles for cancer therapy, International
journal of nanomedicine, 4 (2009) 115.
[241] X.-X. Xia, M. Wang, Y. Lin, Q. Xu, D.L. Kaplan, Hydrophobic Drug-Triggered Self-
Assembly of Nanoparticles from Silk-Elastin-Like Protein Polymers for Drug Delivery,
D

Biomacromolecules, 15 (2014) 908-914.

TE

[242] B. Subia, S. Chandra, S. Talukdar, S.C. Kundu, Folate conjugated silk fibroin nanocarriers
for targeted drug delivery, Integrative Biology, 6 (2014) 203-214.
[243] P. Wu, Q. Liu, R. Li, J. Wang, X. Zhen, G. Yue, H. Wang, F. Cui, F. Wu, M. Yang, Facile
P

preparation of paclitaxel loaded silk fibroin nanoparticles for enhanced antitumor efficacy by
locoregional drug delivery, ACS applied materials & interfaces, 5 (2013) 12638-12645.
CE

[244] M. Chen, Z. Shao, X. Chen, Paclitaxel‐loaded silk fibroin nanospheres, Journal of

Biomedical Materials Research Part A, 100 (2012) 203-210.
[245] N. Vadia, S. Rajput, Study on formulation variables of methotrexate loaded mesoporous
AC

MCM-41 nanoparticles for dissolution enhancement, European Journal of Pharmaceutical

Sciences, 45 (2012) 8-18.
[246] A. Taheri, R. Dinarvand, F.S. Nouri, M.R. Khorramizadeh, A.T. Borougeni, P. Mansoori, F.
Atyabi, Use of biotin targeted methotrexate–human serum albumin conjugated nanoparticles to
enhance methotrexate antitumor efficacy, International journal of nanomedicine, 6 (2011) 1863.
[247] B. Subia, S. Kundu, Drug loading and release on tumor cells using silk fibroin–albumin
nanoparticles as carriers, Nanotechnology, 24 (2013) 035103.

41
 ACCEPTED MANUSCRIPT

PT
RI
SC
NU
MA
Graphical abstract
D
P TE
CE
AC

42