Materials Science & Engineering C 95 (2019) 302–311

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Materials Science & Engineering C

journal homepage: www.elsevier.com/locate/msec

Review

Preparation and biomedical applications of silk fibroin-nanoparticles T

composites with enhanced properties - A review
⁎
Zongpu Xua, Liyang Shib, Mingying Yanga, Liangjun Zhua,
a
Institute of Applied Bioresource Research, College of Animal Sciences, Zhejiang University, Hangzhou 310058, PR China
b
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha 410082, PR China

A R T I C LE I N FO A B S T R A C T

Keywords: Polymer-nanoparticles composites have attracted considerable attention over the years, due to the superior
Silk fibroin combination performance from both materials. As a versatile natural polymer, silk fibroin has been combined
Nanoparticles with various nanoparticles. The introduction of nanoparticles brings many optimized and new functionalities to
Preparation methods silk fibroin, such as mechanical, biological, thermal, electrical, florescent and magnetical properties. This review
Enhanced properties
focuses on the preparation methods and enhanced properties of silk fibroin-nanoparticle composites, especially
Biomedical applications
their biomedical applications.

1. Introduction of nanoparticles (NPs) provides an effective means to obtain target

Polymer-nanoparticles composites are generally regarded as a two-
phase system including matrix and reinforcement, and the two phases
can be combined via different approaches. The composites integrate the
physicochemical and biological properties of both materials, which
have been widely applied in biomedical fields [1–3], including tissue
engineering, drug delivery and biosensing. Polymers work as a matrix
to directly connect with cells or tissues, thus they should be bio-
compatible and biodegradable [4,5]. Among various types of polymers,
naturally derived such as collagen, chitosan, alginate and silk fibroin
attract more and more attention.
Silk fibroin (SF), a natural protein extracted from Bombyx mori
silkworms, contains H-chain, L-chain and glycoprotein P25 [6,7]. In H-
chain 94% of the amino acid sequences are glycine-alanine-glycine-
alanine-glycine-serine (GAGAGS) repeats, and they assembly into
highly crystalline antiparallel β-sheet structure [8,9], endowing SF with
remarkable mechanical properties [10]. And by adjusting the crystal-
linity and β-sheet content, the degradability of SF can be engineered
from weeks to months. SF also contains a considerable percentage of
amino acids with side chains, such as serine, threonine, aspartic, glu-
tamic and tyrosine, which provide reactive sites for chemical and
functional modification [11]. Moreover, SF can be prepared as multiple
forms including films, scaffolds, hydrogels, microspheres and nanofi-
bers, and a great number of in vitro and in vivo studies have proved
their good cytocompatibility and histocompatibility [12–15]. In order
to further improve the performance of SF-based materials, introduction
properties needed in biomedical fields, which depends on NPs species
and the interactions between polymer and NPs [16,17].
NPs can be divided as inorganic/ceramic NPs (silicates, calcium
carbonate, calcium phosphate), metal/metal-oxide NPs (gold, silver,
iron oxide), carbon-based NPs (carbon nanotubes, graphene) and
polymeric NPs (natural, synthetic) [18], and they possess large surface
area, high adsorptive effect and a series of intrinsic nature such as ex-
ceptional mechanical, biological, thermal, electrical, florescent and
magnetical properties. Apart from their application in optoelectronics,
chemocatalysis or cosmetics, NPs have been used as drug delivery
system, antibacterial agents, tissue engineering scaffold fillers and di-
agnostical biosensors [19–25]. However, some drawbacks arise when
they are applied in biomedical areas. The tendency for aggregation
weakened their nanoscale effect, leading to degradation of specific
properties. Another important concern is their potential toxicity [26],
as the large surface area and very small size enable them to directly
interact with, or easily enter into cells and tissues, thus leading to ne-
gative impact on biological structure and function, even permanent
damage. To avoid aggregation and reduce toxicity, introduction of NPs
into polymer matrix is effective [27–29] and the polymer-NPs compo-
sites can be designed and optimized according to different require-
ments.
Due to outstanding chemical versatility and miscibility, SF-NPs
composites with good interfacial relations have been successfully fab-
ricated, and they show enhanced performance on micro- or nanos-
tructures, physicochemical properties and biological response. There

⁎
Corresponding author.
E-mail address: ljzhu@zju.edu.cn (L. Zhu).

https://doi.org/10.1016/j.msec.2018.11.010
Received 14 February 2018; Received in revised form 25 July 2018; Accepted 5 November 2018
Available online 06 November 2018
0928-4931/ © 2018 Elsevier B.V. All rights reserved.
Z. Xu et al.
Fig. 1. Schematic of preparation methods for SF-NPs composites. A: Directly mixing; B: In-situ synthesis via different approaches including using the intrinsic
reducing ability of SF materials, grafting or coating SF materials with functional groups (Ag NPs as an example), and the prepared NPs distributed on the surface of SF
materials; C: Silkworm feeding method, silkworm fed with mulberry leaves sprayed by NPs solution, and NPs-incorporated silk fibers can be naturally spun, where
the NPs mainly distributed inside the SF fibers.
have been some reviews related to polymer-nanoparticles composites
[3,18,27] and SF-based materials for biomedical applications
[11,13–15], but they only mentioned a fraction of SF-NPs composites.
Therefore, in this review we systematically summarize the preparation
methods of various SF-NPs composites, discuss the interactions of SF
and NPs, and highlight the enhanced properties for their biomedical
applications.
2. Preparation methods of SF-NPs composites
Introduction of NPs to SF matrix can be realized by three main
routes: directly mixing, in-situ synthesis and silkworm feeding method,
which are illustrated in Fig.1. For the first, NPs are generally added into
SF solution, then the mixture can be formed as film, nanofiber, scaffold
or hydrogel, and in these composites NPs bind to SF macromolecules by
physical interaction. For the second, SF materials are put into precursor
solution of NPs, which means they work as templates for the in-situ
nucleation and growth of NPs. For the third, silkworms are fed with
diets containing NPs and the SF-NPs composite fibers will be naturally
spun.
2.1. Directly mixing
This approach is easy to manipulate, but NPs tend to aggregate
because of high surface energy. To overcome this problem, mechanical
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stirring or ultrasonic dispersion is commonly used to uniformly dis-
tribute NPs in SF solution. We once added multi-walled carbon nano-
tubes (MWCNTs) into SF solution, and after sonication the mixture
could still keep stable and homogeneous even a month later [30].
Wang et al. prepared a stable graphene oxide (GO) dispersion by
ultrasonication, then added dropwise into SF solution, and obtained SF/
GO composite films via solvent evaporation [31]. SEM images showed
GO sheets were well dispersed and embedded in the SF matrix without
aggregation, indicating that they were compatible. The addition of GO
promoted the formation of the crystal structure in the SF/GO compo-
sites, which was attributed to strong intermolecular forces like hy-
drogen bonds between the functional groups of GO sheets and the polar
groups on the SF molecular chains. With very low concentrations of GO
(≤1.0 wt%), the thermal stability and mechanical properties of com-
posite films, as well as their resistance to enzymatic degradation have
significantly improved.
Surface modification of NPs is also an effective method to avoid
aggregation. For example, a solution of carboxy functionalized CdTe
quantum dots (CdTe QDs) was mixed with SF solution under alkaline
conditions, thus forming a stable SF/QDs hybrid solution [32]. The
CdTe QDs were distributed uniformly in amorphic domains of SF, and
these QDs-doped amorphic domains are in turn connected to the crys-
talline domains. It was also believed that the hydrogen-bonding inter-
actions between the -NH groups of the SF and the –COO– groups on the
QDs played a key role in this process. Similarly, in another study
Z. Xu et al.
Fig. 2. SEM images of original degummed SF fibers (A, C, E) and SF fiber/AgNPs composites (B, D, F). B was fabricated by immersing A into 10 mM AgNO3 solution
for 4 h in the dark [35]. D was fabricated by soaking C into 10 mM AgNO3 solution and irradiating with a 365 nm UV light for 3 h [37]. F was fabricated by soaking
(PAA/PDDA) film–coated E into10 mM AgNO3 solution and irradiating with a 365 nm UV light for 1 h [40]. The density of synthesized AgNPs varied with the Ag+
ions binding sites, as well as the incubating time under different conditions.
modified Laponite (LAP) nanoplatelets with sodium polyacrylate could
be exfoliated and dispersed homogeneously in SF solution, resulting
from the mutual negative charge repulsion [33]. The incorporation of
LAP accelerated the gelation process of SF solution, and the prepared
SF/LAP composite hydrogel displayed thixotropic properties, as it was
injectable. Also, the proliferation and osteogenic differentiation of
primary osteoblasts encapsulated in SF/LAP hydrogel could be sig-
nificantly promoted, indicating that the nanocomposite had great po-
tential for irregular bone defect repair.
Directly mixing NPs and SF solution provides a simple and con-
venient approach to fabricate SF-NPs composite, but rational percen-
tage of NPs was important for biomedical applications, as excessive
amount may lead to reduced function and increased toxicity [30,34].
2.2. In-situ synthesis
In this process SF materials work as reduction templates to mediate
the synthesis of NPs, which can be achieved by their intrinsic nature or
by functional grafting and coating groups. We show this process in
Fig.1B with Ag+ precursor as an example, because Ag as well as Au NPs
are most commonly used for the in-situ synthesis and then further ap-
plied in biomedical fields.
Dong et al. [35] successfully deposited AgNPs on degummed SF
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fibers by simply immersing into AgNO3 solution. They deduced the
exposed tyrosine (Tyr) residue of SF fibers contributed to the in-situ
reduction of Ag (I) to Ag (0). The phenolic groups of Tyr were ionized
during the pretreatment under alkaline conditions, and could capture
Ag+ ions to form Ag nanoclusters. Similarly, the Tyr residues in the SF
molecular chain were also responsible for the reduction of the AuCl4−,
as they were very easily oxidized to display strong electron donating
properties. Zhou et al. [36] prepared core-shell nanostructured gold
colloid-SF bioconjugate by adding SF solution dropwise into HAuCl4
solution. Serving as an effective biotemplate, the oxidized SF molecular
chains attached to the surface of AuNPs and stabilized their mono-
dispersed nature. In addition to use the intrinsic reducing ability of SF,
some researchers synthesized AgNPs with UV light assist. Lu et al. [37]
soaked degummed SF fabrics into AgNO3 solution and put them under
irradiation with a UV light lamp. With the irradiation time increasing,
AgNPs emerged but gradually aggregated into clusters, which were not
distributed uniformly on the SF fiber surface. They speculated the
carboxyl groups on the side chains of SF proteins played critical roles in
Ag+ absorption and reduction. In another study, Ag/SF nanofibers were
prepared by electrospinning the mixture of SF and AgNO3 solution [38].
Then the nanofibers were irradiated with UV light to reduce Ag+ ions,
and round AgNPs of 8 nm in diameter could be formed on silk nanofi-
bers. From the above studies we could find out that the density of Ag+
Z. Xu et al.
ions binding sites on SF materials initially dominated the yield of
AgNPs. In order to increase the Ag+ binding sites, polymer modifica-
tion has been employed [39–41], which means the functional groups at
one end of polymers are grafted onto silk materials and groups at an-
other end bound to Ag+ ions. For example, Meng et al. [40] built a poly
(acrylic acid)/poly (dimethyldially lammonium chloride) (PAA/PDDA)
multilayered structure on degummed SF fibers by alternatively de-
position of two oppositely charged polymers. After 8 rounds of de-
position, SF fibers were fully covered with a layer of polymer film, and
then the fibers were soaked in AgNO3 solution under the treatment with
UV irradiation. Since the abundant carboxyl groups of PAA could
strongly bind with Ag+ ions and assist the conversion of Ag (I) to Ag
(0), high density of AgNPs in good crystalline structure were in-situ
synthesized. Also, the polymer film prolonged sustainable release of
Ag+, providing a long-term antibacterial effect against both Gram-ne-
gative and Gram-positive microorganisms. More interestingly, Co-
henkarni et al. [42] combined the methods of directly mixing and in-
situ synthesis to prepare SF-NPs composite. Briefly, SF solutions with
gold seed NPs were firstly electrospun into nanofibers, and then the
samples were reduced in Au+ solution to form AuNPs doped SF na-
nofibers (SNFAu). After incubation for 3–4 days at room temperature,
AuNPs grew to > 2-fold size of the gold seed NPs and dispersed
throughout the SNFAu. The UV–vis spectrum of SNFAu showed a strong
absorption peak at 525 nm, yet no absorption peaks for SNFseed and SNF
incubated in Au+ solution for 6 days, which indicated the gold seed NPs
were necessary for the formation of AuNPs.
Fig.2 shows the SEM images of SF-AgNPs composites fabricated by
different routes mentioned above, which indicate the density of AgNPs
varied with the Ag+ ions binding sites, as well as the incubating time
under different conditions. Except for AgNPs and AuNPs, other metal-
based NPs including silver iodide (AgI) [43], silver bromide (AgBr)
[44], silver chloride (AgCl) [45], trimanganese tetraoxide (Mn3O4) [46]
and magnesium hydroxide (Mg(OH)2) [47] have been successfully
synthesized on SF fibers by circularly dipping in solutions with different
ions. Furthermore, other forms of SF materials including SF microfibers
[48], SF nanofibers [49], SF hydrogels [50] and SF films [51] can all be
used to regulate the in-situ formation of NPs, indicating the versatility
of SF as a template.
2.3. Silkworm feeding method
Recently, nanoparticle-reinforced silk fibers have been successfully
obtained by directly feeding silkworm with diets containing nanoma-
terials, such as metal NPs (copper [52,53], iron [52], silver [53]),
metal-oxide NPs (titanium dioxide [52,54], iron oxide [55]) and
carbon-based NPs (carbon nanotubes [56,57], graphene [57]). For ex-
ample, Wu et al. [52] fed Bombyx mori silkworm mulberry leaves spread
with three kinds of NPs as Cu, Fe and TiO2, and the metal content
analysis confirmed that a certain amount of NPs could be absorbed by
the posterior silk glands, which then entered the fibroin and ended
inside the SF fibers. More importantly, the incorporated metal NPs
maintained their nanoscale features as an effective reinforcement, thus
leading to significantly improved tensile strength and strain at break. In
another study, silkworm mulberry leaves spray-coated with single-
walled carbon nanotubes (SWNTs) or graphene (GR) solutions were
used to feed Bombyx mori larval [57]. Researchers also found some of
the SWNTs or GR could be incorporated into silk fibers, and the pre-
sence of these nanofillers hindered the transformation of α-helix and
random coils to β-sheet structures, which significantly increased elon-
gation at break and toughness modulus due to more easily movable
chains. However, with excessive SWNTs and GR in the silk, these na-
nofillers may aggregate and act as defects, resulting in low fracture
strength and a low percentage of elongation. As this method is eco-
friendly and easy to handle, it is expected to open up possibilities for
naturally producing functional SF-NPs composites on a large scale.
All the three methods can work effectively to prepare SF-NPs
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composites, but they have their own advantages in view of properties
and applications. Composites prepared from directly mixing perfectly
integrate SF and NPs, so it is easier to perform their functions as a
whole, especially for bulk materials such as scaffold and hydrogel in
tissue engineering. In-situ synthesis demonstrates the role of SF as a
template to mediate the formation of NPs, which can promote the
specific applications of composites related to NPs. Silkworm feeding
method provides a natural way to yield SF fibers doped with NPs, but in
most cases their applications are limited by the fiber form. Therefore,
researchers can choose different methods according to the needs.
3. SF-NPs composites with enhanced properties for biomedical
applications
As a fascinating natural protein with many excellent properties, SF
has been widely used and deeply studied in biomedical fields, from
tissue engineering, drug delivery to biosensing and bioimaging. The
introduction of NPs brings many optimized or new functionalities (in
some ways both can be considered as “enhanced properties”) including
mechanical, biological, thermal, electrical, fluorescent and magnetical
properties, which definitely improve the performance of SF-based bio-
materials. In this part, we listed various SF-NPs composites with en-
hanced properties, and introduced their biomedical applications.
3.1. Enhanced mechanical properties
High mechanical properties of biomaterials play important roles to
influence cell behaviors and hard tissue regeneration [13,58,59], and
SF-based materials with enhanced mechanical properties can better
match the mechanical requirements in biomedical applications. As is
known that SF fibers possess remarkable mechanical properties, but
they must be dissolved into regenerated SF solution to prepare as other
forms, which inevitably disrupted its well-ordered structure and
weaken its mechanics resulting from lower β-sheet content and lower
crystallinity [50,60]. The incorporation of NPs can effectively work as
fillers to reinforce SF materials, and carbon-based NPs such as carbon
nanotubes and graphene oxide are frequently used.
We once used MWCNTs to improve the mechanical properties of SF
film [30], with only 0.5% (w/w) MWCNTs the composites exhibited a
24% increase in the Young's modulus, 34% in the strain at break, 39%
in the ultimate tensile strength and 30% in the yield strength compared
to pure SF film. Also, MWCNTs induced an increase of β-sheet content
from 32.77% to 51.67%, which might contribute to the reinforcement
effect. However, as the percentage of MWCNTs reached to 1.0% (w/w),
the mechanical properties decreased due to aggregation of NPs and
emergence of nanocracks at the MWCNT-SF interface. The same results
appeared in another study for MWCNTs reinforced electrospun SF na-
nofibers [61]. When the addition amount of MWCNTs increased from 0
to 1.0 wt%, the β-sheet content increased from 35.8% to 49.4%, and the
composites showed a 2.8-fold increase in breaking strength, a 4.4-fold
in Young's modulus and a 2.1-fold in breaking energy compared to pure
SF electrospun mats. But the β-sheet content decreased to 25.7% when
the percentage of MWCNTs was up to 1.5 wt%, the mechanical prop-
erties meanwhile rapidly decreased, also resulting from the agglomer-
ates of MWCNTs. In summary, with appropriate amount of CNTs not to
cause critical aggregations, the improved mechanical properties mainly
attributed to the reinforce effect of CNTs, and partially to the increased
β-sheet and crystallinity. What's more, the addition of CNTs did not
influence the biocompatibility of SF materials and multiple types of
cells could achieve better adhesion and proliferation on the SF-NPs
composites, thus illustrating their potential applications as in tissue
engineering.
In some cases, it is suggested the interfacial interactions between
NPs and SF play the dominant role for improved toughness. For ex-
ample, Hu et al. fabricated GO-SF nanocomposite membranes using
spin assisted layer-by-layer technique [62], with the average bilayer
Z. Xu et al.
thickness of around 5.4 nm. When the volume ratio of GO (0.04 wt% in
concentration) came to 23.5%, the tensile modulus, ultimate stress and
toughness of nanocomposites reached to 145 GPa, over 300 MPa and
above 2.2 MJ m−3 respectively, which exceed manyfold than other SF-
based materials. The extraordinary mechanical properties were as-
cribed to the interphase reinforcement mechanism, as the com-
plementary heterogeneous surface functionalities of GO and SF made
them intimately contact by a dense network of various interactions
including hydrogen bonding, polar interactions and hydrophobic in-
teractions, thus promoting the formation of strong molecular interphase
zones. These ultrathin and ultrarobust GO-SF nanocomposite mem-
branes have the potential application in bio-nanosensing devices or
protective molecular coatings.
Other reinforcement fillers include carbon nanoscroll [63], titania
[34], silica [64] and cellulose nanowhiskers [65], which all effectively
enhance the mechanical properties of SF composites and expand their
biomedical applications.
3.2. Enhanced biological properties for bone tissue engineering
SF has good biocompatibility both in vitro and in vivo, and the
introduction of specific NPs can improve the cell-biomaterial interac-
tions or tissue repair outcomes. For example, Hydroxyapatite (HAP) is
not only chemically similar to the minerals in bone, but also shows
good biocompatibility and osteoinductivity [66]. To mimic the struc-
ture and function of bone tissue, SF and HAP composite materials have
been developed for bone tissue engineering. Niu et al. fabricated na-
nofibrous SF scaffolds containing HAP NPs by electrospinning [67].
They found the incorporation of HAP significantly improved the me-
chanical properties of scaffolds, and more importantly rat bone marrow
mesenchymal stem cells (rBMSCs) cultured on SF/HAP scaffolds dis-
played better osteogenic differentiation capability than on pure SF
scaffolds, as both the representative markers of ALP activity at early
stage and calcium nodule at terminal stage of osteogenic differentiation
were effectively facilitated. Furthermore, when growth factor bone
morphogenetic protein 2 (BMP-2) introduced to SF/HAP scaffolds, the
osteogenic differentiation of rBMSCs were further enhanced due to
synergistic effect of HAP and BMP-2, which could be observed in Fig.3.
Silica-based glasses have also been applied for bone regeneration
due to their good osteoconductive and osteoinductive properties [68].
To combine the advantages of both SF and silica NPs, Mieszawska et al.
[69] prepared SF/silica film to study the osteogenic differentiation of
human mesenchymal stem cells (hMSCs). The results showed that the
presence of silica in SF matrix upregulated the gene expression of os-
teogenic markers, leading to high levels of bone sialoprotein (BSP) and
collagen type I (Col I). The SF/silica film also increased the formation of
collagen/calcium phosphate extracellular matrix, indicating the posi-
tive influence of silica on osteogenesis. Moreover, the silica NPs were
found to be biodegradable, as their large surface area facilitated the in
vitro dissolution over time, which was suitable for further bone re-
modeling.
To prevent infection caused by bacterial adhesion is also very im-
portant in bone tissue engineering. Based on the good osteoinductivity
of SF-HAP composites, Ribeiro et al. [70] further modified SF/HAP
hydrogels with in-situ synthesized AgNPs and AuNPs, and the anti-
microbial studies revealed that hydrogels with AgNPs and AuNPs ex-
hibited significant inhibition ability against both gram-positive and
gram-negative bacteria. It is worth noting that the incorporation of
multiple NPs may simultaneously improve multiple biological proper-
ties for SF materials, which can preferably meet the requirements in
clinic.
3.3. Enhanced photothermal properties for tumor and infection treatment
Photothermal properties are based on photo-absorbing nanomater-
ials, which can convert incident light into heat with high efficiency so
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as to induce local hyperthermia, displaying less side effects and im-
proved selectivity [71]. There are many photo-absorbing nanomaterials
including AuNPs, carbon-based NPs and transitional metal dichalco-
genides (TMDCs), and some have been combined with SF to explore
their applications in tumor or infection treatments.
Kojic et al. [72] once proposed a treatment for focal infections based
on AuNPs suspended in an injectable SF hydrogel. The SF hydrogel is a
biocompatible and biodegradable carrier, preventing the aggregation of
AuNPs and ensuring their spatial stabilization at the injection site. The
SF hydrogels with or without AuNPs were subcutaneously injected into
the infected wounds of mice. After exposure to laser for 10 min, the
maximum temperature of the wound with AuNPs (NP + Gel + Laser
group) and without AuNPs (Gel + Laser group) reached 55 °C and 40 °C
respectively. The normalized colony forming units (CFU) demonstrated
that the NP + Gel + Laser group showed a strong antibacterial effect,
with the number of CFU decreasing to about 20% or even complete
eradication of untreated group, while the CFU of Gel + Laser group
only decreased to 80%, suggesting the necessity and importance of
AuNPs. This treatment effectively controlled the bacterial infection, and
avoided the use of high-dose antibiotics and incision-drainage of the
wound.
In another study, Li et al. [73] directly adding MoS2 nanosheets (a
type of TMDCs) dispersion into SF solution, and the SF nanofibrils
formed on the surfaces of the MoS2 improved the stability of MoS2 in
aqueous solution. Also, the prepared SF/MoS2 composites showed
better photothermal performance, as the temperature of hybrid solution
increased 37.8 °C within 5 min of irradiation while the pristine MoS2
dispersion only increased 26.8 °C, which was attributed to the enhanced
scatter of incident light by SF nanofibrils. Therefore the SF/MoS2
composites displayed good photothermal properties because they could
effectively ablate HeLa cells under 808 nm laser within 5 min, with a
significant reduction of cell viability to < 60% and 5% when the con-
centrations of SF/MoS2 were 50 and 100 μg/mL respectively. This
biocompatible SF/MoS2 hybrids exhibited great potential in photo-
thermal therapy for future cancer treatment.
3.4. Enhanced electrical properties for nerve regeneration and
electrochemical biosensor
Although SF is non-conductive, it can be combined with conductive
nanomaterials such as carbon nanotubes [74,75] and AuNPs [76–78] to
create functional biocomposites. Mottaghitalab et al. [75] used SWCNT
to overcome the electrical conductivity limitations of pure silk, as they
prepared SF/SWCNT composite scaffolds for nerve defect repair in rats.
The conductivity endowed by SWCNT support the electrical transmis-
sion, which contributed to the successful connection between the in-
jured nerves and conduits. Moreover, aligned fibronectin (FN) con-
taining nanofibers functioned as chemical cue for cell adhesion and
spreading. After 5 weeks post implantation, newly regenerated nerve
emerged through the conduits, and nerve conduction velocities and
more myelinated axons were observed compared with negative group
(Fig.4), indicating a functional recovery of the injured nerves.
AuNPs have also been used to improve the conductivity of SF ma-
terials. Das et al. [76] first got SF-AuNPs composites via electrospin-
ning, and then the nanofibrous mat was fabricated into tubular conduits
by sheet rolling method. After incorporation of AuNPs the electrical
resistance of SF nanofibers decreased for 25 times, suggesting the en-
hanced conductivity through the SF-AuNPs scaffold. The conduits were
used for long term rat sciatic nerve regeneration, and after 18 months
growth of nerve along the nerve gap was complete. The nerve con-
duction velocity, compound muscle action potential and motor unit
potential of the group implanted with SF-AuNPs conduits were close to
or even higher than the normal values, which indicated the conduits
enhanced myelination and performed good recovery effect for both
structural and functional regeneration of severed sciatic nerves.
Apart from nerve regeneration, AuNPs and SF bioconjugate could be
Z. Xu et al.
Fig. 3. Morphology behaviors (above), ALP staining results (middle) and Calcium nodule staining results (below) of rBMSCs cultured on SF (A, D and G), SF/HAP (B,
E and H) and SF/HAP/BMP-2 (C, F and I) scaffolds [67]. The incorporation of HAP and BMP-2 significantly improved the osteogenic differentiation capability of
rBMSCs, as both the representative markers of ALP activity at early stage and calcium nodule at terminal stage of osteogenic differentiation were effectively
facilitated.
used to modify glassy carbon electrode (GCE) for biosensor fabrication
[77]. In this system, horseradish peroxidase (HRP) could be successfully
immobilized on AuNPs-SF/GCE composites and the direct reduction of
H2O2 was achieved, because AuNPs-SF matrix effectively maintained
the bioactivity of HRP and facilitated the electron transfer between HRP
and electrode surface. The prepared biosensor displayed a quick elec-
trocatalytic response to the reduction of H2O2 with low detection limit
and wide linear range, also it exhibited good reproducibility and long-
term stability. This new class of electrochemical biosensor with the
advantage of simple preparation and convenient operation have the
potential for detection in biological systems.
3.5. Enhanced fluorescent properties for bioimaging
Tracking the distribution or degradation of biomaterials is im-
portant for tissue engineering and drug delivery, and using fluorescent
NPs for bioimaging has gained much attention for non-invasive mon-
itoring in recent years [79]. In one study, 3-mercaptopropionic acid
(MPA)-coated CdTe QDs (MPA-QDs) were mixed with SF solution,
followed by ultrasonication to induce gelation, and SF hydrogels con-
taining QDs (Silk/QDs Gel) were prepared [78]. The incorporation of
QDs into SF hydrogels not only significantly reduced the cytotoxicity,
but also stabilize their fluorescent property. The fluorescence of Silk/
QDs Gel remained stable in PBS (pH 7.4) for > 96 h in vitro. After
subcutaneously injected into mice, the fluorescence of Silk/QDs Gel
could still maintain for 96 h while it was quenched within 24 h for free
QDs. Fig.5 showed the in vivo fluorescence imaging of subcutaneously
injected Silk/QDs Gel at different periods, where relatively stable
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fluorescence signals could be clearly observed from 0 to 4 days. The
QDs-incorporated silk hydrogels with stable and strong fluorescence
have potential application in tracking the degradation and distribution
of SF biomaterials, as well as a potential diagnostic tool.
Nanodiamonds (NDs) have also been used for bioimaging, as the
negatively charged nitrogen vacancy (NV−) center in NDs is a photo-
stable emitter with a bright broad fluorescence band [80]. Khalid et al.
[81] encapsulated NDs within SF nanospheres (ND-SF spheres), and
investigated their fluorescence properties and mobility in fibroblast
cells. The emission properties results showed ND-SF spheres were able
to fluoresce with 2–4 times higher brightness compared to NDs alone,
which could contribute to efficiently isolate the emission of spheres
from the background signals. Also, the biodegradable SF coating in-
creased the mobility and dynamics of NDs inside the cells, thus enabling
long-term tracking and imaging under biological environment.
3.6. Enhanced magnetic properties for drug delivery
Magnetic-sensitive biomaterials have the advantages of noncontact
control and noninvasive damage, which is suitable for site-specific and/
or time-controlled delivery [82,83]. SF-based NPs have been widely
used as drug delivery systems in cancer therapy, due to their good
biocompatibility, tunable biodegradability and controlled drug release
properties [84,85]. In order to achieve tumor-targeted drug delivery in
vivo, Tian et al. [86] prepared doxorubicin-loaded magnetic SF NPs
(DMSs) by introduction of magnetic Fe3O4 NPs (MNPs) in the one-step
potassium phosphate salting-out process. The particle generation and
drug entrapment behavior could be simply regulated by tuning the
Z. Xu et al. Materials Science & Engineering C 95 (2019) 302–311

Fig. 4. Inclusive observation (above), cross sections of regenerated nerves taken from types of nerve conduits with hematoxylin/eosin (H&E) staining (middle) and
with toluidine blue staining (below) after 5 weeks implantation. a) Defected sciatic nerve without nerve guide conduits (control group), b) Implanted SF/SWNT
conduit, c) Implanted SF/SWNT/FN conduit. d, h) Normal nerve, e, i) Negative control, f, j) Defects filled by SF/SWNT conduits, g, k) Defects filled by SF/SWNT/FN
conduits. Wide arrows: myelinated axon. Narrow arrows: Schwann cells [75]. The results showed newly regenerated nerve emerged through the conduits, and more
myelinated axons were observed compared with negative group, indicating a functional recovery of the injured nerves. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. In vivo fluorescence imaging of subcutaneously injected Silk/QD Gel for 96 h. (A–E) Images were taken at 0–4 days post injection. The mice were injected with
Silk hydrogels (left) and Silk/QD Gel (right). (F) Plot of fluorescence intensities from the Silk/QD Gel samples [78]. The incorporation of QDs into Silk hydrogels
stabilize their fluorescent property.

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Fig. 6. Schematic of representative biomedical

applications of SF-NPs composites. a: SF-AuNPs
composite nanofibers for cell spreading and pro-
liferation [42]; b: Doxorubicin-loaded magnetic
SF nanoparticles for drug delivery and tumor
targeting therapy [86]; c: SF-AgNPs composite
fabrics for antibacteria [37]; d: SF-AuNPs com-
posite conduit for nerve regeneration [76]; e: SF-
QDs composite hydrogels for bioimaging [78]; f:
Implantable conformal bio-integrated devices
based on SF and nanoscale electrodes for biomo-
nitoring [89].

Table 1
A brief summary of SF-NPs composites and their potential biomedical applications.
Forms of SF materials Types of NPs Enhanced properties Biomedical applications Ref.

Film GO Mechanical property, thermal stability Biomedical engineering [31]

Fiber and film CdTe QDs Fluorescent property Bioimaging [32]
Hydrogel LAP Osteoinductivity Bone tissue engineering [33]
Scaffold TiO2 Mechanical property, Osseo-integration Bone tissue engineering [34]
Fiber Ag Antibacterial property Antibacteria [37–40]
Nanofiber Au Mechanics, cell-material interactions Tissue engineering [42]
Fiber AgCl Antibacterial property Antibacteria [45]
Nanofiber MWCNT Mechanical property Tissue engineering [61]
Membrane GO Mechanical property Bio-nanosensing [62]
Membrane Silica Mechanical property Biosensor [64]
Scaffold HAP Osteoinductivity Bone tissue engineering [67]
Film Silica Osteoconductivity, osteoinductivity Bone tissue engineering [69]
Hydrogel Au Photothermal property Infection treatment [72]
Nanofibrils MoS2 Photothermal property Tumor treatment [73]
Film SWCNT Conductivity Neuro-regeneration [74]
Scaffold SWCNT Conductivity Nerve defect repair [75]
Scaffold Au Conductivity Nerve regeneration [76]
Membrane Au Conductivity Biosensor [77]
Hydrogel CdTe QDs Fluorescent property Bioimaging [78]
Nanosphere Nanodiamond Fluorescent property Bioimaging [80]
Nanoparticle Fe3O4 Magnetical property Cancer treatment [86]
Nanoparticle Fe3O4 Magnetical property Drug delivery [87]

amount of MNPs in the system. When DMSs were injected via tail vein
to nude mouse bearing subcutaneous tumors, under magnetic field they
were found to accumulate at the tumor sites, thus greatly inhibiting the
growth of tumors. The effective chemotherapy of multidrug resistant
cancer demonstrated that the DMSs expanded the clinical applications
of SF-based NPs, and could serve as a novel drug delivery system for
cancer treatment.
In another study, Chen et al. [87] used Methotrexate (MTX) as a
drug model and prepared MTX-loaded magnetic SF (MTX-Fe3O4-SF)
NPs, and then investigated the penetration effect of drugs under dif-
ferent magnetic fields in comparison with passive diffusion. MTX-
Fe3O4-SF NPs exhibited a mean particle size of 75 nm and a narrow size
distribution, and they could be separated into free NPs by ultra-
sonication. The application of magnetic fields improved the penetration
of drugs across the skin in vitro, as it could induce the movement of
magnetic NPs. And notably, using synergistic combination of stationary
and alternating magnetic fields would generate a massage-like effect on
the skin, thus further enhancing the permeation of the drugs, which
would greatly expand the application of transdermal drug delivery
systems.
4. Conclusions and outlooks
By directly mixing, in-situ synthesis and silkworm feeding method,
various forms of SF-NPs composites have been successful prepared with
potential or practical biomedical applications, which were illustrated in
Fig.6 and summarized in Table 1. On one hand the incorporation of NPs
endowed SF with numerous enhanced properties, on the other hand SF

309
Z. Xu et al.
provided a matrix for NPs to avoid aggregation and reduce toxicity,
thus their combination is a win-win strategy. More interestingly, one
type of NPs can improve different properties in different materials
[47,72,76], and multiple types of NPs can simultaneously improve
multiple properties for specific demands [70]. NPs with further mod-
ifications could also be achieved [42] as in AuNPs doped SF nanofibers
the integrin-binding cell adhesive peptide of RGD could be covalently
bonded to AuNPs through the free thiol on cysteine residues, leading to
promoted cellular spreading and proliferation. Nevertheless, more NPs
does not mean better outcomes both for physicochemical properties and
biological response, suggesting the importance of rational percentage of
NPs in SF materials to keep balance of structure and function. In ad-
dition, more animal studies are needed to evaluated the in vivo bio-
safety and practical effect of such SF-NPs composites, so as to facilitate
their transformation and application from lab to clinic.
As mentioned above, SF has remarkable mechanical properties,
excellent biocompatibility and controllable biodegradability, as well as
chemical versatility, which indicated its great potential for developing
sustainable and “environmentally interactive” materials [88]. Except
for conventional biomedical applications like tissue engineering and
drug delivery, SF-NPs composites can also be designed as implantable
conformal bio-integrated devices for biomonitoring [89] and wearable
sensory electronics for biosensing [90]. Zhu et al. reviewed the utili-
zation of SF in flexible electronic devices [91], which benefit from
exquisite mechanical, optical and electrical properties of SF and open
new avenues for the next-generation biocompatible devices. In the fu-
ture, we believe novel SF-NPs composites will be created by fusion of
such advanced nanotechnology, and will further expand the biomedical
applications.
Acknowledgements
The authors acknowledge the financial support from China
Agriculture Research System (CARS-18-ZJ0501) and Zhejiang
Provincial Natural Science Foundation (LZ17C170002). Z.P.X. also ac-
knowledges the financial support program from Zhejiang University.
We would also like to thank Dr. Wei Tan from University of Colorado at
Boulder and Dr. Doudou Hu from Zhejiang University for revision of the
manuscript.
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