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Folate conjugated silk fibroin nanocarriers for

targeted drug delivery†
Cite this: Integr. Biol., 2014,
6, 203
Bano Subia,a Sourov Chandra,b Sarmistha Talukdara and Subhas C. Kundu*a

Received 9th September 2013,
Accepted 31st October 2013
DOI: 10.1039/c3ib40184g
www.rsc.org/ibiology
Disease treatment processes mainly focus on the development of nontoxic, biodegradable, non-
immunogenic, biocompatible materials capable of controlled and long-term release of biomolecules. In
this work silk protein fibroin from non-mulberry tropical tasar silkworm, Antheraea mylitta, is used to
prepare nanoparticles as a drug delivery system. Folate is a vitamin, which is brought into healthy and
cancerous cells by folate receptors. The efficiency of silk fibroin–folate nanoparticles loaded with
anticancer drug doxorubicin was evaluated by analysing the cell viability, proliferation and endocytosis.
Consequently the effects of pro-inflammatory responses by cytokines such as TNF-a, IL-1b and nitric
oxide were checked by stimulating the macrophages with folate conjugated silk fibroin nanoparticles.
The fibroin–folate nanocarriers are nontoxic, easily taken up by cells and capable of sustained drug
release. Nanoparticles loaded with anticancer drug doxorubicin target cancer cells. The results show
that silk fibroin–folate nanoparticles may serve as promising nanocarriers for different biomedical and
nanotechnology applications in cancer research.

Insight, innovation, integration

Silk fibroin folate conjugated nanoparticulate system is reported for first time. Silk biomaterials have versatile properties such as biocompatibility,
biodegradability, low immunogenicity and non-toxicity. The efficiency of silk fibroin–folate nanoparticles loaded with an anticancer drug are evaluated by
analysing the cell viability, mechanism of cellular uptake, pro-inflammatory responses and pH dependent drug release. This demonstrates the broad
applicability of the silk fibroin–folate nanoparticulate system. The system facilitates interaction with the cells and retention at the site of action for a longer
period of time, which is advantageous for sustained release. The silk fibroin–folate nanoparticulate system leads to new insight for the potentiality of a carrier
for targeted drug delivery.

1. Introduction They should have properties to enhance their circulation time

In the past few years nanotechnology has been used in the
improvement of conventional chemotherapy for cancer treatment.
The fundamental advantage of nanoscale particles is that they
have a greater surface area to volume ratio. Recent advances
suggest that nanotechnology is capable of engineering and
manipulating materials at the nanoscale level. Nanoscale particles
reach at target site at a proper concentration to destroy diseased
cells while minimizing damage to normal cells.1 Nanocarriers
designed to deliver a drug at cancerous cells should be non-
toxic, non-immunogenic and with high drug loading efficiency.
a
Department of Biotechnology, Indian Institute of Technology, Kharagpur, 721302,
India. E-mail: kundu@hijli.iitkgp.ernet.in; Fax: +91-3222-27843
b
Department of Chemistry, Indian Institute of Technology, Kharagpur,
721302, India
† Electronic supplementary information (ESI) available: Video on cellular uptake
of nanoparticles. See DOI: 10.1039/c3ib40184g
in blood and actively target the cancerous cells.2 The surface of
these sub-microscopic particles can be easily modified with
targeting molecules to improve in vivo and in vitro treatment.3
Over the past decades versatile polymeric nanoparticles have
been used for drug delivery systems. Polymeric nanoparticles
have excellent biocompatibility, biodegradability, high drug
encapsulation efficiency, excellent endocytosis efficiency, passive
tumor-targeting, controlled drug release and long circulation
half life.4 Among them natural protein silk fibroin are used in
the field of nanotechnology to modify the interface of nano-
particles and control the growth of particles.5 The silk fibroin
obtained from the Indian tropical tasar silkworm Antheraea
mylitta has been used for the fabrication of nanoparticles. Silk
fibroin from this tasar silk has the versatile properties of a
natural polymer.6 The fibroin nanoparticles are helpful for the
delivery of enzymes and drugs.7 They have several active amino
groups and tyrosine residues that help the conjugation of
drug and other macromolecules.8 Coating or conjugating with

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targeting molecules to the silk fibroin overcomes the major
shortcomings of the nanoparticles by increasing the specificity
of the nanoparticles for cell surface attachment.9 This non-
mulberry fibroin is naturally gifted with RGD sequences which
interact with surface integrin molecules and enhance cellular
attachment.10,11 The addition of RGD with this fibroin is
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required for improving the cell attachment efficiency.
Folic acid/folate is conjugated with fibroin to further
increase the specificity and targeted delivery of nanoparticles.
The fundamental role of folic acid is essential for nucleotide
synthesis that promotes rapidly proliferating cells and tissue.3
This includes advanced stages of cancer, and it is also critical
for the ‘‘1-carbon pathway’’, a key initiating step in the synthesis
of nucleotide precursors as building blocks for DNA synthesis in
the S-phase of the cell cycle.3 Folic acid/folate is often selected as
a model molecular probe for the targeted delivery of drugs to
cancer cells.12 However, active targeting of membrane bound
tumor associated receptors is achieved by the conjugation of
nanoparticles with folate.12 Recently, the folate mediated target-
ing technique has been established as a non-invasive approach
for the detection of cancers. The cancerous cells have an inherent
property of overexpressing folate receptors due to their enormous
requirements for folate.13 Tumor selective targeting is achieved
by combining folic acid conjugates with liposomes/nanoparticles
to enhance their uptake and targeting ability.14 Folate receptors
have high-affinity membrane folate-binding proteins, which
enables them to rapidly bind and be internalized by the process
of endocytosis15 or through a hypothetical pathway involving
endocytosis proposed for targeted delivery.16 Recently it has been
proposed that folate receptor mediated delivery can enhance
cellular uptake, sustained drug release and promote effective
cytotoxicity towards cancer cells both in vitro and in vivo.17
Doxorubicin is used as a model anticancer drug and is a
member of the anthracycline ring antibiotics.18 It is crucial for
the treatment of acute leukaemia, malignant lymphoma and
breast cancer.19 This drug has a narrow therapeutic index and its
clinical use is hampered by several side effects such as cardio-
toxicity and myelosuppression.20 To reduce the side effects of the
drug attempts are being made to deliver the drug to its target
sites by nanoparticle carriers, which are receiving much atten-
tion for targeted drug delivery.
Nanocarriers of silk fibroin–folate–doxorubicin conjugates have
been fabricated as a targeted drug delivery system to minimize the
risk of drug toxicity and increase drug efficacy. It is hypothesized
that doxorubicin conjugated to nanocarriers through linkers could
be readily delivered into cells by an endocytosis mechanism.
Within the acidic endosomal compartment the doxorubicin may
be cleaved in an intact form from the prodrug complex.21 Based on
this hypothesis, we have designed and synthesized a prodrug com-
plex, i.e., silk fibroin–folate–doxorubicin by using folic acid as a
targeting agent. Fibroin is a biocompatible, hydrophobic carrier,
and forms a pH-sensitive linkage with the anticancer drug doxo-
rubicin. The major goal of cancer therapy is to achieve the active
targeting of cancer cells and minimize drug side effects.
In this work, to achieve effective tumor targeting, silk fibroin–
folate nanoparticles are synthesized, characterized and investigated
204 | Integr. Biol., 2014, 6, 203--214
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as a targeted drug carrier system. The doxorubicin release profile is
examined as a function of pH with an emphasis on the acid
cleavability. The tumor cell targeting, endocytosis mechanism of
cellular uptake and antitumor activity of the prodrug are examined.
2. Materials and methods
2.1 Materials
Mature late 5th instar larvae of non-mulberry tropical tasar
silkworm Antheraea mylitta were collected from our experi-
mental farm, folic acid (Sigma-Aldrich), cellulose tubing of
MWCO 12 kDa (Pierce, USA), acetone (Merck), glutaraldehyde,
doxorubicin, DMSO (dimethyl sulfoxide), MTT (3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide), rhodamine B
isothiocyanate (RITC), 2,2 0 -(ethylenedioxy)bis(ethylamine) (EDBE),
N-hydroxysuccinimide (NHS), cytochalasin B, nystatin (Sigma),
sodium azide (Merck) and a live/dead viability/cytotoxicity kit
(Invitrogen), were purchased for these experiments.
2.2 Isolation of silk fibroin protein from Antheraea mylitta
The fully-grown 5th instar larvae were dissected for extraction of the
silk protein fibroin following the earlier described method.6 The
non-bioengineered silk fibroin was isolated from the silkworms
just prior to spinning into cocoon fibers. In brief, the posterior silk
glands were isolated and washed in deionized water to remove
traces of sericin. The glandular tubes were then squeezed with fine
forceps to extrude out the protein. Isolated silk fibroin protein was
dissolved using a mild anionic surfactant SDS following the
standard method. Excess surfactant was removed by dialysis
against water using dialysis membranes (12 kDa). Fibroin
solution was collected and the concentration was determined
by weighing the remaining solid after drying at 60 1C.
2.3 Fabrication of silk fibroin nanoparticles
Silk fibroin protein nanoparticles were prepared by a desolvation
technique using acetone as the desolvating agent. To induce the
desolvation process, 20 ml of acetone were added in a small
container and regenerated silk fibroin solution (2%, w/v) was
added dropwise (50 ml per drop) with constant stirring. After
desolvation, the formation of silk nanoaggregates was visible in
the form of precipitates. Nanoaggregates were then centrifuged at
16 000 rpm for 20 min to collect the precipitates. SF precipitates
were then further purified with repeated centrifugation at
16 000 rpm for 15 min with deionized water. The purified nano-
particle pellets were then redispersed in deionized water and soni-
cated. The nanoparticles were filtered through a 0.45 mm syringe
and lyophilized to obtain freeze-dried nanoparticles for further use.
2.4 Synthesis of silk fibroin–2,2 0 -(ethylenedioxy)-
bis(ethylamine) conjugates (SF–EDBE)
10 mg silk nanoparticles was completely dispersed into 40 ml of
water with the assistance of a bath sonicator for 1 h. Then the
particles were activated with 5 mg EDC–NHS (1 : 1) at pH 6 with
constant stirring for 3 h in the dark. After that 1 ml of EDBE was
added dropwise into the above reaction mixture and the stirring
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was again continued for 10 h. EDBE conjugated SF NPs were
separated out by centrifugation at 4000 rpm for 10 min. Finally
the product was washed with deionized water several times to
remove excess EDBE followed by lyophilization for further use.
2.5 Synthesis of silk fibroin–2,2 0 -(ethylenedioxy)bis-
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(ethylamine)–folate nanoconjugates (SF–EDBE–FA)
10 mg of folic acid was sonicated in 10 ml of 1 : 1 aqueous
DMSO for 30 min. After that the dispersion was activated by using
5 mg EDC–NHS (1 : 1) at pH 6 with vigorous stirring for 3 h in a
closed dark container. After activation, the aqueous dispersion of
folic acid was mixed dropwise into the amine functionalized silk–
EDBE nanoparticles. The pH of the mixture was adjusted to 8 by the
addition of a small amount of pyridine. Stirring was again continued
for 10 h and finally the product (silk–EDBE–FA) was isolated by
centrifugation at 4000 rpm for 10 min followed by washing several
times with deionized water and collection after lyophilization.
2.6 Particle characterization
Folate conjugated silk fibroin nanoparticles were analyzed by
transmission electron microscopy (TEM) for measurements of
size and surface morphology. TEM samples were prepared by
putting 10 ml of diluted nanoparticles after dispersion in
deionized water. The grid containing nanoparticles was air dried
in a dust free environment at room temperature. Then the samples
were analyzed under JEOL-JEM 2100, TEM at 80 kV (voltage). The
average surface charge and polydispersity index were determined
using dynamic light scattering (DLS). For the particle size analysis
nanoparticles were resuspended in deionized water and sonicated.
Particle stability and dispersity were calculated from the poly-
dispersity index and zeta potential and data were collected from
several measurements (n = 3).
2.7 Fourier transform infrared spectroscopy (FTIR)
The conformation transitions of lyophilized silk fibroin, and
folate conjugated silk-fibroin nanopowders were obtained by
the use of Perkin Elmer FTIR spectroscopy in the spectral
region 500–4000 cm1 at the resolution of 4 cm1 in KBr pellet.
All the samples were analyzed in transmission mode.
2.8 Synthesis of silk fibroin–2,2 0 -(ethylenedioxy)bis-
(ethylamine)–rhodamine isothiocyanate (SF–EDBE–RITC)
1 mg of rhodamine isothiocyanate (RITC) was dissolved in 1 : 1
aqueous DMSO and added dropwise into an aqueous dispersion
of silk–EDBE (5 mg in 0.1 (M) sodium bicarbonate solution) with
continuous stirring. The pH of the solution was kept at 8 and
stirring was continued overnight in the dark. Finally SF–EDBE–
RITC was recovered by centrifugation at 4000 rpm for 10 min and
washed several times with deionized water to remove excess RITC.
2.9 Synthesis of folate conjugated silk fibroin–2,2 0 -
(ethylenedioxy)bis(ethylamine)–rhodamine isothiocyanate
labelled nanoparticles (SF–EDBE–RITC–FA)
After conjugation with RITC, SF–EDBE–RITC and folic acid (FA)
were dissolved in 1 : 1 aqueous DMSO separately. Folic acid was
then activated with the addition of 1 : 1 EDC–NHS and the
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mixture was stirred for 3 h in the dark. After that silk–EDBE–
RITC was added dropwise to the above solution and the pH was
adjusted to 8 by the addition of a small amount of pyridine. The
reaction mixture was stored in the dark and stirring was
continued for 10 h. Finally the product (silk–EDBE–RITC–FA)
was isolated by centrifugation followed by washing with deionized
water several times.
2.10 Drug loading and entrapment
2.5 mg Doxorubicin (DOX) was loaded into 10 ml of an aqueous
(deionized water) dispersion of folic acid conjugated silk nano-
particles (25 mg), with mixing along with continuous stirring
for 12 h in the dark. The drug (DOX) loaded silk–EDBE–FA nano-
particles were separated out by centrifugation at 10 000 rpm for
10 min. The particles were then washed thrice with deionized
water to remove excess DOX molecules. Finally the drug loading
was evaluated spectrophotometrically by measuring the absor-
bance at 482 nm of a standard DOX solution and the super-
natant respectively.
2.11 Cumulative drug release
Cumulative drug release from silk fibroin–EDBE–FA was studied
at two different pH values. pH 4.5, which is comparable to the
lysosomal pH of cancerous cells and pH 7.4, the extracellular
physiological pH of normal cells. Briefly, 3 mg of the DOX loaded
SF–EDBE–FA was dispersed in 2 ml of buffer solution and sealed
inside a dialysis bag (molecular cut-off 50 kDa), which was then
subsequently immersed in 50 ml of external buffer solution
(PBS). Dialysis was further continued at 37 1C and drug release
was monitored spectrophotometrically by measuring the absor-
bance of the external buffer at 482 nm. The results of triplicate
test data were used to calculate cumulated drug release.
2.12 Cell culture
Human breast adenocarcinoma cell line (MDA-MB-231) was
used for intracellular localization and cytotoxic studies. Breast
adenocarcinoma cells were maintained inside an incubator at
37 1C with air supplemented with CO2 (5%). The cells were
cultured in DMEM with FBS (10%) and antibiotics (1%) and
incubated for 24 h. Cultures were split when they reached 70–80%
confluence with medium change every 2–3 days in between
culturing.
2.12.1 Intracellular localization. To observe the folate conju-
gated silk fibroin nanoparticles, human breast adenocarcinoma
cells (MDA-MB-231) were incubated with nanoparticles. Briefly,
2  104 cells per well were seeded into 24 well culture plates in
DMEM culture medium (FBS 10% and antibiotics 1%) for 24 h.
After that the growth medium was removed and replaced with
fresh medium containing RITC loaded SF–FA incubated for 48 h.
RITC loaded pure SF nanoparticles were taken as a control.
Specific uptake of RITC labelled folate conjugated silk fibroin
nanoparticles by breast cancer cells (MDA-MB-231) was further
evaluated in a competitive inhibition assay. RITC labelled SF–FA
conjugated nanoparticles were incubated with 2 mM of free folic
acid and incubated for 12 h. Blocking of the folate receptor with
an excess of free folate was analysed by confocal microscopy.
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In the absence of free folate, RITC labelled silk fibroin folate
nanoparticles were taken as control.
2.12.2 Confocal microscopy. Attachment and spreading of
MDA-MB-231 was observed using confocal laser microscopy.
Cells were fixed with 4% formaldehyde for 15 min. The samples
were washed with PBS and treated with 0.1% Triton-X for
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membrane permeabilization. After that the whole constructs
were washed with PBS and treated with 1% bovine serum
albumin (BSA) for 30 min. The cell nuclei were stained with
Hoechst 33342 (5 mg ml1) for 30 min. Samples were imaged
using confocal laser scanning microscopy (CLSM, Olympus FV
1000 equipped with an inverted microscope IX 81, Japan) equipped
with argon (488 nm) and HeNe (534 nm) lasers. Two-dimensional
multichannel image processing was done using FV 1000 Advance
software version 4.1 (Olympus, Japan).
Video image analysis. Movies of SF–FA conjugated nano-
particle uptake analysis by MDA-MB-231 were recorded using
confocal laser microscopy at different time intervals.
2.12.3 Nanoparticle uptake in the presence of endocytosis
inhibitors. Known inhibitors of endocytosis were used to elucidate
the mechanism of cellular uptake of silk fibroin–folate conjugated
nanoparticles. Human breast adenocarcinoma cells were seeded
in 24 well culture plates at the density of 1  104 cells per well and
grown to 70% confluence. The cells were pre-incubated with the
inhibitors, NaN3 (0.1%), nystatin (15 mg ml1), and cytochalasin D
(5 mg ml1) for 1 h. The cells were washed 3 with cold sterile
PBS. Following this, the cells in each well were resuspended in
1 ml DMEM and incubated with folate conjugated nano-
particles (200 mg ml1) for 6 h. The mechanism of endocytosis
was elucidated with confocal laser microscopy.
2.12.4 Cell viability and cytotoxicity. MTT (3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used
to measure the cell cytotoxicity. Human breast adenocarcinoma
cells (MDA-MB-231) were seeded at the desired density 2 
104 cells per well into 24 well plates at 5% CO2 at 37 1C. The
cells were pre-incubated on tissue culture plates (TCP) for 24 h
to allow cell attachment. The culture medium was removed and
replaced with incomplete medium. Following this, nanoparticles
with different concentrations (100, 250 and 500 mg ml1) were
added and incubated for 48 h. After that 100 ml of MTT dye
solution was added (5 mg ml1 MTT in PBS at pH 7.4) in each
well and incubated for 4 h. The MTT solution was then removed
and 500 ml of DMSO was added in each well to solubilize the
formazan crystals. The absorbance of 96 well tissue culture
plates was recorded with a microplate reader at 595 nm.
2.12.5 Cytotoxicity. Doxorubicin conjugated silk fibroin–
folate nanoparticle uptakes were evaluated using cancer cells.
The cancer cells with a density of 2  104 cells per well were
seeded in 24 well tissue culture plates. After 24 h, culture
medium was replaced with incomplete culture medium to starve
the cells. Nanoparticles with different drug concentrations
(0.1, 1 and 10 mg ml1) were added into fresh medium (10% FBS
and 1% antibiotics) and incubated for 48 h in a humidified
atmosphere containing 5% CO2 at 37 1C. After that, an MTT
assay was carried out to evaluate the cytotoxicity of the DOX
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loaded nanoparticles. 100 ml of MTT solution (5 mg ml1 in PBS
pH 7.4) was added in each well plate and incubated for 4 h.
Following this, the MTT solution was removed and 500 ml
of DMSO was added to dissolve the formazan crystals. The
absorbance of samples was measured by a microplate reader
at 595 nm.
2.12.6 Flow cytometry analysis. MDA-MB-231 breast cancer
cells were seeded at a density of 1  106 cells per well and
incubated at 37 1C inside an incubator. The cells were pre-
incubated on TCP for 24 h to allow cell attachment. Culture
medium was removed and replaced with incomplete medium to
starve the cells. Following this cells were incubated with nano-
particles loaded with pure silk fibroin (500 mg ml1), silk fibroin–
doxorubicin (10 mg ml1) and pure doxorubicin (10 mg ml1) for
12 h. Wells without nanoparticles were taken as the control. After
that, the cells were harvested by addition of 1 ml of 0.25%
trypsin–EDTA to the samples. They were then incubated at 37 1C
in an incubator until all cells were detached from the flask. The
trypsinized cell suspension was neutralized with 3 ml complete
medium, followed by centrifugation at 1000 rpm for 10 min. The
cell pellet was resuspended in cold PBS and centrifuged for
10 min at 1000 rpm. To the cell pellet 70% chilled ethanol was
added in shaking conditions and incubated at 4 1C. The cell
pellet was resuspended in 200 ml PBS containing 0.1 mg ml1
RNase and 40 ml of PI solution (1 mg ml1 PI) and incubated for
30 min at 37 1C. The resultant cell suspension was analyzed with
a flow cytometer (FACS Calibur, BD using Cell Quest Pro soft-
ware) at 488 nm excitation and a 560 nm band pass filter for red
fluorescence of propidium iodide.
2.12.7 Live/dead assay. A live/dead assay was performed for
the analysis of cell viability after cellular uptake of nanoparticles.
Pure SF–FA nanoparticles without drug and nanoparticles conju-
gated with DOX drug were analyzed in the live/dead assay. Samples
were stained with calcein and ethidium bromide to visualize the
populations of live and dead cells, after 48 h incubation of nano-
particles. Briefly, nanoparticle encapsulated cells were incubated at
37 1C in PBS containing calcein AM and ethidium homodimer-1
(Molecular Probes) for 20 min to stain viable and non-viable cells,
respectively. The samples were washed thrice with PBS for 10 min
each, and imaged by confocal microscopy.
2.13 Determination of TNF-a, IL-1b and nitric oxide (NO)
production
The immunogenicity of the SF–FA nanoparticles was evaluated
by in vitro quantitative determination of human tumour necrosis
factor alpha (TNF-a), IL-1b and NO concentrations in cell culture
supernatant. Two sets of experiments were designed to analyze
in vitro immune response with different SF–FA concentra-
tions (100, 250 and 500 mg ml1) for long and short term
culture (1 and 7 days). Briefly macrophage cells with density
1  105 cells per well were seeded into TCP and incubated for
48 h. After 48 h of cell seeding, the complete medium was
replaced with incomplete medium. Nanoparticles with concen-
trations 100, 250 and 500 mg ml1 were added into each well to
stimulate the macrophages. The cell supernatant was collected
to analyze TNF-a and IL-1b levels using TNF-a and IL-1b
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quantification kits (Invitrogen). Data accumulations were carried
out at 450 nm and 600 nm using a microplate reader. To quantify
the NO production cell supernatants were collected and incubated
with Griess reagents (Sigma) for 10 min. Data accumulations were
done at 548 nm and 600 nm using a microplate reader.
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2.14 Statistical analysis
Statistical differences were performed using one-way ANOVA.
Data are presented as mean  SD. p-Values o0.05 were con-
sidered as statistically significant.
3. Results
3.1 Surface properties of silk fibroin–folate conjugated
(SF–FA) nanoparticles
Protein based nanoparticles were fabricated by a desolvation
method followed by conjugation with folate by successive steps.
The size and zeta potential of SF–FA conjugated nanoparticles
were determined using transmission electron microscopy and a
dynamic light scattering particle size analyzer respectively. TEM
images show that the particles are monodisperse and o200 nm
in diameter. The particles are almost uniform and spherical in
shape (Fig. 1). The stability of the particles is confirmed from
their zeta potentials. The surface potentials of SF and SF–FA
conjugated nanoparticles are 22  5 and 32  3 respectively.
3.2 FTIR analysis of nanoparticles
The FTIR spectrum of silk fibroin (Fig. 2a) illustrates the
presence of an amide A band (3296 cm1) along with amide I,
Fig. 1 TEM pictographs of (a) silk protein fibroin nanoparticles and (b) silk
fibroin–folate nanoparticles.
Fig. 2 FTIR spectra of (a) pure silk fibroin; (b) silk fibroin–2,2 0 -(ethylenedioxy)bis(ethylamine) nanoconjugates (SF–EDBE); and (c) silk fibroin–2,2 0 -
(ethylenedioxy)bis(ethylamine)–folate nanoconjugates (SF–EDBE–FA).
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II and III bands (1630, 1516, 1231 cm1). No characteristic
peak for carboxylic acid is observed in such silk fibroin as
these are fewer in comparison to the amide groups. Only the
terminal part of the silk fibroin contains a –COOH moiety,
which is used for the conjugation of folic acid. Fig. 2b
reveals the FTIR spectrum of silk fibroin after treatment with
2,2 0 -(ethylenedioxy)bis(ethylamine), in which the peaks for
amide I and II are shifted to higher wavelengths (1652 and
1524 cm1 respectively). An additional broad peak at 3417 cm1
clearly signifies the presence of 11 amine (–NH2) groups
on the surfaces of those particles. Finally Fig. 2c illustrates
the conjugation of folic acid with silk fibroin–EDBE nano-
particles revealing the formation of amide bonds in the
presence of EDC–NHS acting as the coupling agent. The amide
I, II and III bands in silk fibroin–EDBE–FA are recorded at 1637,
1524, and 1239 cm1 respectively. The absence of free amine in
silk fibroin–EDBE–FA suggests that all –NH2 groups are con-
verted to amide bonds after conjugation with the –COOH
groups of FA. A large and broad peak at 3437 cm1 confirms
the presence of –OH groups in SF–EDBE–FA coming from FA
(Fig. 2a–c).
3.3 Cellular uptake and localization of silk fibroin–folate
nanoparticles on cancer cells
In this study, it was hypothesized that cellular uptake of folic
acid is enhanced by conjugation of folic acid with SF nano-
particles. The cellular binding and uptake of rhodamine iso-
thiocyanate (RITC) labelled folate silk fibroin nanoparticles
were analyzed on human breast adenocarcinoma cells by con-
focal laser microscopy. The carboxylic group of folate is
covalently conjugated with the amino group of silk fibroin by
using the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC) coupling method. Uptake efficiencies of RITC labelled
pure silk fibroin nanoparticles and covalently folate conjugated
silk fibroin nanoparticles were analyzed at different incubation
periods (0 and 24 h). RITC labelled pure silk fibroin nano-
particles were chosen as a control (Fig. 3A). However, cellular
uptake of RITC labelled silk fibroin nanoparticles by cancer
cells was analysed after 12 h. It showed that in the presence of
competitive inhibitor (free folate, 2 mM), nanoparticle binding
to the folate receptor is less (Fig. 3B). Whereas the same
nanoparticles effectively bind with cancer cells in the absence
of competitive inhibitor (free folate).
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Fig. 3 (A) Confocal images of human breast adenocarcinoma cells incubated with 1 mg ml1 of RITC labelled nanoparticles (a–d) 0 h; (e–h) RITC
labelled pure silk fibroin nanoparticles; and (i–l) RITC labelled silk fibroin–folate nanoparticles. Represents cell attachment, proliferation and viability at
37 1C. Scale bars represent 50 mm. (B) Confocal laser microscopy images after 12 h incubation of RITC labelled silk fibroin folate nanoparticles with
MDA-MB-231 cells, in (a) excess free folate (2 mM); (b) the absence of free folate. Scale bars represent 50 mm diameter. (C) Blocking the cellular uptake of
silk fibroin–folate nanoparticles by using endocytosis inhibitors. The cells are pre-treated with endocytosis inhibitors for 1 h, followed by addition of silk
fibroin–folate nanoparticles for 6 h (a) control; (b) RITC labelled SF–FA nanoparticles; (c) NaN3; (d) cytochalasin B; (e) nystatin; (f) mean fluorescence
intensity of nanoparticles after treatment with endocytosis inhibitors. Scale bars represent 50 mm diameter.

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3.4 Nanoparticle uptake mechanism
A variety of endocytosis mechanisms operated in cells includ-
ing calthrin-mediated, caveolin-mediated, calthrin or caveolin
independent endocytosis and macropinocytosis. The cells are
pre-incubated with various endocytosis inhibitors and incu-
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bated with silk fibroin–folate nanoparticles. Nanoparticle entry
into cells under these conditions is evaluated by measuring the
fluorescence intensity. To investigate the processes, MDA-MB-
231 cells are pre-incubated in the presence of sodium azide
(energy dependent endocytosis), nystatin (calthrin or caveolin
mediated endocytosis), cytochalasin D (macropinocytosis) and
then treated with SF–FA nanoparticles. An inhibited inter-
nalization is observed after treatment with these inhibitors.
The result shows that the internalization of nanoparticles into
breast cancer cells pre-treated with these inhibitors is compara-
tively less than non-treated cells (Fig. 3C).
3.5 Live/dead assay
To study the biocompatibility of silk fibroin–folate conjugated
nanoparticles and doxorubicin (DOX) loaded SF–FA nano-
particles, live/dead assays were performed on MDA-MB-231 cells.
SF–FA nanoparticles with concentration (100, 250 and 500 mg ml1)
and DOX loaded SF–FA nanoparticles with concentration
(0.1, 1.0 and 10 mg ml1) were used for live/dead assays
respectively. From Fig. 4a–d it is clearly observed that a large
percentage of live cells are observed on SF–FA NPs with
concentration 100, 250 and 500 mg ml1. This indicates that
nanoparticles are nearly non-toxic to the cells (Fig. 4a–d).
Similarly, SF–FA–DOX conjugate nanoparticles are able to
effectively target the cancer cells (Fig. 4e–h). In this study, the
loading ratio of DOX is evaluated as 1.0 mg per mg of SF–FA
nanoparticles. The results suggest that with increasing drug
concentration, cell viability decreases in comparison to non-
treated cells (Fig. 4e–h).
Fig. 4 Live/dead assay on human breast cancer cells incubated with silk fibroin–folate nanoparticles with different concentration: (a) control; (b) 100 mg ml1;
(c) 250 mg ml1; (d) 500 mg ml1 and doxorubicin loaded silk fibroin folate nanoparticles with different drug concentration: (e) control; (f) 0.1; (g) 1.0;
(h) 10 mg ml1 after 48 h of incubation, represents cell attachment, proliferation and viability at 37 1C. Scale bars represent 100 mm.
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3.6 Cytotoxicity assay
In vitro cytotoxicity assays of SF–FA and DOX loaded SF–FA NPs were
carried out on human breast adenocarcinoma cells. The cytotoxi-
cities of folate conjugated silk fibroin nanoparticles with different
concentrations (100, 250 and 500 mg ml1) were evaluated by MTT
assay. The results demonstrate that the cell viability and prolifera-
tion are not significantly affected by different nanoparticle concen-
trations (Fig. 5b). In contrast, cell inhibitory effects were evaluated
with free drug (DOX) and drug loaded nanoparticles (DOX–SF–FA)
with different drug concentration (0.1, 1.0 and 10 mg ml1) on
human breast cancer cells. Cell viabilities were determined by MTT
assay. The MTT assay data more likely suggest a change in the cell
metabolic activity or enzyme activity in the mitochondrial respira-
tory chain in treated cells. Free DOX and DOX–SF–FA nanoparticles
show dose dependent cytotoxicity (Fig. 5c).
3.7 Cell cycle analysis
As shown in Fig. 6, SF–FA–DOX creates cytotoxicity in breast cancer
cells, whereas the effect of SF–FA nanoparticles is almost similar
to that of the control. Doxorubicin creates immediate effects on
breast cancer cells (10 mg ml1), while SF–FA conjugated DOX
creates slow and effective cytotoxicity. Thus, the maximum of the
cells seems to be influenced by the cytotoxic effect of doxorubicin
released from the folate conjugated silk fibroin nanoparticles.
FACS showed that the SubG0 of the apoptotic cells was 0.76%,
4.28%, 3.50%, 25.85% for the control, SF–FA, SF–FA–DOX nano-
particles and pure DOX respectively. On the other hand the
proportion of cells in the G1 phase was 79.47%, 70.23%, 13.21%,
43.21% and in the G2 phase of the cell cycle was 9.03%, 13.55%,
65.27%, and 8.72% respectively in all four groups.
3.8 Cytokine production assay
To analyze the immunogenic response of silk fibroin folate
nanoparticles mouse bone marrow macrophages were used.
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Fig. 5 (a) Schematic representation of the mechanism of action of doxorubicin (DOX) conjugated nanoparticles on cancer cells (modified from
Mansoori et al., 2010, ref. 14); (b) viability of human breast adenocarcinoma cells after 48 h exposure to silk fibroin–folate nanoparticles with different
concentration (100, 250 and 500 mg1 ml); and (c) cytotoxicity effect of doxorubicin loaded pure silk fibroin and silk fibroin–folate conjugated
nanoparticles with different drug concentration (0.1, 1, 10 mg ml1) on human breast cancer cells after 48 h exposure as determined by MTT assay.
The data represent mean  SD (n = 3), * represents statistically significant differences (p o 0.05).

Fig. 6 Flow cytometry analysis of cell phase distribution: (a) control; (b) SF–FA nanoparticles (500 mg ml1); (c) pure DOX (10 mg ml1); (d) SF–FA–DOX
NPs (10 mg ml1); (e) overlay of SF–FA NPs and DOX; (f) overlay of SF–FA–DOX NPs and pure DOX incubated with cancer cells for 12 h, determined by
flow cytometry using FACS Diva software after the cells were labelled by PI preceding RNase treatment and the percentage of apoptosis was calculated.

These macrophages constitute a homogeneous population of
primary cells that respond to proliferate or activating stimuli
under some circumstances by becoming apoptotic. The pro-
inflammatory response of the cells with nanoparticles was
studied by the production of cytokines such as TNF-a, IL-1b and
NO by using ELISA kit. Briefly, an immune response study has
done with SF–FA nanoparticles with concentration (100, 250 and
500 mg ml1) for short and long day culture periods (1 and 7 days).
For nitric oxide production, a macrophage stimulation assay
was observed by using Griess reagents. It clearly indicated that
RAW 264.7 cell stimulation with SF–FA shows minimal immune
response, while lipid polysaccharide (LPS) reflects high cytokine

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Fig. 7 (a) TNF-a production by RAW 264.7 murine macrophages; (b) IL-1b production; and (c) nitric oxide (NO) production. RAW 264.7 cells were
stimulated with silk fibroin–folate (SF–FA) nanoparticles with different concentrations (100, 250, 500 mg ml1). Lipopolysaccharide from Escherichia coli
was added (100 ng ml1) to each experimental set as a positive stimulant and media as a negative control. Error bars represent the standard error of the
mean (n = 3).
production (Fig. 7a–c). LPS was chosen as a positive control for
an inflammatory response due to its known ability to activate
antigen presenting cells.
3.9 In vitro drug release
The administration of chemotherapeutic drugs needs a proper
delivery system to achieve effective killing of cancer cells. The
encapsulation of anticancer drugs with nanoparticles can over-
come the issues and help in localization within a carcinoma and
stimulating the uptake.22 The use of polymeric nanoparticles
as drug delivery vehicles is gaining importance in biomedical
applications. They enable the encapsulation and successful
delivery of drugs with poor aqueous solubility profiles such as
antitumor agents. In vitro release studies were conducted at
pH 7.4 and pH 4.5 by using PBS as a release medium to simulate
the physiological pH and endosomal pH of tumour cells res-
pectively. The release medium shows a typical release profile
followed by a sustained release pattern. Fig. 8 represents the
in vitro cumulative release profile of doxorubicin at different
Fig. 8 Release profile of doxorubicin from doxorubicin–silk fibroin–folate
nanoparticles in PBS solution at pH 7.4 and 4.5 at 37 1C, observed for a
period of 21 days.
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pH values. The patterns reveal that the release profile of
doxorubicin varies with pH. The overall release of the drug is
much higher at pH 4.5 than at pH 7.4.
4. Discussion
Our aim is the conjugation of folate with silk protein fibroin
(SF) to accomplish targeted drug delivery. However, in this work
for effective drug targeting SF is conjugated with amine functional
groups. The folate is conjugated with amine functionalized
nanoparticles by using carbodiimide chemistry by free radical
polymerization.23 Recent studies have shown that the smallest
silk fibroin–folate conjugated (SF–FA) nanoparticles (B200 nm)
with higher zeta potential take advantage of the enhanced
permeability retention effect and circulate in the intercellular
spaces of blood capillaries without any aggregation.24 Spherical
shaped nanoparticles experience faster uptake than rod shaped
nanoparticles.25
However, the cellular uptake and fluorescence of folate
decorated silk fibroin nanoparticles is higher in comparison
to pure silk fibroin (Fig. 3A and Movie S1 and S2, ESI†).
Fluorescent labelled SF–FA nanoparticles get internalized by
the cells through folate receptors.26 The particles reside in the
cytoplasm and perinuclear space of the cells and their fluores-
cent intensity and internalization increases with time. It has
been proposed that folate is often a limiting nutrient in human
serum; up-regulation of high affinity FRs on cancer cells may
enable malignant cells to compete more aggressively for the
vitamin.27 Hence, folate not only increases the retention of the
nanoparticles at the tumour site but also promotes cellular
uptake in drug delivery systems.28 The involvement of folate
cell surface receptors in the binding and cellular uptake of silk
fibroin nanoparticles with cancer cells is validated by the
blocking of folate receptors with excess folic acid. It seems to
be interesting that uptake of the silk fibroin folate nano-
particles is much less in the presence of competitive inhibitor
(free folate). Whereas in the absence of free folate, the binding
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affinity of RITC labelled folate conjugated silk fibroin nano-
particles to the cancer cell is high. On the basis of that we
conclude that excess free folate (competitive inhibitor) blocks
the folate receptors. It inhibits the binding and cellular uptake
affinity of folate conjugated silk fibrin nanoparticles to the
receptor sites of cancerous cells.
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However, endocytosis inhibitors are used for the further
assessment of the cellular uptake mechanism of nanoparticles.
Endocytosis inhibitor, sodium azide, blocks cellular ATP
synthesis.29 In this result a remarkable internalization inhibi-
tion effect is observed. However, the inhibitory internalization of
nanoparticles into breast cancer cells pre-treated with nystatin
and cytochalasin D remains approximately the same.30 On the
basis of the above experimental results, we conclude that the
admission of silk fibroin–folate nanoparticles into breast cancer
cells is predominated by endocytosis (Fig. 3B). It seems that
none of the specific inhibitors leads to greater than 90% inhibi-
tion of internalization.
Biocompatibility and non-toxicity of SF–FA conjugated nano-
particles are the other considerations for targeted drug delivery.
Live/dead and cell viability assays evaluate that nanoparticles
are nearly non-toxic and compatible with cells ( p o 0.05)
(Fig. 4a–d and 5b). However, DOX loaded SF–FA nanoparticles
create toxicity to human breast cancer cells (Fig. 4e–h). It seems
to be interesting that folate conjugated silk fibroin nano-
particles are nearly less toxic to fibroblast cells. While DOX
conjugated silk fibroin folate nanoparticles (DOX–SF–FA) create
cytotoxicity to cancer cells, with a neutral effect on fibroblast
cells (Fig. S4b, ESI†). This might be due to the presence of folate
receptors on cancer cells that promote/increase the cellular
uptake of doxorubicin loaded nanoparticles.
The in vitro cell inhibitory effects of DOX loaded SF–FA
conjugated nanoparticles show superior cytotoxic activity as
compared to free DOX ( p o 0.05). The IC50 value (50%
inhibitory concentration) for DOX loaded nanoparticles is lower
(IC50 0.611  1.05 mg ml1) in comparison to free DOX ( p o 0.05).
SF–FA nanoparticles enhance 3-fold (approximately) cytotoxicity of
DOX in comparison to free DOX. The efficacy of DOX-loaded
SF–FA nanoparticles is due to ready internalization of nano-
particles by endocytosis compared to a passive diffusion mecha-
nism of free DOX.31 However, nanoparticles enhance the uptake
and retention of DOX at the effective site. Fig. 5a shows a
hypothetical representation of the internalization and diffusion
of DOX loaded SF–FA nanoparticles in cancer cells. The cell
viability of human breast adenocarcinoma cells is decreased;
this may be due to apoptosis induction and inhibition of the
cell cycle. It has already been reported that DOX prevents
cancer cell growth by inducing DNA breakage as well as cell
cycle arrest.32 It arrests the cell cycle at the G2/M phase.33 It is
observed that both SubG0/G1 and G2/M phases increase in
DOX loaded NP treated cells with respect to a control at 12 h
incubation of cancer cells as shown in Fig. 6. On the other hand
G1 and G2 phases decrease in a dose dependent way without
significant alteration in the S phase. Pure DOX showed cell
cycle arrest in the G1 phase, whereas DOX loaded SF–FA
nanoparticles arrest the cell cycle in the G2 phase. The results
212 | Integr. Biol., 2014, 6, 203--214
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signify that SF–FA conjugated with doxorubicin more effectively
release the drug as compared to pure DOX. Doxorubicin release
from the SF–FA matrices is most likely due to the presence of
enzymes and pH variations. It accelerates degradation of
matrices and allows the diffusion of DOX from the matrix
and exhibits cytotoxicity.34,35 DOX conjugated nanoparticles
may enhance cell internalization and lead to the delivery of
drug closer to the intracellular site of action.36 These findings
suggest that SF–FA conjugated with doxorubicin might effec-
tively kill cancer cells. Although further studies are needed to
understand the exact mechanism of toxicity of the drug loaded
nanoparticles against the cancer cells. However, more detailed
investigations are needed to further explore the mechanism of
DOX conjugated silk fibroin and DOX conjugated silk fibroin
folate nanoparticles through cellular uptake, intracellular
trafficking, and the cytotoxic mechanism of DOX delivered by
this system.
Interactions of blood occur when drug loaded nanoparticles
are inoculated intravenously and come in contact with macro-
phages. By internalization of exogenous substrate macrophages
could secretes cytokines, chemical messengers for regulating
innate and adaptive immune systems.37 Their concentrations
are low in cells in the absence of exogenous stimuli but become
synthesized de novo when the cell is activated.38 Stimulation of
macrophages with SF–FA nanoparticles failed to respond with
consistently elevated levels of TNF-a, IL-b and NO production
(Fig. 7a–c). Macrophages cultured in the presence of SF–FA
nanoparticles did not up-regulate transcript levels for a wide
range of pro-inflammatory cytokines to any significant degree.39
The maximum levels of TNF-a and IL-b release from macro-
phages are less than 500 pg ml1 and for NO it is o15 mM. It has
been already reported that this level of cytokines would not cause
any inflammatory response and not prevent proliferation of the
cells.40,41 The lower cytokine secretion from SF–FA particles
confirms the absence of any significant inflammatory activity.
It could therefore be concluded that nanoparticles would not
elicit significant macrophage response and it may confirm the
less immunogenic behaviour of the nanoparticles.
Doxorubicin conjugated silk fibroin folate nanoparticles
could be used as a pH-stimulated drug delivery system.42,43
It improves the drug’s half-life in plasma and increases the
drug’s accumulation at a tumour site.44 Though, a lesser
amount of drug is released slowly at pH 7.4 due to poor
solubility of DOX at basic pH, where dissociation of the drug
from nanoparticles is less.45 It is assumed that the DOX release
rate is maximum and quicker at pH 4.5 because of weak
binding between DOX and the carboxylic group of polymer
and the reprotonation of the amino groups of DOX (Fig. 8).
When the environmental pH is lower, polypeptide segments
become hydrophilic because under acidic conditions, H+ in
solution would compete with the hydrogen bond forming group
and weaken hydrogen bond interactions, which make the polymer
more soluble in water.46 Both drug and polymer carry positive
charge at lower pH, which provides the necessary repulsion
between them.47,48 These factors control the higher and prolonged
drug release in the acidic environment of a tumour site and the
This journal is © The Royal Society of Chemistry 2014

Integrative Biology
lower drug release in other tissues. Moreover, there is more
accelerated release inside the endosome/lysosome of tumour
cells. The results showed that nanoparticles could not only
solubilize the poorly soluble drug but also control sustained
drug release. Therefore, a pH-dependent release function is
especially useful for achieving tumour-targeted drug delivery.
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5. Conclusions
Silk fibroin–folate conjugated nanoparticles were fabricated for
targeted delivery to tumour cells. The fabricated nanoparticles
loaded with doxorubicin were taken up by breast cancer cells
via endocytosis. The particles are less than B200 nm in
diameter, stable, non-toxic and less immunogenic to the cells.
The pH dependent release pattern of doxorubicin suggests that
the prepared nanoparticles can serve as effective drug delivery
agents. The experimental observations based on the fabricated
natural protein fibroin–folate conjugated nanoparticles promise
a potential, effective and targeted drug delivery system.
Acknowledgements
We are thankful to Dr Tuli Dey for FACS analysis in this work.
The work is financially supported by the Department of Bio-
technology, through its Bioinformatics SUB-DIC and NER twin-
ning programmes, Govt. of India, New Delhi. The discussions
and suggestions in this work by the expertise from the Indian
Council of Medical Research are thankfully acknowledged.
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214 | Integr. Biol., 2014, 6, 203--214 This journal is © The Royal Society of Chemistry 2014