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Folate conjugated silk fibroin nanocarriers for targeted drug delivery† Bano Subia,a Sourov Chandra,b Sarmistha Talukdara and Subhas C. Kundu*a Disease treatment processes mainly focus on the development of nontoxic, biodegradable, nonimmunogenic, 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

Received 9th September 2013, Accepted 31st October 2013

oxide were checked by stimulating the macrophages with folate conjugated silk fibroin nanoparticles.

DOI: 10.1039/c3ib40184g

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

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nanotechnology applications in cancer research.

The fibroin–folate nanocarriers are nontoxic, easily taken up by cells and capable of sustained drug

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 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 nontoxic, non-immunogenic and with high drug loading efficiency.

a

Department of Biotechnology, Indian Institute of Technology, Kharagpur, 721302, India. E-mail: [email protected]; 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

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They should have properties to enhance their circulation time 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 nanoparticles 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 nonmulberry 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 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 targeting 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 cardiotoxicity 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 attention 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 complex, 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 doxorubicin. 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

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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 experimental farm, folic acid (Sigma-Aldrich), cellulose tubing of MWCO 12 kDa (Pierce, USA), acetone (Merck), glutaraldehyde, doxorubicin, DMSO (dimethyl sulfoxide), MTT (3-(4,5-dimethylthiazol-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 nanoparticle pellets were then redispersed in deionized water and sonicated. 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(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 polydispersity 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 nanoparticles (25 mg), with mixing along with continuous stirring for 12 h in the dark. The drug (DOX) loaded silk–EDBE–FA nanoparticles 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 absorbance at 482 nm of a standard DOX solution and the supernatant 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 absorbance 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 conjugated 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 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 nanoparticle 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 nanoparticles (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-dimethylthiazol-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 preincubated 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 nanoparticles 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 software) 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 conjugated 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 nanoparticles. 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 concentrations (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 concentrations 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. 2.14

Statistical analysis

Statistical differences were performed using one-way ANOVA. Data are presented as mean  SD. p-Values o0.05 were considered 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.

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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 nanoparticles 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 converted 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 nanoparticles. The cellular binding and uptake of rhodamine isothiocyanate (RITC) labelled folate silk fibroin nanoparticles were analyzed on human breast adenocarcinoma cells by confocal 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 nanoparticles 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).

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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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

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3.6

Cytotoxicity assay

A variety of endocytosis mechanisms operated in cells including calthrin-mediated, caveolin-mediated, calthrin or caveolin independent endocytosis and macropinocytosis. The cells are pre-incubated with various endocytosis inhibitors and incubated with silk fibroin–folate nanoparticles. Nanoparticle entry into cells under these conditions is evaluated by measuring the fluorescence intensity. To investigate the processes, MDA-MB231 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 internalization 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 comparatively less than non-treated cells (Fig. 3C).

In vitro cytotoxicity assays of SF–FA and DOX loaded SF–FA NPs were carried out on human breast adenocarcinoma cells. The cytotoxicities 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 proliferation are not significantly affected by different nanoparticle concentrations (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 respiratory chain in treated cells. Free DOX and DOX–SF–FA nanoparticles show dose dependent cytotoxicity (Fig. 5c).

3.5

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 nanoparticles 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.

Live/dead assay

To study the biocompatibility of silk fibroin–folate conjugated nanoparticles and doxorubicin (DOX) loaded SF–FA nanoparticles, 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 nontreated cells (Fig. 4e–h).

3.7

3.8

Cell cycle analysis

Cytokine production assay

To analyze the immunogenic response of silk fibroin folate nanoparticles mouse bone marrow macrophages were used.

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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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 proinflammatory 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

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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 overcome 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 respectively. 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 fluorescent 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 nanoparticles 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 nanoparticles 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. 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 inhibition 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% inhibition of internalization. Biocompatibility and non-toxicity of SF–FA conjugated nanoparticles 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 nanoparticles 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 nanoparticles by endocytosis compared to a passive diffusion mechanism 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

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Integrative Biology

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 effectively 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 macrophages. 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 macrophages 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

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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.

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 Biotechnology, through its Bioinformatics SUB-DIC and NER twinning 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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Folate conjugated silk fibroin nanocarriers for targeted drug delivery.

Disease treatment processes mainly focus on the development of nontoxic, biodegradable, non-immunogenic, biocompatible materials capable of controlled...
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