Functionalized Spider Silk Spheres As Drug Carriers for Targeted

Jun 25, 2014 - and Hanna Dams-Kozlowska*. ,†,§. †. Chair of Medical Biotechnology, Poznan University of Medical Sciences, Poznan 61-866, Poland. ...
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Functionalized Spider Silk Spheres As Drug Carriers for Targeted Cancer Therapy Anna Florczak,†,‡ Andrzej Mackiewicz,†,§,∥ and Hanna Dams-Kozlowska*,†,§ †

Chair of Medical Biotechnology, Poznan University of Medical Sciences, Poznan 61-866, Poland NanoBioMedical Centre, Adam Mickiewicz University, Poznan 61-614, Poland § Department of Diagnostics and Cancer Immunology, Greater Poland Cancer Centre, Poznan 61-866, Poland ∥ BioContract, Poznan 61- 051, Poland ‡

ABSTRACT: Bioengineered spider silk is a biomaterial that combines the properties of self-assembly, biocompatibility and biodegradability with reasonable accessibility and a simple purification procedure. Moreover, genetic engineering enables the functionalization of silk by adding the peptide coding sequences of the desired attribute. Hybrids composed of Her2 binding peptides (H2.1 or H2.2) and bioengineered silk MS1 (based on the MaSp1 sequence from N. clavipes) were designed. Bioengineered silks were expressed in a bacterial system and purified using a tag-free thermal method. The hybrid silks with Nterminal functionalization were bound more efficiently to cells that were overexpressing Her2 than those with the C-terminal fusion. Moreover, the functionalization did not impede the self-assembly property of bioengineered silk, enabling the processing of silk proteins into spheres. The binding domains were exposed on the surface of the spheres, because the functionalized particles specifically bound and internalized into Her2-overexpressing cells. The binding of the functionalized spheres to Her2positive cells was significantly higher compared with the control sphere and Her2-negative cell binding. Silk spheres were loaded with doxorubicin and showed pH-dependent drug release. The silk spheres were not cytotoxic, unless they were loaded with the drug doxorubicin. This study indicates the ability of drug-loaded functionalized spider silk spheres to serve as a carrier for targeted cancer therapy.

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INTRODUCTION

delivery vehicles at a solid tumor site. This deposition takes days to weeks, in contrast to low molecular weight agents, for which the drug retention period is less than a few minutes.2 Active delivery is based on targeting the tumor cells, followed by cell penetration and the intracellular release of the active substance. Accordingly, decorating a drug carrier with ligands that target tumor-associated receptors and tumor-specific membrane molecules is crucial for the specificity and efficiency of this therapy. Numerous types of drug delivery nanosystems are currently being developed, including liposomes, polymer conjugates, polymeric micelles, dendrimers, nanoshells, and protein and nucleic acid−based nanoparticles.1 For active delivery, the ligands that can be added to carrier surfaces include monoclonal

Anticancer agents that are administered systemically at therapeutic doses may cause serious adverse events (SAE). Accordingly, the therapeutic benefit-toxicity balance may not be acceptable for patients. Thus, intensive research is being performed to deliver these toxic agents directly to tumor cells, sparing the other cells in the body. The encapsulation and direct targeting of the toxic drug to the tumor would be very profitable. Two primary strategies can be exploited to deliver a drug into a biological target, namely, passive and active drug delivery.1 Passive drug delivery is based on the accumulation of a drug at the deposition site because of the balance between vascular hemodynamic forces and diffusion mechanisms. In tumors, passive targeted drug delivery also takes advantage of the leaky vasculature of tumor tissues and their poor lymphatic systems. These characteristics enable an enhanced permeability and retention (EPR) effect.2 For a nanoparticle-based delivery system, the EPR effects allow for the enhanced deposition of © XXXX American Chemical Society

Received: April 22, 2014 Revised: June 19, 2014

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CAGTATTGGTACGAAA, H2.1R: 5′-CTAGTTTCGTACCAATACTGCAGCCAATGGCTATCGCCCCAATACATG, and H2.2F: 5′-CTAGCCTGACCGTGAGCCCGTGGTATA, H2.2R: 5′-CTAGTATACCACGGGCTCACGGTCAGG. H2.1F/H2.1R and H2.2F/ H2.2R were annealed, resulting in double-stranded DNA fragments with cohesive ends complementary to those generated by NheI and SpeI. After their ligation, the sequences of the resulting plasmids were confirmed by sequencing at the Adam Mickiewicz University Core Facility in Poznan. Enzymes for the digestion and ligation were supplied by Fermentas (Thermo Fisher Scientific Inc., Waltham, MA) and New England Biolabs, Inc. (Ipswich, MA), respectively. Expressing and Purifying Bioengineered Silks. The plasmids pETNX-MS1, pETNX-H2.1-MS1, pETNX-MS1-H2.1, pETNX-H2.2MS1 and pETNX-MS1-H2.2 were used to transform E. coli strain BLR (DE3; Novagen, Madison, WI). A Bioflo 3000 (New Brunswick Scientific, Edison, NJ) fermentor was used for large scale expression, as formerly reported.20 Silk variants were purified by using the thermal method (called the 80/20), as previously described.20,21 In brief, the bacteria were added to 20 mM HEPES (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid), pH 7.5, and 100 mM NaCl, and the mixture was supplemented with Protease Inhibitor Cocktail (2 mM AEBSF, 1 mM phosphoramidon, 130 mM bestatin, 14 mM E-64, 1 mM leupeptin, 0.2 mM aprotinin, and 10 mM pepstatin A; Sigma, St. Louis, MO) and lysed with 0.2 mg/mL lysozyme (Thermo Fisher Scientific, Inc., Waltham, MA). Next, the lysate was sonicated, treated with DNaseI (Sigma, St. Louis, MO; 0.1 mg/mL), and it was then centrifuged at 50000 × g for 30 min at 4 °C. Soluble bacterial proteins were precipitated by heat denaturation (80 °C for 20 min) and removed by sedimentation at 50000 × g for 30 min at 4 °C. The soluble silk proteins were precipitated with 20% (for MS1, H2.1MS1, MS1H2.1, and MS1H2.2 proteins) or 25% (for H2.2MS1 protein) ammonium sulfate (ICN Biomedicals Inc., Aurora, OH), centrifuged at 10000 × g for 15 min, rinsed with 20 or 25% ammonium sulfate, respectively, and then dissolved in 6 M guanidinium thiocyanate (ICN Biomedicals Inc., Aurora, OH). The resulting proteins were dialyzed against 50 mM sodium borate, pH 8.5 (Sigma, St. Louis, MO). ZelluTrans Regenerated Cellulose Dialysis Tubing with an MWCO of 12000−14000 Da (Carl Roth, Karlsruhe, Germany) was used for the dialysis. The protein concentration was determined by UV spectroscopy at 280 nm with molar extinction coefficients of 22350, 43320, 43320, 29340, and 29340 M−1 cm−1 for MS1, H2.1MS1, MS1H2.1, H2.2MS1, and MS1H2.2 proteins, respectively. SDS-PAGE gel electrophoresis was performed with a 12.5% gel and the gel was stained with colloidal blue (Roti-Blue; Carl Roth, Karlsruhe, Germany). Cell Culture. Her2 overexpressing cell lines, human ovarian cancer cells (line SKOV3), and human breast cancer cells (line SKBR3), as well as Her2-negative human fibroblasts MSU1.1, were used. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% fetal bovine serum (PAA Laboratories GmbH, Pasching, Austria) and 80 μg/ mL gentamycin (KRKA, Novo Mesto, Slovenia). Cells were grown at 37 °C in a humidified atmosphere containing 5% CO2. Preparing Bead-Silk Conjugates. To couple the silk proteins covalently to yellow-green fluorescent beads, the manufacturer’s protocol was applied for carboxylate-modified FluoSphere Fluorescent Microspheres, 0.5 μm (Molecular Probes, Eugene, OR). The carboxylate-modified beads were covalently coupled to MS1H2.1 and MS1H2.2 with water-soluble carbodiimide (EDC; Sigma, St. Louis, MO) and N-hydroxysulfosuccinimide (Sulfo-NHS; Sigma, St. Louis, MO). In brief, 50 μL of beads (2% solids) were washed and suspended in 50 μL of 0.1 M MES, pH 6.0 (2-[morpholino]ethanesulfonic acid) activation buffer. Next, 1 mg of EDC (final concentration 5 mM) and 1.1 mg of Sulfo-NHS (final concentration 5 mM) were added and incubated for 15 min at room temperature in the dark with continuous mixing. After the activation, the buffer pH was increased above 7.0 by washing the beads with coupling buffer (PBS, pH 7.4). Silk proteins were prepared at a concentration of 1 mg/mL in coupling buffer; they were mixed with the activated bead suspension and then the reaction was allowed to proceed for 2 h at room temperature with gentle stirring and protection from light. To quench the reaction, 100 mM glycine was

antibodies or their fragments, proteins, or peptides, nucleic acid ligands (such as aptamers), and small molecules.1 However, all contemporary delivery systems display some limitations, which include the lack of vehicle biodegradation or local and systemic elimination. Silk sutures have been successfully used in medicine for decades.3 Bioengineered spider silk technology was recently developed.4−7 Bioengineered silk is based on the repetitive consensus sequences found in the corresponding native silk. The construction of synthetic silk genes was previously described, and it employs double-stranded oligonucleotides and their subsequent multimerization.8 The size and sequence of the spider silkbased block copolymers that are designed via genetic engineering can be controlled.9 Moreover, bioengineered silk proteins may be further modified to gain new functions. The strategy of constructing hybrid protein at the DNA level combines the sequence encoding bioengineered spider silk, which is responsible for the biomaterial structure, with sequences encoding the polypeptides for functionalization. Recombinant silk proteins containing functional domains, such as the silica formation sequence known as silaffin (an R5 peptide), hydroxyapatite nucleation and crystallization sequence dentin matrix protein 1 (CDMP1), cell-binding peptides, cell penetrating peptides (CPPs), the nucleolin-binding sequence (F3 peptide), tumor homing peptides, antimicrobial peptides, and DNA-binding poly(L-lysine) have been reported.10−17 These bioengineered silk proteins contain tumor-recognizing peptides that could be employed in specific drug delivery for cancer treatment. The amplification of the Her2/neu gene and/or the overexpression of the corresponding protein have been identified in approximately 20−30% of invasive breast carcinomas.18 The combination of Her2 overexpression in tumors and its low expression in normal tissues provides a potential therapeutic window for agents targeting the Her2. Therefore, Her2 has been an attractive model for targeted molecular therapy development.19 The aim of this study was to design and develop a drug delivery system based on spheres of bioengineered spider silk that were functionalized with peptides specific to a given cell. We employed Her2-oriented spider silk spheres as a model system. We constructed, expressed, and purified new hybrid silk proteins that were functionalized with peptides, which bind to Her2. Stable silk particles were obtained by a simple aqueous process triggered by potassium phosphate. Silk sphere binding and drug delivery to the cancer and control cells were investigated. The functionalized spheres targeted and killed the cancer cells when loaded with the cytostatic drug doxorubicin.

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EXPERIMENTAL SECTION

Construction of Expression Plasmids pETNX-MS1, pETNXH2.1-MS1, pETNX-MS1-H2.1, pETNX-H2.2-MS1, and pETNXMS1-H2.2. The expression vector pET30(a)+ (Novagen, Madison, WI) was modified with linker NX, and the pETNX-15X construct was then obtained as previously described.20 A pETNX-15X plasmid carrying synthetic gene 15X comprised 15 repeats of the MaSp1 consensus sequence from Nephila clavipes, which in this study is named MS1. The hybrid constructs were obtained by cloning H2.1 and H2.2 sequences into the NheI restriction site for 5′-terminal functionalization and into the SpeI restriction site for the 3′-terminal functionalization of the MS1 construct (pETNX-H2.1-MS1, pETNX-H2.2-MS1 pETNXMS1-H2.1, and pETNX-MS1-H2.2). The sequences of the synthetic oligonucleotides encoding the H2.1 and H2.2 domains were as follows: H2.1F: 5′-CTAGCATGTATTGGGGCGATAGCCATTGGCTGB

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added for 30 min. Next, the bead-silk conjugates were washed and resuspended in 50 μL of storage buffer (PBS, pH 7.4, with 0.1% azide and 0.5% BSA). H2.1MS1, H2.2MS1, MS1, and Herceptin (anti-Her2 antibody) (Roche, Basel, Switzerland) were conjugated to amine-derivatized beads. First, the amine groups were introduced in the place of carboxyl groups by incubation with hexamethylenediamine (HMDA; Sigma, St. Louis, MO). In brief, 50 μL of carboxylate-substituted beads were mixed with EDC and Sulfo-NHS in activation buffer (0.1 M MES, pH 6.0), as described above, 2.335 mg of HMDA (final concentration 20 mM) was added, and the mixture was incubated for 2 h at room temperature under continuous mixing. After adjusting the pH to 7.4 by washing in coupling buffer (PBS, pH 7.4), the amine-modified beads were coupled to the protein carboxyl groups. For this purpose, 1 mg/mL of protein in activation buffer was incubated with EDC and Sulfo-NHS, as described above. Next, the protein was separated from the excess reducing agent and cross-linker using a PD-10 desalting column (GE Healthcare, Uppsala, Sweden). Finally, 50 μL of activated protein solution and 50 μL of amine-modified bead suspension were incubated for 2 h at room temperature, quenched, washed, and resuspended in storage buffer as described above. Cell Binding Assay for Bead-Silk Conjugates. SKOV3, SKBR3, and MSU1.1 cells were seeded onto a 24-well culture plate (1 × 105 cells/well) 24 h prior to the binding experiments. After a brief sonication, 1 μL of bead-silk conjugate or bead-Herceptin conjugate suspension was added to 1000 μL of culture medium and incubated with the cells for 4 h at 37 °C. The cells were then washed with PBS and the cell nuclei were counterstained with 5 μg/mL 4′,6′-diamidino-2phenylindole (DAPI; Sigma, St. Louis, MO). The cells were washed again after 30 min of incubation, fresh medium was added, and the samples were analyzed with a Leica DMI3000 B fluorescent microscope (Leica, Welzlar, Germany). Image acquisition and analysis were performed with a 20× objective lens and Leica Application Suite (LAS) microscope software. The experiment was repeated three times. Silk Sphere Formation. Silk spheres were formed by inducing nucleation and particle growth by adding potassium phosphate.22,23 In brief, 100 μL of 0.5 mg/mL silk solution was mixed with 1000 μL of 2 M potassium phosphate pH 8.0 (Sigma, St. Louis, MO) by using a pipet. The resulting particles were incubated at room temperature for 12 h. After dialyzing against ultrapure water, the spheres were centrifuged at 10000 × g for 5 min and then redispersed in 50 μL of ultrapure water. For the cell binding assays, the bioengineered silks were first conjugated with the ATTO 647N fluorophore (Sigma, St. Louis, MO) or FITC (Sigma, St. Louis, MO) according to the manufacturer’s protocol and they were then used for sphere formation. The sphere concentration was determined gravimetrically. Silk Spheres: The Loading and Release of Doxorubicin. Doxorubicin HCl (Dox; Adriamycin, Pfizer Inc., New York City, NY) was used as a model drug for loading the silk spheres. The following protocol was used to form the silk spheres that were loaded with Dox: 100 μL of solution containing silk protein at a 0.5 mg/mL concentration and Dox at a 1 mg/mL concentration was mixed with 1000 μL of potassium phosphate (2 M, pH 8.0). After 12 h of incubation at room temperature, the spheres were dialyzed against ultrapure water and then centrifuged for 5 min at 10000 × g. The sphere quantity was determined gravimetrically. The amount of Dox loaded into the spheres was calculated spectrophotometrically by measuring the Dox absorbance at a wavelength of 509 nm. A standard calibration curve for the model drug was used for drug quantification. Loading was determined by using the following equation:

Waltham, MA). A standard calibration curve for the model drug was used for drug quantification. The cumulative amount of Dox released from the spheres was plotted against time. Characterizing the Silk Spheres by Confocal Laser Scanning Microscopy (CLSM). The Dox-loaded silk particles were prepared as described above, resuspended in purified water and then analyzed by CLSM. The spheres were imaged with a Leica TCS SP5 X confocal laser scanning microscope under a 100× objective and a 1.4 N.A. oil immersion lens (Leica, Welzlar, Germany) controlled by Leica Application Suite Advanced Fluorescence (LAS AF) Lite software. An excitation wavelength of 488 nm and an emission wavelength of 590 nm were used for Dox-loaded silk particles. Characterizing Silk Spheres by Scanning Electron Microscopy (SEM). The silk spheres were applied to coverslips (Nunc, Naperville, IL) and allowed to air-dry. After being sputtered with gold under a vacuum, the samples were analyzed with an SEM (JSM 5900LV, JEOL Ltd., Japan) at 10 kV. The particle sizes were determined with ImageJ 1.46r software. Cell Binding Assay and Cellular Uptake of Silk Spheres. Flow Cytometry. The cells were washed with PBS/0.5% BSA and detached with nonenzymatic cell dissociation solution (Sigma, St. Louis, MO). Next, 30 μL of FITC-labeled spheres at a concentration of 10 μg/mL were added to 1 × 105 cells suspended in PBS/0.5% BSA and incubated for 1 h at 4 °C in the dark. After washing three times with PBS, the binding of the spheres to the cells was analyzed with a FACSCanto flow cytometer (BD Biosciences Pharmingen, San Jose, CA) and FACSDiva (v6.1.2) software. Three independent experiments were performed. CLSM. A quantity of 5 × 104 cells/well was plated onto 8-well LabTek chambered cover glasses (Nunc, Naperville, IL) 48 h prior to the binding experiments. A total of 1 μL of ATTO 647N-labeled silk spheres (10 μg/mL) were added and incubated with the cells for 4 h at 37 °C. The cells were then washed with PBS and the cell membranes were stained with FITC-conjugated Concanavalin A (ConA-FITC; Sigma, St. Louis, MO) at a concentration of 50 μg/mL. After 30 min of incubation, the cells were washed, immersed in fresh medium and then analyzed with a Leica TCS SP5 X CLSM (Leica, Welzlar, Germany). Image acquisition and analysis were performed with a 100× objective, a 1.4 N.A. oil immersion lens and Leica LAS AF Lite software. Images of the silk spheres were visualized by using 647 nm excitation and 661 nm emission wavelengths. To visualize the cell membranes, 488 nm excitation and 525 nm emission wavelengths were applied. The experiment was repeated three times. Intracellular Distribution of Silk Spheres over Time. SKBR3 cells (2 × 104 cells/well) were plated onto 8-well Lab-Tek chambered cover glasses (Nunc, Naperville, IL) and cultured for 48 h. Next, 1 μL of ATTO 647N/H2.1MS1 spheres (10 μg/mL) was added and incubated with the cells at 37 °C for the indicated time periods. After washing with PBS, the cells were fixed with 4% paraformaldehyde (PFA; Sigma, St. Louis, MO), and then the cell membranes were stained with ConAFITC at a concentration of 50 μg/mL. After 30 min of incubation, the cells were washed, immersed in PBS, and then analyzed with a Leica TCS SP5 X CLSM (Leica, Welzlar, Germany) as described above. Intracellular Distribution of Dox. A 2 × 104 portion of cells per slide were plated onto a 10-well multitest slide (MP Biomedicals, Solon, OH). After 24 h, 1 μL of Dox-loaded H2.1MS1 spheres (10 μg/mL) were added to the complete culture medium and incubated at 37 °C. After 4 h of incubation, the cells were washed with PBS and fixed with 4% PFA. The cell nuclei were then counterstained with DAPI at a concentration of 1 μg/mL. After 15 min of incubation, the cells were washed with PBS, immersed in mounting medium (Sigma, St. Louis, MO), covered with a cover glass, and then analyzed under an Olympus Scanning Confocal Microscope FV1000 (Shinjuku, Tokyo, Japan) connected to a laser diode at 405 nm and an argon laser. Image acquisition and analysis were performed with a 60× objective, a 1.4 N.A. oil immersion lens and FLUOVIEW Viewer software, ver. 4.1. The nuclei were visualized using 350 nm excitation and 470 nm emission wavelengths. To visualize Dox-loaded spheres and the Dox released from the spheres, 488 nm excitation and 590 nm emission wavelengths were applied. The experiment was repeated three times.

loading = (amount of Dox in the sample)/(amount of sample) The Dox-loaded spheres were then resuspended in 1 mL of phosphate buffer at pH 4.5 and 7.4 and incubated at 37 °C with constant shaking. The solvent was removed periodically from each sample and replaced with fresh PBS that was at an appropriate pH. The Dox concentration in the sampled media was determined by measuring the Dox fluorescence (excitation 480 nm, emission 590 nm). The Dox fluorescence was analyzed with a Victor X3Multimode Plate Reader controlled by PerkinElmer 2030 Workstation software (PerkinElmer, C

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Figure 1. (A) Amino acid sequences of bioengineered spider silk protein (MS1) and functionalized proteins with Her2-binding peptides. The functional domain sequences H2.1 and H2.2 are shown in italics. (B) Analysis of purified silk proteins by 12.5% SDS-PAGE. 1, molecular weight marker (PageRuler, Fermentas); 2, MS1, control spider silk; 3, H2.1MS1 hybrid silk (an H2.1 peptide fused to the N-terminus of MS1); 4, MS1H2.1 hybrid silk (an H2.1 peptide fused to the C-terminus of MS1); 5, H2.2MS1 hybrid silk (an H2.2 peptide fused to the N-terminus of MS1); 6, MS1H2.2 hybrid silk (an H2.2 peptide fused to the C-terminus of MS1). Cytotoxicity Study. A total of 2.5 × 104 cells/well were seeded onto a 96-well plate and incubated overnight. The next day, different concentrations of silk spheres loaded with Dox or control spheres without drug were added to the cell cultures. After 4 h of incubation, cells were washed with PBS and fresh medium was added. The medium without spheres was used as a negative control. After 72 h of incubation, to each well was added 50 μL (5 mg/mL) of MTT reagent (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma, St. Louis, MO); this was then incubated an additional 4 h and then the medium was discarded and insoluble purple formazan was dissolved in 200 μL of dimethyl sulfoxide (Sigma, St. Louis, MO). The absorbance of the colored solution was measured at a wavelength of 570 nm with a microplate reader ELX808IV (Bio-Tek Instruments, Winooski, VT). The mitochondrial function and, by extension, the relative cell viability (%) related to the negative control were calculated by test sample/ negative control × 100%. The experiment was repeated three times in triplicate. Statistics. To analyze the significant differences between MS1, H2.1MS1, and H2.2MS1 treated groups, one-way ANOVA was used. In the case of significance ANOVA (p < 0.05) post hoc tests with Bonferroni correction were performed. The differences were considered significant when p < 0.05.

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MSU1.1 control cells (Her2-negative fibroblasts) were incubated in the presence of bead-silk conjugate variants. The functionalized spider silk conjugates demonstrated substantially higher binding to Her2-positive than to Her2-negative cells (Figure 2). Moreover, the functionalization of the N-terminus of the silk sequences (H2.1MS1 and H2.2MS1) demonstrated a considerably higher binding capacity than functionalization at the Cterminus (MS1H2.1, MS1H2.2). The binding of control protein MS1 to all tested cell lines was negligible. Anti-Her2 antibody (Herceptin) conjugated with beads (positive control) confirmed the Her2 overexpression on SKOV3 and SKBR3 cells (Figure 2). Silk Sphere Formation. The control and functionalized spider silk proteins were subsequently examined for their sphere formation ability. Because the silk variants that were functionalized at the N-terminus exhibited the most efficient binding to the target cells, we selected them for the next step. Functional domains H2.1 and H2.2 did not affect the silk’s potential to form spheres (Figure 3A). Qualitative characterization indicated that all types of silk spheres had a similar morphology. The average sphere size was approximately 400 nm with a size distribution of 444 ± 134, 359 ± 114, and 399 ± 120 nm, for MS1, H2.1MS1, and H2.2MS1, respectively. Silk Spheres: The Loading and Release of Doxorubicin. The ability of silk spheres to serve as a potential drug delivery system was analyzed by using Dox as a drug model. The sphere concentrations were determined gravimetrically and the amount of Dox within the spheres was measured with a spectrophotometer. The final concentration of Dox was 364.6 (±12.3), 338.9 (±12.1), and 357.8 (±34.9) ng per μg of spheres made from MS1, H2.1MS1, and H2.2MS1, respectively. Moreover, the loading of Dox into the spheres was confirmed by confocal microscopy (Figure 3C). SEM analysis indicated that the morphology of Dox-loaded spheres was slightly modified when ̈ spheres (Figure 3B and A, respectively). compared with naive Dox-loaded spheres were more spherical. The average size and distribution of Dox-loaded spheres were 365 (±123), 357 (±136), and 357 (±122) nm for MS1, H2.1MS1, and H2.2MS1, respectively. The in vitro Dox release experiment was performed in PBS at a pH 4.5 and 7.4. As shown in Figure 4, an approximately 60% Dox release was observed at pH 4.5 over 24 h, and an approximately 80% cumulative Dox release occurred within 15 days for all sphere variants. The release rate at pH 4.5 was faster than it was at

RESULTS

Expressing and Purifying Recombinant Spider Silk Proteins. Figure 1A shows the amino acid sequences of the five spider silk variants as follows: (i) H2.1MS1 and MS1H2.1 carrying a Her2-binding domain named H2.1 with the sequence MYWGDSHWLQYWYE,24 (ii) H2.2MS1 and MS1H2.2 carrying a Her2-binding domain named H2.2 with the sequence LTVSPWY,25 and (iii) control MS1 without any functional domain. The yields for recombinant silk proteins after purification differed depending on the protein (MS1, 45.8 mg/ L; H2.1MS1, 22 mg/L; MS1H2.1, 6.375 mg/L; H2.2MS1, 4.25 mg/L; and MS1H2.2, 3.85 mg/L). The protein yield was expressed as the amount of purified protein per liter of culture medium. An SDS-PAGE analysis of MS1, H2.1MS1, MS1H2.1, H2.2MS1, and MS1H2.2 showed bands corresponding to molecular weights of approximately 39, 41, 41, 40, and 40 kDa, respectively (Figure 1B). The apparent molecular weights of the silk proteins were consistent with expected values. Cell Binding Assay for Bead-Silk Conjugates. To determine the cell binding potential of functionalized spider silk, we covalently coupled the proteins to the fluorescent beads. SKOV3 and SKBR3 cells that were overexpressing the Her2 and D

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Figure 2. Cell binding assay with silk-bead conjugates using Her2-overexpressing cells SKOV3 (A), SKBR3 (B), and Her2-negative control cells MSU1.1 (C). The cells were incubated for 4 h at 37 °C with beads conjugated with functionalized protein variants or controls: positive control, herceptin and negative control, nonfunctionalized silk (MS1). The binding efficiency was analyzed by fluorescent microscopy; green, silk-bead conjugates; blue, cell nuclei labeled with DAPI; scale bar, 50 μm.

pH 7.4. The cumulative release of Dox after 15 days of incubation at pH 7.4 was 21.3 ± 1.2%, 20.5 ± 0.4%, and 21.8 ± 0.3% for MS1, H2.1MS1, and H2.2MS1 spheres, respectively (Figure 4). Cell Binding and Cellular Uptake of Silk Spheres. The FITC-labeled spheres were incubated with SKOV3, SKBR3, and MSU1.1 cells and analyzed by flow cytometry (Figure 5). The binding of functionalized spheres (H2.1MS1 and H2.2MS1) to Her2-overexpressing cells was 8−10 times significantly higher than the binding of the control spheres (MS1). There was an average of 9% nonspecific binding to fibroblasts (MSU1.1) for all tested sphere variants. Moreover, the binding of functionalized spheres H2.1MS1 and H2.2MS1 to cancer cells was significantly higher (7-fold) than to fibroblasts. Figure 5B shows the examples of flow cytometry analysis for silk spheres binding to the cells.

The results were confirmed by confocal microscopy (Figure 6). As shown in Figure 6, functionalized spheres targeted and effectively entered the cells. The spheres made of H2.1MS1 and H2.2MS1 silks were internalized by cells that overexpressed Her2 (SKOV3 and SKBR3), but not by control cells (MSU1.1). Internalized spheres were distributed in the cytoplasm, but they did not penetrate to the nucleus. Moreover, the signal from fluorescently labeled spheres decreased with the time of incubation (Figure 7). SKBR3 cells were incubated in the presence of ATTO 647N/H2.1MS1 spheres at 37 °C for the indicated periods of time. Pictures were taken from different samples to avoid the loss of fluorescence upon continuous light excitation. As shown in Figure 7, the fluorescently labeled spheres were hardly visible after 48 h of incubation. E

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Figure 3. Morphology of drug-free (A) and Dox-loaded (B, C) silk spheres made of MS1, H2.1MS1, and H2.2MS1 proteins. Spheres analyzed by (A, B) scanning electron microscopy (scale bar 1 μm) and (C) by confocal microscopy; red, Dox-loaded spheres; scale bar, 10 μm.

Figure 4. Cumulative drug release from silk spheres. Dox-loaded MS1, H2.1MS1 and H2.2MS1 spheres were resuspended in PBS at pH 4.5 and 7.4 and incubated at 37 °C with constant shaking. The Dox released at the indicated time points was determined spectrofluorometrically. The cumulative amount of Dox released from the spheres was plotted against time.

Cytotoxicity of Dox-Loaded and Control Spheres. SKOV3, SKBR3, and MSU1.1 cell lines were maintained in the presence of different concentrations of spider silk spheres loaded with Dox (MS1-Dox, H2.1MS1-Dox, and H2.2MS1-Dox) and control spider silk spheres without the drug (MS1, H2.1MS1, and H2.2MS1). The drug-loaded spheres made of functionalized spider silk reduced significantly higher the viability of Her2overexpressing cancer cells when compared with the Dox-loaded spheres made of control MS1 silk (Figure 8A,C). The observed cytotoxicity was dependent on the concentration of silk spheres. There was no difference in the cytotoxicity of Her2-negative cells in the presence of drug-loaded functionalized or control spheres (Figure 8E). The highest concentrations of Dox-loaded spheres caused toxicity in the MSU1.1 cells, but the cell viability was approximately 90% for all tested samples at a sphere concentration of 1.88 μg/mL. The same concentration of both functionalized silk spheres reduced cancer cell viability by up to 40% (Figure 8A,C). The differences were significant.

Figure 5. Flow cytometry analysis of cell binding by functionalized silk spheres. Her2-overexpressing cells (SKOV3 and SKBR3) and control cells (MSU1.1) were incubated with spheres made of functionalized silk variants (H2.1MS1 and H2.2MS1) and control silk (MS1) conjugated with fluorochrome (FITC). (A) The binding of hybrid spheres to the cells overexpressing Her2 was compared with the control protein without the functional domain and with Her2-negative cells. The mean percentage and (±SD) of at least three independent experiments for each cell/sphere combination are shown; ***indicate statistical significance (p < 0.001). (B) Examples of the flow cytometry analysis. Control: nontreated cells.

The observed cytotoxicity resulted from drug activity, because the bioengineered functionalized and control spider silk spheres did not reduce cell viability over the tested concentration range (Figure 8B,D,F). F

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Figure 6. Binding and cellular uptake of the silk spheres. SKOV3, SKBR3, and MSU1.1 cells were plated on 8-well chambered cover glasses and treated with spheres made of fluorescently labeled control MS1 and functionalized spider silk proteins H2.1MS1 and H2.2MS1. The cell membranes were counterstained with ConA-FITC. Red, the spheres made of MS1, H2.1MS1, and H2.2MS1 proteins conjugated with ATTO 647N; green, the cell membranes stained with ConA-FITC; scale bar, 10 μm.

Figure 7. Confocal microscopy of the intracellular distribution of silk spheres by the time of incubation. SKBR3 cells were incubated with H2.1MS1 spheres at 37 °C for the indicated periods of time. Red, spheres made of H2.1MS1 proteins conjugated with ATTO 647N; green, cell membranes stained with ConA-FITC; scale bar, 10 μm.

Moreover, the release of Dox from the spheres inside the cells was analyzed by confocal microscopy. As shown in Figure 9, a large fraction of Dox molecules was accumulated in the nuclei. Figures 5 and 6 show that the silk spheres did not penetrate into the nucleus; thus, the Dox observed there (Figure 9) must be released from the spheres before entering the nucleus.

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concept and feasibility of the system are based on the following six major findings: (1) bioengineered, functionalized spider silk proteins can be produced in a bacterial expression system and purified using methods based on the properties of natural spider silk without any additional Tag sequences, (2) receptor binding peptides (H2.1. and H2.2) can be fused to the silk sequence without an additional peptide linker, and they are functional, (3) the binding efficiency of N-terminally functionalized silk was higher compared with C-terminus functionalized silk, (4) the functional domains do not impede the self-assembly property of

DISCUSSION

Here, we present a novel biovehicle system for the targeting and delivery of active substances into cancer cells. The proof of G

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Figure 8. Cytotoxicity study by MTT assay. Her2-overexpressing cells SKOV3 (A, B), SKBR3 (C, D), and control cells MSU1.1 (E, F) were cultured in the presence of the silk spheres loaded with Dox (MS1-Dox, H2.1MS1-Dox, and H2.2MS1-Dox; A, C, E) and control silk spheres without Dox (MS1, H2.1MS1, H2.2MS1; B, D, F). The % of the MTT reduction was calculated in reference to nontreated control cells. The results are expressed as the mean of three independent experiments and the error bars show the standard deviation; ***indicates statistical significance with p < 0.001, **p < 0.01, and *p < 0.05.

Figure 9. CLSM images of SKBR3 cells incubated with Dox-loaded H2.1MS1 spheres at 37 °C for 4 h. (A) The nuclei stained with DAPI (blue), (B) Dox-loaded spheres and Dox released from the spheres (red), (C) merge, the colocalization of the nucleus and Dox. The scale bar represents 10 μm.

The MS1 was functionalized with two Her2 binding peptides, namely H2.1 and H2.2, that internalize into cells upon binding to Her2.24−26 The H2.1 and H2.2 peptides were fused to the N- and C-terminus of MS1 by using a short two AA linker (which was derived from the restriction site) to minimize the void sequence. The experiment for binding soluble proteins indicated that the fusion did not abolish the activity of H2.1 and H2.2 domains. Moreover, variants with the N-terminal modification displayed better binding than variants with a C-terminal fusion. The

the silk, (5) spheres made of functionalized silk bind and are internalized into target cells, and (6) the drug-loaded spheres made of functionalized silk effectively kill target cells. We previously demonstrated that the thermal denaturation method for purifying 15mer bioengineered spider silk protein (MS1) yielded not cytotoxic, not immunogenic, and free of endotoxin silk protein.20 Accordingly, we employed the same purification method in this study of functionalized spider silk variants. H

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ized spheres was different from that of nontreated cells; upon the massive uptake of spheres, the cell size increased and normalized with time. The signal of the internalized spheres disappeared with time, which may suggest that the silk spheres were degraded within the cells and thus, the cell morphology returned to its initial state. Moreover, a considerable quantity of Dox molecules was accumulated in the nucleus. Because the silk spheres were observed mostly in the cytoplasm and did not penetrate the nucleus, the Dox deposited in the nucleus was released from the spheres before entering the nucleus. Drug release studies showed that, at pH 4.5, the Dox released from silk spheres significantly increased. All these observations suggest that the cell uptake of silk spheres may follow endocytosis, which would lead to the accumulation of spheres in endosomes (pH 6.0) and then in lysosomes (pH 4.5). The lysosomal microenvironment may not only increase the Dox release (pH 4.5), but the proteolytic enzymes present in the lysosome may also degrade the spheres, which could further enhance the drug release. Another group showed that the inhibition of endocytosis and lysosomal function suppressed the activity of a drug carried by the LTVSPWY (H2.2) peptide.26 However, the process of sphere uptake and the fate of the silk carrier inside the cells require more extended study. The degradation of spheres within cells is a very important factor in the development of a targeted drug delivery strategy. This strategy may prevent the accumulation of the intact drug carrier and eliminate potential toxicity. The silk spheres were not cytotoxic, which is consistent with numerous studies.13,20,37 However, the Dox-loaded spheres effectively killed cells, and the most important finding is that the functionalized spheres had significantly higher cytotoxicity than the control spheres. Seib et al. showed particles made of silkworm silk for anticancer drug delivery.28 Since the silk-based particles did not have any functional domains, they would interact with any cell type (due to nonspecific interactions). Their accumulation in the tumor tissue can be passive due to the enhanced permeation and retention effect (EPR). The phenomenon of EPR is based on leaky blood vessels and reduced lymphatic drainage of the tumor vasculature. Since our bioengineered spheres are functionalized, their accumulation in the tumor tissue can be both passive and active. Accordingly, the dose of administered drug-loaded spheres may be reduced. The lower dose of drug-loaded particles, the lower systemic toxicity. The flow cytometry and microscopy analyses indicated the high selectivity of functionalized spheres. The specificity of selected peptides (H2.1 and H2.2) was examined previously.24−26 Our results showed that these peptides can be fused to bioengineered silk and processed into spheres, and this type of system can serve as a targeted drug delivery platform. Numata et al. proposed a system of silk-based complexes with tumor-homing peptides for tumor-specific gene delivery.15,17 Although the basic concept of using functionalized silk proteins is similar, we have focused on constructing a system based on functionalized silk containing the shortest redundant sequence possible. We intend to employ the silk property to purify the protein and to assemble a structure (such as a sphere, film, scaffold, etc.). Thus, the functional domain should be as short as possible. The foreign sequence may not only impair the silk property but may also cause toxicity and immunogenicity, which are of great importance for in vivo applications.

Shadidi and Sioud group fused the H2.2 peptide at the C-termini of GFP by using a 3AA linker (GGG).25 It is possible that a longer linker is needed for the C-terminal functionalization of silk to preserve the binding potential of the fused peptide. The next issue was whether the fused domains affect the silk properties. A high concentration of potassium phosphate triggers the self-assembly of silk proteins into spheres.22 The protein and phosphate concentrations and the mixing method affect the morphology of spheres.22,27 Spheres made of control and functionalized silk variants were similar, indicating that the binding domains did not interfere with the self-assembly property of silk. Producing spheres at a low silk concentration and a high concentration of potassium phosphate by pipet mixing created particles of a relatively small diameter; however, the particles had a wide size distribution. The application of a micromixing chamber combined with high-pressure pumps should improve the morphology of spheres with regard to their size and size distribution.22 Employing ultrafiltration membranes of a defined pore size is another method of obtaining uniform spheres.28 The size and surface characteristics of the drug carriers play a key role in their pharmacokinetics. They influence on blood opsonization processes. Various nanoparticles larger than 200 nm activate more efficiently the complement system and thus are cleared from the blood faster than their smaller counterparts.29 Moreover, endocytosis, the major method of particle uptake, favors particles from 80 to 150 nm.30 Thus, sphere production optimization is required to obtain smallersized, uniform particles. Moreover, since the proof of concept and feasibility of the system have been indicated, a better physical and chemical characterization of the spheres is needed. These issues are currently being addressed in a separate study. The loading efficiency and release profile of Dox were similar for all silk variants, indicating that the binding domains did not affect these processes. The Dox release was similar to the one obtained by Seib et al.28 The release was affected by the pH, and a low pH (4.5) enhanced drug release from silk particles. This finding applies to several silk particles of different origins (silkworm, bioengineered ADF4).28,31,32 For particles made of silkworm silk, the loading and release of Dox were explained (in part) by electrostatic interactions.28 Silkworm silk particles were negatively charged at pH 7.4, which could be attractive for positively charged Dox. In our study, the silk variants were positively charged during the loading process (pH 8). The isoelectric points of MS1, H2.1MS1, and H2.2MS1 were 10.83, 10.27, and 10.69, respectively. Both components (silk and Dox) had the same charge. However, Hofer et al. observed the loading of positively charged spheres made of bioengineered silk eADF4 with positively charged molecules such as FITC-lysozyme and FITC-BSA.31 The above studies suggest that not only electrostatic forces are involved in drug-silk interactions. Moreover, the release of the drug is a complex phenomenon and depends on the properties of both a matrix and a drug (silk and Doxorubicin). The pH dependency of Dox release was also shown in different delivery systems. It was explained that higher release/diffusion rate of Dox at lower pH results from its basic nature and in consequence higher solubility at lower pH.33−36 Another study was required to show that beyond the structural changes, the functional domains are exposed on the sphere surfaces and they preserve the binding capacity. Indeed, the binding domains remained functional and were not only specifically targeted, but most of them (except the largest particles) were also internalized into cells that were overexpressing Her2. The morphology of cells containing internal-

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CONCLUSION The targeted-delivery concept goes beyond standard therapy because it recapitulates some of the advantages of topical drug I

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(14) Numata, K.; Kaplan, D. L. Silk-based gene carriers with cell membrane destabilizing peptides. Biomacromolecules 2010, 11, 3189− 95. (15) Numata, K.; Reagan, M. R.; Goldstein, R. H.; Rosenblatt, M.; Kaplan, D. L. Spider silk-based gene carriers for tumor cell-specific delivery. Bioconjugate Chem. 2011, 22 (8), 1605−10. (16) Widhe, M.; Johansson, U.; Hillerdahl, C. O.; Hedhammar, M. Recombinant spider silk with cell binding motifs for specific adherence of cells. Biomaterials 2013, 34 (33), 8223−34. (17) Numata, K.; Mieszawska-Czajkowska, A. J.; Kvenvold, L. A.; Kaplan, D. L. Silk-based nanocomplexes with tumor-homing peptides for tumor-specific gene delivery. Macromol. Biosci. 2012, 12 (1), 75−82. (18) Witton, C. J.; Reeves, J. R.; Going, J. J.; Cooke, T. G.; Bartlett, J. M. Expression of the HER1−4 family of receptor tyrosine kinases in breast cancer. J. Pathol. 2003, 200 (3), 290−7. (19) Baselga, J.; Swain, S. M. Novel anticancer targets: revisiting ERBB2 and discovering ERBB3. Nat. Rev. Cancer 2009, 9 (7), 463−75. (20) Dams-Kozlowska, H.; Majer, A.; Tomasiewicz, P.; Lozinska, J.; Kaplan, D. L.; Mackiewicz, A. Purification and cytotoxicity of tag-free bioengineered spider silk proteins. J. Biomed Mater. Res., Part A 2013, 101 (2), 456−64. (21) Huemmerich, D.; Helsen, C. W.; Quedzuweit, S.; Oschmann, J.; Rudolph, R.; Scheibel, T. Primary structure elements of spider dragline silks and their contribution to protein solubility. Biochemistry 2004, 43 (42), 13604−12. (22) Lammel, A.; Schwab, M.; Slotta, U.; Winter, G.; Scheibel, T. Processing conditions for the formation of spider silk microspheres. ChemSusChem 2008, 1 (5), 413−6. (23) Slotta, U. K.; Rammensee, S.; Gorb, S.; Scheibel, T. An engineered spider silk protein forms microspheres. Angew. Chem., Int. Ed. 2008, 47 (24), 4592−4. (24) Urbanelli, L.; Ronchini, C.; Fontana, L.; Menard, S.; Orlandi, R.; Monaci, P. Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage. J. Mol. Biol. 2001, 313 (5), 965−76. (25) Shadidi, M.; Sioud, M. Identification of novel carrier peptides for the specific delivery of therapeutics into cancer cells. FASEB J. 2003, 17 (2), 256−8. (26) Wang, X. F.; Birringer, M.; Dong, L. F.; Veprek, P.; Low, P.; Swettenham, E.; Stantic, M.; Yuan, L. H.; Zobalova, R.; Wu, K.; Ledvina, M.; Ralph, S. J.; Neuzil, J. A peptide conjugate of vitamin E succinate targets breast cancer cells with high ErbB2 expression. Cancer Res. 2007, 67 (7), 3337−44. (27) Lammel, A. S.; Hu, X.; Park, S. H.; Kaplan, D. L.; Scheibel, T. R. Controlling silk fibroin particle features for drug delivery. Biomaterials 2010, 31 (16), 4583−91. (28) Seib, F. P.; Jones, G. T.; Rnjak-Kovacina, J.; Lin, Y.; Kaplan, D. L. pH-dependent anticancer drug release from silk nanoparticles. Adv. Healthcare Mater. 2013, 2 (12), 1606−11. (29) Moghimi, S. M.; Hunter, A. C.; Murray, J. C. Nanomedicine: current status and future prospects. FASEB J. 2005, 19 (3), 311−30. (30) Sahay, G.; Alakhova, D. Y.; Kabanov, A. V. Endocytosis of nanomedicines. J. Controlled Release 2010, 145 (3), 182−95. (31) Hofer, M.; Winter, G.; Myschik, J. Recombinant spider silk particles for controlled delivery of protein drugs. Biomaterials 2011, 33 (5), 1554−62. (32) Lammel, A.; Schwab, M.; Hofer, M.; Winter, G.; Scheibel, T. Recombinant spider silk particles as drug delivery vehicles. Biomaterials 2010, 32 (8), 2233−40. (33) Qi, J.; Yao, P.; He, F.; Yu, C.; Huang, C. Nanoparticles with dextran/chitosan shell and BSA/chitosan coredoxorubicin loading and delivery. Int. J. Pharm. 2010, 393 (1−2), 176−84. (34) Sanson, C.; Schatz, C.; Le Meins, J. F.; Soum, A.; Thevenot, J.; Garanger, E.; Lecommandoux, S. A simple method to achieve high doxorubicin loading in biodegradable polymersomes. J. Controlled Release 2010, 147 (3), 428−35. (35) Subedi, R. K.; Kang, K. W.; Choi, H. K. Preparation and characterization of solid lipid nanoparticles loaded with doxorubicin. Eur. J. Pharm. Sci. 2009, 37 (3−4), 508−13.

application, namely, high local concentration and low systemic exposure. Cancer stands out as a disease most likely to be treated by targeted drug delivery. We developed the system based on functionalized silk that would target breast cancer specifically. The targeted delivery of drug molecules can increase the therapeutic index of chemotherapeutic agents against tumor cells, simultaneously reducing the toxicity in normal tissues. However, the targeted drug delivery concept is not limited to the treatment of cancer, but it has much broader applications.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +48 61 88 50 874. Fax.: +48 61 85 28 502. E-mail: hanna. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.F. was supported by the International Ph.D. Projects Programme of Foundation for Polish Science operated within the Innovative Economy Operational Programme (IE OP) 20072013 within European Regional Development Fund.

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REFERENCES

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