PtBz SFNs Dalton Trans 2015.pdf

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PAPER

Cite this: DOI: 10.1039/c5dt00378d

Received 27th January 2015,Accepted 9th March 2015

DOI: 10.1039/c5dt00378d

www.rsc.org/dalton

Antitumor properties of platinum(IV)prodrug-loaded silk broin nanoparticles

A. Abel Lozano-Prez,a Ana L. Gil,b Sergio A. Prez,b Natalia Cutillas,b Hajo Meyer,c

Mnica Pedreo,b Salvador D. Aznar-Cervantes,a Christoph Janiak,c Jose Luis Cenisa

and Jos Ruiz*b

Platinum(IV) complexes take advantage of the exclusive conditions that occur within the tumor to carry

out their cytotoxic activity. On the other hand, silk broin has natural properties which make it very inter-

esting as a biomaterial: high biocompatibility, biodegradability, low immunogenicity, high cellular pene-

tration capacity and high reactive surface. Herein we report the preparation of silk broin nanoparticles

(SFNs) loaded with the hydrophobic Pt(IV) complex cis,cis,trans-[Pt(NH3)2Cl2(O2CC6H5)2] (PtBz). Only a

small fraction of the loaded PtBz is released (less than 10% after 48 h). PtBzSFNs trigger strong cytotoxic

eects against human ovarian carcinoma A2780 cells and their cisplatin-resistant variant A2780cisR cells.

Interestingly, PtBzSFNs are very cytotoxic (nanomolar IC50 values) toward the triple negative breast

tumor cell line MDA-MB-231, and also toward SK-BR-3 and MCF-7, while maintaining an excellent

selectivity index.

1. Introduction

Platinum(II) drugs are eective anticancer agents against manymalignancies including testicular, ovarian, bladder, and non-small-cell lung cancer.1 In clinics, cisplatin (CDDP) is one ofthe most widely used anticancer drugs, which can bind togenomic DNA. However, like most of the other small-moleculeanticancer drugs, its clinical use is limited by its high toxicityand severe side-eects,2,3 usually nephrotoxicity and neurotoxi-city, as well as incidences of Pt associated drug resistance.4

Surrounded by six ligands in an octahedral coordinationenvironment, the hydrophobic platinum(IV) metal centre PtBz(Fig. 1) is more inert towards substitution reactions than plati-num(II). Upon reduction in the tumor cells by several intra-cellular reducing agents such as glutathione, metallothionein,cysteine, and ascorbic acid, two axial ligands are released anda hydrophilic divalent CDDP species is generated.5,6

Nanoparticles have drawn particular attention in recentyears because they can facilitate the delivery of therapeutic

drugs taking advantage of nanosized porous tumor vascula-tures coupled with poor lymphatic clearance and slow venousreturn in these tissues, i.e. the enhanced permeability andretention (EPR) eect.714 Nanocarrier-based delivery of plati-num compounds is one such area involving intense researcheort and may allow the development of the next generation ofplatinum chemotherapy.1526

On the other hand, natural polymers such as proteins andpolysaccharides represent a very promising biomedical plat-form because of their renewability, nontoxicity, biocompatibil-ity, and biodegradability.2732 One such polymericnanoparticle is the silk fibroin protein,3335 which shows lowimmunogenicity.36

Silk fibroin protein obtained from a domesticated mulberrysilkworm Bombyx mori is a protein based biomacromoleculecomposed of 5507 amino acids consisting of repeatedsequences of six residues of (Gly-Ala-Gly-Ala-Gly-Ser)nrepeats.37 Silk fibroin has been widely used in blood vessel,skin, bone, cartilage and nervous tissue scaolds.3843 SFNs

Fig. 1 Chemical structure of (a) cisplatin CDDP and (b) hydrophobicplatinum(IV) prodrug PtBz.

Electronic supplementary information (ESI) available: Additional figuresregarding characterization of NPs and biological study details. See DOI: 10.1039/c5dt00378d

aInstituto Murciano de Investigacin y Desarrollo Agrario y Alimentario (IMIDA),

E-30150 La Alberca, Murcia, SpainbDepartamento de Qumica Inorgnica, Universidad de Murcia and Instituto

Murciano de Investigacin Biosanitaria IMIB-Arrixaca, E-30071 Murcia, Spain.

E-mail: [email protected]; Fax: +34 868 88 41 48; Tel: +34 868 88 74 55cInstitut fr Anorganische Chemie und Strukturchemie, Universitt Dsseldorf,

D-40204 Dsseldorf, Germany

This journal is The Royal Society of Chemistry 2015 Dalton Trans.

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loaded with some antitumor drugs such as paclitaxel,29 metho-trexate,34 doxorubicin,36,44 and CDDP45 (chemically combinedvia coordinate bonds) have been recently reported.

Here we disclose novel Pt(IV)-SFNs which exert strong cyto-toxic eects in the CDDP resistant cells A2780cisR and a panelof breast cancer cell lines. These constructs have been furtherstudied for cell cycle arrest and apoptosis. We choose an inerthydrophobic Pt(IV) analog of CDDP, PtBz (Fig. 1),46 in order toavoid CDDP deactivation by possible covalent interactions withsome aminoacids of fibroin.

2. Materials and methodsReagents, materials and apparatus

Silk fibroin was obtained from silkworms reared in the seri-culture facilities of IMIDA (Murcia, Spain). Cisplatin was pro-vided by Alfa Aesar. PBS 1 stock solution was from BDH.Purified water (18.2 M cm at 25 C) was obtained from theMillipore Direct-Q1 ultra-pure water system, Billerica, MA.MTT assay kit was from Promega (Madison, USA). Solventswere dried by the usual methods. DMSO was obtained fromScharlau. All chemicals and solvents were of analytical gradeand were used without further purification. cis,cis,trans-[Pt(NH3)2Cl2(O2CC6H5)2]

46 (PtBz) and SFNs30 were prepared usingprocedures described elsewhere.

Platinum analyses were undertaken using an atomicabsorption spectrometer (model 800, Perkin-Elmer, Shelton,USA) equipped with a Zeeman-eect background correctionsystem, a transversely heated graphite tube atomizer and anAS-800 autosampler. The autosampler was not used, and thesamples were pipetted manually into the atomizer. Pyrolyticgraphite platforms in pyrolytically coated tubes were obtainedfrom the same manufacturer (part number B050-4033). Argon(99.999% of purity) at 250 mL min1 was used as inert gas, exceptduring atomization, when it was stopped. A platinum hollowcathode lamp (Perkin-Elmer) was used as the radiation source.Probes were injected at a volume of 40 L into regular graphitewall tubes. All the measurements were done in triplicate.

Infrared spectra of the synthesized complex PtBz, unloadedSFNs, and PtBzSFNs were compared (Fig. S1). Measurementswere carried out on a Bruker TENSOR 37 IR spectrometer atambient temperature in the range of 4000500 cm1. Samples(2 mg) were mixed with 198 mg of KBr (Sigma-Aldrich,Steinheim, Germany) and ground into a fine powder using amortar and pestle before compressing it into a disc. All thesamples were analyzed in transmission mode. Nitrogen phys-isorption isotherms were measured on a Quantachrome Nova4000e. The surface area of the particles was determined byvolumetric nitrogen sorption measurements at 77 K. Dynamiclight scattering (DLS) and zeta potential measurementswere carried out with a Malvern Zetasizer Nano Z and aMastersizer Malvern 2000E. For the determination of the SFNand PtBzSFN sizes and their size distributions the drysamples were redispersed to a concentration of 1 mg mL1 in1.5 mL doubly deionized water via ultrasonication for 30 min.

The dispersions were investigated by DLS to yield a hydro-dynamic radius for SFNs in the range of 80400 nm with anaverage of 133 22 (standard deviation ). The PtBzSFNs havea broader radius distribution of 80800 nm with an average of173 57. Transmission electron microscopy measurementswere carried out on a Zeiss 902 at ambient temperature. Scan-ning electron microscopy measurements were carried out on a1430 VP at ambient temperature. Light microscopy pictureswere taken with a Carl Zeiss Axiophot 2 and a Zeiss Plan-Neofluar 63 lens with a 630-time magnification. Powder X-raydiraction (PXRD) patterns of all the samples were measuredat ambient temperature on a Bruker D2 Phaser using a flatsample holder and Cu K radiation ( = 1.54182 ). Ramanmeasurements were carried out on a Bruker MultiRam with asolid sample at 900 mW laser intensity and an aperture of6 mm. UV measurements were carried out on a ShimadzuUV-2450 UV spectrometer at ambient temperature with a con-centration of 1 mg mL1. ESI mass (positive mode) analyseswere performed on a HPLC/MS TOF 6220. The isotopic distri-bution of the heaviest set of peaks matched very closely withthat calculated for the formulation of the complex cation.

Preparation of platinum(IV)-loaded nanoparticles (PtBzSFNs),and the drug loading content (DLC) and eciency (E)

Platinum(IV)-loaded nanoparticles (PtBzSFNs) were preparedby an incubation method. 25 mg of dried SFNs were sus-pended with 1 mL of platinum(IV) complex solution, from 1 to10 mg mL1, in DMSO in Eppendorf tubes, sonicated for4 cycles of 15 s at 30% power with pulses 15 s ON and 15 sOFF, gently agitated at 103 rpm for 72 h, and protected fromlight. The loaded particles were collected by centrifugation at18 000g for 15 min. The quantity of the loaded Pt(IV) prodrugwas determined by an indirect method by AAS from the super-natant and SFNs were washed with 500 L MilliQ waterDMSO(15 : 1) and with 500 L MilliQ water. Each phase was sonicatedand centrifuged again under the above conditions. All batcheswere prepared in triplicate. PtBzSFNs were diluted to thedesired concentration immediately before use according to theequivalent dose of the platinum prodrug.

The drug loading content and eciency were presented byeqn (1) and (2), respectively:

DLC % Weight of the drug innanoparticlesWeight of the nanoparticles

100 1

E % Weight of the drug innanoparticlesWeight of the feeding drugs

100 2

Release of the drug from PtBzSFNs

The release rate of the platinum complex from the silk fibroinnanoparticles in phosphate buered saline (PBS 1, pH 7.4)was evaluated. A suspension of PtBzSFNs (25 mg) in waterwas confined (15 mL) into a semipermeable Slide-A-LyzerDialysis cassette (3.5 kDa MWCO, Pierce-Thermo Scientific)and dialyzed against 600 mL PBS (pH 7.4) at 310 K. At a pre-determined time, an aliquot of 500 L of the external PBS was

Paper Dalton Transactions

Dalton Trans. This journal is The Royal Society of Chemistry 2015

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removed, and the platinum content was determined by AAS.After sampling, an equal volume of fresh PBS was immediatelyadded to the external medium.

Samples were diluted to the calibration range (10200 gL1) with PBS. Each assay was performed in triplicate. Theamount of Pt(IV) released from the nanoparticles was expressedas a percentage of the total prodrug loaded in the nanoparti-cles and was plotted as a function of time.

Material preparation for fluorescent silk fibroin nanoparticles

Preparation of FITC (fluorescein isothiocyanate) labeled SFNswas based on the coupling reaction between the isothiocyanategroup of the FITC and the primary amino group of the silkfibroin. Reaction conditions were set up following the protocoldescribed by the supplier of the FITC. Briefly, 100 mg of FITCin 10 ml of DMSO was slowly added to 10 ml of 1 mg mL1

SFN suspension in borate buer (50 mM, pH 8.5). The coup-ling reaction was allowed to proceed for 1 h at room tempera-ture protected from light. FITC labeled fibroin nanoparticleswere then recovered using centrifugation (18 000g for 40 minat 4 C). To remove the unconjugated FITC, the labelled SFNswere subjected to repeated cycles of washing with PBS 1 (pH7.4) and centrifugation until no fluorescence (ex 490 nm;em 520 nm) was detected in the supernatant. The FITC conju-gated nanoparticles were then dialyzed in a Slide-A-LyzerDialysis cassette (3.5 kDa MWCO, Pierce-Thermo Scientific) for3 days in the dark against 4 L of distilled water; the water wasreplaced on a daily basis. Labelled SFNs were refrigerated andprotected from light until its use.

Materials, cell lines and culture conditions

Three breast cancer cellular phenotypes were evaluated asMCF-7 (estrogen receptors), MDA-MB-231 (no estrogen recep-tors) or SK-BR-3 (overexpressed HER2/c-erb-2 gene) and theywere grown in Dulbeccos modified Eagles medium (DMEM)without phenol red to contain 1000 mg L1 glucose sup-plemented with 10% (v/v) final concentration of heat-inacti-vated fetal bovine serum (FBS, Sigma, USA), 2 mML-glutamine, 2 mM sodium pyruvate and antibiotics (penicil-lin/streptomycin) at 310 K under a humidified atmospherecontaining 7.5%10% CO2. Human ovarian carcinoma A2780and A2780cis cell lines were grown in RPMI-1640, sup-plemented with 10% (v/v) final concentration of heat-inacti-vated fetal bovine serum (FBS, Sigma, USA), 2 mM L-glutamineand antibiotics (penicillin/streptomycin). Both cell lines weremaintained at 310 K under a humidified atmosphere containing5% CO2. This cisplatin-resistant cell line has been developed bychronic exposure of the parent cisplatin-sensitive A2780 cellline. In order to retain resistance to CDDP, it was added 1 Mevery two subcultures. EA.hy926 cell lines were grown in Dulbec-cos modified Eagles medium (DMEM) without phenol red con-taining 1000 mg L1 glucose supplemented with 10% (v/v) finalconcentration of heat-inactivated Fetal Bovine Serum (FBS,Sigma, USA), 2 mM L-glutamine, 2 mM sodium pyruvate andantibiotics (penicillin/streptomycin) at 310 K under a humidi-fied atmosphere containing 7.5%10% CO2.

LLC-PK1 cell lines were grown in M199 medium sup-plemented with 3% Fetal Bovine Serum (FBS, Sigma, USA),2 mM L-glutamine and antibiotics (penicillin and streptomycin)at 310 K under a humidified atmosphere containing 5% CO2.

Breast cancer cell lines, EA.hy926 cell lines and LLC-PK1cell lines were obtained from American Tissue Culture Collec-tion (ATCC, U.S.A.) and ovarian carcinoma cell lines wereobtained from European Collection of Animal Cell Culture(ECACC, Salisbury, U.K.).

Before and after experiments, all cell lines were mycoplasma-free, as determined by the Hoechst DNA stain method.47

In all cases, cells from 80% confluent monolayers wereremoved from flasks using a 0.25% trypsin solution (SigmaAldrich), centrifuged at 200g for 10 min and washed twice withPBS, and finally the cell pellet was diluted with completemedium. The medium was changed twice every week.

Cytotoxicity assays

Cell proliferation was evaluated by the MTT assay (3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). MCF-7,MDA-MB-231, SK-BR-3, A2780, A2780cis and LLC-PK1 wereplated in 96-well sterile plates at a density of 5 103 cells perwell with 200 L of medium and then incubated for 24 h. Afterattachment to the culture surface, cells were incubated withvarious concentrations of loaded nanoparticles (PtBzSFNs)diluted by adding 50 L of the culture medium for 72 h at310 K. For the MTT assay, the medium was removed by pipet-ting, then 200 L of the corresponding medium for each cellline was added and 50 L of a 5 mg mL1 MTT solution wasadded and left at 310 K for 4 hours in the dark. Finally, thissolution was removed from each plate, and then 100 L ofDMSO was added and shaken for 5 min at 120 rpm (always inthe dark) before the measurement. The absorbance wasmeasured at 560 nm for the MTT assay using a FluorstarOmega spectrophotometer.

The eects of complexes are expressed as corrected percen-tage inhibition values according to the following equation

% inhibition 1 TC

100

where T is the mean absorbance of the treated cells and C isthe mean absorbance in the controls.

The inhibitory potential of compounds was measured bycalculating concentrationpercentage inhibition curves; thesecurves were adjusted to the following equation:

E Emax1 IC50

C

n

where E is the percentage inhibition observed, Emax is themaximal eects, IC50 is the concentration that inhibits 50% ofmaximal growth, C is the concentration of compounds testedand n is the slope of the semi-logarithmic doseresponsesigmoid curves. This non-linear fitting was performed usingSigma Plot 11.0 software.

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For comparison purposes, the cytotoxicity of cisplatin wasevaluated under the same experimental conditions. All com-pounds were tested in three independent studies with quadru-plicate points. The in vitro studies were performed at theSupport Service for Experimental Sciences of the University ofMurcia (Murcia, Spain).

All SFN and cisplatin studies were performed in water and inall the experiments measurements were corrected with a watercontrol. Due to the low aqueous solubility of PtBz, it was dis-solved in DMSO first and then serially diluted in completeculture medium such that the eective DMSO content was 0.4%.

TEM sample preparation

One million cells per well were cultured on a 6-well plate andleft for 24 h at 37 C. Afterwards, 1 M of the drug was addedand left again for 24 h at the same temperature. The mediumwas then added to a Falcon tube. Cells were removed fromthe wells with trypsin and the resulting suspension was put inthe Falcon tube together with the medium. This was fol-lowed by centrifugation (200g, 10 min), discarding the super-natant. Cells were fixed with glutaraldehyde (2.50%) for 1.5 hand then washed with a washing liquid (cacodylate buer) bymeans of centrifugation (under the same conditions). Increas-ing concentrations (30, 50, 70, 90%) of ethanol were added tothe cells over 10 min. Subsequently, absolute ethanol and, atthe same time, copper(II) sulphate were added over10 minutes. To complete the inclusion method, subsequentadditions of propylene oxide in epoxy resins at increasing con-centration were made. Finally, the capsules were left in theepoxy resin overnight and then cut into micrometer dimen-sions, and sections of 7080 nm were collected on meshcopper grids. The sections were imaged on a ZEIS EM10 TEM.Neither osmium tetroxide nor uranyl acetate was added duringthis process to ensure that contrast images were only due tothe added metal drug.

Cell cycle arrest assays

Cell cycle arrest studies were performed on MCF-7,MDA-MB-231, and SK-BR-3 breast cancer cells and A2780 andA2780cis ovarian cancer cells. Typically, 1.5 105 cells wereseeded in a 6-well plate with DMEM or RPMI medium (5 mLper plate). Cells were allowed to fix to the plate by incubationfor 24 h at 310 K (7.510% or 5% CO2). Then, nanoparticles(PtBzSFNs) were added at final concentrations similar to theirrespective IC50 values in the dierent plates. One well for eachcell line was untreated, for use as a control. The cultures wereincubated for an additional 24 h under the same conditions asdescribed above. At this point, the medium was removed andstored in 15 mL Falcon tubes. Immediately, the fixed cellswere treated with 1 mL of trypsin for 4 minutes at 310 K andthen 1 mL of DMEM or RPMI medium containing FBS wasadded to stop the enzymatic action. The resulting 2 mL wereadded to the 15 mL Falcon tube. In this way both the poss-ible floating cells and adherent cells were considered for theassay. Cells were centrifuged (250g, 10 min) and the precipi-tated cells were washed with 2 mL of PBS. After another cycle

of centrifugation under the same conditions, the supernatantwas removed and the cells were resuspended in 200 L of PBS.Subsequently, 1 mL of a PBS (30%)ethanol (70%) mixed solu-tion was added to the cells. The solution was kept in ice for30 minutes. The supernatant was eliminated by centrifugation(under the same conditions). The cells were again resuspendedin 800 L of PBS. Finally, 100 L of RNase solution (1 mgmL1) and 100 L of propidium iodide solution (400 g mL1)were added. After stirring the resulting suspension was incu-bated at 310 K for 30 min in the dark. The stained cells wereanalyzed using a Becton-Dickinson FACScalibur flow cyto-meter. In each case 30 000 events were acquired.

Apoptosis experiments

For apoptosis determination assays, 1.5 105 cells were typi-cally seeded in a 6-well plate. Breast and ovarian cancer cellswith compounds and their control were incubated, collectedand washed twice with PBS as described above (no PBSethanol mixture was used in this case). After removing thePBS, 40 L of a solution containing Annexin V and PI(Annexin-V-Fluos from Roche) and 160 L of incubation buer(HEPES 10 mM, NaCl 140 mM, CaCl2 5 mM pH = 7.4) wereadded to the cell pellet. Cells were resuspended in this solu-tion and left at room temperature in the dark for 15 min. 200L of PBS was added immediately before the measurements.These were carried out using a Beckman Coulter Epics XL flowcytometer, registering the emission at wavelengths of 620 and525 nm for PI and Annexin V, respectively. In each case, 10 000events were acquired.

Results and discussionPreparation and characterization of PtBzSFNs

The PtBzSFNs were generated using a suspension of lyophi-lized SFNs, obtained from Bombyx mori,31 with a PtBz46 solutionin DMSO, dispersed by sonication and then agitation for 72 h.

The loaded particles were collected by centrifugation. Theentrapping of the prodrug was optimized to maximize boththe loading and eciency. The Pt content in the PtBzSFNswas measured from the supernatant by an indirect method byatomic absorption spectrometry (AAS). The determination ofthe sizes of the NPs was done by dynamic light scattering(DLS). Drug loading in over 15 independent experiments withdierent batches of nanosilk gave up to 4.1% PtBz loading anda maximum drug uptake eciency of 23%. By using 10 mgmL1 (or more) PtBz concentration non-nanometric sized par-ticles were obtained. Optimally loaded NPs were generated byusing 5 mg mL1 of PtBz. These PtBzSFNs are of adequatesize to exploit the EPR eect and they were characterized(Table 1) by DLS, zeta potential measurements, transmissionelectron microscopy (TEM), scanning electron microscopy(SEM), powder X-ray diraction (XRD), and IR and Ramanspectroscopy.

For the determination of the SFN and PtBzSFN sizes andtheir size distributions the dry samples were redispersed to a



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concentration of 1 mg mL1 in 1.5 mL doubly deionized watervia ultrasonication for 30 min. The dispersions were investi-gated by DLS to yield a hydrodynamic diameter for SFNs in therange of 80 400 nm with an average of 133 22 nm (stan-dard deviation ) (Fig. 2). The PtBzSFNs have a broader dia-meter distribution of 80 800 nm with an average of 173 57(Fig. 2). The hydrodynamic diameter from DLS is considerablylarger than the primary particle size from TEM and SEMmeasurements (see below). This is apparently due to agglo-meration eects.

The FT-IR spectra of SFNs and PtBzSFNs (see Fig. S1 andS2) exhibit characteristic absorptions for a water insoluble-sheet conformation of the silk proteins. Vibrations at1631 cm1 for amide I (CvO stretching), at 1528 cm1 foramide II (secondary NH bending) and at 1236 cm1 for amideIII (CN and NH functionalities) are in agreement with litera-ture data.30 The small amount of platinum complex comparedwith silk nanoparticles makes its infrared signals barely per-ceptible (Fig. S1 and S2).

The TEM pictures (Fig. 3 and S3) of SFNs and PtBzSFNsshow no obvious dierences. No dierence in shape or sizewas monitored in both samples. We think that DLS coulddierentiate between primary particles and their agglomeratesbut this may also be due to the redispersion. At higher TEMresolutions primary particles of 50 10 nm size can beobserved. SEM investigation of SFN and PtBzSFN samplesdepicted in Fig. S4 gave roughly the same results as the TEMpictures. At the highest resolution (Fig. S4 bottom row) the

primary particle diameter can be determined to about 50 nmwhich is in agreement with the TEM measurements. Again nodierence could be observed in the shape of the nanoparticlesfor both samples. The macroscopic visual appearance of thesamples is slightly dierent, however. A sample of SFNs lookslike gravel and the PtBzSFN has a fibre morphology whenviewed with a naked eye or light microscope (Fig. S5). Thesespherical NPs have an overall moderate negative net charge of19 8 mV (values very close to those of SFNs; see Table 1).The surface area of the particles was determined from volu-metric nitrogen sorption measurements at 77 K to give a BETsurface of 56 m2 g1 for SFNs and 60 m2 g1 for PtBzSFNs.Both values are identical within experimental error which isabout 20 m2 g1. Also a BET surface of about 50 m2 g1

corresponds to the outer surface of a fine powder.Powder X-ray diraction (PXRD) analysis illustrated that the

XRD curves of SFNs and PtBzSFNs had the characteristic scat-tering pattern of the -sheet crystalline silk fibroin (Fig. S6).They show a major peak at 2 = 20.2 and two shoulders at9.9 and 24.3 in both loaded and unloaded SFNs. There is nosignificant dierence between the powder patterns of SFN andthe PtBzSFN samples.30 The Raman spectra of SFNs andPtBzSFNs (Fig. S7) showed exactly the same absorptions asreported in the literature for SFNs.30 The ratio values of theI852/I830 (Rtyr) bands were 1.29 and 1.45 for the lyophilizedPtBzSFNs and the unloaded SFNs, respectively (Fig. S7). Thedierence of Rtyr values

30,35,48 led to the conclusion that thehydrogen bonds involving the tyrosyl residues in the loadednanoparticles were weaker than those in the empty SFNs, butstronger than those in the lyophilized aqueous silk. In otherwords, the tyrosine residues around SFNs were more exposedthan those around the PtBzSFNs. UV-spectra of both SFNsand PtBzSFNs showed a broad absorption in the range of

Table 1 Comparative values for the particle size, polydispersity (PDI), -potential and mobility of SFNs and PtBzSFNs formed by 5 mg mL1 drugfeed (water at 25 C, n = 5 and accumulation times = 100)

Sample Diameter width (nm) PDI -potential SD (mV) Wall zeta potential (mV) Mobility (m cm V1 s1)

SFNs 133 22 0.11 20 11 35.2 1.6 0.9PtBzSFNs 173 57 0.36 19 8 24.4 1.5 0.6

Fig. 2 Dynamic light scattering experiment of SFNs (black) and thePtBzSFNs (red) in 1.5 mL doubly deionized water with the resulting dis-tribution of the hydrodynamic diameter.

Fig. 3 TEM pictures of SFNs (a) and the PtBzSFNs (b).



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270285 nm (Fig. S8). No significant change in the absorptionspectra upon PtBz modification of SFNs was observed.

For targeted delivery, drug payload should only be releasedat the site of its intended target. In order to study the releaseproperties of the NPs, aliquots of PtBzSFN suspension weredialyzed against phosphate buered saline (PBS) at 37 C over10 days. The release of platinum, measured by AAS in this andsubsequent experiments, during dialysis is presented in Fig. 4as the fraction of the entrapped platinum released over time.The results show that only 8.5% of platinum is released during48 h keeping the loading constant during the rest of the time.This is in line with the poor water solubility of the drug. Inaddition, to identify the nature of the release products ofPtBzSFNs, a study by HPLC-MS in water was also undertaken(no PBS used). The unique fraction where platinum peaks wereobserved showed clusters at 543.00 and 420.97 correspondingto [PtBz + H]+ and the loss of a benzoate group from PtBz+,respectively (Fig. S9). Altogether, these findings suggest thatmost of PtBz could reach the tumor tissue, in spite of its pooraqueous solubility profile.

SFN cell internalization

As in a previous assay for the cytotoxicity study, human endo-thelial cells EA.hy926 were incubated for 3 h with fluoresceinisothiocyanate (FITC) labelled SFNs and then studied byoptical fluorescence microscopy. FITC-labelled SFNs are equi-valent to non-labelled SFNs in size distribution and cyto-toxicity. Cell penetration of the labelled nanoparticles can bemonitored easily by its intense fluorescence. These images(Fig. 5) show significant cell internalization of FITC-SFNs.

PtBzSFN antitumor activity

The cytotoxicity of PtBzSFNs was evaluated (Table 2) toward apanel of human cancer cell lines representative of epithelialovarian carcinoma cells A2780 and A2780cisR (acquired resist-ance to cisplatin) and several breast cancer cells: SK-BR-3

(HER2+), MCF-7 (ER+, PR+) and the triple-negativeMDA-MB-231 (ER, PR, no HER2 overexpression), where cis-platin is not very active. The PtBzSFNs trigger strong cytotoxiceects, i.e. nanomolar IC50 values in all the tumor cell linesassayed, being much more active than cisplatin in all of them:over 20 times as sensitive to cisplatin A2780 or over 70 times inMCF-7. Free platinum SFNs were not cytotoxic in A2780 andMCF-7 cell lines after 72 h. These results suggest that PtBzSFNs are able to overcome the resistance mechanisms ofovarian and most breast cancer cells, including the triple nega-tive MDA-MB-231. Furthermore, the cytotoxicity of PtBzSFNs

Fig. 5 Dierent images of the cellular uptake of FITC-labeled silk nano-particles by endothelial cells (EA.hy926) after incubation for 2 h at 310 Kunder a 7.5% CO2 atmosphere in a CO2 incubator. (A) Cells under brighteld; (B) cells under uorescence and (C) composition of (A) and (B); (D)cells under phase contrast; (E) cells under uorescence and (F) compo-sition of (D) and (E). (A) to (C) at 4 magnication, scale bar = 500 m;(D) to (F) at 40 magnication, scale bar = 100 m).

Table 2 IC50 values [M] after 72 h of incubation of CDDP, PtBz andPtBzSFNs prepared by using 5 mg mL1 of PtBz expressed as obtained

Cell line CDDP PtBz PtBzSFNs

A2780 2.06 0.30 0.031 0.020 0.09 0.01A2780cisR 7.60 0.22 0.34 0.00 0.78 0.24MCF-7 22.70 1.11 0.86 0.04 0.31 0.02MDA-MB-231 >25 0.59 0.01 0.39 0.15SK-BR-3 7.20 0.19 0.17 0.01 0.34 0.03LLC-PK1 4.12 0.13 1.62 0.03 4.20 0.22

Fig. 4 Release of platinum from PtBzSFNs in PBS (310 K).



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toward the non-tumorigenic renal cells LCC-PK1 is similar tothat of cisplatin and less than that of PtBz. The cell selectivityfactors (SF = IC50 for LLC-PK1/IC50 for A2780 cells) for thePtBzSFNs (SF = 46.7) are higher than that of CDDP (SF = 2.0).

By using TEM, it seemed likely that we would observe plati-num derived contrast in sections of cancer cells treated withthe PtBzSFNs studied here if their deposition is sucientlylocalized in cell organelles. A2780 cells were exposed to 1 Mof SFNs or PtBzSFNs for 24 h, and the treated and controlcells were fixed and embedded in epoxy resin. Ultrathin sec-tions were observed under the TEM (Fig. 6). Neither uranylacetate nor OsO4 was used for staining both the control cellsand the treated cells. Any additional contrast observedbetween the control cells and the treated cells is due to celluptake of SFNs or PtBzSFNs.

Sections of cells treated with 1 M of PtBzSFNs show morecontrast in comparison with those from control cells in thenucleolus and the internal nuclear membrane (Fig. 6E).Several cells displayed the morphological changes associatedwith apoptosis (Fig. 6E and F).

To understand the eect of PtBzNFSs on cell growth weexamined their eect on the cell cycle by FACS (FluorescenceActivated Cell Sorter) analysis. Treatment of A2780 and

A2780cisR cell lines with 0.5 M PtBzSFNs (approximately theIC50 value for the NPs with respect to A2780 cells) for 24 h(Fig. 7) showed a significant increase of these cells in theS and G2 phases, with a corresponding reduction of those inG0/G1, as observed also in cisplatin. The emergence of a tetra-ploid cell population unable to divide occurred (Fig. 7B). Cellcycle analysis of MCF-7, MDA-MB-231 and SK-BR-3 cancer cellswas also undertaken (Fig. S10). Apoptotic studies were alsocarried out with all cell lines. Exposure of phosphatidylserine(PS) was followed by a flow cytometric assay with the AnnexinV-FLUOS apoptosis detection kit (Roche). Propidium iodide(PI) was also applied to stain necrotic or late apoptotic cells.The fluorescence intensity was measured for each cell by flowcytometry. The results for MDA-MB-231 cells are shown inFig. 8. The four typical quadrants identifying the living (lower

Fig. 6 Images of A2780 cells after 24 h of exposure to: (C) and (D)10 M of SFNs; (E) and (F) 10 M of PtBzSFNs; in (E) early apoptosis isobserved; in (F) late apoptosis is observed; the inset in (C) shows mag-nications. (A) Control; (B) SFNs in the intercellular space.

Fig. 7 Cell cycle analysis of A2780 and A2780cisR human ovariancancer cells after 24 h at 310 K. (A) and (C) FL2 histograms for negativecontrols: cells untreated (A2780 and A2780cisR, respectively); (B) and(D) FL2 histograms for cells exposed to PtBzSFNs, the concentrationused was 0.5 M.

Fig. 8 Flow cytometry analysis of MDA-MB-231 human breast cancercells after treatment with PtBzSFNs as detected using annexin V/PI.Density plots for (A) untreated cells (control) and for (B) cells treatedwith 1 M (24 h) PtBzSFNs. The experiments were performed intriplicate.



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left quadrant D1, not or less stained cells), the early apoptotic(lower right quadrant D2, only the annexin-V stained cells), thenecrotic (upper-left quadrant D4, only PI stained cells) and thelate apoptotic (upper right quadrant D3, cells stained withboth fluorescent dyes) cells appear in these diagrams. As canbe seen clearly PtBzSFNs induce a high incidence of latestage apoptosis in MDA-MB-231 cells. Similar results wereobserved in the rest of cells assayed (ESI, Fig. S11 and S12).

4. Conclusions

In summary, we have successfully generated SNFs loaded witha hydrophobic platinum(IV) prodrug, PtBz. Only a small frac-tion of the loaded PtBz is released (less than 10% after 48 h).PtBzSFNs are very cytotoxic (nanomolar IC50 values) towardthe ovarian cell lines A2780 and A2780cisR, and several breasttumor cell lines SK-BR-3, MCF-7 and MDA-MB-231, over-coming drug resistance to cisplatin. Interestingly, the PtBzSFNs are poorly active against the non-tumorigenic renal cellsLLC-PK1. The cell selectivity factors (SF = IC50 for LLC-PK1/IC50 for A2780 cells) for the PtBzSFNs (SF = 46.7) are higherthan that of CDDP (SF = 2.0). Therefore, loading on SFNs canameliorate the delivery of the poorly soluble PtBz and reduceits toxicity towards non-tumorigenic cells. TEM images showedhigh electron density in the nucleus of A2780 cells. PtBzSFNscause cell cycle arrest in the S and G2 phases and induce latestage apoptosis.

Acknowledgements

This work was supported by the Spanish Ministerio de Econo-ma y Competitividad and FEDER/ERDF (ProjectSAF201126611) and by Fundacin Sneca-CARM (Project15354/PI/10). COST CM1105 for providing opportunities of dis-cussion and Dr Ignacio Lpez for AAS assistance are alsoacknowledged. Dr A. Abel Lozano-Prezs research contract waspartially supported (80%) by the FEDER/ERDF of the Region ofMurcia 20072013.

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PtBz SFNs Dalton Trans 2015.pdf