Journal of Inorganic Biochemistry 120 (2013) 54–62

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Journal of Inorganic Biochemistry

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Reversibly PEGylated nanocarrier for cisplatin delivery

Ekaterina Stoyanova a,b, Violeta Mitova a, Pavletta Shestakova c, Agnieszka Kowalczuk d, Georgi Momekov e,Denitsa Momekova e, Andrzej Marcinkowski d, Neli Koseva a,⁎a Institute of Polymers, Bulgarian Academy of Sciences, Acad. Georgi Bonchev Str. Bl.103, 1113 Sofia, Bulgariab Faculty of Chemistry and Pharmacy, Sofia University “St. Kliment Ohridski”, 1 James Bourchier Blvd, 1164 Sofia, Bulgariac Institute of Organic Chemistry with Centre of Phitochemistry, Bulgarian Academy of Sciences, Acad. Georgi Bonchev Str. Bl. 9, 1113 Sofia, Bulgariad Centre of Polymer and Carbon Materials, Polish Academy of Sciences, 34M. Curie-Sklodowskie,j 41–819 Zabrze, Polande Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Str., 1000 Sofia, Bulgaria

⁎ Corresponding author. Tel.: +359 2 979 6630; fax:E-mail addresses: ajfa_jest@abv.bg (E. Stoyanova), m

(V. Mitova), psd@orgchm.bas.bg (P. Shestakova), akowa(A. Kowalczuk), gmomekov@pharmfac.acad.bg (G. Momdmomekova@yahoo.com (D. Momekova), koseva@poly

0162-0134/$ – see front matter © 2013 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.jinorgbio.2012.12.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 August 2012Received in revised form 10 December 2012Accepted 12 December 2012Available online 21 December 2012

Keywords:Reversible PEGylationCisplatin conjugateDiffusion NMRDrug delivery

A star-shaped copolymer bearing a shell of poly(ethylene glycol) (PEG) chains was designed as a carrier ofcisplatin. The proposed strategy was based on synthesis of a PEGylating agent and the incorporation ofcisplatin as a reversible linker for PEG modification of the star macromolecules. The attachment of PEG chainsto the stars and their release under physiological conditions, as well as the changes in particle size andmobility upon drug loading, was evidenced by diffusion ordered NMR spectroscopy (DOSY). The results dem-onstrated that PEGylation reduced inter-stars cross-linking and increased the stability of the nanocolloidalsolution. The formation of PEG shell resulted in higher drug payload and improved drug release profile ofthe nanoconjugates. The in vitro bioassay in a panel of human tumor cell lines confirmed that the PEGylatedconjugates exhibited superior growth inhibitory activity compared to the cisplatin-loaded nonPEGylatedcarrier.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

PEGylation is a term that refers to modification with poly(ethyleneglycol) (PEG) via covalent binding, non-covalent entrapment or adsorp-tion of PEG onto an object. The biological inertness and hydrophilicityare the basis of the successful in vivo applications of PEG as a deliveryplatform [1]. PEGylated molecules and colloidal drug carriers haveprolonged circulating half-life and altered tolerability profile whichaffords more convenient dosing schedules to patients [2–5].

The choice of a PEGylation strategy depends on many factors,i.e. the parameter to be modified, the function and the structure ofthe entity (drug molecule or colloidal carrier), the availability of reac-tive groups, etc. Two alternative approaches are being explored— thepermanent modification through a stable covalent binding of PEGstrands to the agent (i.e. drug, peptide or protein) or the particle sur-face, and the releasable PEGylation via customized linkers that arehydrolytically/enzymatically labile or sensitive to certain stimuli. Areview on the design of reversible linkers has been published [6].The reversible PEG modification is an advantageous delivery platformthat provides both regeneration of the active agent and control overthe pharmacological behavior of the entity. Recent examples are

+359 2 870 03 09.itova@polymer.bas.bglczuk@cmpw-pan.edu.plekov),mer.bas.bg (N. Koseva).

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polyamidoamine dendrimers loaded with doxorubicin and PEGylatedvia acid-sensitive cis-aconityl linkage and acid-insensitive succiniclinkage. The conjugates with a stable PEG shell released negligibledrug at any tested pH condition and were less cytotoxic than theconjugate with reversibly attached PEG chains [7].

In the present study PEGylation has been envisioned to enhance thephysical and functional attributes of a cisplatin nanocarrier. Our recentpaper [8] has reported the results on the design of a core–shell typestar polymer and its evaluation as a delivery vehicle of cisplatin. Themacromolecular carrier possesses a branched hydrophobic interiorand covalently attached poly(acrylic acid) arms. This construct displaysa combination of key features as cisplatin carrier such as high density ofcarboxylate ions that are able to reversibly conjugate with the drug, ahigh drug payload, stability in distilled water on storage and sustainedrelease of the agent under physiological conditions. Despite all these ad-vantageous features some limitations of the star carrier were observed.Upon loading the increase of the drug amount resulted in larger particleformation and, consequently, the particle size distribution broadened.Though the drug mass fraction in the loaded particles was about 45%,just one-third of the conjugated drug was released over a period of9 days. Further increase of the ratio of cisplatin to the coordinatinggroups of the carrier reduced the stability of the colloidal solution.Therefore, we undertook the present investigations aimed at creatinga PEG shell reversibly attached to the star macromolecules in order toallow increase of the drug loading capacity and stability of the systemwithout hampering the sustained release profile of the platinum com-plexes. Different analytical methods such as diffusion ordered NMR

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spectroscopy (DOSY), infrared spectroscopy (IR), atomic forcemicrosco-py (AFM), zeta potential measurement, electrothermal atomic absorp-tion spectrometry (ETAAS) and inductively coupled plasma atomicemission spectrometry (ICP AES) were applied for characterization ofthe PEGylated drug systems.

2. Materials and methods

2.1. Materials

p-(Iodomethyl)styrene was synthesized from p-(chloromethyl)styrene via the Finkelstein reaction [9]. tert-Butyl acrylate (Aldrich,98%) was distilled over CaH2 prior to use. The initiator: α,α′

azobis(isobutyronitrile) (Fluka, >98%) was recrystallized fromdiethyl ether. Trifluoroacetic acid (Aldrich, 99+%) was used as re-ceived. Cis-dichlorodiamminoplatinum(II) (cisplatin) (99.9+%)was purchased from Sigma-Aldrich. Monomethoxy PEG 2000(Fluka) was dried via azeotropic distillation with toluene. Pyridine(Fluka) was dried above anhydrous KOH. Succinic anhydride, anhy-drous dimethylformamide and silver nitrate were supplied bySigma-Aldrich and 15-crown ether-5 (С10Н20О5) by Fluka. These re-agents were used as received. Dialysis membranes with molecularweight cut off (MWCO) 1000, 3500 and 12000–14000were suppliedby SpectraPor.

2.2. Synthesis of star polymer with poly[p-(iodomethyl)styrene] core andpoly(acrylic acid) arms

The synthesis of the precursor star polymers with poly[(p-iodomethyl)styrene] (PS) core and poly(tert-butyl acrylate) arms(PSPtBuA) was carried out using iodinemediated controlled radical po-lymerization as described by Kowalczuk-Bleja et al. [10]. A copolymerproduct of Mn=56 800 g/mol was prepared.

1H NMR (C6D6, 300 MHz): δ 1.2–1.6 ppm (CH3), 1.6–1.9 (CH2),3.7–4.7 (CHI and CH2I), 5.2, 5.9 and 6.3 (CH2=,–CH=), 6.6–7.4(CHarm).

The hydrolysis of PSPtBuA polymer was carried out withtrifluoroacetic acid in a five-fold molar excess of acid with respect toester groups. The procedure is described in [8]. The hydrolyzed copoly-mer was dialyzed to remove lowmolar mass products using SpectraPormembrane with MWCO 1000 g/mol. Subsequently water was evapo-rated and the obtained product was dried under vacuum. It wasassigned as PSPA.

1H NMR (D2O, 600 MHz), δ ppm: 7.2–6.6 (H-atoms from thebenzene rings), 2.4–1.9 (CH in the chains) and 1.9–1.2 (CH2 in thechains).

2.3. Preparation of а carboxylate derivative of PEG

Succinic anhydride (0.168 g; 1.68×10−3 mol) was added tomelted monomethoxy PEG 2000 (3.055 g; 1.53×10−3 mol). Understirring pyridine (0.136 ml; 0.133 g; 1.68×10−3 mol) was addedand the mixture was stirred for 24 h at 70 °C. After cooling to roomtemperature, the product was dissolved in deionized water andNaOH was added until pH reached 11. Then water was evaporatedunder reduced pressure. The obtained product (PEG-COONa) waspurified via crystallization in acetone and dried under reducedpressure.

The 1H NMR (D2O, 600 MHz) δ ppm: 4.17–4.11 (m, CH2OOC),3.76–3.42 (m, H-atoms from the oxyethylene units), 3.26 (s, OCH3),2.52–2.47 (t, OOCCH2CH2COONa), 2.37–2.32 (t, OOCCH2CH2COONa)IR, cm−1: 2883 (νas(CH2)), 2862 (νs(CH2)), 1734 (ν (C_O in estermoiety)), 1101 (ν(C\O)).

2.4. Preparation of а PEG–cisplatin conjugate

Silver nitrate (0.033 g; 1.9×10−4 mol) dissolved in dimethyl-formamide (DMF) (2.5 ml) was added to a solution of cisplatin(0.060 g; 2.0×10−4 mol) in DMF (2.5 ml). The mixture was stirredfor 24 h in darkness at room temperature and then filtered fromAgCl. PEG-COONa (0.354 g; 1.66×10−4 mol) was dissolved in DMF(2 ml) and 15-crown ether-5 (С10Н20О) (33 μl; 1.66×10−4 mol)was added. Both solutions of the activated reagents were mixed andstirred at room temperature for 24 h. Then, DMF was evaporatedunder reduced pressure.

In order to remove the unreacted cisplatin, the crude productwas re-dissolved in acetone. The obtained solution was filtered, andthen thefiltratewas stored overnight in a freezer. The precipitated poly-mer conjugate was filtered and washed with cold acetone. Finally, theproduct was dried under vacuum, keeping the temperature below30 °C. The yield of conjugation reaction (69%) was assessed throughquantitative determination of platinum(II) by ICP-AES. The productwas assigned as PEG–cisplatin.

The 195Pt NMR (D2O, 600 MHz) δ ppm: −2089.IR, cm−1: 2883 (νas(CH2)), 2860 (νs(CH2)), 1732 (ν (C_O in ester

moiety)), 1616 (δ(NH3)), 1101 (ν(C\O)).

2.5. NMR study of PEGylation and cisplatin loading of the star-shapedcopolymer

Star polymers with PS core and poly(acrylic acid) arms (PSPA)(0.013 g, 4×10−7 mol)was dissolved in 0.5 ml D2O andpHwas adjust-ed to 7.3 by addition of NaOD. PEG–cisplatin (0.009 g, 2.8×10−6 mol ofPEGylating agent)wasdissolved in 0.5 mlD2O andadded to the solutionof the star copolymer. Themixture was stirred for 24 h at room temper-ature. It was twice diluted with D2O to obtain a PSPA concentration of6.5 mg/ml and DOSY spectrum was measured. Then the solution wasdivided into two portions of 1 ml.

To one portion NaH2PO4/Na2HPO4/NaCl was added to obtain phos-phate buffered saline solution which was incubated at 37 °C. Aftercertain periods of time DOSY spectra were measured.

Cisplatin was added to the other portion stepwise: 2 mg cisplatinat the first step, two portions of 1.3 mg cisplatin and another portionof 1 mg cisplatin at the second, third and fourth steps of loading. Ateach loading step the system was stirred until the turbid yellowishsolution became transparent and afterwards colorless. Then 1H NMRand DOSY spectra were measured.

2.6. Loading of the PEGylated star copolymers with cisplatin

Firstly, the PEGylated star copolymer was obtained. The PEG–cisplatin product (0.080 g; containing 2.38×10−5 mol of PEGylatingagent) and star copolymer (0.112 g; 3.40×10−6 mol) were separatelydissolved in deionized water. Then, both reagents were mixed and pHof the solution adjusted to 7.3. The total volume of the solution was8.6 ml. The mixture was stirred at room temperature for 24 h.

2.3 ml of the above aqueous solution that contained PEGylatedstars bearing 3.87×10−4 mol COO− groups was added to cisplatin(0.0387 g; 1.29×10−4 mol) in 17 ml deionized water. The molar ratioof cisplatin to carboxylate groups was 1:3. The pH of the solution wasadjusted to 8.0, followed by stirring for 48 h at room temperature. Un-bound cisplatin and PEG derivative were removed by dialysis againstdeionized water for 48 h using membrane with MWCO 12000–14000.

The second portion of 2.3 ml of the PEGylated carrier was loadedin a similar manner using greater amount of cisplatin (0.0464 g;1.54×10−4 mol), i.e.molar ratio cisplatin to carboxylate groups 1:2.5.

The remaining solution of the PEGylated star copolymer was usedin AFM and zeta potential measurements.

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For comparison a loading experimentwith the original starswas alsoperformed at a feeding molar ratio [COO−]:[cisplatin]=3 following theprocedure described above.

2.7. Determination of the PEGylation degree of the star copolymer

The water was evaporated from a determined volume of the dia-lyzed solution containing loaded PEGylated star copolymer and D2Owas added to the solid residue. The mixture was heated for 1 h inwater bath and then known amount of benzoic acid as a referencewas added to the solution. Then 1H NMR spectrum was measured.

2.8. Cisplatin release from the loaded stars

The release of cisplatin from the copolymer carrier in phosphatebuffered saline (10 mM PBS, pH 7.4, 0.14 M NaCl) was studied bydialysis method using a membrane with MWCO 12000–14000. Asolution (10 ml) of the loaded carrier with known platinum drugconcentration was placed into a dialysis bag and dialyzed againstphosphate buffered saline (200 ml) at 37 °C and gentle stirring.Aliquots of 10 ml were taken from the solution outside of the dialysisbag at defined time periods and fresh phosphate buffered saline of thesame volume was added. The concentrations of platinum(II) com-plexes present in the dialysate aliquots were measured and the con-centration of Pt(II) released from the stars was expressed as aaccumulative percentage of the total Pt(II) available and plotted as afunction of time.

2.9. Analytical methods

All NMR spectra were measured on Bruker Avance II+600 NMRspectrometer using 5 mm direct detection dual broadband probe, witha gradient coil delivering maximum gradient strength of 63 G/cm. Theexperiments were performed at a temperature of 293 K. 1H NMR spec-tra were acquired with 32 K time domain points, spectrum width of9600 Hz and 128 scans. The DOSY measurements were performedwith copolymer samples dissolved in NaOD/D2O (pH=8) at concentra-tion of 6.5 mg/ml. The experimental conditions were described in ourprevious paper [8]. The spectra were processed with an exponentialwindow function (line broadening factor 5) and 16 K data points in F2dimension and 1 K data points in the diffusion dimension, using thefitting routine integrated in Topspin2.1 package. The evaluation of thediffusion coefficients was performed by fitting the sum of the columnsalong the chemical shift of each signal in the DOSY spectrum with theGaussian distribution curve.

Assuming spherical shape approximation the apparent hydrody-namic radius, Rh, of the polymer particles can be estimated usingthe Stokes–Einstein equation and the obtained value of the diffusioncoefficient:

Rh ¼ kT6πηD

ð1Þ

where k is the Boltzmann constant, T is the temperature (K) and ηis the solvent viscosity. In the present experiment: η(D2O)=1.2518×10−3 Pa s at 293 K (NIST, USA).

The molar mass and the dispersity of the precursor polymers withpoly(tert-butyl acrylate) arms (PScorePtBuAarm) was determined byGPC with a differential refractive index detector (Dn-2010 fromWGE Dr. Bures) and a multiangle light scattering detector (DAWNEOS from Wyatt Technologies). The experimental conditions weregiven elsewhere [8].

For the atom force microscopy (AFM) analyses, a multimode instru-ment equipped with a NanoScope 3D controller (MultiMode, Veeco In-struments Inc., USA) operating in tapping mode in air with standard125 μmsingle-crystal silicon cantilevers (Model TESP; Veeco Instruments

Inc., USA)was used. Thepiezoelectric scanner had a scan range of approx-imately 10×10 μm2. The aqueous solutions were spin-coated onto micawafers in air at 1500 rpm for 8 min. All samples were imaged at roomtemperature and measured 24 h after coating.

The concentrations of platinum(II) present in the dialysate or in theloaded polymer solutions were measured using electrothermal atomicabsorption spectrometry (Perkin Elmer AAS Zeeman3030with graphitefurnace HGA 600) or inductively coupled plasma. Zeta potential wasmeasured on Zetasizer NanoZS,Malvern Instruments Ltd (UK) equippedwith HeNe gas laser 632.8 nm. IR spectra were recorded on IRAffinity-1(Shimadzu) Fourier transform infrared (FTIR) spectrophotometer withMIRacle Attenuated Total Reflectance Attachment.

2.10. Cell lines and culture conditions

The cell lines used in this study namely HL-60 (acute myelocyteleukemia), K-562 (chronic myeloid leukemia) and HUT-78 (T-cell lym-phoma) were purchased from the German Collection of MicroorganismsandCell Cultures (DSMZGmbH, Braunschweig, Germany). The cellsweremaintained as suspension cultures in a controlled environment, using10% fetal bovine serum (FBS) and 2 mM L-glutamine supplementedRPMI-1640 liquidmedium, in cell culture flasks, housed at 37 °C in an in-cubator ‘BB 16-Function Line’ Heraeus (Kendro, Hanau, Germany) withhumidified atmosphere and 5% CO2. The exponential growth of the cellcultures was maintained by removal of cellular suspension and supple-mentation with fresh medium aliquots two or three times weekly.

2.11. Growth inhibition assessment (MTT-dye reduction assay)

The growth inhibitory activity of free cisplatin or its PEGylatednanoconjugates against human tumor cell lines was tested using thestandard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-mide (MTT)-dye reduction assay as previously described by Mosmann[11] with some minor modifications [12]. The method is based on thereductive biotransformation of the yellowMTT dye to a violet formazanproduct via the mitochondrial succinate dehydrogenase in viable cells.In brief, exponentially growing cellswere seeded in 96-wellmicroplates(100 μl/well) at a density of 1×105 cells per ml and incubated for 24 hat 37 °C. Thereafter the cells were treated with serial dilutions of eitherthe free drug or its nanoconjugates for 72 h or 120 h. After the exposureperiod 10 μl aliquots of a MTT solution (10 mg/ml in phosphate buff-ered saline (PBS)) were added to each well. The microplates were fur-ther incubated for 4 h at 37 °C and the MTT-formazan crystals weredissolved through addition of 100 μl/well 5% solution of formic acid in2-propanol. TheMTT-formazan absorption was determined using a mi-croprocessor controlledmicroplate reader (Labexim LMR-1) at 580 nm.For each treatment group a set of 8 wells was used and all experimentswere run in triplicate.

2.12. Data processing and statistics

The cell survival data were normalized as percentage of theuntreated control (set as 100% viability). The statistical processingof biological data included a two sided Student's t-test wherebyvalues of p≤0.05 were considered as statistically significant.

3. Results and discussion

3.1. Synthesis of a star copolymer with a releasable PEG shell

The preparation of the PEGylating agent involved several syn-thetic steps (Fig. 1). The first step was the synthesis of methoxyPEG2000 succinate from poly(ethylene glycol) monomethylether witha molar mass 2000 g/mol and succinic anhydride (SuA). The 1HNMR spectrum of the product PEGCOONa (Suppl. 1) showed that thePEG carboxylation proceeded quantitatively and the low molecular

Fig. 1. Synthesis of the PEGylating agent.

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side products, i.e. succinic acid or pyridinyl succinate, were removedfrom the purified product.

The next step involved complexing of the PEG chains bearing achelating carboxylate moiety at the polymer terminus with a drugmolecule. The reaction was performed in anhydrous DMF. Both reac-tants were activated. 15-Crown ether-5 was added to complex the so-dium counterion of the carboxylate terminus of PEG, while cisplatinwas activated with silver nitrate. The latter was added in a 5% molardeficiency to the molar equivalent amount of drug to minimize theexchange of the two chloride ligands in the drug with carboxylateions. The unbound cisplatin was removed from the product throughdissolving of the PEG-drug conjugate in acetone and filtering off theundissolved solid. Both 195Pt NMR and IR spectra (Suppl. 2) of the pu-rified product evidenced cisplatin bound to the PEG chain. The yieldof the reaction was assessed through quantitative determination ofplatinum(II) using ICP-AES. It was found that 69% of PEG chainswere complexed with cisplatin.

The synthetic procedure for the preparation of the star polymer witha branched poly[(p-(iodomethyl)styrene] core and poly(acrylic acid)arms was described in detail earlier [8,10]. A hyperbranched productwith a degree of branching 0.36 and molar mass Mn=2400 g/molwas obtained using self-condensing polymerization of p-(iodomethyl)styrene. It contained one double bond [13,14] and in average up to 10active iodomethyl or iodomethine groups [15] and was used as amultifunctional initiator of the livingpolymerization of tert-butyl acrylate.Star shaped macromolecules with arms bearing active ester functionali-tieswere obtained. GPC-MALLSmeasurement of the copolymer displayedthe following characteristics: monomodal distribution of molar masses,Mn=56 800 g/mol and Mw/Mn=1.84. Having in mind that the corecould carry up to ten arms, the average degree of polymerization of thearms was 42.5. Further, the acid hydrolysis of the active ester groups ofthe precursor star copolymer yielded macromolecular structures with abranched hydrophobic interior and hydrophilic shell from poly(acrylicacid) chains (Fig. 2). The copolymer is assigned as PSPA. Based on thetotal removal of the ester groups according the 1H NMR spectral data,

Fig. 2. The route to PEGylated star

the molar mass of PSPA was calculated Mn=33 000 g/mol. The size ofthe core–shell type star particles was evaluated by diffusion orderedNMR spectroscopy (DOSY). The method enables determination of thetranslation diffusion coefficient,D, which afterwards can be used to calcu-late the apparent hydrodynamic radius, Rh, of the particle according toStokes–Einstein equation, assuming spherical shape. The mean Rh valueof the PSPA particles measured by DOSY was 11.4 nm. Fig. 3a displaysthe DOSY spectrum of the star copolymer.

The PEGylation of the PSPA was performed in an aqueous solution.The molar ratio of PEG–cisplatin to the copolymer arms was 0.7,i.e. the mass fraction of the stabilizing shell to be 30 wt.% of thePEGylated particles. The pH value of the solution was adjusted to 7.3.The reaction medium afforded conditions the second chloride ligandin the drug moiety to be substituted with a carboxylate ligand fromthe star arms. The attachment of PEG chains to the stars was evidencedby NMR DOSY experiment. In the DOSY spectrum (Fig. 3b) PSPAand PEG fragments give signals with equal diffusion coefficients(7.94×10−12 m2/s), corresponding to the newly formed particles withan apparent hydrodynamic radius, Rh, of 21.6 nm. The product wasassigned as PSPA–PEG. It possessed arms consisting of poly(sodiumacrylate) and PEG blocks linked by a platinum(II) bridge. The size ofthe PEGylated stars was almost twice as big as that of the nonPEGylatedparticles. This significant increase of the hydrodynamic volume of thebranched macromolecules was an expected effect. An increase of 3 to4-fold in molecular radius was observed about PEGylated hemoglobinwith ten 5 kDa PEG chains per protein [16].

Unbound PEG is also seen in the DOSY spectrum of the PEGylatedsystem (Fig. 3b). The reasonwas that the PEGylating agent was not sep-arated from the uncomplexed PEGCOONa with cisplatin. PEG chainsthat did not bind to the stars were removed via dialysis after drug load-ing of the particles. Therefore, the efficiency of the PEGylation reactionwas evaluated by NMRmeasurement after carrier loading and purifica-tion as described in the experimental. A reference substance (benzoicacid) was added to the star solution for the quantitative determinationof the tethered PEG chains. A grafting efficiency of 86% was achieved. It

polymer–cisplatin conjugates.

Fig. 3. DOSY spectra of: (a) PSPA, (b) PSPA–PEG, (c) PSPA–PEG after 24 h in saline buff-ered solution pH=7.4 at 37 °C.

58 E. Stoyanova et al. / Journal of Inorganic Biochemistry 120 (2013) 54–62

was calculated that the PEG shell of each star macromolecule was com-posed of six chains and presented 28 wt.% of the PEGylated particleweight. The neutral shell decreased the zeta potential of the particlesfrom −56 mV for the original stars to −39 mV after PEGylation.

The PEGylated particles were visualized by atomic force microsco-py (AFM). An AFM image of PSPA–PEG is shown in Fig. 4. Spherical inshape particles are observed. Moreover, two concentric regions are

clearly seen which is attributed to the presence of inner and outershells of the branched macromolecules built up from different poly-mers. Particle dimensions in horizontal direction calculated fromthe image are in the range 46–50 nm which is in agreement withthe determined size from the DOSY NMR data. Some broadeningobserved in the features could be ascribed to tip convolution effects.Additionally, flattering of the particles caused by electrostatic repul-sion between the negatively charged polyacrylate arms could bealso assumed.

DOSYwas also used to study the release of the hydrophilic shell fromthe PEGylated stars in physiological saline. Fig. 3c represents the DOSYspectrum of the system measured 24 h after addition of NaH2PO4/Na2HPO4/NaCl to obtain phosphate buffered saline solution and incuba-tion at 37 °C. The spectrum clearly shows that PEG and PSPAmacromol-ecules have different diffusion coefficients. The diffusion coefficient ofthe PEGylated particles is much higher (1.23×10−10 m2/s) than thoseof the particles after incubation (1.51×10−11 m2/s). This result provesthe PEG chains' release from the star macromolecules. Moreover, theDapp of the PSPA corresponds to the Dapp measured in an aqueous solu-tion of the stars prior PEGylation.

3.2. Loading of the PEGylated star copolymer with cisplatin

The in situ PEGylated star macromolecules were loaded with cis-platin under similar conditions as applied for nonPEGylated stars[8]. In that previous study the changes of the star size and mobilityupon drug complexation were followed by NMR study. The DOSYspectral data undoubtedly indicated that at low degrees of loadingparticle size decreased, while the increase of drug load favored starcoupling via ligand exchange with drug molecules. The result wasappearance of larger particles and broadening of the particle size dis-tribution. Thereon, in the present work the carrier was PEGylatedwhich aimed at steric hindering the cross-linking of the stars uponloading with cisplatin.

Based on our previous experience, changes in PEGylated starmacro-molecules mobility and size with increasing amount of incorporateddrug were followed by DOSY measurements. Drug loading was doneby stepwise addition of cisplatin to a solution of PEGylated macromole-cules in D2O. At the first step and feeding ratio [COO−]:[cisplatin]=12.5:1 the DOSY spectrum of the particles clearly indicated that theirhydrodynamic radii decreased to 12.9 nm, which is almost twice assmall compared to the unloaded stars (Fig. 5a). The drug amount wasfurther increased to a ratio of [COO−]:[cisplatin]=7.6:1. The DOSYspectrum showed that the particles shrink additionally Rh=11.4 nm(Fig. 5b). The third drug loading step ([COO−]:[cisplatin]=5.5:1)led to further decrease of the particle Rh to 7.86 nm (Fig. 5c). At thisstep a significant signal broadening was observed indicating someintra-particle arm cross-linking, however larger particles were notdetected in the DOSY spectra as it was observed for the nonPEGylatedcarrier with a similar payload [8]. Further increase of the drug quantityby adding a fourth portion of cisplatin to the solution did not showconsiderable changes in the size of the loaded PEGylated particles. Theaverage Rh measured from the DOSY spectrum was 7.0 nm (Fig. 5d).These results clearly demonstrate that the PEG shell indeed hindersthe inter-stars cross-linking and stabilized the drug loaded particles.

The abovefindings encouraged us to obtain PEGylated particleswithdrug payload higher than the one achieved for the nonPEGylated carrier[8]. Drug loading of the PEGylatedmacromolecules was done under thefollowing conditions: cisplatin concentration — 2 mg/ml; temperature22 °C; pH 8 and reaction time 48 h. Two loading experiments wereperformed applying molar ratio [COO−]: [cisplatin]=3:1 and 2.5:1and the two products obtained were assigned as PSPA–PEG–Pt1 andPSPA–PEG–Pt2, respectively. As monitored by a naked eye the turbidyellow mixture became transparent and colorless over a period longerthan 24 h, therefore, the time for drug loading of the PEGylated carrierwas doubly increased in comparison with the time needed for the

Fig. 4. AFM image of the PEGylated stars on mica surface.

59E. Stoyanova et al. / Journal of Inorganic Biochemistry 120 (2013) 54–62

original stars. The unbound drugwas removed by dialysis against deion-ized water using a membrane with MWCO 12000–14000 for 48 h.Under these experimental conditions drug loading efficiency about80%was achieved (Table 1) in the two loading experiments. The amountof the immobilized cisplatin was determined to be 42 wt.% and 46 wt.%of the mass of the loaded particles PSPA–PEG–Pt1 and PSPA–PEG–Pt2,respectively. For comparison with the nonPEGylated stars the drugfractions were calculated against the mass of the original star andcorrespondingly the following values were obtained: 51 wt.% and55 wt.%. The aqueous solutions of the conjugates were stable for

Fig. 5. DOSY spectra of PSPA–PEG particles loaded with cisplatin at feedin

more than three months which exceeded considerably the stability ofthe nonPEGylated system. That means the attachment of PEG chainscontributes to the increase of both loading capacity and solution stability.

An AFM image of PSPA–PEG–Pt2 is shown in Fig. 6. Spherical inshape particles were observed. Their dimensions calculated fromthe AFM images in horizontal direction are in the range from 26 nmto 33 nm, while in the Z-direction from 6 nm to 10 nm. Themeasuredvalue of Rz is in a good agreement with results obtained by DOSYexperiment. Most probably, the observed size broadening in thehorizontal direction resulted from tip convolution effects.

g ratio [COO−]:[cisplatin]=(a) 12.5:1, (b) 7.6:1, (c) 5.5:1, (d) 4.5:1.

Table 1Data about the star copolymer loading with cisplatin in an aqueous solution at a drugconcentration of 2 mg/ml, temperature 22 °C, pH 8 and incubation time 48 h.

Sample Feedingmolar ratio[COO]:[cisplatin]

Loadingefficiency(%)

Drug massfraction inPEGylatedparticles(wt.%)

Drug massfraction inoriginal stars(wt.%)

PSPA-Pt 3 82±3 – 51PSPA–PEG–Pt1 3 81±4 42 51PSPA–PEG–Pt2 2.5 79±4 46 55

60 E. Stoyanova et al. / Journal of Inorganic Biochemistry 120 (2013) 54–62

Drug conjugation resulted in further decrease of the zeta potentialof the particles. The measured value for PSPA–PEG–Pt2 was−23 mV.

Fig. 7. Release of Pt(II) complexes from drug loaded star copolymers in phosphatebuffered saline (pH 7.4, 0.14 M NaCl) at 37 °C: (●) PSPA–Pt (data taken from [8]);(△) PSPA–PEG–Pt1; (■) PSPA–PEG–Pt2.

3.3. Release of platinum(II) complexes in physiological saline

The release of the platinum(II) complexes from the loadedPEGylated stars was evaluated under the same experimental condi-tions used for the nonPEGylated particles [8]. The obtained release pro-file (Fig. 7) followed similar sustained manner of drug release from thePEGylated carrier, therefore, similar conclusions could be made as inthe case of unmodified stars. In all cases, no clearly expressed initialburst effect was observed. During the first 8 h of incubation only 4%of the immobilized drug was released. This could be an indicationthat the whole amount of drug was complexed to the copolymer via li-gand exchange. For that reason, no platinum release was evident in dis-tilled water on storage and its replacement with the physiologicalsolution was essential for the release process, which proceeded as aninverse ligand substitution reaction. Moreover, it is apparent that therelease kinetics of the platinum(II) complexes was not hampered bythe attached PEG strands demonstrating that they were also releasedin the same dependent manner.

PEGytation of the stars slightly increased the percentage of the drugreleased from the carrier. Indeed, during the first 24 h the amount ofdrug released from the PEGylated conjugates exceeded that releasedfrom the nonPEGylated carrier. Probably, it is a contribution of cisplatinincorporated in the stars as reversible linker of the PEG shell and local-ized on the surface of the stars. The PSPA–PEG–Pt2 conjugate releasednearly 40% (against 30 % for the nonPEGylated stars) of the loadeddrug over a period of 12 days. This effect in combination with theother important contribution of PEGylation, i.e. the higher drug loadingcapacity of the stars achieved, allowed increase in the total amount ofthe released drug. It is also worth mentioning that the observedsustained release of the Pt(II) complexes has a great advantage for thepassive drug targeting to solid tumors because of the prolonged timeperiods known to be required for macromolecular drugs to accumulatein solid tumors through the bloodstream [17].

Fig. 6. AFM image of loaded PSPA–PE

3.4. Growth inhibitory activity

The growth inhibitory activity of cisplatin-loaded nanoconjugatesvs. the free drug was investigated in a panel of three human tumorcell lines with distinct cell type and origin, namely K-562 (chronicmyeloid leukemia), HL-60 (acute myeloid leukemia) and Hut-78(T-cell lymphoma). Cells were continuously exposed for 72 h or 120 hto serial dilutions of the agents investigated corresponding to cisplatinconcentrations in the range from 3.125 μmol/l to 100 μmol/l for thefree drug, and from 25 μmol/l to 200 μmol/l for the nanoconjugates.Thereafter the cell viability was assessed using the MTT-dye reductionassay. The bioassay data were fitted to sigmoidal concentration–response curves (Fig. 8) and the corresponding IC50 values, i.e. the con-centrations yielding half-maximal suppression of cellular viability werecalculated by non-linear regression (Table 2).

As expected on the basis of the drug-release monitoring studiesthe dose–response curves for the nanoconjugates were shifted tohigher concentrations, and the IC50 values were higher compared tothose of the free drug. In all cell lines the extended treatment periodand hence the intensified exposure of cells was associated withan increase in the in vitro growth inhibitory activity of both thenanoconjugates and the nonimmobilized cisplatin. Throughout thebioassay testing PSPA–PEG–Pt2 proved to exert superior growth in-hibitory activity as compared to PSPA–PEG–Pt1, which could beregarded as an outcome of the higher drug payload and more pro-nounced release of the platinum complex from the PSPA–PEG–Pt2conjugate. A comparison with the growth inhibition data obtainedfor the nonPEGylated carrier showed that PEGylation did not

G–Pt2 particles on mica surface.

Fig. 8. Growth inhibitory effects of (●) free cisplatin; (▼) PSPA–Pt; (■) PSPA–PEG–Pt1; (▲) PSPA–PEG–Pt2 in a panel of human tumor cell lines as determined by the MTT-dyereduction assay.

61E. Stoyanova et al. / Journal of Inorganic Biochemistry 120 (2013) 54–62

compromise the pharmacological effect of the system, just the oppo-site— the IC50 values determined for the PEGylated conjugates were in-variably lower than those for the nonPEGylated system. Data about thegrowth inhibitory activity of PEG–cisplatin against the same threehuman tumor cell lines are provided in Suppl. 3.

The non-loaded PSPA carrier proved to be devoid of cytotoxicityin a wide range of concentrations (0.0156–0.5 mg/ml) causing lessthan 10% cell growth inhibition at the highest concentration eval-uated (Data not shown). In a recent study it was found that PSPAdid not activate complement system in blood and had no apoptotic

Table 2IC50 values (μM) of cisplatin as free drug or as PEGylated or nonPEGylated nanoconjugates

Treatment series IC50 (μM)

HUT-78[a] HL-60[

72 h 120 h 72 h

Cisplatin 3.1±1.1 2.3±0.7 6.4PSPA–Pt 46.9±2.1 24.4±1.7 89.6PSPA–PEG–Pt-1 35.9*/§±1.4 13.3*/§±1.7 68.4*/§

PSPA–PEG–Pt-2 29.4*/§±2.1 17.0*/§±2.0 57.6*/§

[a]T-cell lymphoma; [b]acute myeloid leukemia; [c]chronic myeloid leukemia. *Significant diffthe non-pegylated system PSPA–Pt at the same treatment duration (p≤0.05) (Student's t-t

or necrotic effect on cultured human neurons at a star concentrationof 100 μmol/ml [18].

4. Conclusions

A feasible PEGylation procedure for cisplatin nanocarrier was pro-posed. The carrier possessed star-shaped geometry with a highlybranched core and covalently attached linear arms of polyacrylic acid.The functionality of the carrier and the property of cisplatin to undergoligand exchange with carboxylate ions defined the choice of linker.

against three human tumor cell lines after 72 h or 120 h exposure.

b] K-562[c]

120 h 72 h 120 h

±1.1 2.9±1.2 9.2±2.1 3.5±1.8±4.1 41.7±2.8 154.2±6.2 66.7±2.3±3.4 28.8*/§±1.7 136.4*/§±4.7 47.2*/§±4.1±2.9 24.1*/§±2.2 110.1*/§±7.9 38.4*/§±2.3

erence vs. cisplatin at the same treatment duration (p≤0.05); §significant difference vs.est).

62 E. Stoyanova et al. / Journal of Inorganic Biochemistry 120 (2013) 54–62

Cisplatinwas successfully used as a reversible linker for PEGmodificationof the star macromolecules. PEG shell was stable in deionized watersolution and released under physiological conditions over a period of24 h. The design of releasable multi-PEGylated format versus a few lon-ger PEG chains has its rationale in viewof achieving prolonged blood res-idency time and reduced risk of nonspecific accumulation of the carrierin the body. In addition, the PEGylation of the carrier enabled increasein drug loading capacity and solution stability on storage. The PEGylatedconjugates displayed sustained manner of Pt(II) complex release with-out initial burst effect. They proved to exert enhanced growth inhibitoryactivity compared to the nonPEGylated system which can be attributedto the higher drug payload and improved drug release profile.

AbbreviationsPEG poly(ethylene glycol)DOSY diffusion ordered NMR spectroscopyAFM atomic force microscopyETAAS electrothermal atomic absorption spectrometryICP AES inductively coupled plasma atomic emission spectrometryGPC-MALLS gel permeation chromatography with multiangle laser

light scatteringMWCO molecular weight cut offPBS phosphate buffered salineMTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium

bromideSuA succinic anhydrideD translation diffusion coefficientRh apparent hydrodynamic radiusPSPA copolymer with a poly[(p-(iodomethyl)styrene] core and

poly(acrylic acid) armsPSPA–PEG PEGylated copolymer carrierPSPA–PEG–Pt loaded with cisplatin PEGylated copolymer carrier

Acknowledgements

The support by the NSF of Bulgaria (Contract No DO 02-198/2008and DRNF 02/13) is highly acknowledged. The authors are grateful toDr. Irina Karadjova for the help with ETAAS and ICP AES experiments.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jinorgbio.2012.12.005.

References

[1] J. Harris, R. Chess, Drug Discov. 2 (2003) 214–221.[2] F. Veronese, Biomaterials 22 (2001) 405–417.[3] M. Hamidi, A. Azadi, P. Rafiei, Drug Deliv. 13 (2006) 399–409.[4] S. Stolnik, Pharm. Res. 11 (1994) 1800–1808.[5] G. Kaul, M. Amiji, Pharm. Res. 19 (2002) 1061–1077.[6] D. Filpula, H. Zhao, Adv. Drug Deliv. Rev. 60 (2008) 29–49.[7] S. Zhu, M. Hong, L. Zhang, G. Tang, Y. Jiang, Y. Pei, Pharm. Res. 1 (2010) 161–174.[8] A. Kowalczuk, E. Stoyanova, V. Mitova, P. Denkova, G. Momekov, D. Momekova, N.

Koseva, Int. J. Pharm. 404 (2011) 220–230.[9] A.S. Gozdz, Polym. Bull. (Berl.) 4 (1981) 577–582.

[10] A. Kowalczuk-Bleja, B. Sierocka, J. Muszyński, B. Trzebicka, A. Dworak, Polymer 46(2005) 8555–8564.

[11] T. Mosmann, J. Immunol. Methods 65 (1983) 55–63.[12] S. Konstantinov, H. Eibl, M. Berger, Br. J. Haematol. 107 (1999) 365–380.[13] S. Gaynor, S. Edelmann, K. Matyjaszewski, Macromolecules 29 (1996) 1079–1081.[14] M. Weimer, J. Frechet, I. Gitsov, J. Polym. Sci. A Polym. Chem. 36 (1998) 955–967.[15] A. Kowalczuk-Bleja, B. Trzebicka, H. Komber, B. Voit, A. Dworak, Polymer 45

(2004) 9–18.[16] K. Vandegriff, M. McCarthy, R. Rohlfs, R. Winslow, Biophys. Chem. 69 (1997) 23–30.[17] Y. Matsumura, H. Maeda, Cancer Res. 46 (1986) 6387–6392.[18] R. Donev, N. Koseva, P. Petrov, A. Kowalczuk, J. Thome, World J. Biol. Psych. 12

(Suppl. 1) (2011) 44–51.