Acta Biomaterialia xxx (2015) xxx–xxx

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Advancing the delivery of anticancer drugs: Conjugated polymer/ triterpenoid composite Katarzyna Krukiewicz a, Tomasz Jarosz a, Jerzy K. Zak a, Mieczyslaw Lapkowski a, Piotr Ruszkowski b,⇑, Teresa Bobkiewicz-Kozlowska b, Barbara Bednarczyk-Cwynar c,⇑ a b c

Department of Physical Chemistry and Technology of Polymers, Silesian University of Technology, M. Strzody 9, 44-100 Gliwice, Poland Department of Pharmacology, Faculty of Pharmacy, Poznan University of Medical Sciences, Rokietnicka 5, 60-806 Poznan, Poland Department of Organic Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland

a r t i c l e

i n f o

Article history: Received 28 August 2014 Received in revised form 25 February 2015 Accepted 5 March 2015 Available online xxxx Keywords: Conducting polymer Poly(3,4-ethylenedioxythiophene) Oleanolic acid Drug delivery Anticancer activity

a b s t r a c t Exemplifying the synergy of anticancer properties of triterpenoids and ion retention qualities of conjugated polymers, we propose a conducting matrix to be a reservoir of anticancer compounds. In this study, poly(3,4-ethylenedioxythiophene), PEDOT, based matrix for electrically triggered and local delivery of the ionic form of anticancer drug, oleanolic acid (HOL), has been investigated. An initial, one-step fabrication procedure has been proposed, providing layers exhibiting good drug release properties and biological activity. Investigation of obtained systems and implementation of modifications revealed another route of fabrication. This procedure was found to yield layers possessing a significantly greater storage capacity of OL , as evidenced by the 52% increase in the drug concentrations attainable through electro-assisted release. Examination of the biological activity of immobilised and released OL molecules proved that electrochemical treatment has negligible impact on the anticancer properties of OL , particularly when employing the three-step procedure, in which the range of applied potentials is limited. PEDOT/OL composite has been demonstrated to be a robust and cost-effective material for controlled drug delivery. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Chemotherapy is one of the most widely utilised procedures for treating cancer [1]. Despite its many merits, the application of this treatment is limited by severe toxic side effects of anticancer drugs on healthy tissues [2,3]. Efforts are being made to tackle this issue by developing more benign drugs [4]. Recent reports, however, show that it is also possible to overcome this challenge by exploiting the potential of local drug delivery systems (DDS) for the deployment of anticancer agents [5]. The highlight of a localised DDS approach is the possibility of implanting drug-releasing devices directly at the tumour site. Proceeding this way, it is possible to minimise both systemic exposure and side effects of chemotherapy [6]. Several approaches to the development of such systems have heretofore been reported, utilising chitin microparticles [7], biodegradable polymeric ⇑ Corresponding authors. Tel.: +48 61 854 66 74 (B. Bednarczyk-Cwynar). E-mail addresses: [email protected] (K. Krukiewicz), [email protected] (T. Jarosz), [email protected] (J.K. Zak), [email protected] (M. Lapkowski), [email protected] (P. Ruszkowski), [email protected] (T. Bobkiewicz-Kozlowska), [email protected] (B. Bednarczyk-Cwynar).

microspheres [8], poly(D,L-lactide-co-glycolide) wafers [9], poly[bis(p-carboxyphenoxy)propane-sebacic acid] copolymer discs [10] as well as other, intravenous delivery systems, based on nanoparticles and polymers [11,12]. All of the above share a common mechanism of drug delivery – the spontaneous release of bioactive molecules from the matrix upon its bio-assisted decomposition. Conjugated polymers, possessing ion-exchange properties, are considered promising materials for use as drug reservoirs in drug delivery systems [13]. In contrast to the physical entrapment [14], conjugated polymers allow controlled, reversible electrostatic immobilisation. The mechanism of this process relies on the fact that conducting polymers, depending on their oxidation state, undergo a charging–discharging process and adopt positive or negative charges. These charges draw ions of opposite charge into the polymeric matrix, binding them via Coulomb interactions. Therefore, they are able to immobilise anionic drugs during oxidation (doping) and release them in the process of reduction (dedoping). The controlled immobilisation/release mechanism is highly desired, however, the development of implantable drug delivery systems necessitates all of the device constituents to be fully

http://dx.doi.org/10.1016/j.actbio.2015.03.006 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Krukiewicz K et al. Advancing the delivery of anticancer drugs: Conjugated polymer/triterpenoid composite. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.03.006

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biocompatible. Although biocompatibility is not inherent to conjugated polymers, some among them, such as polypyrrole [15] and poly(3,4-ethylenedioxythiophene) (PEDOT) [16], exhibit this trait. Consequently, conducting polymers have been used in the design of novel electrochemical drug release devices [17]. Lira et al. [18] described a polyaniline-based device able to release safranin as a result of electric pulse. The amount of released drug was found to be dependent on the oxidation state of polyaniline which was modified through the application of potential. ValdesRamirez et al. [19] developed a microneedle-based drug delivery system, in which controlled release of multiple therapeutic agents was realised by the use of a polypyrrole (PPy) nanoactuator. PPy in its reduced state obstructed the flow of the therapeutic solution through the needle; the oxidation of PPy was followed by its contraction and the facilitation of flow. Nevertheless, a significant issue connected with the use of conducting polymers as drug delivery devices is the method for altering the redox state of the polymer. The majority of reported, electrically triggered delivery systems, based on conjugated polymers, rely on percutaneous electrodes to deliver electric current. The necessity of ensuring physical electrical contact with polymer matrix significantly hindered the practical use of conducting polymers. This issue, however, has been resolved by Gao et al. [20], who found an effective way to change the redox state of polymer remotely. Dexamethasonesaturated polypyrrole platforms were designed to be sensitive to local, pulsatile electromagnetic field. The remote control of oxidation state of the polymer provided the framework for an electrically-triggered device that can be implanted into the body without requiring any physical contact with the surroundings for operation. Superior chemical and electrochemical stability is the primary highlight of PEDOT, when compared to other conjugated polymers [21]. Heretofore, there have been several reports concerning the use of PEDOT matrices for electrochemical immobilisation and electrically triggered release of biomolecules. These have utilised pure PEDOT [22], as well as its composites with carbon nanotubes [23] and polystyrene sulphonate [24]. In each case, doping/dedoping of PEDOT was exploited for controlled drug release, evidencing the versatility of this mechanism of immobilisation [25]. Triterpenoids are one of the most promising groups of anticancer compounds. These compounds are secreted by living tissues of numerous higher plants, granting them exceptionally good availability. To exemplify, oleanolic acid (HOL) can be isolated from more than 1600 plants species, including common flora, such as olive, mistletoe, lavender, rosemary, clove and many others [26]. The prime highlight of HOL is its anticancer activity in various stages of tumour development, including inhibition of tumour promotion, invasion and metastasis. This quality has been reported in a number of cell lines, including HeLa [27], L1210, K562 and HL-60 [28], HepG2, Hep3B, Huh7 and HA22T [29]. Furthermore, numerous tests have evidenced the anti-diabetic [30,31], antiviral [32,33], antibacterial [34], antifungal [35], anti-inflammatory [36–39], anti-allergic [39], wound-healing [40], nephroprotective [41], diuretic [42] and hepatoprotective [43] activity of oleanolic acid. Whereas its anticancer activity is of prime importance, this impressive array of secondary, beneficial qualities may aid in combating possible post-chemotherapy complications. Encouraged by these superior traits, numerous derivatives of oleanolic acid have been synthesised and were reported to exhibit anticancer activity against many cancer cell lines, e.g. PC3, A549, BGC-823 [44], MCF-[44,45], KB, HeLa [45], Hep-G2 [46], CCRF-CEM, CCRF-VCR1000, CCRF-ADR5000 [47]. Therefore, should the systems designed for delivery of HOL achieve success, it would be valuable to explore triterpene-based DDSs. In this study we describe a robust and cost-effective method to fabricate PEDOT/OL composite layers for drug delivery. Two

procedures of immobilisation have been proposed: immobilisation during the process of polymerisation and post-polymerisation modification. The latter was found to yield layers possessing a significantly greater storage capacity of OL . Examination of the biological activity of immobilised and released OL evidenced that electrochemical treatment has negligible impact on the anticancer properties of OL , particularly when employing the three-step procedure, in which the range of applied potentials was limited. PEDOT/OL composite was found to exhibit good properties in terms of controlled release, along with maintaining biological activity of the embedded drug, making it a promising candidate for further development and commercial application. 2. Materials and methods 2.1. Materials Diethyl ether (Chempur, analytical grade), sodium hydroxide (Chempur, analytical grade), ethyl alcohol (Chempur, analytical grade), hydrochloric acid (Chempur, analytical grade) were used to isolate oleanolic acid. Trichloroacetic acid 99% (SigmaAldrich), acetic acid 99% (Chempur), sulphorhodamine dye (SigmaAldrich), Tris buffered saline (SigmaAldrich), aqua pro injectione (Baxter), HeLa human cell line (ECACC), KB cell line (human nasopharynx carcinoma) (ECACC), A-549 cell line (ATCC), Streptomycin (Polfa), Penicilinum (Polfa), PBS buffer tablets (SigmaAldrich), Fetal Bovine Serum (FBS) (BecktonDickinson), dimethyl sulphoxide (DMSO) – (SigmaAldrich), Dulbecco’s Modified Medium (DMEM) – (Sigma Aldrich), RPMI-1640 Medium (SigmaAldrich) were used to perform anticancer activity tests. 3,4-ethylenedioxythiophene (EDOT) (Sigma–Aldrich, >97%), lithium perchlorate (Sigma–Aldrich, >95%, ACS grade), potassium chloride (Avantor, analytical grade), dipotassium hydrogen phosphate (Avantor, analytical grade), potassium dihydrogen phosphate (Avantor, analytical grade), ethyl alcohol 99.8% (Avantor) and sodium hydroxide (Avantor, analytical grade) were used as received. Grade 1 (R > 10 MX cm 1) deionised water was employed as solvent for all prepared solutions. 2.2. Instrumentation Büchi apparatus was used to determine the melting point of oleanolic acid. Varian Gemini 300 VT apparatus was used to record 1 H, 13C and DEPT spectra of isolated oleanolic acid. Laminair Heraeus Class II, Microplate reader Tecan sunrise, Thermomixer eppendorf, CO2 Incubator New Brunswick 170R, Ultra Low Temperature Freezer New Brunswick C340 Premium, Centrifuge Sigma 1–6, Laboratory Analytical Scale (Radwag), Centrifuge Z 326K (Hermle) were used to perform anticancer activity tests. Immobilisation and release processes were carried out in a standard three-electrode setup, employing a platinum foil working electrode (1 cm2), Ag/AgCl reference electrode and a glassy carbon counter electrode, therefore, all potentials in this work are reported in relation to Ag/AgCl. Electrochemical measurements were performed on a CH Instruments 660c Electrochemical Workstation. Spectroscopic measurements were executed on a Hewlett Packard 8453 UV–Vis Diode Array Spectrophotometer. 2.3. Methods 2.3.1. Preparation of oleanolic acid Oleanolic acid was isolated from waste-product obtained during production of mistletoe extract. This waste product, in a form

Please cite this article in press as: Krukiewicz K et al. Advancing the delivery of anticancer drugs: Conjugated polymer/triterpenoid composite. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.03.006

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of water–ethanol dense suspension, was filtered off, dried and crumbled. 220 g of such material was extracted continuously with 1500 ml of diethyl ether. Next, 180 ml of 1 M NaOH solution was added and shaken out. The formed residue was filtered out, washed with water and dried. Next, it was dissolved with ethanol, boiled with active carbon, filtered out and acidified with HCl. The formed crystals were filtered out, washed with cold ethanol, dried and recrystallised from ethanol to give white needles of 3b-hydroxyolean-12-en-28-oic acid (about 50 g), C30H48O3, mol. mass 456.71 g mol 1, m.p. 308 – 310 °C. The purity of the obtained triterpene was proven on the basis of spectral data which were in accordance with literature reports [48]. Sodium oleanolate (NaOL, Scheme 1) has been prepared in full adherence to the procedure earlier described by Tong et al. [49]. 2.3.2. One-step procedure for the fabrication of PEDOT/OL The one-step procedure involved the simultaneous electrochemical polymerisation of EDOT and modification of the incipient polymer layer with OL via the doping mechanism established for conjugated polymers. Solutions utilised in this procedure comprised EDOT, OL and LiClO4 dissolved in deionised water. Immediately prior to fabrication of the composite, the solution was sonicated for 30 min. Deposition of the composite layer was realised on Pt foil (1 cm2) through performing 50 potential cycles in the range of 0.0 V  1.1 V, at a scan rate of 0.1 V s 1 (Fig. SI.1a and b). 2.3.3. Three-step procedure for the fabrication of PEDOT/OL The three-step procedure comprised electropolymerisation of EDOT in 1.0 M LiClO4, followed by deep dedoping of the polymer layer and oxidative immobilisation of OL . The sample solution, containing 10 mM EDOT and 0.1 M LiClO4 in deionised water, was subjected to 30 min sonication prior to electrochemical polymerisation. Polymerisation was carried out on Pt foil (1 cm2) realised via 50 potential cycles in the potential range of 0.0 V  1.0 V, at a scan rate of 0.1 V s 1 (Fig. SI.1c). Dedoping of the polymeric deposit was performed in an aqueous 0.1 M LiClO4 solution, at a constant working electrode potential of 0.7 V over the span of 10 min. Immobilisation of OL featured potentiostatic oxidation of the polymer at 0.8 V in a 30 mM aqueous solution of NaOL for the duration of 10 min. Chronoamperometric curves registered for both dedoping and immobilisation stages are presented as Supplementary information (Fig. SI.2). 2.3.4. Procedure for composite conditioning Prior to investigation of OL release, the fabricated composite layers were subjected to conditioning in a pH 6.5 phosphate buffered saline solution (PBS) comprising 0.15 M KCl, 0.006 M K2HPO4 and 0.001 M KH2PO4 for 10 min. The goal of such treatment was to remove traces of the monomer (EDOT) as well as

Scheme 1. Chemical structure of sodium oleanolate (NaOL).

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non-immobilised OL . This was particularly important for the spectroscopic investigation of OL release from the composite, as the absorption of EDOT partially overlaps signals originating from OL . Preliminary experiments performed for a pristine PEDOT matrix have shown that this procedure is sufficient for purging traces of unreacted monomer. 2.3.5. Procedure for drug release study The release of OL from the PEDOT/OL composite, submerged in PBS, was realised in a 2 mm optical path cuvette (Hellma Analytics, type No. 100-QS). The electrodes were positioned beyond the optical path, so as to allow following the process in situ by time-resolved UV–Vis spectroscopy. Electro-assisted (active) drug release was performed with the application of a potential of 0.5 V, whereas spontaneous (passive) drug release was carried out in open circuit conditions. Spectral snapshots of the system were taken every minute and the evolution of absorption at 254 nm was exploited for the determination of OL concentration in solutions, as this wavelength corresponds to the maximum of a distinct absorption peak of OL (Fig. SI.3). Concentrations of OL have been determined via the calibration curve method (Fig. SI.4) and related to the surface area of the polymer-coated electrode (1 cm2). A linear relationship was observed for NaOL concentrations between 0.01 and 1 mM, with a linear correlation coefficient between absorbance and NaOL concentration of 8.39  10 2 dm3 mmol 1, with an estimated relative error of 3.0%. 2.3.6. Biological activity of released OL In the biological experiments, HeLa, KB and A-549, cancer cell lines were subjected to the procedure that was described in our earlier experiments [45], using the OL solutions obtained via drug release from composite layers. The protein-staining sulphorhodamine B (SRB, Sigma–Aldrich) microculture colorimetric assay, developed by the National Cancer Institute for in vitro antitumour screening, was used in this study to estimate the cell number by providing a sensitive index of total cellular protein content, being linear to cell density. 2.4. Statistical analysis Heteroscedastic t tests were used to assess the statistical significance of the results of drug release experiments. Relationships between experimental data were investigated using regression methods, with the significance of the Pearson product–moment correlation coefficient being tested using a standard t distribution approach. 3. Results and discussion 3.1. One-step fabrication of PEDOT/OL Fabrication of PEDOT/OL composite involved the electrochemical polymerisation of EDOT in the presence of NaOL. The use of cyclic voltammetry results in layer-by-layer deposition of PEDOT, wherein OL anions are coulombically bound to cationic species present within the growing polymer deposit. The general scheme of this process is presented in Fig. 1a, whereas selected CV curves registered during polymerisation are shown in Fig. 1b. Electropolymerisation was carried out at potentials corresponding to the onset of the oxidation peak of EDOT, so as to avoid oxidative degradation of NaOL. The development and evolution of broad redox bands with consecutive potential cycles indicates that a conducting layer is being deposited, consistent with reports of EDOT polymerisation [50]. The effect of varying the EDOT:OL ratio in

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Fig. 2. Concentration of OL released, during a representative experiment, from PEDOT/OL obtained via one-step fabrication method as a function of time for active release (black squares) and passive release (red dots).

Fig. 1. One-Step fabrication of PEDOT/OL : general scheme and visualisation of the process (a), CV curves recorded during electrochemical polymerisation of 10 mM EDOT in 0.1 M LiClO4 aqueous solution in the presence of 10 mM NaOL (b).

the polymerisation mixture was investigated. Consequently, a 1:1 ratio was found to be optimal in terms of drug content in the polymer and feasibility of preparation. 3.2. Release of OL from PEDOT/OL composite layers The role of OL , immobilised within the composite, is that of a dopant (counter-ion) – balancing the positive charges present on the oxidised PEDOT chains. In order to release immobilised drug, it is sufficient to apply negative voltage to the composite. The schematic representation of an electrically triggered drug release from the composite is depicted in Scheme 2. Highly doped conjugated polymers have been reported to undergo spontaneous dedoping [51] when soaked in an electrolyte solution, which can potentially lead to the release of OL from the composite. In order to measure the magnitude of this effect, drug release was followed in electro-assisted (active) and spontaneous (passive) modes (Fig. 2). During both passive and electro-assisted release of OL , a concentration plateau is observed, indicating that the systems

Scheme 2. General scheme and visualisation of the process of electrically triggered release of OL from PEDOT/OL composite.

approach their respective equilibria. The burst release of drug during the initial application of potential was avoided through the careful optimisation of the applied reduction potential – sufficient to initiate the process of release but high enough to maintain control over it [52]. The concentrations achieved after 10 min of release, when concentration changes for both systems become negligible, are respectively 0.22 (±0.08) mM and 0.72 (±0.11) mM for the passive and electro-assisted OL discharge. The difference in attainable OL concentrations (approximately 1:3) implies that in the case of passive release only OL located in the vicinity of the composite/electrolyte interface may be discharged. This is consistent with polymer self-dedoping, as only OL dopants located in the vicinity of the composite surface are prone to diffuse into the solution. During electro-assisted release, the application of potential serves to both facilitate the composite dedoping process and shift the equilibrium state of PEDOT towards a fully electro-neutral state. In light of the above, when developing implants for specific operating conditions, extreme care must be taken in optimising the thickness of the active composite layer. This step is crucial as fabrication of thin layers may significantly reduce drug capacity of the implant and, therefore, its efficiency as drug reservoir. Conversely, the use of thick layers introduces a ‘‘dead volume’’ in which OL is immobilised, but cannot be released. In order to alleviate this issue, a three-step composite fabrication procedure was devised, relying on the phenomenon of secondary doping [18].

3.3. Three-step fabrication of PEDOT/OL composite The main goal of developing this procedure was to resolve issues related to thickness of the active layer of PEDOT/OL composite. Conversely, this approach allowed a number of lesser issues to be avoided as well, such as the fact that the presence of NaOL in the polymerisation solution increases the potential necessary to induce electropolymerisation (Fig. SI.1a and b). This may lead to overoxidation of both NaOL and PEDOT, which may interfere with the biocompatibility of potential implants. Therefore, it is beneficial to separate the preparation of the composite and the drug immobilisation, giving rise to the three-step fabrication procedure, according to Scheme 3. The first stage of the procedure comprises the electrochemical polymerisation of EDOT in an aqueous solution of LiClO4

Please cite this article in press as: Krukiewicz K et al. Advancing the delivery of anticancer drugs: Conjugated polymer/triterpenoid composite. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.03.006

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Scheme 3. General scheme and visualisation of the process of three-step fabrication of PEDOT/OL composite.

Fig. 3. Concentration of OL released, during a representative experiment, from PEDOT/OL matrix obtained via three-step fabrication method as a function of time for active release (black squares) and passive release (red dots).

(Fig. SI.1c). The perchlorate dopant introduced during polymerisation is subsequently removed from the polymer film via reduction of the polymer from the oxidised state to the ground, electro-neutral state (Fig. SI.2a). Drug immobilisation is realised afterwards, when the dedoped polymer is re-oxidised in a saturated aqueous solution of NaOL (Fig. SI.2b). Comparison of the CV registered for one-step fabrication of PEDOT/OL (Fig. SI.1b) and the one for three-step fabrication reveals slightly elevated polymerisation currents in the latter case (Fig. SI.1c). This is attributed both to the different structure of polymer films as well as to their different intrinsic conductivities, resulting from the incorporation of differing populations of ions. Utilising the three-step method of fabricating the PEDOT/OL composite it was possible to lower the maximum potential applied during polymerisation by 0.1 V, limiting any possible overoxidation of both PEDOT and NaOL. Moreover, the separation of polymerisation and immobilisation processes resulted in lifting the constraints on the maximum concentration of NaOL solutions utilised in the latter process. This allowed a saturated NaOL solution (approx. 30 mM) to be used in contrast to the 10 mM NaOL used

Fig. 4. Release of OL from PEDOT/OL synthesised via 1-step and 3-step procedures in different modes (passive and active, respectively). The release experiments were carried out in PBS for 10 min. Error bars indicate the standard error of the mean (n = 3 for passive release and n = 7 for active release).

in the one-step approach. Higher concentration of NaOL enhances the process of drug incorporation due to the increased probability of NaOL entrapment into PEDOT chain. This leads to the increase in the drug storage capacity of the final PEDOT/OL composite. The release of OL from the PEDOT/OL composite, fabricated via the three-step procedure, was followed by time-resolved UV– Vis spectroscopy (Fig. 3). The conditions developed for the onestep procedure were strictly maintained to enable comparison between the two types of composite layers. Conversely, the concentration plateau observed earlier (Fig. 2) is also present, indicating that the systems approach their respective equilibria. The concentrations achieved after 10 min of release are 0.27 (±0.11) mM and 1.94 (±0.25) mM for the passive and electro-assisted OL discharge, respectively. Electro-assisted and passive drug release was carried out for composite layers obtained via both fabrication procedures. In the case of passive release, qualitatively similar release properties have been obtained for the two types of composites: 0.23 (±0.13) mM for 1-step fabrication and 0.33 (±0.19) mM for 3-step fabrication

Please cite this article in press as: Krukiewicz K et al. Advancing the delivery of anticancer drugs: Conjugated polymer/triterpenoid composite. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.03.006

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via active release from both types of composite layers was found to be statistically significant (p < 0.05). The concentrations of OL achieved during active release differed sharply: 0.92 (± 0.11) mM for 1-step fabrication and 1.42 (±0.25) mM for 3-step fabrication procedure (Fig. 4). The concentration of OL produced by release from the composite fabricated via the three step procedure was 52% higher than in the case of the system obtained by the one-step procedure. The deviations in drug concentrations obtained in consequent experiments, conducted in the same conditions, arise primarily from the differences in the structure of the composite layers. This variation, in turn is inherent to the process of potentiodynamic electrochemical polymerisation. The greater variance for three-step procedure samples is attributed to the introduction of additional fabrication steps, utilising potentiostatic methods, known to be prone to interferences originating from capacitative effects. 3.4. Biological activity of released OL

Fig. 5. Cytotoxic activity data for OL immobilised and released from PEDOT/OL matrices (passive release – black squares, active release from 1-step matrix – red dots, active release from 3-step matrix – blue triangles) and for NaOL reference solutions (violet triangles) examined against: HeLa cells (a), KB cells (b) and A-549 cells (c). The centre of each ‘‘crosshair’’ shows the mean values for cell viability and attained OL concentration, whereas the length of the horizontal and vertical lines represents the standard deviations across all the experiments constituting a given group.

procedure, the differences being statistically insignificant, consistent with the postulated nature of the passive release process. Conversely, the difference between concentrations of OL obtained

Having demonstrated and examined the immobilisation and release of OL from PEDOT/OL composite in a controlled manner, it is crucial to determine whether the resultant OL solutions maintain their anticancer properties. In order to measure the effect and efficacy of the released OL , in vitro studies were conducted with cervical (HeLa), oral (KB) and lung (A-549) cancer cells. Examined OL solutions were obtained as a result of drug immobilisation and ten minute release from PEDOT/OL matrices. For reference purposes, NaOL solutions (0.5 mM, 1.0 mM, 1.5 mM) that were not subjected to electrochemical stimuli were prepared and examined. The resulting cytotoxic activity data for all OL solutions is presented in Fig. 5. Each group of data points presented in Fig. 5 has been supplemented with a ‘‘crosshair’’, whose position indicates the mean values for cell viability and attained OL concentration, whereas the length of the horizontal and vertical lines represents the standard deviation across all the experiments constituting a given group. Alternatively, the data constituting Fig. 5 can be presented for each drug release method, rather than for each of the investigated cell lines (Fig. SI.5). Although varying concentrations of OL have been achieved for each of the drug release methods, a correlation between cell viability and OL concentration can be observed and is similar for each of the cell lines. All OL solutions for which the calculated OL concentration was higher than 0.25 mM, i.e. solutions obtained as a result of active release, demonstrated anticancer activity against HeLa, KB and A-549 cells. In the case of passive release, in the absence of electrical stimuli, the amount of released drug was significantly smaller, sufficiently so, that it was not biologically active against cancer cells. The biological activity of samples decreased with the decrease of samples’ concentration in a reciprocal way – the same trend was observed for all cancer cells used. The biological activity against cervical, oral and lung cancer cells is lower for OL subjected to electrochemical treatment than for the reference NaOL solutions. The highest anticancer activity was shown by OL solutions obtained after electrically triggered release from PEDOT/OL matrix synthesised via three-step procedure. 4. Conclusions In an attempt to design a new material for local drug delivery systems, a conjugated polymer/triterpenoid composite has been described and demonstrated to be a robust and cost-effective system. An initial, one-step fabrication procedure provided layers exhibiting good drug release properties, with the drug retaining its anticancer activity. Investigation of obtained systems and

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implementation of modifications revealed another route of fabrication. The application of this procedure resulted in the formation of layers possessing a significantly greater storage capacity of OL , as evidenced by the 52% increase in the drug concentrations attainable through electro-assisted release. Exploration of the parameter space in the case of the fabrication and drug release processes may serve to increase the storage capacity of OL within the composite even further. Significant improvements, however, are expected to result from increasing the active surface of the composite layer, via its deposition on highly porous or hyperbranched substrates. Examination of the biological activity of immobilised and released OL molecules showed a negligible impact of electrochemical treatment on the anticancer properties of OL , particularly when employing the three-step procedure, in which the range of applied potentials was limited. Summarising, the PEDOT/OL composite was found to exhibit competitive properties in terms of controlled release, along with maintaining biological activity of the embedded drug, making it a promising candidate for further development and possible commercial application. Disclosures The authors declare no conflicts of interest. Acknowledgements Parts of this research were supported by the research project 2012/07/N/ST5/01878 funded by the Polish National Science Centre. Katarzyna Krukiewicz is a scholar in SWIFT project POKL.08.02.01-24-005/10 which is co-financed by European Union within European Social Fund. Tomasz Jarosz is a scholar supported by the ‘‘Doktoris—scholarship program for an innovative Silesia’’, co-financed by European Union within European Social Fund. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1–3 and 5 are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio. 2015.03.006. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2015.03. 006. References [1] Guo Y, Chu M, Tan S, Zhao S, Liu H, Otieno BO, et al. Chitosan-g-TPGS nanoparticles for anticancer drug delivery and overcoming multidrug resistance. Mol Pharm 2014;11:59–70. [2] Kobayashi M, Wood PA, Hrushesky WJ. Circadian chemotherapy for gynecological and genitourinary cancers. Chronobiol Int 2002;19:237–51. [3] Lv S, Li M, Tang Z, Song W, Sun H, Liu H, et al. Doxorubicin-loaded amphiphilic polypeptide-based nanoparticles as an efficient drug delivery system for cancer therapy. Acta Biomater 2013;9:9330–42. [4] Curigliano G, Spitaleri G, Fingert HJ, Braud F, Sessa C, Loh E, et al. Drug-induced QTc interval prolongation: a proposal towards an efficient and safe anticancer drug development. Eur J Cancer 2008;44:494–500. [5] Matsusaki M, Akashi M. Functional multilayered capsules for targeting and local drug delivery. Expert Opin Drug Deliv 2009;6:1207–17. [6] Fung LK, Saltzman WM. Polymeric implants for cancer chemotherapy. Adv Drug Deliv Rev 1997;26:209–30. [7] Nsereko S, Amiji M. Localised delivery of paclitaxel in solid tumors from biodegradable chitin microparticle formulations. Biomaterials 2002;23:2723–31. [8] Langer R. Polymer implants for drug delivery in the brain. J Control Release 1991;16:53–60.

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Please cite this article in press as: Krukiewicz K et al. Advancing the delivery of anticancer drugs: Conjugated polymer/triterpenoid composite. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.03.006

triterpenoid composite.

Exemplifying the synergy of anticancer properties of triterpenoids and ion retention qualities of conjugated polymers, we propose a conducting matrix ...
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