Partitioning of doxorubicin into Langmuir and Langmuir–Blodgett biomimetic mixed monolayers: Electrochemical and spectroscopic studies

Journal of Electroanalytical Chemistry xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem

Partitioning of doxorubicin into Langmuir and Langmuir–Blodgett biomimetic mixed monolayers: Electrochemical and spectroscopic studies Dorota Nieciecka a, Agata Królikowska b, Aleksandra Joniec a, Pawel Krysinski a,⇑ a b

Laboratory of Electrochemistry, Faculty of Chemistry, University of Warsaw, Pasteura 1, Warsaw 02-093, Poland Laboratory of Intermolecular Interactions, Faculty of Chemistry, University of Warsaw, Pasteura 1, Warsaw 02-093, Poland

a r t i c l e

i n f o

Article history: Available online xxxx Keywords: Biomimetic membranes Langmuir–Blodgett technique Doxorubicin Anthracycline drugs

a b s t r a c t The partitioning of doxorubicin, a potent anticancer drug, was examined in relation to the composition and structural properties of the biomimetic molecular films. We have systematically studied the passive permeation of doxorubicin across the two-component monolayers of different organization, following the drug interactions with the hydrophilic part of the monolayer and then, with its hydrophobic moiety, separately. We investigated independently these two types of the interactions, likely competing in a native membrane, respectively for the Langmuir monolayers on an aqueous subphase and for Langmuir–Blodgett monolayers on gold, exposed to the drug in an aqueous solution. This gave us a unique possibility to monitor the drug interactions with different regions of biomimicking film. The overall picture emerging from these results suggests that the hydrophobicity of monolayer interior allows for the penetration and accumulation of the drug, while its organization controls the rate and amount of doxorubicin molecules through such films. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction The partitioning of anthracycline drugs into the hydrophobic membrane moiety is the basis for their passive transport across the biological membranes. It is also a prerequisite of micelle-based drug delivery systems [1,2] and can be used for the evaluations of structure and organization of lipid artificial mono- and bilayer systems [3,4]. Doxorubicin (DOX) is an anthracycline, cytostatic antibiotic, which is widely used in the treatment of a wide variety of cancers, such as breast carcinoma, lung carcinoma, acute leukemia, liver and several soft-tissue sarcomas [5]. However, the clinical use of doxorubicin is somehow limited due to its cardiotoxicity and other side-effects [6,7]. Doxorubicin targets the DNA molecules, primarily intercalating their double strands with reversible, non-covalent binding, inhibiting this way the duplication and transcription to mRNA [8] and causing the cell death. The relatively flat condensed ring system intercalates into the B-form of the DNA helix with specificity towards guanine–cytosine base pairs [9]. The drug molecule is also known as inhibitor of topoisomerase II-DNA complex [10,11] causing the formation of breaks in the DNA strands. Unfortunately other modes of DOX side effects may include cardiotoxicity [12,13] and production of reactive oxygen species (ROS) [14].

⇑ Corresponding author. Tel.: +48 22 8220211x389; fax: +48 22 8225596. E-mail address: [email protected] (P. Krysinski).

Before addressing its target, doxorubicin molecules have to penetrate through the cellular membrane into the cell and partitioning of doxorubicin into the hydrophobic moiety of lipid bilayer can determine its passive transport across the cell membrane. This membrane, due to the hydrophobicity of its interior strongly influences the permeation of polar molecules such as anthracyclines. Generally, two different mechanisms have been proposed for the permeation of various molecules across lipid membranes: (i) the diffusion through defects or transient pores in the membrane or/and (ii) partitioning into the hydrophobic core of the bilayer with subsequent diffusion to the opposite side. There exists relatively large literature describing the various possible pathways as to how doxorubicin and other anthracyclines pass across the membrane into the cell. Generally, there appears to be some disagreement with respect to the prevailing interaction mode of DOX and model membranes during the drug penetration. Based on their experimental results, some researches claim that the hydrophobic interactions play major role [15,16], while others provide evidence on strong doxorubicin binding to anionic phospholipids in model membranes [17,18]. Inadequate drug delivery to the tumor cells is now recognized as the key factor that increases their anticancer drug resistance [19]. The effect of the limited drug distribution within tumor on its therapeutic index cannot be neglected [20], therefore a special attention should be paid to the studies of the interactions responsible for the efficient drug transport across the systems mimicking liposomal membranes.

1572-6657/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jelechem.2013.03.004

Please cite this article in press as: D. Nieciecka et al., J. Electroanal. Chem. (2013), http://dx.doi.org/10.1016/j.jelechem.2013.03.004

2

D. Nieciecka et al. / Journal of Electroanalytical Chemistry xxx (2013) xxx–xxx

In our previous study on alkanethiol self-assembled monolayers [21] we have shown the effect of terminal groups on the passive penetration of doxorubicin through such monolayers. We demonstrated that DOX molecules adsorbed on the polar head groups of lipids hinder further drug penetration across the membrane. In the present work we have extended our experiments on mixed, two-component monolayers. First, we monitored the physico-chemical behavior and contact potential difference of Langmuir films, depending on the presence of doxorubicin in a subphase. Subsequently, after the Langmuir–Blodgett transfer of the monolayers onto a solid surface we used cyclic voltammetry, QCM, SPR and SERRS to evaluate the amount and mode of the drug penetrating/adsorbing onto the surface of biomimetic films. This novel concept provided us with a unique insight separately into drug interactions with the charged and hydrophobic fragments of the biomimetic film, which can shed more light on topic of the DOX partitioning, closely related to its efficiency as therapeutic agent. An effect of the monolayer structural quality on the monolayer permeability toward DOX was also studied to clarify this issue. 2. Materials and methods All reagents were of the highest quality available commercially and were used without further purification. Octadecanethiol (C18SH, 98%), dihexadecyl phosphate (DHP, 97%), octadecylamine (C18NH2), doxorubicin hydrochloride, iron(II) cyanide, potassium salt (98%), and lithium perchlorate (99.9%) were purchased from Aldrich. Chloroform (99.8% purity) was obtained from Chempur (Poland). Aqueous solutions were prepared with milli-Q water (resistivity 18.2 MX cm) obtained from the Millipore system. 2.1. Monolayer preparation Mixed monolayers of octadecanethiol:octadecylamine (C18SH:C18NH2) and octadecanethiol:dihexadecyl phosphate (C18SH:DHP) were prepared by mixing stock solutions of each compound in chloroform to obtain a desired molar ratio of each constituent in the spreading solution. In these monolayers the octadecanethiol molecules served as anchors of a mixed monolayer after its transfer onto the gold substrates [22,23]. In order to evaluate the lowest possible amount of octadecanethiol in the mixed monolayer, yet providing their stability after the monolayer transfer onto gold, the surface pressure-area isotherms were obtained first for each molar ratio and the excess area (Aexc) was evaluated as a measure of miscibility of monolayer components. Langmuir monolayers were spread from 15 ll of appropriate chloroform solution (2 mg/ml) applied on the aqueous subphase in a NIMA Technol. Langmuir trough (Model 611) equipped with a surface potential sensor (Kelvin Probe SP1, NFT GmbH). After the solvent evaporation (ca. 10 min.), the monolayer was continuously compressed at a rate of 30 cm2/min, to obtain pressurearea isotherms in an isotherm cycling mode set for 2 cycles of compression–decompression. This allowed us to obtain at least two isotherms, to evaluate the reproducibility and monolayer type. For the case of Langmuir–Blodgett transfer, immediately before spreading the chloroform solution at the air/water interface, cleaned gold substrates were immersed into the subphase. Then the monolayer forming solution was applied, chloroform was allowed to evaporate and the monolayer was compressed to a desired target pressure that was kept constant (PC-controlled) during the upstroke Langmuir–Blodgett transfer. The temperature of experiments was 24 °C

2.2. Electrochemistry Electrochemical measurements were carried out with a PC-controlled model 660C Electrochemical Workstation (CH Instruments, Inc., USA), using a small-volume three-electrode cell with a Pt mesh as counter electrode. All potentials are quoted versus the Ag,AgCl|1 M KClaq reference electrode. Gold electrodes (Au, 99.99%) were prepared by melting a tip of a 0.5 mm diameter wire to form a gold ball that was subsequently flat-pressed yielding circular planar polycrystalline electrodes suitable for Langmuir–Blodgett experiments. Prior to further use, the electrodes were annealed in the flame and next electrochemically polarized in 0.1 M HClO4 within the potential window of 0.35 to +1.45 V with a scan rate 0.1 V/s until a stable voltammogram was obtained, characteristic for clean, polycrystalline gold surface [24]. 2.3. Surface Plasmon Resonance (SPR) SPR experiments were carried out with an Autolab Esprit/ Springle system (Eco Chemie B.V., The Netherlands), operating at a fixed 670 nm wavelength and variable angle of incidence of the p-polarized light beam on the SPR substrate. Gold-coated glass disks provided by the manufacturer were used as SPR sensors. The SPR sensor was cleaned by rinsing with ethanol and then dried. The sensor surface was modified as described above (Section 2.1), placed in the SPR cuvette with the sample loading/injection system, and used for further in situ experiments, where the SPR minimum (minimum in reflected intensity) was recorded versus the angle of laser beam reflected from gold. In a kinetic measurement mode, molecular adsorption on the SPR sensor were followed by plotting the SPR angle (angle shifts Dh) over time. The measured SPR angle shifts were converted into mass adsorption using a correlation factor of ca. 120 mdeg per 1 ng/mm2. [25] 2.4. Surface Enhanced Raman Spectroscopy (SERS) 2.4.1. SERS active substrate preparation SERS active substrates were prepared employing electrochemical roughening procedure, involving electrochemical oxidation and reduction cycling (ORC). The polycrystalline silver plates were roughened in a conventional three-electrode cell, using 0.1 M KCl as an electrolyte solution. Five subsequent cycles in which the potential was varied from +300 to 300 mV (versus Ag/AgCl/1 M KClaq electrode, which served as a reference electrode), at a sweep rate of 5 mV s1 were applied. Pt sheet was employed as a counter electrode. The procedure was always terminated in the negative vortex potential (a potential corresponding to reduction peak maximum was held for ca. 30 s) to provide the fullest-possible reduction of the oxidized surface. The ORC-roughened silver electrodes were milky-brown, when exhibiting the best SERS activity. Roughened silver substrates were rinsed thoroughly with the ultrapure water just after roughening procedure and shortly stored in it, prior to further adsorption of the analyte. Silver colloid for the collection of reference SERS spectrum of doxorubicin (DOX) was prepared according to a procedure described by Leopold and Lendl [26], employing hydroxylamine (NH2OH) under alkaline conditions as a reducing agent. For SERS measurement 1 cm3 of silver colloid was mixed with 20 ll of 105M DOX aqueous solution, activated with 40 ll of 0.5 M KNO3. 2.4.2. Raman (SERS) measurements details SERS spectra of doxorubicin on the chemically modified solid Ag substrates were collected in the backscattering configuration with a Labram HR800 (Horiba Jobin Yvon) confocal microscope system, equipped with a Peltier-cooled CCD detector (1024  256 pixel). A

Please cite this article in press as: D. Nieciecka et al., J. Electroanal. Chem. (2013), http://dx.doi.org/10.1016/j.jelechem.2013.03.004

D. Nieciecka et al. / Journal of Electroanalytical Chemistry xxx (2013) xxx–xxx

diode pumped, frequency doubled Nd:YAG laser provided excitation radiation of 532 nm, with the total power below 0.5 mW at the sample. The confocal pinhole size was set to 200 lm and the holographic grating with 600 grooves/mm was used. The calibration of the instrument was performed using a 520 cm1 Raman signal of a silicon wafer. For SERS measurements utilizing colloidal silver suspensions a cuvette holder and quartz cuvette (1 cm optical path length) were used. For typical SERS spectrum utilizing colloidal silver nanoparticles 2 scans were collected with integration time of 50–60 s. The SERS spectra on the Ag electrode were obtained using a 50 magnification Olympus objective and accumulating 1–3 scans, ranging from 30 to 300 s. 2.5. Quartz crystal microbalance (QCM) Quartz crystal microbalance studies were performed with Eureka QCM system (BIOAGE s.r.l., Lamezia Terme, Italy). For the case of QCM sensors, the overall negative resonant frequency shift allowed us to evaluate the total monolayer mass deposited on the surface. The relation of frequency shift and deposited mass was assumed to follow Sauerbrey relation [27]:

Df ¼ 

2f02 Dm Aðlq qq Þ1=2

ð1Þ

where Df is the frequency shift (Hz); Dm mass change (g); A electrode area (cm2); qq the density of quartz (2.65 g cm3); lq shear modulus (2.947  1011 g cm1 s2). 2.6. Spectroscopic ellipsometry The monolayer thickness after transfer onto the gold substrates was evaluated by means of ellipsometry (Sentech SE850 spectroscopic ellipsometer, Sentech Instruments GmbH, Germany). The analysis and modeling of the obtained ellipsometric parameters W and D within the spectral range: 400–800 nm were carried out with use of SpectraRay 2 Program (Sentech Instruments GmbH, Germany).

3

C18NH2 and DHP molecules, acting as a diluent, should provide an increased flexibility of obtained monolayers on the solid substrate. Below the molar ratios of 1:2 and 1:1 for C18SH:C18NH2 and C18SH:DHP, respectively, both types of mixed monolayers show condensed-like behavior with relatively high collapse pressures (up to 40 mN/m), indicating that the interactions between the alkyl chains of monolayer components provide proper organization on the aqueous surface, inhibiting the early collapse induced by the presence C18SH. Above these ratios one can observe a drastic decrease of the collapse pressure towards the value characteristic for octadecanethiol, C18SH (down to 13 mN/m). We also examined miscibility of such monolayers by a quantitative analysis of the excess area (Aexc) at two surface pressures (10 mN/m and 30 mN/m). The excess area can be represented as a difference of the average area per molecule of a two-component, mixed monolayer, with that of an ideal mixed system [31,32]:

Aexc ¼ A12  Aid ¼ A12  ðx1 A1 þ x2 A2 Þ

ð2Þ

where A12 and Aid are the mean experimental and ideal areas per molecule of the mixed monolayer at a given surface pressure, respectively, while x1 and x2 are the mole fractions of component 1 and 2, respectively, of A1 and A2 area per molecule of a pure compound 1 and 2, respectively, at the same surface pressures. When the binary mixture of 1 and 2 form an ideal mixed monolayer or these components are totally immiscible, Aexc will be zero and A12 will scale linearly with x1 (or x2) at a given surface pressure. Any deviation from this behavior would indicate non-ideally mixed monolayers. Thus, Fig. S2 (SM) shows the area per molecule graphs as a function of composition of mixed C18SH:C18NH2 and C18SH:DHP monolayers on a water subphase at surface pressure of 10 mN/m It is important to note however, that while for 10 mN/m the graph covers the total range of mole fraction of C18SH, for the case of 30 mN/m, one could get only up to x = 0.5, because of the early collapse for higher content of octadecanethiol. Taking into consideration the above results, all experiments on mixed, two-component monolayers reported below, were carried out with 1:2 and 1:1 M ratios of C18SH:C18NH2 and C18SH:DPH, respectively, for the case of air/water interface and after the Langmuir–Blodgett transfer for the case of ellipsometry, QCM, SPR, SERS and electrochemical experiments.

3. Results and discussion

3.2. Influence of doxorubicin on Langmuir monolayers

3.1. Behavior of mixed monolayers on water subphase; miscibility of the two-component monolayers

The behavior of pressure-area isotherms of mixed monolayers on free water surface were compared with those spread on an aqueous solution of 1  105 M doxorubicin (Fig. 1A–D). For this purpose, we included also the contact potential difference (CPD) measurements, as shown in this figure. The CPD value can be used to assess the value of the so-called ‘‘effective’’ normal component of dipole moments (l,eff) [33] of molecules forming the monolayer and, for the case of at least partially ionized monolayer, a double-layer potential drop, the latter being most frequently described with the Gouy–Chapman theory:

The surface pressure-area per molecule (P-A) isotherms for mixed C18SH:C18NH2 and C18SH:DHP at various C18SH molar fractions at 24 °C on a water subphase are shown in Fig. S1A and B (Supplementary Material, SM). As the amount of C18SH increases in mixed monolayers, all isotherms show a decrease of the liftoff values of mean molecular area. This is particularly visible for the case of C18SH:DHP monolayers. The shapes of P-A isotherms for pure C18NH2 and DHP suggest that such monolayers can be considered as condensed monolayers. However, these molecules, upon Langmuir–Blodgett transfer on gold surface, do not form stable, bound monolayers. Such monolayers can be formed by a selfassembly of alkanethiol molecules [28,29], but for the case of pure C18SH molecules their hydrophobic/hydrophilic balance does not allow for the formation of well-packed Langmuir monolayer on a free water surface, since the compression isotherm can reach up to 12 mN/m of surface pressure only. Therefore, we investigated mixed monolayers with the amount of C18SH allowing for the formation of miscible, well-packed monolayer that can be subsequently transferred onto a gold surface [30]. The alkanethiol molecules were expected to provide a stable chemical bond, while

Dv ¼

l? Aer eo

þ wGCh

ð3Þ

where Dv is the surface potential, A is the area per molecule at a given surface potential, er and eo are the relative dielectric constant and the permittivity of free space, respectively, and wG-Ch is the electric diffuse layer potential. According to the Demchak–Fort model [34], the normal component of the dipole moment contains the contributions due to the reorientation of water molecules in the presence of the monolayer, normal component from the hydrophilic headgroups at the interface and normal component of the dipole moment from the hydrophobic tails of the monolayer-forming molecules. The latter contribution was expected to be unaffected by

Please cite this article in press as: D. Nieciecka et al., J. Electroanal. Chem. (2013), http://dx.doi.org/10.1016/j.jelechem.2013.03.004

4

D. Nieciecka et al. / Journal of Electroanalytical Chemistry xxx (2013) xxx–xxx

A

B

500

1.6x10 -6

300

0.0

Δχ /mV

J/ (μA/cm2 )

8.0x10

-8.0x10 -7 -1.6x10

2

400

-7

1

-6

-2.4x10 -6

1

200

100

4

-3.2x10 -6

0

-1.0

-0.8

-0.6

-0.4

-0.2

0.15

0.0

0.20

C

D 300

40

2

0.35

0.40

2

200

1

Δχ /mV

Π / (mN/m)

0.30

1

250 30

0.25

A pm / nm 2

E /V

20

150 100 50

10

0 -50

0 0.0

0.5

1.0

1.5

A pm /

2.0

2.5

3.0

nm 2

-100

0.0

0.5

1.0

A pm /

1.5

2.0

2.5

nm 2

Fig. 1. Langmuir compression isotherms and contact potential differences; Panel A and B: (1) C18SH:C18NH2 molecules on an aqueous subphase, (2) C18SH:C18NH2 molecules on an aqueous subphase with 1  105 M doxorubicin; Panel C and D: (1) C18SH:DHP molecules on an aqueous subphase, (2) C18SH:DHP molecules on an aqueous subphase with 1  105 M doxorubicin.

the presence of doxorubicin in the subphase, unless some penetration of this drug into the monolayer occurs. As can be seen from Fig. 1a and b, the presence of doxorubicin in the subphase causes an increase of mean area per molecule for the case of C18NH2:C18SH mixed monolayers by ca. 5–7 A2 and almost twofold increase of CPD value, as compared to pristine aqueous subphase, suggesting some penetration and adsorption of the drug on such monolayer. For this type of system, for the case of pure aqueous subphase, but also with DOX present (vide infra), it is difficult to visualize the diffuse layer and to estimate its contribution. Taking into account the pKa value of octadecylamine (pKa = 10.6, [35]) and its surface concentration of 3.6  1011 mol/cm2 under our experimental conditions (or number of molecules in contact with pure water, N = 2.2  1013), its almost total protonation hardly affects the ionic concentration resulting from the autoprotolysis of water at pH 6.5–7. Even for the case of DOX with pKa = 8.2, its presence in a total concentration of 1  105 mol/ dm3, yields a dissociation degree of ca. 0.02. This means that the concentration of ionic forms (cationic DOX or chloride anions) is ca. 2.5  107 mol/dm3, barely exceeding the ionic concentration of H+ and OH ions originating from the self-dissociation of pure water. As already noted by Oliveira and Bonardi [34], the calculations based on the Gouy–Chapman theory lead to poor results for fully ionized monolayer, overestimating the diffuse layer contribution

(calculation presented by these authors gave a value of 200 mV on pure water, pH ca. 5.8, for expanded monolayer of stearylamine). Therefore, in the description of this interfacial region, we are inclined to think that the Helmholtz model of a layer of hydroxyl ions compensating the charge of protonated octadecylamine monolayer is more appropriate. If so, this layer should be rather included within the solvation layer of reoriented water molecules (Demchak–Fort model [33]) next to the hydrophilic headgroups of a monolayer. Nevertheless, the adsorption of positively charged DOX moieties within the monolayer interface should increase the surface potential of the interfacial region. More pronounced changes can be seen for the case of mixed C18SH:DHP monolayers. The P-A isotherms changed from condensedlike behavior to a liquid-like with evident phase transition plateau. However, we cannot exclude the formation of domains due to the strong electrostatic interactions between DOX molecules and DHP headgroups. With the same amount of molecules spread on the interface as for the case of pristine aqueous subphase, we could see an increase of mean molecular area by more than 10 A2. Also, the CPD value of ca +300 mV for mixed monolayer spread on pure water phase, was diminished by ca. 30% when doxorubicin was present (1  105 M) in the subphase. Similar reasoning as for the case of C18SH:C18NH2, can be applied for the case of a mixed monolayer containing dihexadecylphosphate (pKa = 5.6–6.5, [36]). Under the experimental

Please cite this article in press as: D. Nieciecka et al., J. Electroanal. Chem. (2013), http://dx.doi.org/10.1016/j.jelechem.2013.03.004

5

D. Nieciecka et al. / Journal of Electroanalytical Chemistry xxx (2013) xxx–xxx

conditions of our work, at surface concentration of 2.7  1011 mol/cm2, (N = 1.7  1013), its dissociation is somehow smaller than that of octadecylamine (ca. 80%) and therefore does not contribute to the amount of ions present in an aqueous phase (without or with 1  105 mol/dm3 DOX, vide supra). Therefore, we think that the observed change in the isotherm shape reflects the drug penetration into the monolayer, whereas a suppression of a positive CPD value reflects DOX strong adsorption that compensates the charge of a monolayer. Our observations correspond well with the results found in the literature, where some penetration of doxorubicin was found for the case of POPC (b-palmitoyl-c-oleylphosphatidylcholine) and POPC-DPPA (b-palmitoyl-c-oleyl-phosphatidylcholine-1,2-dipalmitoylphosphatidic acid) Langmuir monolayers [37] and, for the case of charged layers, an additional drug adsorption on their surface [37,38]. At present however, considering the Demchak–Fort model, we cannot resolve, which contributing layer in this model (perhaps all) was more affected. Nevertheless this change is substantial, and requires further studies which are beyond the scope of the present work. Having gained some experimental insight into the interactions of doxorubicin with hydrophilic and charged region of biomimetic monolayer system, we now turn into the investigations of the drug interactions with the hydrophobic part of such systems. And so, the next section of this work presents our results for mixed monolayer of C18SH:C18NH2 and C18SH:DPH after their transfer via the Langmuir–Blodgett technique onto the surface of gold (SPR sensors, QCM sensors and pure gold electrodes). Thus, the monolayers present now their hydrophobic chains toward the aqueous phase. For all cases reported below, the monolayer transfer was done at 40 mN/m surface pressure controlled mode, with transfer ratios between 0.87 and 1. For the case of ellipsometric and quartz crystal microbalance experiments, the same QCM crystals were used. 3.3. Ellipsometry of mixed L–B monolayers The monolayer thickness after Langmuir–Blodgett transfer onto gold surface of QCM sensor was measured with use of ellipsometry. This technique measures the change in polarization state of light reflected from a sample. The results are expressed in terms of ellipsometric parameters W and D, where W is the amplitude component upon reflection and D is the phase shift of the complex reflectance ratio. Both W and D are related to the ratio of Fresnel reflection coefficients of polarized light. Fig. S3a (in Supplementary Material) presents the ellipsometric parameters W and D of mixed C18SH:NH2 and C18SH:DHP monolayers deposited with Langmuir–Blodgett technique on gold surface, as a function of wavelength at fixed 700 angle of incidence, and fits with Levenberg–Marquardt algorithm. The analysis and modeling of the obtained ellipsometric parameters W and D within the spectral range: 400–800 nm with use of SpectraRay 2 Program (Sentech Instruments GmbH, Germany), allowed us to obtain mixed monolayer thickness equal to 2.3 nm ± 0.1 nm for SH:DHDP and 2.6 nm ± 0.1 nm for SH:NH2 (refractive index for monolayer was set to 1.561 for both monolayers) which is in an excellent agreement with literature data [39].

that the molar ratio of molecules after transfer is the same as that on the surface of Langmuir trough before the transfer, we could calculate the area occupied by molecule in the monolayer after their transfer and compare it with similar information derived from the pressure-area P-A isotherms. These results are presented in Table 1. The ellipsometric and QCM data both provided us with an initial information about the molecular organization of mixed monolayers on the gold surface. Comparison of these data shows that the transfer of mixed monolayers onto the surface of gold does not affect the mean molecular area of monolayer-forming molecules. This means, that the monolayers preserved their molecular organization, achieved in Langmuir trough. Then, after optimizing the Langmuir–Blodgett transfer conditions, we used such prepared monolayer-modified substrates as electrodes in the subsequent electrochemical experiments. 3.5. Electrochemical characterization of mixed monolayers The integrity and compactness of L–B monolayers after their transfer on gold were verified by means of cyclic voltammetry in the presence of hydrophilic, kinetically facile redox probe – K4Fe(CN)6. Cyclic voltammograms for monolayer-modified electrodes were recorded in 0.1 M LiClO4 aqueous supporting electrolyte containing 1 mM K4Fe(CN)6 with scan rate 100 mV/s (Fig. S3b). Curve 1 presents the diffusion-controlled, reversible redox reaction of iron(III) hexacyanoferrate on bare gold electrode, whereas curves 2 and 3 present CVs on gold modified with C18SH:DHP and C18SH:C18NH2 monolayers, respectively. Exponential shape on curves 2 and 3 suggests that now the redox reaction is kinetically controlled, and so we conclude that the deposited monolayers are well organized, without pinhole defects, blocking the penetration of hydrophilic redox probe of ca. 0.5 nm diameter. Only these types of monolayers were used in the subsequent experiments with doxorubicin. 3.6. Interaction of DOX with Langmuir–Blodgett monolayers on gold Monolayer-modified electrodes were immersed into an aqueous solution of 1  10–5 M doxorubicin in 0.1 M LiClO4 as supporting electrolyte, at pH 5.6. The choice of measurement conditions balancing between the monolayer stability and the value of pHdependent formal potential of doxorubicin was explained in our earlier work [21]. 3.6.1. Adsorption of DOX onto mixed monolayers: voltammetric studies In order to monitor the time evolution of doxorubicin voltammetric response, cyclic voltammograms were recorded every 30 s after immersion of monolayer-modified electrode, until the stable CV curves were obtained, suggesting the maximum saturation of monolayer with the drug. Fig. S4 shows the development of CV curves in time (time intervals as noted in figure caption) for such modified electrodes. The time of drug adsorption/partitioning on/ into mixed C18SH:C18NH2 and C18SH:DHP monolayers is relatively short. Essentially, already after one minute the monolayer is

3.4. Quartz crystal microbalance, QCM studies of mixed monolayers The same crystals were subsequently used to measure the frequency shift, with respect to the base frequency, as a result of Langmuir–Blodgett transfer of mixed monolayer. The monolayer formation and its transfer in this part of experiments was always carried out in the absence of DOX in the subphase. From these data the mass of a monolayer deposited on gold was calculated using the Sauerbrey equation. Knowing the substrate area and assuming

Table 1 Area per molecule for the two types of mixed monolayers at the same surface pressure. Monolayer

Maximum pressure (mN/m)

Area per molecule (nm2)  result from PA isotherm

Area per molecule (nm2)  result from QCM

C18SHAC18NH2 C18SHADHP

40 40

19.6 29.5

20.5 31

Please cite this article in press as: D. Nieciecka et al., J. Electroanal. Chem. (2013), http://dx.doi.org/10.1016/j.jelechem.2013.03.004

6

D. Nieciecka et al. / Journal of Electroanalytical Chemistry xxx (2013) xxx–xxx

equilibrated with the drug molecules in the solution. For these types of monolayers, the time required to reach equilibration is ca.10 times shorter than that for the case of self-assembled alkanethiol single-component monolayers, reported previously [21], suggesting much faster adsorption/partitioning of doxorubicin into two-component monolayers. This is most likely due to the fact that in the present case the monolayers are of mixed nature and obtained via the Langmuir–Blodgett transfer. We think that this can result in less compact, more loosely packed structure of such films, with more molecular freedom of hydrophobic tails due to the lower number of anchoring thiol groups. It is also worth to notice that the potential of cathodic peak for the case of C18SH:C18NH2 is shifted more negatively (ca. 50 mV) as compared to C18SH:DHP, showing that the energy barrier imposed by the first type of monolayer is larger than for the latter. This shift reflects differences in the Gibbs free energies of charge transfer between DOX and gold electrode in the presence of different monolayers. And again, we think that this result is mainly due to a more fluid-like structure of C18SH:DHP monolayer as compared to C18SH:C18NH2. Therefore, to our opinion, such films are more analogous to the natural biological membranes. Once the monolayer achieved its maximum drug load, the cyclic voltammograms were recorded at different scan rates. The resulting voltammograms are shown in Fig. 2A and B. At low scan rates this approach revealed two distinct reduction peaks that can be assigned to the quinone moiety of doxorubicin. As in our previous work [21] we assign the cathodic peak at less negative potentials (ca. 0.610 V) to the drug being reduced at the possible collapse sites of mixed monolayers, even though for the case of C18SH:DHP monolayer it appears to grow faster with scan rate increase than that at more negative potentials (at ca. 0.690 V). Finally, at scan rates above 100 mV/s both cathodic peaks merge into one, accompanied by its anodic counterpart at ca. 0.620 V. For the case of C18SH:DHP monolayer one can get an impression of surface-confinement-like behavior of doxorubicin undergoing reduction. Our attempts to verify this by scaling the cathodic peak current values linearly with scan rate or square root of scan rate failed (data not shown), therefore we can only state that in the presence of doxorubicin in an aqueous electrolyte solution there are two distinct pathways of drug reduction due to its penetration through the mixed monolayers reported here. All the above results show that even though our mixed monolayers are practically impermeable to hydrophilic redox probe (cf. Fig. S3b), however doxorubicin molecules, being more hydrophobic, with flat condensed ring system, can penetrate through such monolayers due to the relatively loose organization of molecules in the monolayer.

A

2.0x10

-6

3.6.2. Adsorption of doxorubicin onto/within the monolayers: SERRS, QCM and SPR studies 3.6.2.1. SERRS studies. To verify the presence of DOX and the mode of its binding with the monolayer (adsorption onto the monolayer surface versus penetration within the monolayer), the surface-enhanced resonance Raman scattering technique (SERRS) was employed. SERRS is a combination of surface-enhanced Raman scattering (SERS) and resonance Raman (RR) technique, the latter utilizes the excitation wavelength near the electronic transition in the surface attached molecule (here: DOX) This approach overcomes the obstacle of the decaying surface enhancement with a distance provided by a molecular spacer (here: mixed monolayers). SERRS provides additional 103–104 enhancement in comparison to standard SERS experiment. Therefore it is possible to observe the vibration modes of the molecule adsorbed/penetrating the monolayer coated surface; however this procedure is limited to the molecules containing a chromophore, like DOX. The additional advantages of SERRS are its high sensitivity (down to 108 M) and selectivity (probing only chromophore group). The VIS spectrum of DOX exhibits absorption maximum around 480 nm [40], however due to our equipment limitations, we could use 532 nm excitation wavelength for SERRS spectra collection. The 532 nm laser that was used in our Raman system was still in the range of the electronic absorption band of DOX molecules. The limitation of SERRS spectroscopy is that only these metals exhibiting the surface plasmon resonance (SPR) in the visible region can be considered as an electrode (support) material. In practice, this limits the choice to Ag or Au substrates. Gold substrates provide high biocompatibility and easiness of the electrochemical studies in a large potential window [41]. On the other hand, even though silver is considered as toxic and unstable, it exhibits higher surface enhancement and SPR behavior from near UV to infrared [42], as compared to gold. In contrast, the wavelength dependence of the SPR for gold requires the use of exciting wavelengths in red or even infrared region in order to obtain SERS activity, which makes this metal unsuitable for combining SERS and RR for the case of chromophores absorbing beyond this region. Regrettably, DOX is absorbing mostly in the blue and violet region and exhibits only some weaker RR effect at green wavelengths. A few strategies were demonstrated in the literature to overcome this obstacle. The group of van der Zwan introduced the use of silver electrode, polished to a mirror-like appearance prior to the oxidation–reduction cycle (ORC) roughening procedure, allowing to successfully perform both cyclic voltammetry and SERRS experiment on the same substrate [43]. Hildebrandt’s group designed a hybrid Ag/Au support, providing successful transfer of plasmonic properties of Ag to

B

0.0

J/ (μA/cm2 )

J/ (μA/cm2 )

0.0

-2.0x10

2.0x10 -6

-6

1

-4.0x10-6

-2.0x10 -6 1

-4.0x10 -6 -6.0x10 -6

5

-8.0x10 -6 -6.0x10

5

-6

-8.0x10-6

-1.0x10 -5 -1.2x10 -5

-0.8

-0.6

-0.4

E/V

-0.2

0.0

-0.8

-0.6

-0.4

-0.2

0.0

E/V

Fig. 2. Cyclic voltammogram of DOX in 0.1 M LiClO4 with DOX concentration 1  105 M; Panel A: C18SH:C18NH2 monolayer; Panel B: C18SH:DHP monolayer recorded for different scan rates: (1) 9 mV/s, (2) 16 mV/s, (3) 36 mV/s, (4) 64 mV/s and (5) 100 mV/s.

Please cite this article in press as: D. Nieciecka et al., J. Electroanal. Chem. (2013), http://dx.doi.org/10.1016/j.jelechem.2013.03.004

7

D. Nieciecka et al. / Journal of Electroanalytical Chemistry xxx (2013) xxx–xxx

upper gold layer, securing the high SERRS sensitivity, while displaying the electrochemical stability and higher biocompatibility of gold [44]. Still, in this work we simply correlate the partitioning of DOX within the examined monolayers deduced from SERRS experiment performed on silver substrates, with the information gathered from the electrochemical experiments on flat gold supports, assuming similarity of the studied monolayer system for both substrates. Hence, the two-component monolayers on roughened silver were prepared using the same procedure as for gold substrate (Langmuir–Blodgett transfer). Prior to the SERRS measurements we immersed the mixed monolayer-coated, SERS active Ag electrodes into DOX aqueous solution of 1  105 M with subsequent collecting the SERRS spectra every minute for 1 h, in order to monitor the DOX penetration/ adsorption onto the biomimetic film. The representative SERRS spectra for C18SH:DHP and C18SH:C18NH2 taken after the time required to attain constant Raman intensity are shown respectively in Fig. 3a and b. Both spectra present the most intense bands around 450 cm1, 1210 cm1, 1240–1265 cm1 and a broad band at 1310–1345 cm1. The doublet at 1210 and 1240 cm1 can be attributed to the d(CAOAH) vibrations (dihydroxyanthraquinone residue), different in symmetry, denoted as A1 and B1 accordingly

[41]. A shoulder around 1265 cm1 marked slightly exclusively for the C18SH:C18NH2 monolayer is due to the ring stretching mode [41]. A broad band above 1300 cm1 can be ascribed to the coupling of the m(CAO) component (around 1310 cm1) and ring stretching: m(CAC) component (around 1340 cm1) [36,41]. The band around 450 cm1 is due to C@O deformation [36,41]. Another quite intense band located around 1580 cm1 is due to the m(C = C) mode of the aromatic ring [40,45]. This latter band is strongly overlapping with the Raman mode around 1635 cm1, which corresponds to C@O stretching vibration for hydrogen bonded carbonyl groups [40]. Another very strong band is a broad feature centered around 1440 cm1, for which the ring stretching is believed to contribute mainly (C@C and CAC stretches of the aromatic carbons) [40,45]. Another less pronounced bands observed around 345 cm1, 620 cm1, 820 cm1 and 915 cm1 can be ascribed to skeletal deformation modes [45]. SERRS mode around 985 cm1 can be related to the ring-breathing mode [40,45]. Due to the superimposition of SERS and RR enhancement, SERRS bands of DOX practically overwhelm the SERS signal of the monolayer. Selective enhancement of the Raman modes for DOX chromophore is possible due to the local amplification of the electric field (electromagnetic enhancement) and modification of the Raman polarizability tensor (chemical enhancement) for the mole-

1600

1444

1400

1210

1240 1309 1336

1200

1576 1590

c

1152 992

Relative intensity

1000

411 462 800

1265

1241 1210

346

1581

1328

451

600

1437

823

619

347

913

1635

985

b

1211 1240

1440

448

400

817

622

345

912

1577

1325

987

1634

200

a

0 300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500

1600

1700

Raman Shift (cm-1) Fig. 3. SERRS spectra (after reaching stable intensity) of 1  105 M DOX aqueous solution in contact with the ORC roughened silver, coated with preformed: (a) C18SH:DHP and (b) C18SH:C18NH2 monolayers. (c) SERRS spectrum of DOX on silver colloid given for comparison. All spectra were excited with 532 nm laser and scaled and shifted for the clarity of presentation.

Please cite this article in press as: D. Nieciecka et al., J. Electroanal. Chem. (2013), http://dx.doi.org/10.1016/j.jelechem.2013.03.004

8

D. Nieciecka et al. / Journal of Electroanalytical Chemistry xxx (2013) xxx–xxx

cules near the metal nanostructures. The range of surface enhancement is short (a few nanometers) and decays rapidly with the distance from the metal. The Raman intensity is expected to decrease as (a/r)12, where a is a radius of the silver island and r is a distance of the observed point to the center of this island [46]. It was also demonstrated that the enhancement for the SERRS signal of DOX on silver is mostly governed by a short-range mechanism (chemical one) and hence the Raman enhancement factor decreases by more than ten-fold when the molecule is 5 Å from the metal surface [47]. The molecular thickness of the monolayers can be approximated with the one the octadecanethiol. Assuming the structure of the C18SH self-assembled monolayer being typical for alkanethiols on Au(1 1 1), with a chain tilted off the surface normal by ca. 30° and 1.27 Å incremental chain length per methylene unit [48] and Au–S distance of 2 Å [49] the organic film thickness is more than 20 Å. This is in nice agreement with the thicknesses determined with ellipsometry (see Section 3.3), giving 23 Å ± 1 Å for C18SH:DHP and 26 Å ± 1 Å for C18SH:C18NH2 mixed monolayers. Clearly, the presence of the SERRS modes characteristic for DOX precludes the adsorption of the drug at the monolayer surface without subsequent penetration, as at this distance the surface enhancement no longer operates efficiently to give a decent spectrum. Therefore, the first information derived from the SERRS spectra is that for both C18SH:DHP and C18SH:C18NH2 monolayers, the molecules of doxorubicin partitioned into the biomimetic film, locating themselves in close proximity of the metal surface. Due to the SERRS short range, the most enhanced will be the modes of those molecular fragments that are closest to the metal. Moreover, according to the surface selection rules, the electric field at the metal surface is strongly anisotropic (the component normal to the surface is significantly larger than the tangential one) and hence the Raman modes with the polarizability tensor component normal to the surface will exhibit the strongest enhancement [50]. Therefore the estimation of an average orientation of DOX molecules with respect to the metal surface from SERRS spectrum is possible. The orientation of anthracyclines in phospholipid monolayers and bilayers was studied with SERRS spectroscopy by Heywang et al. [37,51,52]. They examined the orientation of the anthracyclines integrated into the biomimetic layer spontaneously adsorbing/penetrating the phospholipid (bi)layers prior to the Langmuir–Blodgett or Langmuir–Schäfer transfer onto the Ag surface. In order to determine the orientation of the anthracyclines they compared their SERRS spectra for the planar lipid (bi)layers deposited on the silver coated prism with the respective RR spectra of aqueous solutions of the studied compounds. Unfortunately, the collection of RR spectrum of DOX is impossible with 532 nm excitation, due to a strong fluorescence. Therefore we must refer to the literature RR spectrum of DOX, collected with 457.9 nm laser [40,53]. On the other hand, it is known that the maximum intensity of the given RR band is dependent on the excitation line energy (the so-called excitation profiles can be determined as plots of intensity of the RR signal as a function of energy of incident light). Therefore, the comparison of the relative intensities between SERRS spectrum taken at 532 nm and RR spectrum at 457.9 nm for DOX must be done skeptically. More importantly however, a shift of the original position of the bands for DOX in aqueous solution upon the drug interactions with the mixed monolayers should provide an important information on the nature of these interactions. The doublet character of the RR band around 450 cm1 disappears almost completely upon DOX interaction with the monolayers examined here. The RR doublet at 1215/1245 cm1 undergoes a redistribution of its intensities and downshifts to 1210/1240 cm1 for SERRS experiment. Instead of the band at 1445 cm1 with a shoulder at 1420 cm1 seen in RR spectrum, one broad and intense SERRS band occurs around 1440 cm1. Also the doublet observed

at 1308 and 1345 cm1 for RR for DOX in the solution, merges into one broad band centered around 1325 cm1 for the drug interacting with biomimetic films. The ring vibration modes at 994 and 1587 cm1 undergo substantial downshift to 985 and 1580 cm1, respectively. The qualitative changes of the SERRS spectral pattern described here, as compared to RR of DOX in aqueous solution, are common for both studied monolayers. A downshift of the d(CAOAH) bands (doublet above 1200 cm1) suggests this moiety is affected by the drug penetration through the monolayer (bond strength weakening [54]). Moreover, the significant intensity of these two modes, together with the one around 450 cm1, ascribed to the C@O deformation implies the presence of OH  O@C molecular fragment in close proximity to the silver surface. One could suspect even a direct interaction of the dihydroxyantraquinone residue with Ag surface, especially that SERRS m(C@O) mode is shifted by 5–8 cm1 toward the lower wavenumbers (comparing to the solution) [40,45]. To verify this, we tried to collect the SERRS spectrum of the DOX molecules adsorbed directly on a roughened silver electrode, but unfortunately it resulted in drug decomposition upon the enhanced electromagnetic field, evidenced by SERS band characteristic of amorphous carbon. Therefore, in order to acquire SERRS spectrum characteristic of direct interactions of the drug with silver, we recorded also the spectrum of DOX in contact with the suspension of colloidal silver nanoparticles (NPs), produced by the reduction of silver ions with hydroxylamine. This spectrum is shown in Fig. 3c. The obtained spectral pattern is comparable with this observed for DOX interacting with mixed monolayers, but still displays some differences. First of all the doublets around 450 cm1 and 1440 cm1 are maintained for DOX interacting with Ag NPs. However, the overall intensity pattern is somewhat changed when comparing the discussed spectra (spectra a and b with spectrum c in Fig. 3), naming only the smaller intensity of the bands around 450 cm1, 1440 cm1 and 1575 cm1 and slightly higher enhancement of the band around 990 cm1 and 1150 cm1 for the drug interacting with silver colloid. Since the wavenumber shifts (comparing to RR spectrum of DOX in the solution [40,53]) also seem to be smaller for the drug in contact with colloidal Ag NPs, we can conclude that a stronger disturbance of the drug structure is due to the monolayer penetration of DOX. The differences observed in the relative intensity of the SERRS bands for DOX interacting with biomimetic films and with silver NPs imply different geometry of adsorption in these two experimental systems [37]. We also attempted to evaluate the orientation of the DOX molecules embedded into the mixed monolayers studied here. It was shown that SERRS excitation profile for DOX adsorbed directly on silver does not match the electronic transition in the adsorbate, but rather follows the SPR band of the SERS active substrate [47]. Therefore, we attempted to rationalize the altered relative intensities in SERRS spectrum of monolayer bound DOX comparing it to the drug RR spectrum in the solution. In the case for DOX at monolayer-coated silver (for both types of the monolayers) the ring breathing and stretching modes are enhanced (at 985 cm1 and 1575 cm1, respectively), so we can suppose (vide supra) that the aromatic rings are located close to the silver electrode [37]. The B1 mode of d(CAOAH) around 1240 cm1 gains intensity for SERRS spectrum of DOX in comparison to the solution, which suggests a decrease in symmetry upon DOX penetration into the monolayers [45]. This effect seems to be stronger for the monolayer containing DHP, as the intensity ratio of I1240cm-1 (B1)/I1210cm-1 (A1) is equal to 0.98 and 0.87 for C18SH:DHP and C18SH:C18NH2, respectively. Taking into account our electrochemical results, where we considered C18SH:DHP monolayer as more loosely packed, this may imply deeper penetration of DOX into such monolayers and stronger interactions with Ag surface, affecting the original molecular symmetry of DOX for this monolayer. The affected intensity ratio of these two bands,

Please cite this article in press as: D. Nieciecka et al., J. Electroanal. Chem. (2013), http://dx.doi.org/10.1016/j.jelechem.2013.03.004

9

D. Nieciecka et al. / Journal of Electroanalytical Chemistry xxx (2013) xxx–xxx

together with the changed intensity distribution for two other doublets (around 450 cm1 and above 1300 cm1), and the downshifted positions of the d(CAOAH) bands and ring vibrations (see discussion above) suggest that the most affected part of DOX molecule during its penetration into the mixed monolayers, is the chromophore involved in the intramolecular hydrogen OH  O bonds. This part of a molecule is also situated in close proximity to the silver surface. However, some interactions of the OH  O@C molecular fragment with silver in the proximity to the substrate cannot be totally ruled out, and therefore a ‘‘flat’’ configuration of the chromophore, with the aromatic rings parallel to the substrate should also be considered. And yet, as discussed in the papers by Heywang et al. [37,51,52], the basic signature of such parallel orientation is an increase of intensity of the bands related to the ring stretching, particularly the bands at 1260 cm1 and 1410 cm1. As seen in Fig. 3a and b, this is not the case for the two systems studied here. For our case, the observed SERRS spectral pattern for DOX contacting the preformed mixed monolayers is also different than that for anthracyclines with the entire chromophore oriented perpendicular to the surface of metal [51]. Consequently, we may

3.6.2.2. QCM and SPR studies. In order to evaluate the amount of doxorubicin interacting with mixed monolayer systems we carried out quartz microbalance studies in a flow-injection mode. The results are presented in Fig. S5A (Supplementary Material). As was discussed above, we used the Sauerbrey equation [27] that relates the negative frequency shift of QCM resonator with the mass

1.2x10-6 6.0x10-7

5.0x10 -7

0.0

4.0x10 -7

-6.0x10-7

3.0x10 -7

-1.2x10

I /A

J/ (μA/cm2 )

A

state that for both examined organic films, DOX molecules exhibit tilted orientation. The sugar moiety of the drug is expected to be exposed away from the metal surface, mostly due the steric hindrance. A residual contribution of the ring stretching mode around 1260 cm1 is visible in the case of C18SH:C18NH2 monolayer, hence slightly less tilted orientation (with respect to the surface) of DOX can be predicted for this type of mixed monolayer. It is somewhat surprising, as this is the C18SH:DHP, which we believe to be more loosely packed. However, this is presumably the amount of the interacting DOX, which is decisive for the drug orientation when penetrating the monolayer, and this amount is lower for C18SH:C18NH2 according to the electrochemical, QCM and SPR results. (see next sections).

1

2.0x10 -7

-6

1.0x10 -7

4

-1.8x10-6

0.0

-2.4x10

0

-6

50

100

150

200

scan rate [mV/s]

-0.8

-0.6

-0.4

-0.2

0.0

E/V

B 8.0x10-7

2.0x10 -6

0.0

1.5x10 -6

I /A

J/ (μA/cm2 )

1.6x10-6

-8.0x10-7

1.0x10 -6

1

-1.6x10-6

5.0x10 -7

-2.4x10-6

4

0.0 0

-3.2x10

50

100

150

200

250

300

scan rate /(mV/s)

-6

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

E/V Fig. 4. Cyclic voltammogram of DOX on modified electrodes after rinsing and moving to electrolytes; recorded with different scan rate 1–9 mV/s, 2–36 m, 3–64 mV/s, 4– 100 mV/s. Panel A: C18SH:C18NH2 monolayer; Panel B: C18SH:DHP monolayer.

Please cite this article in press as: D. Nieciecka et al., J. Electroanal. Chem. (2013), http://dx.doi.org/10.1016/j.jelechem.2013.03.004

10

D. Nieciecka et al. / Journal of Electroanalytical Chemistry xxx (2013) xxx–xxx

deposited on its surface. Assuming the same molar ratio after the Langmuir–Blodgett transfer as on the free surface of the trough, this approach yielded the values equal to 4.9  10–11 mol/cm2 and 6.0  10–11 mol/cm2 for the case of C18SH:C18NH2 (1:2, M:M) and C18SH:DHP (1:1, M:M), respectively. The QCM experiments were followed by the surface plasmon resonance experiments (Fig. S5B and C), that gave us somehow lower values of doxorubicin load (1.1  10–11 mol/cm2 and 2.3  10–11 mol/cm2, yet still, as we expected the value for the case of C18SH:DHP mixed monolayer is higher as compared to C18SH:C18NH2, confirming less compact organization of the system containing dihexadecyl phosphate molecules. 3.6.3. Elution from the monolayer: voltammetric studies After saturation of the monolayer with antibiotic molecules, the electrode was removed from the solution of doxorubicin, rinsed with water and then transferred to pure electrolyte solution (0.1 M LiClO4, pH 5.6). Under such conditions the cyclic voltammograms with different scan rates have been recorded (Fig. 4). After plotting the peak current dependence on the scan rate, being linear in the range of low scan rates, the amount of drug adsorbed onto the monolayer was calculated. This evaluation yielded a value of C = 1.3  10–12 mol/cm2 for C18SH:C18NH2 monolayer and C = 3.2  10–12 mol/cm2 for the membrane with phosphate groups (C18SH:DHP). The obtained surface concentration is ca. 10 times smaller than that obtained from the QCM and SPR measurements. Such difference between the results obtained with different techniques may be explained by the presence of doxorubicin in the solution during the QCM and SPR measurements. Thus, these measurements reflect both: the drug adsorbed and partitioned onto/into the monolayers. For the case of drug elution experiments, the CV curves were recorded with no DOX present in the electrolyte solution, after a thorough washing of the electrode with water. Therefore, part of the drug molecules adsorbed onto such membranes was removed. This confirms the ease of drug penetration through the hydrophobic part of biomimetic monolayers. Moreover, the consecutive cyclic voltammograms recorded over an extended period of time, show doxorubicin molecules still present within the monolayers, supposedly being permanently incorporated within their moieties. 4. Conclusions In the present work we monitored the anthracycline drug, doxorubicin, penetration through mixed, two-component monolayers. First, we monitored the physico-chemical behavior and contact potential difference of Langmuir films, depending on the presence of doxorubicin in a subphase. Subsequently, after transfer of such monolayers via the Langmuir–Blodgett technique onto a solid surface we used cyclic voltammetry, QCM, SPR and SERRS to evaluate the amount of the drug penetrating/adsorbing onto the surface of biomimetic films. This approach gave us a unique possibility to monitor the drug interactions with different regions of biomimicking film. During the first type of experiments, the drug was interacting with hydrophilic head-groups the monolayers, whereas during the latter, doxorubicin was interacting with well-organized hydrophobic tails. Our results obtained with electrochemical and spectroscopic studies including SERRS, suggest a relatively facile drug adsorption/penetration through the obtained Langmuir–Blodgett monolayers. Our experiments also show that despite its good solubility in water, the drug can be incorporated permanently in the monolayer. The overall picture emerging from these results suggests that the drug adsorbs easily on the hydrophilic part of a monolayer. The hydrophobic part allows for the penetration and accumulation of the drug within the monolayer

moiety, while its organization controls the rate and amount of DOX partitioning through such films. Acknowledgments This work was supported by Project PSPB-079/2010 – Grant from Switzerland through the Swiss Contribution to the enlarged European Union. Ellipsometric measurements were carried out using the equipment of the Center for Preclinical Research and Technology, co-financed by the European Fund for the Regional Development within the framework of the Operational Programme Innovative Economy 2007–2013. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.jelechem.2013.03.004. References [1] S.C. Owen, D.P.D. Chan, M.S. Shoichet, Nano Today 7 (2012) 53–65. [2] M. Hruby, C. Konak, K. Ulbrich, J. Control Release 103 (2005) 137–148. [3] A. Wieckowska, E. Jablonowska, E. Rogalska, R. Bilewicz, Phys. Chem. Chem. Phys. 13 (2011) 9716–9724. [4] E. Jablonowska, A. Wieckowska, E. Rogalska, R. Bilewicz, J. Electroanal. Chem. 660 (2011) 360–366. [5] D.A. Gewirtz, Biochem. Pharmacol. 57 (1999) 727–741. [6] A.M. Rahman, S.W. Yusuf, M.S. Ewer, Int. J. Nanomedicine 2 (2007) 567–583. [7] C. Batist, Cardiovasc. Toxicol. 7 (2007) 72–74. [8] P. D’Arpa, L.F. Liu, Biochim. Biophys. Acta 989 (1989) 163–177. [9] J.B. Chaires, J.E. Herrera, M. Waring, Biochemistry 29 (1990) 6145–6153. [10] G. Capanico, F. Zunino, in: B. Pullman, J. Jortner (Eds.), Molecular Basis of Specificity in Nucleic Acid–Drug Interactions, Kluwer Academic, The Netherlands, 1990, pp. 167–176. [11] M. Duguet, C. Lavenot, F. Harper, G. Mirambeau, A.M. de Recondo, Nucleic Acids Res. 11 (1983) 1059–1075. [12] K. Nawara, P. Krysinski, G.J. Blanchard, J. Phys. Chem. A 116 (2012) 4330–4337. [13] P.K. Singal, N.N. Iliskovic, New Engl. J. Med. 339 (1998) 900–905. [14] B.K. Singha, E.G. Mimnaugh, Free Radical Biol. Med. 8 (1990) 567–581. [15] L. Dupou-Cezanne, A.M. Santereau, J.F. Tocanne, Eur. J. Biochem. 181 (1989) 695–702. [16] L. Gallois, M. Fiallo, A. Laigle, W. Priebe, A. Garnier-Suillerot, Eur. J. Biochem. 241 (1996) 879–887. [17] F.A. De Wolf, R.W.H.M. Staffhorst, H.P. Smits, M.F. Onwenzen, B. de Krujiff, Biochem. 32 (1993) 6688–6695. [18] F.A. De Wolf, M. Maliepaard, F. Van Dorsten, I. Berghuis, K. Nicolay, B. de Krujiff, Biochim. Biophys. Acta 1096 (1991) 67–80. [19] R. Grantab, S. Sivananthan, I.F. Tannock, Cancer Res. 66 (2006) 1033–1039. [20] A.I. Minchinton, I.F. Tannock, Nat. Rev. Cancer 6 (2006) 583–592. [21] D. Nieciecka, P. Krysinski, Langmuir 27 (2011) 1100–1107. [22] R. Bilewicz, M. Majda, J. Am. Chem. Soc. 113 (1991) 5464–5466. [23] K. Slowinski, R. Bilewicz, M. Majda, Chem. Anal. 40 (1995) 329–339. [24] P. Krysinski, A. Zebrowska, B. Palys, Z. Łotowski, J. Electrochem. Soc. 149 (2002) 189–194. [25] X. Su, H.F. Teh, K.M. Aung, Y. Zong, Z. Gao, Biosens. Bioelectron. 23 (2008) 1715–1720. [26] N. Leopold, B. Lendl, J. Phys. Chem. B 107 (2003) 5723–5727. [27] G. Sauerbrey, Z. Phys. 155 (1959) 206–222. [28] R. Bilewicz, T. Sawaguchi, R.V. Chamberlain, M. Majda, Langmuir 11 (1995) 2256–2266. [29] P. Krysinski, M.R. Moncelli, F. Tadini-Buoninsegni, Electrochim. Acta 45 (2000) 1885–1892. [30] R. Bilewicz, M. Majda, Langmuir 7 (1991) 2794–2802. [31] M. Grzelakowski, Design at nano scale: biomimetic block copolymers for polymers- protein hybrid material, PhD thesis, University of Basel, 2009. [32] T.-H. Chou, C.-H. Chang, Langmuir 16 (2000) 3385–3390. [33] P. Dynarowicz-Ła˛tka, A. Dhanabalan, O.N. Oliveira Jr., Adv. Colloid Interface Sci. 91 (2001) 221–293. [34] O.N. Oliveira Jr., C. Bonardi, Langmuir 13 (1997) 5920–5924. [35] Morton Rosoff, Nano-surface Chemistry, Marcel Dekker, Brooklyn, New York, 2001. p. 86. [36] A.M. Carmona-Ribeiro, S. Hix, J. Phys. Chem. 95 (1991) 1812–1817. [37] C. Heywang, M. Saint-Pierre-Chazalet, M. Masson, J. Bolard, Langmuir 13 (1997) 5634–5643. [38] M.H. Gaber, M.M. Ghannam, A.A. Said, W.A. Khalil, Biophys. Chem. 70 (1998) 229–233. [39] S. Se˛k et al., J. Am. Chem. Soc. 131 (2009) 6439–6444.

Please cite this article in press as: D. Nieciecka et al., J. Electroanal. Chem. (2013), http://dx.doi.org/10.1016/j.jelechem.2013.03.004

D. Nieciecka et al. / Journal of Electroanalytical Chemistry xxx (2013) xxx–xxx [40] M. Manfait, L. Bernard, T. Theophanides, J. Raman Spectrosc. 11 (1981) 68–74. [41] C. Leger, P. Bertrand, Chem. Rev. 108 (2008) 2379–2438. [42] E.C. Le Ru, P.G. Etchegoin, Principles of Surface-Enhanced Raman Spectroscopy and Related Plasmonic Effects, Elsevier, Oxford, United Kingdom, 2009. [43] D. Millo, A. Ranieri, W. Koot, C. Gooijer, G. van der Zwan, Anal. Chem. 78 (2006) 5622–5625. [44] J.-J. Feng, U. Gernert, M. Sezer, U. Kuhlmann, D.H. Murgida, C. David, M. Richter, A. Knorr, P. Hildebrandt, I.M. Weidinger, Nano Lett. 9 (2009) 298–303. [45] G. Smulevich, A. Feis, J. Phys. Chem. 90 (1986) 6388–6392. [46] T.M. Cotton, in: R.J.H. Clark, R.E. Hester (Eds.), Spectroscopy of Surfaces, John Wiley and Sons Ltd., New York, 1988 (Chapter 3). [47] K. Sokolov, P. Khodorchenko, A. Petukhov, I. Nabiev, G. Chumanov, T.M. Cotton, Appl. Spectrosc. 47 (1993) 515–522.

11

[48] C.D. Bain, E.B. Troughton, Y.-T. Tao, J. Evall, G.M. Whitesides, R.G. Nuzzo, J. Am. Chem. Soc. 111 (1989) 321–335. [49] D.M. Alloway, M. Hofmann, D.L. Smith, N.E. GruhnA, L. Graham, R. Colorado Jr., V.H. Wysocki, T.R. Lee, P.A. Lee, N.R. Armstrong, J. Phys. Chem. B 107 (2003) 11690–11699. [50] M. Moskovits, J.S. Suh, J. Phys. Chem. 88 (1984) 5526–5530. [51] C. Heywang, M. Saint-Pierre Chazalet, M. Masson, A. Garnier-Suillerot, J. Bolard, Langmuir 12 (1996) 6459–6467. [52] C. Heywang, M. Saint-Pierre-Chazalet, M. Masson, J. Bolard, Biophys. J. 75 (1998) 2368–2381. [53] L. Angeloni, G. Smulevich, M.P. Marzocchi Absorption, Spectrochim. Acta A 38 (1982) 213–217. [54] Y. Nonaka, M. Tsuboi, K. Nakamoto, J. Raman Spectrosc. 21 (1990) 133–141.

Please cite this article in press as: D. Nieciecka et al., J. Electroanal. Chem. (2013), http://dx.doi.org/10.1016/j.jelechem.2013.03.004