Doxorubicin

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Electrochemical Triggered Dissolution of Hydroxyapatite/Doxorubicin Nanocarriers Ori Geuli, Maya Miller, Avia Leader, Lijie He, Naomi MelamedBook, Edit Tshuva, Meital Reches, and Daniel Mandler ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00011 • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019

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ACS Applied Bio Materials

Electrochemical Triggered Dissolution of Hydroxyapatite/Doxorubicin Nanocarriers Ori Geuli1, Maya Miller1, Avia Leader1, Lijie He1, Naomi Melamed-Book2, Edit Y. Tshuva1 Meital Reches 1 and Daniel Mandler1* . 1Institute

2The

of Chemistry, the Hebrew University of Jerusalem, Jerusalem 9190401, Israel.

Bio-Imaging Unit, The Alexander Silberman Institute of Life Sciences, Hebrew University,

Jerusalem 9190401 , Israel. Keywords: hydroxyapatite, doxorubicin, nanoparticles, electro-responsive Abstract The controlled release of drugs by an external stimulus is of pivotal interest and importance as a means of increasing administration efficacy. Accordingly, many responsive systems have been developed based on primarily pH, temperature and light changes. Here, a novel electrochemical triggered release of Doxorubicin (Dox) loaded hydroxyapatite (HAp) nanoparticles (NPs) system is presented. Dox is loaded onto HAp NPs by producing a stable dispersion in DMSO. The DoxHAp NPs are electrophoretically deposited on a stainless steel (S.S) surface. The adsorbed DoxHAp NPs are released either by applying a moderate electrochemical potential pulse or upon scanning the potential. Two mechanisms were proposed. The first by which the positive potential 1 Environment ACS Paragon Plus

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induces the desorption of the Dox-HAp NPs. Alternatively, the positive potential could drive the oxidation of water and generation of protons, which cause the dissolution of the Dox-HAp NPs. In-situ characterization techniques, such as, atomic force microscopy (AFM) and confocal microscopy were used to gain insight on the release mechanism. All measurements allude to the electrochemically driven dissolution of the Dox-HAp NPs and release of the embedded drug. Invitro antitumor activity against both HT-29 and A2780 cancer cells revealed that the efficacy of the released Dox was not significantly affected by the electrochemical process. We believe that the electrochemically triggered release of NPs could be applied to many other responsive systems.

1. Introduction Doxorubicin (Dox) is one of the most highly efficient anti-cancer drugs, which has been vastly used for the past three decades.1 Although, Dox has been unquestionably proved as an efficient anti-cancer agent, it causes severe side effects, such as cardiac disorders and liver and renal toxicity.2 Dox administration by conventional chemotherapy is based on intravenous intake, which has several significant limitations; systemic toxicity, insufficient amounts of the drug at the tumor site and lack of controlled release of the drug.3-4 Therefore, local, targeted and controlled release of Dox in the vicinity of the tumors could be of utmost importance for efficient anti-cancer therapy. Local release of therapeutic agents into tumor regions has gained much attention due to the substantial advantages over conventional intravenous chemotherapy. Namely, withdrawal of cancer cells can be achieved in lower concentrations of drugs, systemic toxicity can be drastically diminished, and controlled release of the drug can ensure efficient diffusion of the drug into the

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cancer cells.5 Several approaches have been developed to locally delivering of Dox, such as micelles,6-8 liposomes,9 nanoparticles,4,

10-12,

nanofibers,13 and nanogels.14-16 The use of

nanosystems has been vastly employed in multidiscipline biomedical fields, such as bio-imaging, biosensing, and drug nanocarriers.17-18 Biocompatible nanoparticles (NPs), such as magnetite (Fe3O4), calcium phosphates and other biodegradable materials have been extensively utilized as nano-carriers for the local drug delivery of Dox.18-21 Kayal et al reported on Dox-loaded polyvinyl alcohol coated magnetite NPs with improved targeted drug delivery based on an external magnetic field.4 Betancourt and co-workers studied the synthesis of Dox-loaded poly lactic-co-glycolic acid (PLGA) NPs by nanoprecipitation method.22 They showed a pH-dependent release of Dox in acidic pH by accelerated degradation of the polymer. Recently, Ding et al reported on a notably improved anti-tumor activity of Dox by synergistically combining docetaxel into acid-sensitive copolymer.23 Hydroxyapatite (HAp, Ca5(PO4)3OH) has been widely utilized as a biomaterial due to its superior bioactivity, biocompatibility, and non-toxicity.24 In addition, HAp has been extensively studied as an efficient drug carrier because of its capacity to adsorb various functional groups, such as carboxylic acids, amines and phosphates among others.25-26 Therefore, employing HAp as efficient nano-vehicles for Dox delivery is considered highly appealing. Zheng et al used PLGA-HAp nanocomposite fibers as a nanocarrier for sustained release of Dox.27 Kundu and co-workers examined in-vivo the efficiency of HAp NPs as a nanocarrier for Dox. They showed that HAp NPs loaded with 52% wt of Dox exhibited prolonged drug elution and good cancer cell suppression with minimal liver toxicity.26 Controllable drug release can be triggered by an external stimuli, such as pH, light, temperature, magnetic field and electric pulse.28-31 Accordingly, various external stimuli-responsive systems 3 Environment ACS Paragon Plus


using HAp as a drug carrier for Dox have been developed.19, 32-35 For example, Li et al reported the pH-responsive system of mesoporous HAp NPs loaded with high entrapment efficiency of Dox.36 Other pH-responsive systems based on citrate-HAp NPs were also reported.26, 37 Shin et al reported on a thermal-responsive system based on poly(N-isopropylacrylamide)-HAp hybrid gel. He showed a positive thermo-responsive sustained drug release over a month.38 The main essence of utlizing electrochemistry as the driven force for releasing drugs is the ability to control on the elution of the pharmaceutical agents by alternating the potential on the electrode surface. Unlike pH-responsive systems, where the drug-release is dependent on the pH of the environment, in electrochemical-responsive systems, the pH change is limited to electrode surface. This feature provides an efficient apporach to design precise systems, where on-demand tunable release of active materials is required. However, we are not aware of the electrochemically triggered release of Dox from HAp carrier. should be noted that electrochemically responsive systems for the release of drugs including Dox have been reported.28, 39-44 For example, Ge et al. reported the electro-responsive release of drugs from conductive polypyrrole nanoparticles.45 Liu and co-workers described the release of vitamin B12 from chitosan-montmorillonite composite by electrochemical stimulation.46 We have recently shown that Dox could be electrochemcially released by applying a positive potential to a graphene oxide based electrode, which caused the generation of protons and lowered the pH on the surface.42 Here, we report, for the first time, on a novel electrochemical-responsive system of Dox-HAp NPs. Our approach, which is shown schematically in Scheme 1, involves the adsorption of Dox onto synthesized HAp NPs (Dox-HAp NPs) followed by their electrophoretic deposition on an electrode surface. We show that the release of Dox can be controlled by applying a mild positive potential (≥ 2 V) to the electrode, which is coated with Dox-HAp NPs. The release mechanism 4 Environment ACS Paragon Plus

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was studied and we conclude that is based on decreasing the pH by the electrochemical pulse, which causes dissolution of the HAp NPs. Hence, both the loading as well as the release of Dox are electrochemically controlled. We monitored the release of Dox by spectrophotometry as well as by in-situ measurements using atomic force microscopy (AFM) and confocal microscopy. Scheme 1.

2. Experimental Section 2.1. HAp NPs synthesis 4.722 g of Ca(NO3)2 (ACS EMSURE®, Merck) was dissolved in 18.0 mL of deionized water (18.3 M cm, EasyPure UV, Barnstead, Dubuque) by stirring. The pH of the solution was adjusted to 12 by adding 0.6 mL ammonium hydroxide (25%, Baker Analyzed®, J.T Baker) and 17.4 mL of water. 1.584 g of (NH4)2HPO4 (BioUltra≥99.0%, Sigma) was dissolved in 30 mL water while stirring. The pH of the solution was adjusted to 12 by adding 15.0 mL of concentrated ammonium hydroxide and another 19.0 mL of deionized water. The diammonium phosphate solution was slowly added dropwise using a separatory funnel to the calcium nitrate solution while vigorously stirring. The slow addition resulted in a turbid suspension. After the addition was completed, the suspension was boiled for 1 h. Then, it was cooled to room temperature and left until the pH decreased to 7 (after ca. 3 days) as a result of ammonia evaporation. The precipitated NPs were washed with water and centrifuged at 10,000 rpm for 5 min. This was repeated three times. The HAp NPs precipitate was collected and freeze-dried. The HAp NPs were characterized by X-ray diffraction (XRD, Bruker, D9 Advance), X-ray photoelectron spectroscopy (XPS, Axis Ultra), high-resolution scanning electron microscopy (XHR-SEM, FEI Magellan 400L), and high



resolution transmission scanning electron microscopy (HR-TEM, Tecnai F20 G2). All XRD results were compared to the ICSD (Inorganic Crystal Structure Data) files. 2.2. Dox-HAp loading and electrophoretic deposition 0.5 mg/mL of Doxorubicin HCl (98%, Astatech inc.) was dissolved in DMSO (J.T Baker, ACS reagent). 0.25% (wt/v) of synthesized HAp NPs were added to the Dox solution. Stable Dox-HAp dispersion was obtained after 60 min sonication using a standard ultrasonic bath. Polished stainless steel (S.S) 216 L (Mashaf LTD, Israel) were cleaned in ethanol under ultrasonic agitation for 10 min prior to deposition. Coating of S.S was achieved by electrophoretic deposition (EPD) using a DC power supply (Major Science, Mini 300) in a conventional two-electrode cell. Both cleaned S.S substrates with the same dimensions (1.5 cm2) were used as cathode and anode. The two electrodes were placed parallel where the distance between them was approximately 5 mm. For each experiment, a fresh 13 mL dispersion of Dox-HAp NPs was used. EPD was carried out in two sets; either a constant potential (30 V) was applied for a certain duration (1-10 min), or different potentials (30-80 V) were applied for 5 min. each. After deposition, the substrates were dried at 50° C for 1 hr in the dark. The deposit was determined by weighting the substrate before and after deposition by a microanalytical balance (Mettler Toledo, XP26). The total amount of deposited drug was calculated by dissolving a known amount of Dox-HAp NPs with HCl (0.5 M) and determining the eluted Dox by spectrophotometry. 2.3. Coating characterization The coated substrates were analyzed by XRD (2θ = 5–70 at step size of 0.02 deg sec–1). Highmagnification images of the coated surfaces were acquired by XHR-SEM and AFM (Nanoscope 3100). Low-magnification images of Dox-HAp coatings were obtained by fluorescence microscopy (Olympus BX60). The thickness of the samples was measured by SEM cross-section


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images obtained by Focused ion beam (FIB). Infrared reflection absorption spectroscopy (IRAS) spectra were recorded with a Bruker Vertex 70v spectrometer in the reflection mode (80˚) using a liquid-nitrogen-cooled mercury cadmium telluride (MCT) detector. The samples were scanned 1023 times with 1 cm‒1 resolution. The roughness of the coatings was measured by AFM.

2.4. Electrochemical release of Dox and spectrophotometric analysis Inspections of the release mechanism of Dox from HAp coated S.S were carried out in a homemade three-electrode cell based on 3 mL plastic cuvette (Thermo-Fischer). The working and counter electrodes were HAp-Dox coated and uncoated, S.S with the same exposed area (0.85 cm2), respectively. The reference electrode was silver wire (long). The aqueous solution contained 0.5 mL of 5 mM acetic acid (pH≈4.7). The resistance of the solution was measured after applying 4 V vs. Ag wire for 10 min with a fresh S.S electrode (with the same dimensions), using a computer software. The electrochemical release of Dox was carried out using a potentiostat (CHI 750B, CH Instruments) in a conventional three-electrode cell. Platinum wire (35 mm long) was used as a counter electrode, Ag/AgCl (KCl sat.) as a reference electrode, and Dox-HAp coated S.S as a working electrode. Therefore, all potentials are quoted vs. this reference electrode. The aqueous solution contained 10 mM buffer acetate (pH 4.6) and 0.1 M NaNO2. The Dox release measurements were performed by two sets of potential steps: 0-2 V for 3 min or 2 V for different duration. Aliquots (3.5 mL) of the aqueous solutions were spectrophotometrically analyzed by UV-Vis spectrophotometry (Shimadzu UV-3101PC) using a 10 mm transmitted path length glass cuvette. Spectrophotometric quantification was obtained by using a calibration graph of Dox of known concentrations (0-10 g/mL) at 481 nm.



2.5. In-situ confocal microscopy measurements In-situ investigation of the Dox release was conducted by confocal microscope (Leica SP5 upright confocal) equipped with a 40X dipping objective. A petri-dish (8 cm diameter) to which Ag and Pt wires were attached was used as an electrochemical cell. The Dox-HAp coated S.S was used as a working electrode. The petri dish was filled by 15 mL of liquid (10 mM buffer acetate and 0.1 M NaNO2). The scanned surface area by the confocal microscope was 387.5 m2 and the scanning distance of the substrate to the objective was 4-40 m. Cyclic voltammetry (1 mV sec−1) was scanned between 0.5-2.5 V and back to 2 V while acquiring the confocal microscopy images. 2.6. In-situ AFM study: Surface properties and AFM images were obtained in QITM mode using JPK NanoWizard® 3 AFM (JPK instruments, Berlin, Germany) with manufacturer provided software. Silicon cantilever NSC18/Al BS (Micromash, Germany) with a resonance frequency of 75 kHz and a spring constant of 2.8 N/m was applied to acquire the images. All the experiments were conducted in the liquid phase, were the tip was dipped inside approximately 10 mL of 10 mM buffer acetate (pH 4.6) containing 0.1 M NaNO2. The scan size was 1µm2 with a 128×128-pixel resolution. The root mean square (rms) measurements of the surface tension were analysed from the cross section of the slope images by JPK Data processing software at selected area (375X344 nm), which was monitored during the experiment. The changes in the height were also calculated by JPK Data processing software, based on the changes of the height profile across 1m long vertical line. The electrochemical set-up was similar to the one at 2.6. 2.7. In-vitro cytotoxicity tests Cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described on HT-29 human colon and A2780 human ovarian


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cancer cells obtained from ATCC Inc.47 The MTT assay was employed as following: starting from 0.6 x 106 cells in growth medium (containing 1 % penicillin antibiotics, 1 % L-glutamine, 10 % fetal bovine serum, and 88 % RPMI-1640 medium, all purchased from Biological Industries Inc.), were seeded into 96-well plate and allowed to attach for overnight. A stock solution of Dox electrochemically released into buffer acetate (10 mM) at pH 4.6 (containing 0.1 M NaNO2) at 15 mg/L concentration was used for preparation of solutions with concentration ranging from 7.5 mg/L to 0.015 mg/L. The 200 µL medium in each well was discarded and replaced by 100 µL of fresh medium, following addition of 100 µL of buffer acetate containing doxorubicin at 10 different concentrations. The control wells were treated similarly with buffer acetate at pH 4.6 (without Dox) and set as 100 % cell viability. Positive control was employed and measured as 1 mg Dox dissolved in 50 µL DMSO or buffer acetate at pH 4.6, diluting 20 µL of obtained solution with 180 µL medium, adding 10 µL of resulted solution into 200 µL of the above solution of cells in the medium, giving final concentration of up to 12.5 mg/L in medium containing 0.5% of DMSO or buffer acetate pH 4.6, tested at 10 different concentrations. The control wells were treated similarly with medium containing 0.5% DMSO or buffer acetate solution at pH 4.6 and set as 100 % cell viability. The plates were incubated for 3 days (37 ˚C, 5% CO2 atmosphere), MTT (≥ 97.5%, HPLC; Sigma–Aldrich; 0.1 mg in 20 µL) was added, and the cells were incubated for additional 3 h. The MTT solution was removed. The cells were dissolved in isopropanol (200 µL) and the absorbance was measured at 550 nm by a microplate reader, Spark10M, Tecan. Each measurement was repeated at least 3X3 times: three repeats per plate, all repeated at least three times on different days (at least nine repeats altogether). Relative IC50 values were determined by nonlinear regression of variable-slope (four parameters) model and are presented as mean ± SD values. The concentration of Ca2+ ions in the release media after the electrochemical dissolution of different



Dox-HAp films was determined by using Inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7500 Cx). 3. Results and Discussion HAp NPs were synthesized based on the precipitation reaction between calcium and phosphate precursors in alkaline solution. Figure S1 shows TEM (A) and SEM (B) images of the synthesized HAp NPs. It can be seen that the particles have elongated structure, which is in agreement with previous reports.48-50 The average size of the NPs based on TEM is 30±8 nm. In addition, TEMSAED (Figure S1C) analysis shows clear spots in a diffuse ring, which is a typical diffraction pattern of polycrystalline material as reported elsewhere.51 XPS element analysis (Table S1) shows that the powder is composed of Ca, P, O and C. The Ca/P ratio is 1.64, which is close to the Ca/P ratio of stoichiometric HAp (1.67). We believe that the presence of carbon is mostly due to carbonate, which is known to replace the hydroxyl group in HAp. This was also confirmed by carrying out microanalysis of the HAp NPs, vide-infra. XRD (Figure S1D) was conducted in order to verify the purity of the HAp phase. The XRD pattern shows the characteristic diffraction peaks of HAp phase (ICSD-PDF2 file 01-084-1998).52 No other calcium phosphate phases are detected. The calculated degree of crystallinity is approximately 70% based on XRD as reported elsewhere.53 The average crystalline size is 229 Å according to the Scherrer equation.54 Dox loading onto HAp NPs was conducted in DMSO solution. Firstly, Dox was readily dissolved in DMSO, followed by the addition of HAp NPs. Stable dispersion and Dox loading were obtained by intensive sonication as can be seen in Figure S2. It has been reported that HAp has high affinity towards molecules with different functional groups resulting in strong and high loading adsorption.55-57 Previously, we reported on the incorporation of different antibiotics such as, gentamicin and ciprofloxacin into HAp NPs.58 Dox is a tetracyclic molecule, containing three 10 Environment ACS Paragon Plus

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planar and aromatic hydroxyanthraquinone rings, which act as a chromophore, and one nonplanar, nonaromatic ring attached to an aminoglycosidic residue. The four cyclic groups enable the intercalation with DNA, and therefore inhibit tumor cells proliferation. The unique structure of Dox, which contains several functional groups, such as -C=O, -OH, -NH2, -OR, -COOR, enhances its adsorption onto HAp, either by hydrogen bonding or through electrostatic interactions.27, 59 In addition, Dox strongly chelates di- and trivalent metal ions.60 Using stable dispersions is mandatory when EPD is utilized as the driving force for NPs deposition. DMSO was employed as a solvent due to its excellent solubility properties and good dispersibility of HAp. It has been reported that DMSO can interact with Ca2+ to form [Ca(DMSO)x]2+ species,61 which therefore increases the stability and dispersibility of the HAp NPs.62 Figure 1 A-B shows images of the Dox-HAp coatings on S.S, which were obtained upon applying 30 V for 1, 2.5 and 5 min, respectively. Increasing the amount of Dox-HAp NPs that electrophoretically deposits, increases the fluorescence as can be seen by fluorescence microscopy (Figure 1A). Figure 1B shows SEM (top view and cross section) images of Dox-HAp coating as a function of deposition time. Clearly, increasing the deposition time results in thicker layers of Dox-HAp NPs deposit. Comparison between fluorescence microscopy and SEM shows good agreement in which thicker layers exhibit amplified fluorescence. Table 1 summaries the properties of Dox-HAp coatings deposited at 30 V for different times (1, 2.5 and 5 min). While the thickness was determined from FIB, the weight of the deposit was measured by microanalytical balance and the amount of drug was determined spectrophotometrically after dissolving the coating (see Experimental Section). Table 1.



Obviously, the rate of the NPs deposition is dependent on the applied potential and time. Since Dox is adsorbed on the HAp NPs surface, its amount increases almost linearly with time (Figure S3). The calculated deposition rate of Dox-HAp is 2.07±1.07 µg cm−2 sec−1, while the deposition rate of Dox is 0.12±0.05 µg cm−2 sec−1. The rate of film thickening (from FIB) is 8±2 nm sec−1. The EPD does not affect the shape of the particles as can be seen from the high resolution SEM (Figure 1B IV) and AFM (Figure 1C) images. It is evident that the coating maintains the nanoparticulate shape of the Dox-HAp NPs. Figure S4A shows the roughness (Ra) of the coatings as a function of deposition time measured by AFM. Interestingly, the surface roughness increases with deposition time. This indicates that the layers of the nanoparticles do not grow homogeneously. Cracks that can be seen in a larger scale may contribute to the roughness (Figure S4B). Figure 1D shows FTIR spectra of pure HAp and Dox-HAp coatings for different deposition times. The peaks at 560, 588, 875, 960, 1010, 1098, 1160 cm−1 are attributed to PO43− and HPO42− species. The peaks at 630 and 3570 cm-1 are assigned to the OH− group of HAp and the peaks at 1420 and 1450 cm−1 are attributed to CO32− adsorption.63 The latter is due to the substitution of OH− by carbonate during the synthesis of the NPs. In fact, the microanalysis of the HAp NPs shows 0.41.4% of carbon, which is attributed to carbonate. All the typical peaks of HAp are shown in all Dox-HAp coatings. Careful analysis of the Dox-HAp coatings shows the presence of the Dox peaks. Specifically, the peaks at 1210, 1236 and 1285 cm−1 are assigned to C-O vibrations (of Dox), while those at 1582 and 1617 cm−1 are due to the carbonyl groups of the anthracene ring.19 The small peak at 1725 cm−1 may be attributed to N-H bending vibrations, whereas the peaks at 2851 and 2929 cm−1 are the methylene symmetric and asymmetric vibrations.4, 27, 59 In general, the


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intensity of the peaks of Dox is amplified as a function of deposition time, which indicates that more Dox is deposited with time. Figure 1E shows the XRD pattern of Dox-HAp coatings for different deposition times (1, 2.5, 5, 7.5 and 10 min) at 30 V. The two intense peaks are assigned to the substrate, S.S (marked as #). The small diffraction peaks (marked as *) are attributed to HAp. Only the intense peaks at (002), (211), (112), (310) and (222) planes are seen. The amorphous nature and the low intensity of the peaks may be associated with Dox adsorption on HAp NPs surface, which characterizes a conjunction of organic substances onto ionic crystals.4, 59 Figure 1. The elution of Dox from HAp NPs is based either on an electrochemically driven reaction or through an electrical field induced process. HAp is the most stable and the least soluble of all calcium phosphates (Ksp≈10−58).64 In acidic environment, i.e. pH60 V due to the fact that the films were no longer linearly dependent on the deposition potential, and therefore, figure 3A1, may reflect the thickness of the film.


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The leveling off as a function of deposition time (Figure 3A2) can be explained similarly. Namely, under longer deposition times, the film is thicker and therefore, less affected by the potential and pH change at the electrode-film interface. Interestingly, in the time frame of the release (3 min), approximately 20% of the deposited Dox is eluted (based on Table 1). The next step was to examine the release parameters and their influence on Dox elution (Figures 3B1-2). In these experiments, the deposition parameters, i.e., potential and time, were fixed (30 V and 5 min). Figure 3B1 shows the release of Dox as a function of applied potentials. Clearly, by applying higher potentials, the release of Dox is enhanced, presumably due to the higher oxidation rate of water and generation of protons. The levels of Dox at 0 V indicate the release of the drug that is potential independent. Figure 3B2 shows that the amount of Dox that is released varies quite linearly with the time of release. Figure S6 shows the release percentage of Dox as a function of time and applied potential. Clearly, high concentrations of eluted Dox were obtained under higher potentials and longer time. This indicates that the release of Dox from HAp coating is potential and time dependent. Figure 3. All the above results clearly show that the release of Dox can be controlled electrochemically and is caused by the pH change. The latter drives the dissolution of the Dox-HAp NPs. Further insight of this process was obtained by in-situ release measurements as described in the following section. In-situ monitoring of Dox release was performed by monitoring the fluorescence of the Dox-HAp coating as a function of potential using confocal microscopy. The fluorescence intensity of DoxHAp coating on the S.S surface as a function of time was recorded, while scanning the potential at 1 mV/s. Figure 4A shows a series of confocal images at different potentials (note that the



potential and time are interchangeable). Figure 4B shows the change of fluorescence (red curve) and current density (black curve) as a function of the applied potential. It can be seen, that the fluorescence of the surface does not change as long as the potential is less positive than 1.5 V. This correlates nicely with the increase of the anodic current due to water oxidation. The threshold potential that causes the fluorescence to decrease is ca. 1.52 V, which is in accordance with Figure 4B (red curve), where high increase in the oxidation currents is observed. Furthermore, the fluorescence at the S.S surface continues to decay (and eventually disappears) even after scanning back the potential (switching potential at 2.5 V). Additional video of the surface fluorescence is provided in the supplementary section as Video S1. This unique experiment clearly shows that the release of Dox is potential dependent, in which the HAp coating dissolves under local acidic environment. Figure 4. High-resolution investigations on the morphology of Dox-HAp coatings as a function of applied potential were carried out by a series of in-situ AFM experiments. First, we tried to monitor the structural properties of the coating, namely the height (nm) and the surface tension (nN/m) during a potential scan (1 mV sec−1) between 0.5-1.0 V vs. Ag wire. The measured area was 1x1 m2. The height profile was measured by drawing a vertical line (1 m) across the measured area before scanning the potential. At a specific time, the height profile was measured. Figure S7 shows the methodology for calculating the height (z-axis). The surface tension was measured by selecting a specific area 375x344 nm2, which was monitored during the potential scanning (shown in Figure S8). Figure 5A-B shows the dependence of the height and the surface tension on the applied potential, respectively. It can be seen, that the measured height was stable up to 0.8 V, where at


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higher potentials it was significantly changed. On the other hand, the measured tension was gradually changed as function of applied potential, where at a potential>0.8 V, the surface tension was significantly changed, probably due to generation of protons. The results clearly support our previously described findings, where dissolution of the Dox-HAp coatings occurs at potentials sufficiently positive to generate protons by water oxidation. Scanning at potentials >1 V was impossible due to O2 bubbles generation, which interfered with the scanning of the AFM tip. In the second in-situ AFM experiment, we acquired images of the Dox-HAp coating after applying pulses for different duration. Specifically, we applied pulses of 1.75 V for different durations (30, 15, 60, 120 and 600 sec). Figure 5B shows a set of AFM 3D imagings after applying pulses for different durations. The first imaging (top left) was acquired before applying any potential. The observed nanoparticulate structure of the Dox-HAp coating is in agreement with Figure 1A-C. Before applying any potential, the AFM tip was elevated above the solution level to avoid any attachment of O2 bubbles to the tip surface. After applying a pulse, the AFM tip approached the coated surface and an image was recorded. It can be easily seen that the Dox-HAp was drastically impaired upon applying a 30 sec pulse of 1.75 V, where the nanoparticulate structure of the coating was significantly damaged. Further pulses resulted in further deterioration of the coating, where after accumulated 765 sec of applying 1.75 V, the Dox-HAp nanoparticulate structure could not be detected. These in-situ AFM experiments can support our proposed mechanism, whereby Dox is released from HAp coating because of HAp dissolution by an electrochemical reaction of water oxidation. Another perspective of the surface modifications can be seen by the corresponding AFM images (Figure S9), which is also in agreement with the suggested mechanism. Figure 5.



Assessment of the anti-tumor activity of the electrochemically released Dox is crucial to verify the efficacy of Dox after exposure to high voltage and acidic environment. The cytotoxicity tests were investigated on HT-29 human colon cancer cells by the MTT assay as previously described.47 Dox was electrochemically released from Dox-HAp coating to obtain 15 mg/L of Dox stock solution (calibrated by spectrophotometry). The negative control solution contained 1:1 (v/v) growth medium and release media without Dox and was set as 100% cell viability. As a positive control, Dox powder was dissolved in the same release media. Figure 6A shows the therapeutic efficacy of the electrochemically released (ER) Dox as compared with pure Dox. Clearly, the EPD of DoxHAp NPs followed by electrochemically release did not impair the anti-tumor activity of the drug. The relative IC50 of ER-Dox and that of pure Dox were calculated based on nonlinear regression of variable-slope (four parameters) model. We found that the IC50 was 0.8±0.5 and 0.6±0.1 µM for the ER and pure Dox, respectively. Dose-response curves of ER and pure Dox on HT-29 cells are shown in Figure S10. The therapeutic activity of the ER Dox is slightly deficient as compared with pure Dox, mostly pronounced in the reduced maximal inhibition value (ca. 52 vs 71%). We assume that this finding might be attributed to the presence of Ca2+ in the release solution as a result of HAp dissolution. As evidence, we analyzed the concentration of calcium ions by using ICP-MS. Figure S11 shows the concentration of Ca2+ after the electrochemical dissolution of different Dox-HAp films (deposited at 40 V for different times). It can be clearly seen that prolong deposition times generate higher concentrations of Ca2+ due to the dissolution of higher amounts of HAp, which is in agreement with Figure 1 and Table 1. In order to further elucidate our assumption, where the reduced toxicity could be associated with the presence of Ca2+, we conducted an additional cytotoxicity test on A2780 human ovarian cancer cells. Figure 6B indicates that higher concentrations of ER Dox are required to induce similar therapeutic activity


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as compared with pure Dox. Specifically, only at high concentration (7 M), relatively similar therapeutic efficacy was achieved for both pure Dox and ER Dox. In addition, the calculated IC50, based on dose-response curves (Figure S12) for ER Dox was higher than pure Dox (0.8±0.4 and 0.2±0.1 M, respectively). The similar therapeutic efficacy of ER Dox on different cancer cell lines, could be definitely attributed to the considerable amounts of Ca2+ in the release solution. Calcium as an important regulator for cell signaling has a vital role in cellular processes, can indeed affect the efficacy of Dox. Chen et al. showed a suppressive effect of Ca2+ on Dox cytotoxicity via interactions with vacuolar ATPase (V-ATPase) regulators, which results in higher cell survival, compared to pure Dox treatment. Calcium has been reputed as a regulator for the activation of VATPase, which could mediate a cellular rescue mechanism to counteract against Dox by changing the pH within subcellular compartments that ultimately limits the administration of Dox by the cancer cells.69 In addition, higher concentration of extra-cellular calcium can interfere with the activation of voltage-gated calcium channel, which is associated with the overexpression of membrane P-glycoproteins (Pgp). Overexpression of glycoproteins in cancer cells is one of the main mechanisms of multi-drug resistance to different chemotherapeutic drugs 70. The relatively reduced toxicity of our system is in agreement with other reported HAp-Dox based systems, where higher cell proliferation was obtained in comparison with pure Dox. The relatively high (ca. 29%) cell viability observed also for the pure Dox in 7 M is associated with the resistance of HT-29 cancer cells to Dox, where lower cell viability (≥9%) was achieved for A2780 cell line at the same concentration. Although, relatively inferior efficacy was observed for ER Dox compared with pure Dox, the capability to locally deliver Dox in a controlled practice, could provide high concentration of Dox on demand in the vicinity of the tumor, while preventing the systemic toxicity of intravenous administration.



Figure 6. 4. Conclusions For the first time, an electrochemically triggered release system of Dox loaded onto HAp NPs is described. Dox-HAp NPs coatings were prepared by dispersing HAp NPs in DMSO, followed by Dox addition. The Dox loaded HAp NPs were deposited on stainless steel by electrophoresis. Different loading of Dox-HAp NPs on S.S was achieved by alternating the deposition time and potential. The Dox as well as the HAp NPs were released from the coating upon applying moderate potentials. The electrochemically released of Dox and HAp was monitored by conductometry, spectrophotometry and by in-situ confocal microscopy and AFM. Between the two proposed mechanisms, one driven by the electrical field while the other by an electrochemical reaction, all data pointed to the second option. We conclude that the electrochemically driven release of Dox embedded nanoparticles proceeds via the anodic oxidation of water, which causes the local and transient decrease of pH on the electrode surface, and therefore, to the dissolution of the HAp nanoparticles and release of Dox. Unlike, other pH-responsive systems, where the release of the drugs is dependent on the pH of the environment, here, the pH change is limited to electrode surface. In-situ AFM and confocal microscopy measurements clearly enabled determining a threshold potential for the release of Dox that is superimposed with water oxidation. Finally, the biological anticancer activity of the released Dox was confirmed against HT-29 and A2780 cancer cells and found to be almost intact. Electrochemistry has been so far only seldom used for triggering the release of drugs. It has never been used for the release of drug loaded NPs, which are drug carriers. The electrochemical release of drug loaded NPs is more attractive than releasing the drug only as it will allow in the future to


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design systems where drug carriers will be electrochemically released and carry the embedded drug to the target.

Figure 1. A. Fluorescence microscopy (I-III) and B. SEM (I-IV) images of Dox-HAp coating after (I) 1, (II) 2.5 and (III) 5 min of deposition at 30 V. Insets show cross-section SEM images of the coating. IV is a magnified image of III. C. AFM image of Dox-HAp coating. D. FTIR spectra of pure HAp coating and Dox-HAp coatings for different deposition times at 30 V. E. XRD patterns of Dox-HAp coatings for different deposition times at 30 V. # Stainless steel peaks, * HAp peaks.



Figure 2. A. Resistance measurements and B. Current transients upon applying 4 V vs. Ag wire to bare (blank) and Dox-HAp electrophoretically coated (30 V for 1, 2.5 and 5 min) S.S electrodes in a non-stirred solution of 5 mM acetate buffer, pH 4.7. The resistance of the solutions described in A was measured after 10 min of applying the potential.

Figure 3. Spectrophotometric measurements of Dox elution as a function of: A. deposition (1, 2) and B. release (1, 2) parameters (potentials and time, respectively). Insets show the corresponding spectra.


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Figure 4. A. in-situ confocal images of Dox-HAp coating as a function of applied potential. B. Fluorescence intensity measurements of Dox-HAp coating and the corresponding cyclic voltamogram (1 mV/sec) of the electrochemical system (Dox-HAp coated S.S as working electrode, Pt wire as counter electrode and Ag wire as reference electrode in 0.1 M NaNO2 and 10 mM buffer acetate (pH 4.6)).



A

B

Figure 5. A. in-situ AFM calculated surface height (nm) and tension (nN/m), black and red plot, respectively. B. A series of in-situ AFM 3D imagings after applying pulses of 1.75 V vs. Ag wire for different durations.


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Figure 6. Cytotoxicity studies of A. HT-29 and B. A2780 cancer cells treated with increasing concentrations of electrochemically released (ER) Dox and pure Dox as positive control.

Scheme 1. Schematic illustration of synthesis, deposition and release of Dox from HAp NPs predeposited on an electrode surface.



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Applied

Deposition time

Thickness

voltage (V)

(min)

(m)

(mg cm−2)

(g cm−2)

1

0.30±0.03

0.19±0.02

11.01±0.16

2.5

1.61±0.08

0.27±0.01

17.77±0.18

5

2.50±0.05

0.34±0.01

20.29±0.65

30

Deposit weight Deposited drug

Table 1. Summary of coating properties for different deposition times.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS website. Elaborated characterization of HAp NPs, such as SEM, TEM, SAED, XRD and XPS. Graphs of different coating parameters (weight, roughness, Dox concentration) as a function of deposition time. Table of electrical parameters of different release solution. Calibration graph and the corresponding absorbance spectrum of Dox solutions. Release graphs of Dox as a function of different release parameters, applied voltage and time. In-situ confocal imaging video of Dox-HAp coating during cyclic voltammetry. In-situ AFM methodology of height and surface tension. 3D AFM imaging of Dox-HAp coated S.S after applying 1.75 V for different durations. Dose-response curves of electrochemically released and pure Dox on HT-29 and A2780 cancer cells. Detection of Ca2+ ions in the release solution as a function of deposition time at 40 V by ICP-MS. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.


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Graphical Abstract


Doxorubicin

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