Electrically Responsive, Nanopatterned Surfaces for Triggered

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Electrically Responsive, Nanopatterned Surfaces for Triggered Delivery of Biologically Active Molecules into Cells Mo-Yuan Shen,†,∥ Sivan Yuran,‡,∥ Yaron Aviv,‡,∥ Hailemichael Ayalew,†,§ Chun-Hao Luo,† Yu-Han Tsai,† Meital Reches,*,‡ Hsiao-Hua Yu,*,†,§ and Roy Shenhar*,‡ †

Institute of Chemistry, Academia Sinica, 128 Academia Road Sec. 2, Nankang, Taipei 115, Taiwan Institute of Chemistry and the Center for Nanoscience and Nanotechnology, Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel § Taiwan International Graduate Program (TIGP), Sustainable Chemical Science and Technology (SCST), Academia Sinica, Taipei 115, Taiwan ACS Appl. Mater. Interfaces Downloaded from pubs.acs.org by STOCKHOLM UNIV on 12/19/18. For personal use only.

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ABSTRACT: Polyelectrolyte multilayers (PEMs) assembled layer-by-layer have emerged as functional polymer films that are both stable and capable of containing drug molecules for controlled release applications. Most of these applications concentrate on sustained release, where the concentration of the released molecules remains rather constant with time. However, high-efficiency delivery requires obtaining high local concentrations at the vicinity of the cells, which is achieved by triggered release. Here, we show that a nanopatterned PEM platform demonstrates superior properties with respect to drug retention and triggered delivery. A chemically modified block copolymer film was used as a template for the selective deposition of poly(ethylene imine) and a charged derivative of the electroactive poly(3,4-ethylenedioxythiophene) together with a drug molecule. This nanopatterned PEM shows the following advantages: (1) high drug loading; (2) enhanced retention of the bioactive molecule; (3) release triggered by an electrochemical stimulus; (4) high efficacy of drug delivery to cells adsorbed on the surface compared to the delivery efficacy of a similar concentration of drug to cells suspended in a solution. KEYWORDS: block copolymers, nanopatterning, polyelectrolyte multilayers, tissue engineering, drug releasing, cell viability

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INTRODUCTION Efficient delivery of biologically active ingredients, ranging from growth factors, therapeutic molecules, to genetic matters holds the key to program cell functions and viability. To achieve efficient delivery, polymer thin films and coatings were among the most successful and promising tools. They have been applied to promote surface-mediated delivery in drugeluting implants/stents,1,2 cell reprogramming for regenerative medicine,3,4 and controlled release of active therapeutic ingredients.5 Considering all the engineering and biology aspects required, polyelectrolyte multilayer (PEM) films built by layer-by-layer techniques have emerged because of their advantageous features of easy fabrication, tunable material compositions, and stability for ingredient storage and controlled release. When the PEMs were applied for programming cell functions/viability, they demonstrated sustained release of encapsulated ingredients from the surface-initiated decomposition at the cells/materials interface.6−8 For cellular applications, it would be critical to integrate PEM techniques with other advanced material features to achieve higher delivery efficiency, larger active© XXXX American Chemical Society

ingredient loading, better stability in aqueous solutions, and different release profiles. Recently, it was demonstrated that the introduction of nanostructural features into materials provides spatial− temporal control over cell functions.9 Notably, specific cell− substrate interactions observed on three-dimensional micro/ nanostructures provided the topographic cues regulating the cell spreading morphology, thereby promoting the level of cell differentiation for stem cells,10,11 enhancing the transfection efficiency of the cells with targeted gene expression,12,13 and improving the capturing efficiency of circulating tumor cells for noninvasive blood biopsies.14,15 Among the approaches used for the fabrication of nanopatterned substrates, block copolymers (BCPs) provide a simple, economical, and versatile platform. Owing to microphase separation, block copolymer materials are inherently structured and exhibit periodic morphologies such as Received: September 7, 2018 Accepted: December 6, 2018

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DOI: 10.1021/acsami.8b15308 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. Components and scheme of the layer-by-layer assembly of PEDOTS/PEI/Dox on the nanopatterned xBCP substrate.

Figure 2. SEM images (a, d), SFM height images (b, e), and cross sections (c, f) of the patterned substrate before (a−c) and after (d−f) deposition of the first PEDOTS layer. SEM images were taken at 26° tilt angle. The SFM cross sections represent an average over 40 adjacent scan lines (800 nm × 150 nm box). (g) The XPS data of the block copolymer film (blue), the nanopatterned template (xBCP) formed after reaction with DIB (red), and the film after deposition of the first PEDOTS layer (black). (h) Cell proliferation and viability data on different substrate interfaces (data represent averages of 3 repetitions).

alternating lamellae, hexagonally packed cylinders (made of the short block surrounded by a matrix consisting of the long

block), and spheres in a matrix. The typical periodicity of these structures is in the range of a few to a few hundred B

DOI: 10.1021/acsami.8b15308 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 3. (a, b) Quantification of the amount (in nanomole per cm2 of film) of stored Dox in the polyelectrolyte films assembled on different substrates (P2VP homopolymer (xP2VP) vs block copolymer (xBCP)) via its release in a 1 mL phosphate buffer saline (PBS) using (a) sonication or (b) application of 20 cyclic voltammetry sweeps from −0.2 to 0.8 V at 0.1 V/s rate. (c, d) Leakage test, performed by incubation of 3.5-bilayer PEMs for different time intervals: (c) amount of Dox remaining in the multilayers after each interval, quantified after subsequent sonication into a fresh 1 mL of PBS and (d) cumulative amount of Dox released into a 1 mL PBS after each interval. (e) Quartz crystal microbalance (QCM) experiment, showing the buildup of the layers and their disassembly. Numerical labels denote the process steps: (1) introduction of 1 wt % PEI solution; (2) washing with water; (3) introduction of 1 wt % PEDOTS solution; and (4) disassembly upon the application of 10 voltage cycles from −0.2 to 0.8 V at 0.1 V/s rate. Washing cycles following deposition were associated with a slight increase in frequency, owing to the desorption of weakly associating polyelectrolytes. (f) Quantification of Dox released from the multilayer assembled on the nanopatterned substrate into 1 mL of PBS after different number of voltage cycles from −0.2 to 0.8 V at 0.1 V/s rate. Measurements were taken in situ immediately after the completion 1, 5, 10, and 20 cycles. Data shown in (a-d, f) represent averages of 3 repetitions.

nanometers, dictated by the total length of the copolymer. In

Recently, Shenhar and co-workers have demonstrated the utilization of surface patterns made of polystyrene-blockpoly(2-vinyl pyridine) (PS-b-P2VP) for the assembly of nanopatterned PEMs.16−19 In this approach, the P2VP domains are cross-linked after the formation of the surface pattern by reaction of the pyridine units with 1,4-diiodobutane

thin films, such morphologies translate to surface patterns, where standing lamellae (i.e., lamellae oriented normal to the substrate) and lying cylinders give rise to a striped pattern and standing cylinders and spheres lead to a dotted pattern. C

DOI: 10.1021/acsami.8b15308 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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number of live cells by about 65% compared to the number of cells cultured on the xP2VP substrate, demonstrating the biocompatibility of PEDOTS.26−29 Interestingly, the PEDOTS coating on the xP2VP domains of the xBCP template diminishes the effect of the presence of the PS domains, despite the fact that microscopy data indicate a highly selective deposition on the xP2VP domains. Assembly and Quantification of Doxorubicin inside the Polyelectrolyte Multilayers. Polyelectrolyte multilayers consisting of 3.5-bilayers incorporating Dox were assembled on the homogeneous xP2VP and on the nanopatterned xBCP substrates. After the initial PEDOTS layer was deposited, we first tried to absorb the Dox by dipping the substrate into Dox solutions after each polyelectrolyte deposition step. However, only limited amount of Dox was absorbed onto the PEDOTS or the PEI top layer. To increase the Dox loading within the PEMs, layers of Dox were spin-coated after each polyelectrolyte deposition. Probing Dox fluorescence in the films (λex = 488 nm)30 shows increased emission from the films after deposition of 3.5 bilayers containing Dox (Figure S1). Quantification of Dox inside the layers at different stages of the multilayer buildup was performed by releasing Dox into the solution using two independent techniques, namely, sonication and application of voltage scans (Figure 3a,b; see Experimental Section). Both techniques yielded rather similar values, showing a consistent increase in the amounts of stored Dox with the number of bilayers on both types of substrates. Yet, the amounts of Dox stored in the nanopatterned multilayers are considerably higher than those in the multilayers assembled on the laterally homogeneous xP2VP substrates. The observation that the multilayers assembled on the homogeneous xP2VP substrate stored considerably less Dox compared to the amount stored in the nanopatterned xBCP substrate is rather surprising; considering the areas available for polyelectrolyte assembly on both substrates (i.e., the xP2VP domains in the nanopatterned substrates compared to the entire substrate in the xP2VP homopolymer), one would expect the opposite. Explaining this behavior requires considering the differences between the two types of substrates. The main differences are the corrugation and coexistence of both hydrophilic and hydrophobic domains at the surface of the xBCP template compared to the low surface roughness and chemical homogeneity of the xP2VP film. During the last stages of the spin-coating process, the corrugated surface of the nanopatterned template traps the receding solution in the trenches. As the solvent continues to evaporate, dewetting from the PS domains further confines the remaining solution to the side walls of the PEMs domains assembled on the xP2VP domains. These features do not exist in the chemically and topographically homogeneous xP2VP/ PEM substrate, hence only a smaller fraction of the Dox remains on the film, possibly owing to the existence of sporadic surface defects. Incubation of both 3.5-bilayer Dox-containing films in 1 mL of phosphate buffer saline (PBS; pH 7.5) for extended periods of time revealed that multilayers assembled on the unpatterned homopolymer substrate were considerably less stable in terms of retaining the Dox, which leaked out from the multilayer within ∼12 h (Figure 3c,d, red curves). In comparison, the multilayer assembled on the xBCP retained its stored Dox for prolonged time, with less than 5% Dox lost after 3 days of continuous incubation (an average leakage rate of 1.6% per day; Figure 3c,d, blue curves). We propose two possible

(DIB), which also quaternizes the pyridines. This treatment renders the cross-linked P2VP domains (denoted as “xP2VP” hereafter) positively charged and hence amenable for layer-bylayer assembly of polyelectrolytes. The polymer film (termed “xBCP” throughout this manuscript) hence presents alternating neutral and positively charged domains (the PS and xP2VP domains) and can thus serve as a template for the preparation of nanopatterned PEMs. Besides the nanostructure features, integration of stimulation responsiveness should enable controlling the release profile of encapsulated ingredients from the PEM films. PEM disassembly could be triggered by an external stimulus, leading to “on demand” release.20−22 In the present study, we have adapted the fabrication approach for the creation of electrically active, nanopatterned PEMs to enable triggered drug release by including a sulfonate-functionalized poly(3,4-ethylenedioxythiophene) (PEDOTS)23−25 as the negatively charged polyelectrolyte (Figure 1). The biocompatible poly(ethylene imine) (PEI) was used as the positively charged polyelectrolyte. Additionally, we incorporated doxorubicin (Dox) as a model drug molecule in the nanopatterned PEM. The main idea is that the application of a positive voltage would oxidize the PEDOTS, rendering it positively charged, and the electrostatic repulsion between this polymer and the PEI will destabilize the PEM, causing it to disassemble and release the drug. Cells directly adsorbed on this nanopatterned functional surface would experience a large local concentration of the drug. The following sections describe the basic characterization of the nanopatterned functional PEM from the materials as well as the bioengineering perspectives, and the demonstration of triggered drug release from this substrate.

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RESULTS AND DISCUSSION Nanopatterned Substrate Characterization. Figure 2a−f shows the scanning electron microscopy (SEM) and scanning force microscopy (SFM) images of the cross-linked, nanopatterned block copolymer template before and after the deposition of the first PEDOTS layer. A strong increase in height contrast (from ca. 4 to ca. 15 nm) indicates that the PEDOTS adsorbed specifically to the positively charged xP2VP domains. The X-ray photoelectron spectroscopy (XPS) measurements performed on the block copolymer film, the cross-linked template (xBCP), and the template after the deposition of the first PEDOTS layer (Figure 2g) shows a decrease in the intensity of the nitrogen peak after the reaction with DIB, which is attributed to the conversion of the surface pyridine groups into alkylated pyridinium ions. Additionally, a strong decrease in the intensity of the iodine peak as well as an evolution of a sulfur peak is noted after PEDOTS deposition, which corroborates the displacement of iodide anions with the PEDOTS during the electrostatic self-assembly process. Lastly, water contact angle measurements show that the PEDOTS layer renders the substrate more hydrophilic (static contact angle decreased from 66.0 ± 0.3° on the xBCP template to 49 ± 2° on the xBCP-PEDOTS surface). Biological testing of cell proliferation and viability were performed on the different substrates (Figure 2h). Cells did not proliferate on the PS substrate but adhered nicely on the xP2VP substrate. The amount of cells on the xBCP substrate was about half that on the xP2VP substrate, an intermediate value between PS and xP2VP substrates, reflecting the areal fraction occupied by the xP2VP domains in the xBCP template. However, deposition of PEDOTS increased the D

DOI: 10.1021/acsami.8b15308 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 4. Fluorescence images of a live/dead assay of fibroblast cells cultured for 2 days on PEDOTS/PEI multilayers that were assembled on the block copolymer template: (a) without Dox; (b) without Dox, after 20 cycles of electrochemical stimulation; (c) with Dox, without electrochemical treatment; and (d−f) with Dox, after 1, 5, and 10 cycles of electrochemical stimulation. The scale bar in (f) applies to all images.

explanations for such a behavior. The first relates to our previous observation that polyelectrolytes deposited over the interface between the PS and xP2VP domains fold back onto the xP2VP domains during the drying stage.16,17,19 This possibly helps in encapsulating the adsorbed Dox molecules within the PEM and retaining them for prolonged times. The second explanation relates to the possible leakage of the Dox from defect points in the PEM caused by dust particles. Whereas a single defect point may, in principle, drain an entire continuous multilayer, only a few domains would be affected by it in a nanopatterned PEM, which consists of isolated domains. Although we cannot provide direct evidence to support these explanations, the ability to store and retain high amounts of bioactive molecules is a clear advantage of the nanopatterned devices. Stimulated Release of Encapsulated Doxorubicin. Direct evidence into the mechanism of release was obtained by quartz crystal microbalance (QCM) experiments performed on a PEDOTS/PEI multilayer assembled on both xP2VP- and xBCP-coated QCM resonator (Figure 3e). Initially, multilayer buildup is demonstrated by the decrease in the resonator frequency every time a new polyelectrolyte solution is injected. Washing cycles resulted in a small increase in the frequency owing to the desorption of nonspecifically adsorbed polyelectrolytes. Application of 10 voltage cycles (from −0.2 to 0.8 V at 0.1 V/s rate) caused an abrupt increase in the frequency, reaching the original level, indicating a complete disassembly

of the multilayer. We observed that the multilayer built on xP2VP disassembled more rapidly compared to PEM assembled on the xBCP, which also supports our previous findings on the increased stability of PEMs on nanopatterns. Figure 3f shows the extent of Dox released to the solution after different number of cycles of cyclic voltammetry sweeps from −0.2 to +0.8 V at 0.1 V/s rate. Saturation is reached already after 10 scans (200 s), suggesting that the release of all the stored Dox occurs already upon the electrochemically induced destabilization of the PEM, whereas complete disassembly is accomplished over a longer time (Figure 3e). The rate of release is comparable to that measured with other electroactive PEM systems reported in the literature,20 and considerably faster than with non-electroactive PEMs.4 The viability of NIH3T3 fibroblast cells cultured on nanopatterned multilayers was probed (Figure 4). The cells were cultured for 2 days on the PEM, the multilayer was then subjected to electrochemical treatment, and live/dead assay was performed after 6 additional hours of culturing. Figure 4a,b shows that the cells assembled on the nanopatterned PEI/ PEDOTS multilayer that did not contain Dox thrive even when voltage scans were applied to the PEM. This indicates that the electrochemical treatment itself does not harm the cells adsorbed on the PEM. The cells adsorbed on a nanopatterned multilayer that contained Dox also thrived as long as no voltage was applied (Figure 4c), in accordance with our previous findings on the ability of the nanopatterned E

DOI: 10.1021/acsami.8b15308 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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led to the formation of lying P2VP cylinders in the PS matrix, which gave rise to surface patterns of alternating stripes of ca. 36 nm width. Films were cross-linked with DIB for 42 h at 75 °C. These conditions led to quaternization of ca. 22% of all pyridine rings (in the volume sampled by an X-ray photoelectron spectroscopy beam) and degree of cross-linking of ∼16%.19 Fresh films were dipped for 10 min in PEDOTS (10 mM repeat unit concentration, prepared in ultrapure water, 0.055 mSiemens/cm conductivity), rinsed in ultrapure water, and dried by spinning at 2000 rpm for 30 s followed by nitrogen blowing. Preparation of PEMs on Gold Electrodes. xP2VP and xBCP films were alternatingly immersed in 1 wt % aqueous solutions of PEDOTS and PEI. Each immersion cycle was performed for 15 min and followed by rinsing with deionized water and then introduction of Dox (by spin coating a 1 mg/mL solution at 2000 rpm for 30 s). The deposition of the last layer (PEDOTS) was followed by drying under nitrogen flow. The fabricated electrodes were stored under 4 °C. Electrochemical Setup and Disassembly Conditions. The electrochemical cell consisted of an Ag/AgCl reference electrode and a Pt counter electrode and was connected to a potentiostat station (Autolab, Metrohm). The gold electrode coated with Dox-containing 3.5-PEDOTS/PEI bilayer was immersed into PBS (10 mM, pH 7.5) and connected as the working electrode. Cyclic potential sweeps from −0.2 to +0.8 V were applied at a scan rate 0.1 V/s. QCM Study of Multilayer Buildup and Disassembly. Goldcoated QCM sensors were cleaned by RCA-1 procedure (NH4OH/ H2O2/H2O = 1:1:4) at 80 °C for 15 min, rinsed with deionized water, and dried under nitrogen flow before use. The cleaned QCM sensors were coated with either P2VP or BCP films, annealed, reacted with DIB, and then coated with the first PEDOTS layer. A coated sensor was mounted into an electrochemical cell and connected as the working electrode; a leakless miniature Ag/AgCl electrode was used as the reference electrode. Polyelectrolyte solutions and deionized water were introduced at a constant flow rate of 50 μL/min. The experiments were started by running deionized water on the chip, and each solution was introduced after the resonance frequency stabilized. For the disassembly process, the solvent was first changed to PBS (10 mM, pH 7.5) until a stable frequency measurement was obtained and then 10 cyclic potential sweeps were applied. The QCM-D signal of the disassembly process was acquired during continuous buffer flow. Quantification of the Amount of Stored Doxorubicin. Fluorescence images of Dox inside the multilayers were taken using an Olympus BX53 microscope at 488 nm excitation. The amount of Dox stored in the 3.5-bilayer films was quantified by releasing the Dox into 1 mL PBS using either 30 min sonication or electrochemical scans (see above). The amount of Dox released was quantified using absorption spectroscopy (λmax = 481 nm; ε481 = 10 410 M−1 cm−1).30 Data represent averages of 3 repetitions. Quantification of the Amount of Doxorubicin Released upon Electrochemical Treatment. Electrodes coated with 3.5bilayer films were immersed into 1 mL PBS in a quartz cuvette together with a Ag/AgCl reference electrode and a Pt counter electrode. The amount of Dox released after 1, 5, 10, and 20 cycles was quantified in situ by UV−vis spectroscopy immediately after the number of cycles was applied. Leakage Test. Electrodes coated with 3.5-bilayer films were immersed into 1 mL PBS for 0, 6, 12, 24, 48, and 72 h. The samples were then removed from the solution, sonicated in PBS as described above, and the concentrations of Dox in both types of solutions were analyzed by absorption spectroscopy. Data represent averages of 3 repetitions. Cell Culturing. NIH3T3 mice fibroblast cells were cultured for 2 days on the coated electrodes in the Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C under 5% CO2 atmosphere. The samples were subjected to experiments when cell coverage reached 85−90% confluence. Cell Viability on Coated Electrodes. Cell viability before and after multilayer disassembly was performed using live/dead cell double staining kit (purchased from Merck/Sigma-Aldrich), which

multilayer to retain the stored Dox (Figure 3c,d). For comparison, cells adsorbed on the Dox-containing PEM assembled on xP2VP did not survive (Figure S3), owing to Dox leakage from such multilayers (Figure 3c,d). Applying a single voltage scan resulted in ∼5% cell death (Figure 4d); additional scans annihilated the entire population (Figure 4e,f). These experiments demonstrated that cell death occurred only because of the triggered release of Dox. It is noted that the effect of the released drug on the cells is rather quick; much longer time was needed for the drug to penetrate the cell membrane when the cells were incubated with a similar concentration of drug in the solution (Figure S4). This could be attributed to the presence of high local concentrations of the released Dox in the vicinity of the cells, but it may also relate to a certain change in membrane permeability induced by the applied voltage.

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CONCLUSIONS In this work, we have presented a new platform based on a nanopatterned polyelectrolyte multilayer that enables triggered drug delivery to adsorbed cells. The nanopatterned multilayer is furnished by a hierarchical construction approach, where a microphase-separated block copolymer film serves as a template for the selective deposition of the functional components. The multilayer consists of an electroactive polyelectrolyte, which inverts its charge upon the application of voltage and thus leads to multilayer disassembly and release of an embedded drug. One of the most interesting and nontrivial attributes of the nanopatterned multilayer is its ability to retain the drug better than the corresponding homogeneous (i.e., nonpatterned) multilayer. This ability is explained by the different average conformation of polyelectrolytes when adsorbed on nanopatterned substrates, which may assist in encapsulating the drug in the multilayer, and by the isolation of nanopatterned PEM domains, which reduces leakage from defect points caused by dust particles. The other important attribute of our system is the relatively high efficacy of drug delivery to cells adsorbed on the surface compared to the delivery efficacy of similar concentration of drug to cells suspended in a solution. Two reasons may account for this behavior: a high local concentration of the drug, which is released in close vicinity to the cells, and a possible enhancement in cell permeability caused by the application of voltage. Overall, we have developed a delivery platform that efficiently encapsulates high loading of biologically active ingredients and controllably releases them upon the application of an external electrical stimulation. Utilizations of this platform technology for cell reprogramming, therapeutic implants, and tissue engineering are currently underway.

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EXPERIMENTAL SECTION

Preparation of Block Copolymer Templates. Silicon wafers coated with 5 nm titanium adhesion layer and 45 nm gold were precleaned in sulfuric acid-NoChromix (purchased from SigmaAldrich) bath overnight and then rinsed with triply distilled water. Thin films of PS, P2VP, and PS-b-P2VP were prepared by spin-casting from chloroform solutions on each substrate at 3000 rpm for 30 s. The polymer solution concentrations were adjusted to yield film thicknesses in the range of 29−30 nm (determined by ellipsometry before annealing). The block copolymer films were solvent annealed in a closed Petri dish under saturated chloroform vapor for 25 min under ambient conditions. Microphase separation in the BCP films F

DOI: 10.1021/acsami.8b15308 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces simultaneously stains viable and dead cells with green and red fluorescence tags, respectively. The stain solutions were added at 37 °C to the cell-covered electrode for 15 min, and images were then taken using a fluorescence microscope (Olympus BX53) with an excitation wavelength at 488 and 545 nm to differentiate the viable/ dead cells. The data represent averages of 3 repetitions. Triggered Drug Release and Viability Assay. Doxorubicin was released from the 3.5-bilayer-coated gold electrode covered with cultured cells using the same conditions described above. After the electrochemical treatment, 2 mL of DMEM was added into the chamber and culturing was continued for additional 6 h at 37 °C under CO2 atmosphere. The electrodes were then rinsed with PBS three times and stained with live/dead assay kit to determine the amount of viable and dead cells. The data represent averages of 3 repetitions.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b15308.

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Experimental details; fluorescence images of Doxcontaining multilayers (Figure S1), SEM and SFM characterization of the block copolymer film (Figure S2), live/dead assays on PEM assembled on the xP2VP substrate (Figure S3), and control experiment results of cell incubation with Dox (Figure S4) (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (M.R.). *E-mail: [email protected] (H.-H.Y.). *E-mail: [email protected] (R.S.). ORCID

Mo-Yuan Shen: 0000-0002-5220-6225 Meital Reches: 0000-0001-5652-9868 Roy Shenhar: 0000-0002-0631-1542 Author Contributions ∥

M.-Y.S., S.Y., and Y.A. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by The Hebrew UniversityAcademia Sinica collaboration grant on nanoscience and Technology. The research activity in Academia Sinica was also supported by the Ministry of Science and Technology (MOST) of Taiwan (MOST-106-2628-M-001-001-MY3 and MOST-106-2627-M-001-011).

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DOI: 10.1021/acsami.8b15308 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX