Electrolyte solution-assisted electrospray deposition for direct coating and patterning of polymeric nanoparticles on non-conductive surfaces

Chemical Engineering Journal 379 (2020) 122318

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Electrolyte solution-assisted electrospray deposition for direct coating and patterning of polymeric nanoparticles on non-conductive surfaces Seong Jin Lee1, Sang Min Park1,2, Seon Jin Han, Dong Sung Kim

T

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Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Pohang, Gyeongbuk 37673, South Korea

H I GH L IG H T S

G R A P H I C A L A B S T R A C T

of electrolyte solution in • Utilization electrospray deposition. deposition of electrosprayed • Direct nanoparticles on non-conductive surfaces.

enhancement in the deposi• Dramatic tion efficiency of the electrosprayed particles.

of nanoparticles on • Patterning micro-scale region on 2D and 3D

a

curved surfaces.

surface functionalization to • Selective guide cell patterning and alignment.

A R T I C LE I N FO

A B S T R A C T

Keywords: Electrospray deposition Nanoparticle-coating Micro-patterning Electrolyte solution Non-conductive surface

Though electrospray deposition has significant impacts on various fields of energy, display, sensor, and biomedical engineering due to its strength in achieving functional coating or patterning of nano/microparticles on a substrate, most of the electrospray deposition techniques have been applicable only to electroconductive substrates, which limits the potential applications of the electrospray deposition. Here, we report a novel electrolyte solution-assisted electrospray deposition (ELED) process, which facilitates direct deposition of nano/microparticles on various substrates including a non-conductive surface. Beyond the direct surface coating, the ELED enabled to pattern nanoparticles on a selected micro-scale region on 2D flat and even 3D curved surfaces by simply modulating the surface wettability followed by selectively positioning electrolyte solution. Strikingly, the ELED showed the dramatic enhancement in the deposition efficiency of the electrosprayed particles on the nonconductive surface compared to conventional electrospray deposition process with a metal collector. The individual controllability of both the diameter of nanoparticles and the thickness of the deposited nanoparticle layer was achieved by adjusting the polymer concentration, the flow rate, the applied voltage, and electrospraying time. Lastly, we demonstrated one example of biomedical engineering applications of the ELED by patterning the NIH/3T3 fibroblasts and promoting alignment of C2C12 myoblasts on line patterns of cell-friendly polycarprolactone nanoparticles on a cell-repellent polydimethylsiloxane substrate.

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Corresponding author. E-mail address: [email protected] (D.S. Kim). 1 These authors contributed equally to this work. 2 Present address: School of Mechanical Engineering, Pusan National University, 2, Busandaehak-ro 63bon-gil, Geumjeong-gu, Busan 46241, South Korea. https://doi.org/10.1016/j.cej.2019.122318 Received 29 April 2019; Received in revised form 18 July 2019; Accepted 22 July 2019 Available online 23 July 2019 1385-8947/ © 2019 Published by Elsevier B.V.

Chemical Engineering Journal 379 (2020) 122318

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conductivity was able to generate a concentrated electric field toward it, thereby selectively depositing electrospun nanofibers on the substrate, which is crucial for coating and patterning of nano/microparticles through electrospray deposition. Here, we report, for the first time, the utilization of an electrolyte-solution collector in the electrospray deposition process to achieve surface coating or patterning of nano/microparticles directly on a non-conductive surface and we have named this novel electrospray deposition process ‘electrolyte solutionassisted electrospray deposition’ (ELED). With changing the wettability of a non-conductive surface by oxygen plasma treatment without or with a mask, the electrolyte solution could cover the entire or selective region of the substrate, respectively. Subsequent electrospray deposition allowed electrosprayed particles to be deposited on the surface of electrolyte solution, thereby forming nano/microparticle coating or pattering on the substrate. With the experimental validation and numerical simulation, we revealed the role of electrolyte-solution collector, which attracted the electrosprayed particles toward it positioned on the non-conductive surface, thereby enabling the direct coating or patterning of electrosprayed particles on the non-conductive surface. Furthermore, it was also found that the electrolyte-solution collector dramatically improved the coating efficiency of the electrospray deposition. The diameter of the electrosprayed nanoparticles and the thickness of the deposited electrosprayed nanoparticle layer were found to be individually controlled by changing the processing parameters of the ELED including polymer concentration, flow rate, applied voltage, and electrospraying time. Furthermore, we investigated the influence of the space between adjacent line patterns of the electrolyte solution on the patterning performance of electrosprayed nano/microparticles. The feasibility of the newly developed ELED technique was demonstrated by functionalizing the surface of a cell-repellent polydimethylsiloxane (PDMS) substrate with line patterns of cell-friendly polycarprolactone (PCL) electrosprayed nanoparticles, which allowed the selective cell patterning of NIH/3T3 fibroblasts and the enhanced cell alignment of C2C12 myoblasts on the PDMS substrate.

1. Introduction Electrospray has been of great significance in various fields of energy, display, sensor, and biomedical engineering due to not only its ability to produce nano/microparticles with various materials but also its controllability in the size and shape of the nano/microparticles [1–4]. Particularly, electrospray deposition, which is a coating technique based on the electrospray process, has been considered as one of the effective methods to achieve coating or patterning of nano/microparticles. Along with the merits of conventional macro-scale coating techniques that have been ubiquitously used in numerous applications from daily life to industry, the electrospray deposition has offered additional advantages that stem from the distinct nano/micro-scale properties. For example, electrospray deposition of fluoropolymer and silicon rubber nanoparticles enabled to fabricate superhydrophobic surfaces used for oil-water separation and anti-icing coating [5]. Furthermore, the coating of biocompatible polymeric or protein nanoparticles created a biologically active surface for cell culture [6,7]. Likewise, electrospray deposition has been used in wide applications, such as scaffolds for tissue regeneration, actuators, biosensors, gas sensors, and light-emitting devices, by producing functional surfaces or patterns [8–12]. Electrospray deposition techniques in coating and patterning nano/ microparticles have actively advanced until recently. The basic configuration of electrospray deposition generally consisted of a metal capillary-connected syringe, a syringe pump, a voltage supplier, and a metal collector, which produced the coating of electrosprayed particles over the whole surface area of the metal collector, while it failed to control the deposition of electrosprayed particles in a locally patterned form. To achieve the patterning of nano/microparticles, additional components, such as a mask or an additional power supply, should be employed in the basic electrospray deposition configuration. Early studies have adopted a mask to create the pattern of nano/microparticles using electrospray deposition. The mask entirely blocked the deposition of electrosprayed particles on the masking region on a substrate while allowing to generate a nano/microparticle pattern on the blanking region on the same substrate. However, during the process, many of highly charged electrosprayed particles were unnecessarily deposited on the top surface of the mask, and the previously accumulated, highly charged particles on the mask induced electrostatic repulsive forces with the upcoming electrosprayed particles, making it difficult to precisely control the shape of the electrosprayed particle pattern [10,13]. To fabricate the nano/microparticle pattern more effectively, researchers have tried to manipulate the electric fields between the metal capillary and the collector such as by utilizing a patterned electrode as a grounded electrode [14], applying a voltage directly to an electroconductive mask [15], and using an ion-induced electrostatic lens [16]. These approaches were mostly based on inducing a concentrated electric field on an electroconductive substrate to exert electrostatic forces on the electrosprayed particles, thereby selectively depositing the particles on the desired region of the substrate. Although there have been many advances in the electrospray deposition techniques for coating or patterning of nano/microparticles, the substrates have been majorly limited to electroconductive templates, which hampered applications of electrospray deposition. Given that nonconductive substrates are ubiquitous and have been widely used in various fields including biomedical engineering and energy harvesting, it is highly desirable to develop a technique for coating or patterning of electrosprayed particles directly on the non-conductive surfaces [17,18]. In our previous work, we introduced an electrolyte-solution collector as an alternative to a metal collector in the electrospinning process to pattern an electrospun nanofiber mat directly on a nonconductive surface [19,20]. In this electrospinning process, the fluidic nature of the electrolyte solution enabled facile positioning and removal of a ‘temporary’ collector, and furthermore, its electrical

2. Experimental section 2.1. Preparation of PCL solutions and electrolyte solution PCL with average molecular weights (Mn) of 45,000, chloroform, methanol, tetrabutylammonium bromide (TBAB) and potassium chloride (KCl) were purchased from Sigma-Aldrich (USA) and used as provided. Three different solutions with 0.25, 0.5, and 0.75 wt% PCL were prepared by dissolving PCL pellets in a mixed solvent of chloroform and methanol (3:1 by volume). Furthermore, 0.1 wt% TBAB was added to the PCL solutions for the increase in electrical conductivity, which is known to reduce the diameter of the electrosprayed particles down to the nano-scale [21]. For complete dissolution of the polymer solution, magnetic stirring was conducted for 12 h at room temperature. As an electrolyte-solution collector, 0.1 M KCl solution was prepared and utilized for electrospray deposition.

2.2. Substrate preparation A flat PDMS substrate was prepared by mixing PDMS monomer and curing agent (Sylgard 184 Silicone Elastomer Kit, Dow Corning, USA) at a ratio of 10:1 by weight and subsequently baking it in a convection oven at 65 °C for at least 4 h. A 3D curved poly (methyl methacrylate) (PMMA, Acryl Choika, Korea) substrate was prepared by a laser cutter (IS350, INNOSTA, Korea). To evaluate the wettability of the prepared substrates, a contact angle (CA) of a sessile water droplet (5 μl) on the substrate was measured by an automatic CA measurement system (SmartDrop, Femtobiomed, Korea).

2

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of the substrate and the grounded metal electrode were designed to be 1.5 and 3.0 cm, respectively, with the same thickness of 0.2 cm. The same experimental conditions including the applied voltage (28 kV) and the metal-capillary-to-collector-system distance (20 cm) were utilized in the numerical simulations.

2.3. Electrolyte solution-assisted electrospray deposition (ELED) Two different types of masks of a patterned dicing tape and a PDMS mask, both of which were used as a mask for oxygen plasma treatment, were utilized depending on the space between adjacent line patterns. A dicing tape (DS Semicon, Korea) was patterned by the laser cutter with various shapes including line and gear-train patterns, the letter M, and the university emblem. A PDMS line-patterned mask was fabricated by photolithography and subsequent soft-lithography based on the protocol in previous literature [22]. Both the width of the line pattern and the space between the adjacent line patterns had the same dimension of 20, 60, 100, 200, and 500 μm. The line patterns of 20, 60, 100, and 200 μm were produced with PDMS line-patterned masks, whereas the pattern of 500 μm was made with a patterned dicing tape. The patterned dicing tape or a PDMS line-patterned mask was attached to the prepared flat PDMS substrate, and subsequently, the wettability of the blanking region was rendered to be more hydrophilic by oxygen plasma generated by a plasma system (CUTE, Femto Science, Korea) at a constant power (50 W) for 30 s. The 3D curved PMMA substrate for the ELED was prepared under the same processing condition as the flat PDMS substrate. By virtue of the fluidic nature of electrolyte-solution collector, the 0.1 M KCl solution was then selectively deposited on the hydrophilically rendered surface region on the PDMS substrate, which was to be used as a grounded collector in the following electrospray process. For the case of entire coating of electrosprayed particles on the PDMS substrate, the oxygen plasma treatment was conducted on the whole PDMS substrate without a mask, which allowed the 0.1 M KCl solution to entirely cover the PDMS surface. The PCL solution was then loaded into a 5-ml gastight syringe (Hamilton, USA) and fed through a 23-gauge metal capillary placed 20 cm above the electrolyte-solution collector at a constant flow rate by a syringe pump (KDS200, KD Scientific, USA). A high voltage between the metal capillary and the electrolyte solution was applied to perform the electrospray deposition process. The flow rate and high voltage were set to 0.2 ml h−1 and 28 kV, respectively, unless specified. To investigate the influence of the flow rate and applied voltage on the diameter of electrosprayed particles, we conducted the ELED experiments by changing the flow rate from 0.2 to 1.0 ml h−1 and the applied voltage from 22 to 28 kV. The ELED process was conducted in a labmade glove box in which the humidity was maintained with a humidistat. The ELED was conducted at relative humidity of 50–60% and a temperature of 20–25 °C.

2.5. Characterization of electrosprayed PCL nanoparticles After sputter coating of Pt on the electrosprayed PCL nanoparticles, the morphology of PCL nanoparticles was examined using scanning electron microscopy (SEM, SU6600, Hitachi, Japan). The diameter of the PCL nanoparticles was evaluated by using ImageJ (NIH, USA) based on the SEM images. The PCL nanoparticle was regarded as a complete sphere, and at least, over 50 PCL nanoparticles in the SEM image were chosen to yield an average diameter of the electrosprayed PCL nanoparticles. To measure the thickness of the deposited PCL nanoparticle layer, the PCL nanoparticle-coated PDMS substrate was entirely immersed into uncured PDMS. After curing the PDMS for 12 h, the cured PDMS with the PCL nanoparticle-coated PDMS substrate was crosssectioned. Cross-sectional images were captured by an optical microscope (Nikon Eclipse80i), and the thickness of the deposited PCL nanoparticle layer was evaluated by using ImageJ from the cross-sectional images [20]. 2.6. Evaluation of the patterning performance of electrosprayed PCL nanoparticles To evaluate the patterning of electrosprayed PCL nanoparticles according to the space between adjacent line patterns of electrolyte solution, we produced line patterns with different widths and spaces of 20, 60, 100, and 200 µm by the ELED process and examined the electrosprayed patterns through the SEM images. To quantitatively evaluate the patterning performance of electrosprayed nanoparticles by the ELED, we converted the SEM images into the binary images, and then measured the area fraction of satellite electrosprayed particles on the space between the adjacent line patterns by using ImageJ. 2.7. NIH/3T3 fibroblast cell culture and live/dead assay The PDMS substrate with line patterns of PCL nanoparticles was prepared to demonstrate NIH/3T3 cell patterning based on the difference in cell affinities between PDMS and PCL. The line patterns of electrosprayed PCL nanoparticles on the PDMS substrate, which were patterned by the ELED, were sterilized with UV for at least 2 h prior to cell seeding. NIH/3T3 cells were then seeded on the entire PDMS substrate including both the line patterns of PCL nanoparticles and the pristine PDMS surface at a density of 10,000 and 50,000 cells cm−2. After that, the cells were cultured for 2 h in an incubator for adhesion on the substrate. Thereafter, the entire PDMS substrate was washed out with 1× phosphate-buffered saline (PBS) to remove unattached cells. NIH/3T3 cells were cultured in high glucose Dulbecco’s minimal essential medium (DMEM, Hyclone, USA) with 10% fetal bovine serum (Hyclone, USA) and 1% penicillin/streptomycin (Gibco, USA). After 3 days, NIH/3T3 cells were treated with calcein AM (green) and ethidium homodimer-1 (red) to distinguish live cells from dead cells. Fluorescent images were acquired using a phase contrast inverted fluorescence microscope (Nikon TS100F, Japan).

2.4. Numerical simulation The numerical simulation of the electric field between a metal capillary and various types of collector systems was conducted by COMSOL Multiphysics software (Version 5.0, USA) [23]. Four different types of the collector system models, consisted of (1) a non-conductive substrate (PDMS; εr = 2.8) on a grounded metal (Al) electrode which represents a conventional system, (2) grounded electrolyte solution (0.1 M KCl solution) covered on a non-conductive substrate (PDMS), (3) grounded electrolyte solution (0.1 M KCl solution) selectively positioned on a non-conductive substrate (PDMS), and (4) highly charged nanoparticles deposited on a non-conductive substrate (PDMS) on a grounded metal (Al) electrode, were applied to the simulations. To numerically simulate the electric field condition after a short period of electrospray deposition on the non-conductive surface, it was assumed that the highly positively charged electrosprayed nanoparticles were entirely deposited on the top surface of the non-conductive substrate, which was regarded as a thin layer with a high surface charge (the case of collector system model (4)). In this case, we chose 10−7 C as a value of the total surface charge after the short period of electrospray deposition time because an electric current for electrospray deposition process was measured at a few hundred nanoamperes [15]. The width

2.8. C2C12 myoblast cell culture and immunofluorescence staining C2C12 myoblasts were cultured on the prepared substrate with the line patterns of electrosprayed PCL nanoparticles following the same procedure for the patterning of NIH/3T3 fibroblasts, except for a cell density of 50,000 cells cm−2. To examine the morphology of the cultured C2C12 cells, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.3% Triton X-100 3

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Fig. 1. Electrolyte solution-assisted electrospray deposition (ELED). (a) Schematic of sequential experimental process including tape masking, plasma treatment and electrospray deposition of nano/microparticles on a non-conductive surface. (b) Characterization of contact angle differences between bare and plasma-treated PDMS substrates. (c) Patterning of electrolyte solution on the plasmatreated non-conductive PDMS substrate. (d) Selective deposition of electrosprayed nanoparticles in micro-scale. All scale bars are 3 mm.

electrosprayed particles and the grounded electrolyte solution. Touching the small region of the electrolyte solution with a hydrophilic clean tissue enabled us to readily remove the whole electrolyte solution from the deposition region without affecting the deposited nanoparticles. By removing the electrolyte solution, the pattern of electrosprayed particles was directly deposited on the surface of non-conductive substrate. In addition to the surface patterning, the surface coating of electrosprayed particles on the non-conductive substrate could be also achieved with the present ELED by simply eliminating the mask in the whole process. Without the mask, the entire surface of the substrate could be rendered more hydrophilic by oxygen plasma treatment, and the electrolyte solution would cover the whole surface of the substrate. The subsequent electrospray deposition attained the surface coating of electrosprayed particles on the substrate. In this study, PCL and PDMS were selected as a polymer nanoparticle material for electrospray deposition and a non-conductive substrate material, respectively, owing to their wide applications in biomedical fields. Given that the trace amount of remaining electrolyte solution might affect the cell behaviors, we have chosen the KCl solution as an electrolyte-solution collector in that KCl is one of the components in high glucose Dulbecco’s minimal essential media (DMEM) for cell culture. Thus, even if the trace amount of the electrolyte solution remained after the ELED process, it was expected to induce a slight change in the KCl concentration in the DMEM media which was negligible. From a different point of view, the electrolyte-solution collector could contain cell-favorable factors, such as growth factors or drugs, and thus, the remaining electrolyte solution might positively influence on the cell behaviors including cell growth. In our previous report, we revealed that when the concentration of the electrolyte solution was in the range from 10−8 to 3 M, the generated electric field toward the electrolyte solution collector was the same with the metal collector during the electrospinning process [23]. Given that the electrospraying process is fundamentally the same with the electrospinning process, we expected that the concentration of the KCl solution did not significantly influence on the performance of the ELED process. But, one of important observation was that the ELED with a higher concentration of the KCl solution was found to induce the crystallization of KCl in the pattern of the electrosprayed PCL particles, we decided to utilize the KCl solution with a low concentration of 0.1 M. Fig. 1b shows the

in PBS for 15 min at 4 °C followed by washing with 1× PBS. The cells were then blocked with a solution of 1% bovine serum albumin for 1 h at room temperature. And then, the cells were stained with tetramethylrhodamine (TRITC)-conjugated phalloidin (Sigma, USA) at a dilution of 1:200 in PBS for 1 h. After washing with PBS, the cells were stained with DAPI (Molecular probes, USA) at a dilution of 1:300 in DI water for 10 min at room temperature. After washing with DI water, the cells were finally observed by a phase contrast inverted fluorescence microscope (Nikon TS100F, Japan).

3. Results and discussion 3.1. Electrolyte solution-assisted electrospray deposition (ELED) Fig. 1a shows the sequential process of the ELED for the direct coating and patterning of electrosprayed particles on a non-conductive substrate. The first step was positioning a mask on the substrate and performing the oxygen plasma treatment. Though the dicing tape was utilized as a mask in this study, other materials could be used as a mask as long as they have sufficient adhesion with the substrate and masking performance for oxygen plasma. The major role of the mask was preventing the tape-attached (masked) region of the substrate from being exposed to oxygen plasma, thereby maintaining the pristine wettability of the substrate, while rendering the tape-unattached (blanked) region of the substrate to be more hydrophilic. Due to the wettability difference between the plasma-exposed and unexposed regions of the substrate, the electrolyte solution could be selectively positioned only on the more hydrophilic region and eventually patterned with the same shape of the blanking region in the mask. In this stage, the surface treatment techniques other than oxygen plasma treatment capable of selectively modifying the wettability of the surface, such as microcontact printing, lithography, and UV exposure, can be also applied. The patterned electrolyte solution was then connected to the ground of the high voltage supplier to play a role of the grounded collector. The application of a high voltage between the metal capillary and the electrolyte solution made the electrospraying polymer solution ejected into nano/microparticles. The electrosprayed particles were mostly deposited on the patterned electrolyte solution due to the electrostatic attractive forces between the positive surface charge of the 4

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Fig. 2. Characterization of the electrosprayed particles. (a) Magnified SEM images of electrosprayed particles with different concentrations of PCL solution. (b) Relationship between the diameter of the electrosprayed particles and concentration of polymer solution. (c) Processing window of the ELED for (1) production of only nano-sized particles, (2) production of nano/micro-sized particles, and (3) unstable electrospraying. (d) Diameter of electrosprayed particle with respect to flow rate and applied voltage. (e) Relationship between the thickness of the deposited particle layer and process time of electrospray deposition. All scale bars are 5 µm.

electrosprayed droplets generated through the metal capillary during the electrospray process, which in turn resulted in the decreased diameter of final PCL nanoparticles after the evaporation of the solvent in the charged droplets during their flight toward the grounded collector [24]. Other crucial parameters, which significantly affect the diameter of electrosprayed particles, were the flow rate and the applied voltage. We first determined a proper processing window for both flow rate and applied voltage to produce (1) nano-sized electrosprayed particles, (2) nano/micro-sized electrosprayed particles, and (3) unstable electrospraying by the ELED process. In general, to produce only nano-sized particles, the flow rate should be lower than 0.6 ml h−1 while the applied voltage should be higher than 25 kV (Fig. 2c). As the flow rate increased and the applied voltage decreased, the diameter of electrosprayed particles became large, which made it difficult to obtain nanosized particles, and the distribution of their diameters become wider. When the applied voltage was down to critical point, the electrospraying process became unstable, producing unexpectedly large-sized particles due to the unstable Taylor cone with dripping or pulsating behaviors [25]. Thus, we examined the Taylor cone at the tip of the metal needle according to the changes of the flow rate and the applied voltage, and found a borderline for separating “stable electrospraying” and “unstable electrospraying” as shown in Fig. 2c. The previous research has well organized the relationship between the diameter of electrosprayed PCL particles and both parameters of the

wettability change of the PDMS surface before and after oxygen plasma treatment. The CA of the pristine PDMS was measured to be ~108°, while the oxygen plasma-treated PDMS had the CA of ~18°. Owing to the CA difference, the 0.1 M KCl electrolyte solution was selectively located on the oxygen plasma-treated region of 500-μm-width line patterns (Fig. 1c). While the PDMS and PMMA substrates were exclusively subjected to the ELED in this study, a wide range of nonconductive substrates would be applicable to this process, given that the patterning of electrolyte solution on various substrates through the selective oxygen plasma treatment has been extensively demonstrated in the previous works [19]. The subsequent electrospray deposition process produced line patterns of PCL nanoparticles on the PDMS substrate, which replicated the shape of the electrolyte solution pattern (Fig. 1d). The ELED allowed individual control of the diameter of the PCL nanoparticles and the thickness of the deposited PCL nanoparticle layer. The SEM images in Fig. 2a show the PCL nanoparticles on the PDMS substrate generated by the ELED with different PCL concentrations of the prepared PCL solutions. The lower the concentration of the PCL solution was, the smaller the PCL nanoparticles produced as shown in Fig. 2b. The diameter of the PCL nanoparticles was varied from a few to several hundreds of nanometers, and it was around 400 nm with the 0.25% PCL solution. It is likely that the lower concentration of PCL solution reduced the weight fraction of PCL in the charged 5

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flow rate and the applied voltage [26], though they only produced micro-sized particles by the conventional electrospraying process owing to the relatively high concentration of PCL solution. In this regard, we tried to fabricate nano-sized electrosprayed PCL particles by reducing the concentration of the PCL solution, increasing the applied voltage, and decreasing the flow rate in the ELED process. Even for the case of the fabrication of nano-sized electrosprayed particles, we could observe a similar relationship between the diameter of electrosprayed particles and both parameters through the SEM images (Fig. S1). We found that the lower the flow rate and the higher the applied voltage were, the smaller the diameter of the electrosprayed particles by the ELED became (Fig. 2d). The thickness of the deposited PCL nanoparticle layer could be also modulated by changing the electrospraying time. To examine the relationship between the thickness of the deposited PCL nanoparticle layer on the non-conductive substrate and the electrospraying time in the ELED process, three different electrospraying times of 30, 60 and 90 min were selected. As shown in Fig. 2e, the thickness of electrosprayed PCL nanoparticle layer was varied within a few micrometers and linearly proportional to the electrospraying time. It is generally known that, in the conventional electrospray deposition process, the surface charge of the deposited electrosprayed particles hampered the deposition of the next coming electrosprayed particles due to the high electrostatic repulsive forces [27], which retarded the deposition efficiency of the electrosprayed particles on the substrate as time passed. In the case of model 1, the number of deposited nanoparticles was too low to form a particle layer even at 90 min of electrospraying. In this regard, we could not measure the thickness of the deposited nanoparticle layer for model 1. Strikingly, the ELED did not show the recognizable decrease in the deposition efficiency of the electrosprayed particles on the substrate with the increase in the electrospraying time. This implies that the remaining charge of the previously deposited electrosprayed particles on/in the electrolyte-solution collector on the PDMS substrate did not greatly influence on the deposition of the next coming electrosprayed particles. It was conjectured that the high surface charge of the deposited electrosprayed particles would be relaxed through contact with the grounded electrolyte solution, and thus, the coming electrosprayed particles would be readily deposited on the top of the previously deposited electrosprayed particles without experiencing electrostatic repulsive forces.

PDMS substrate through the selective oxygen plasma treatment, a line pattern of electrosprayed PCL nanoparticles was achieved (Fig. 3b-(i)). The concentrated electric field toward the patterned 0.1 M KCl electrolyte-solution collector allowed the electrosprayed PCL nanoparticles to be selectively deposited on the electrolyte solution region, forming the line pattern of PCL nanoparticles. Interestingly, Fig. 3b-(ii, iii) exhibited that almost all of the electrosprayed nanoparticles were selectively deposited only on the electrolyte solution region, whereas none of the nanoparticles were observed on the pristine PDMS region without the electrolyte solution. We have numerically investigated the mechanism behind the coating and patterning of electrosprayed particles through the present ELED by comparing with the conventional electrospray deposition process that was performed with a non-conductive substrate placed on a metal collector. The deposition of electrosprayed particles on the collector was mainly governed by the electrostatic forces acting on the high positive charge of the electrosprayed particles under the electric field generated between the metal capillary and the collector system [14]. The direction of electrostatic force is coincide with that of the electric field, and thus, evaluating the electric field enabled us to indirectly estimate the deposition behavior of electrosprayed particles. Fig. 4a shows a general numerical simulation result of the electric field between the metal capillary and the collector system. In the simulations, four different types of the collector system models were evaluated: (1) the PDMS substrate placed on the grounded Al electrode (Fig. 4b-(i)), (2) the grounded 0.1 M KCl solution covered on the PDMS substrate (Fig. 4b-(ii)), (3) the grounded 0.1 M KCl solution selectively positioned on the PDMS substrate (Fig. 4b-(iii)) and (4) the electrosprayed particle layer with the highly positive charge deposited on the top surface of the PDMS substrate placed on the grounded Al electrode (Fig. 4b-(iv)). The model (1) in Fig. 4b-(i) represented the electrospray deposition for coating of electrosprayed particles on a non-conductive substrate on a metal ground in the absence of the electrolyte solution, which was corresponding to the collector system of the experiment in Fig. 3a-(i). In this case, the collector system was able to focus the electric field toward the PDMS surface because a higher dielectric constant of PDMS compared to the surrounding air generated a concentrated electric field toward the PDMS surface. For the case of the model (2), a strongly concentrated electric field was generated toward the 0.1 M KCl solution on the PDMS substrate (Fig. 4b-(ii)). Furthermore, the patterned 0.1 M KCl solution collectors, which were grounded, on the PDMS substrate were able to make a concentrated electric field towards the individual collector region as indicated in Fig. 4b-(iii). These results were demonstrated in our previous work of adopting the electrolyte solution as a grounded collector in electrospinning to produce patterned nanofiber mats on a non-conductive surface [20]. We found that the electrolyte-solution collector has an intriguing capability to focus the electric field toward it similar to a grounded metal collector. The electric field simulation results implied that the concentrated electric field toward the individual grounded collector facilitated the deposition of the electrosprayed PCL nanoparticles on the collector system. In spite of the numerical result of the electric field in the model (1) which simulated the collector system of the experiment in Fig. 3a(i), the experimental result exhibited the retarded deposition of electrosprayed PCL particles on the PDMS surface, which cannot be expected from the concentrated electric field in the numerical simulation. This retarded deposition of the electrosprayed particles on the PDMS surface could be explained by the electrostatic repulsive forces between the next coming and the previously deposited positively charged electrosprayed PCL particles. After performing a short period of electrospray deposition, highly positively charged electrosprayed particles were deposited on the PDMS surface and they prevented the coming electrosprayed particles from deposition. This implication could be numerically demonstrated by the model (4), which showed the electric field directing outward from the PDMS surface on which the positively

3.2. Comparison between ELED and conventional electrospray deposition in the coating and patterning of electrosprayed particles on a non-conductive substrate The role of the electrolyte-solution collector in the coating and patterning of the electrosprayed particles on the surface of a non-conductive substrate was demonstrated by electrospray deposition experiments on the PDMS substrate. The electrospray deposition of PCL nanoparticles was conducted for 15 min both on (1) the PDMS substrate placed on an Al plate collector and (2) the PDMS substrate on which the 0.1 M KCl electrolyte-solution collector was placed. While the electrospray deposition on the PDMS substrate with the grounded Al plate resulted in only a few PCL nanoparticles deposited on the PDMS substrate (Fig. 3a-(i)), when we placed the grounded 0.1 M KCl solution on the oxygen plasma-treated PDMS substrate, a lot of electrosprayed PCL nanoparticles were successfully deposited on the entire surface of PDMS substrate as shown in Fig. 3a-(ii). Though the electrospray deposition time was the same for both cases, the number of deposited PCL nanoparticles on the PDMS substrate showed a great difference, which suggested that the utilization of the electrolyte solution as a grounded collector could significantly promote the deposition efficiency of electrosprayed particles on the non-conductive substrate. Besides the surface coating of electrosprayed particles, the ELED also provided an effective way to pattern the electrosprayed particles on the surface of non-conductive substrate. By patterning the 0.1 M KCl solution on the 6

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Fig. 3. (a) SEM images of electrosprayed particles on the non-conductive surface without electrolyte solution (a-(i)) and with electrolyte solution (a-(ii)). Scale bars are 5 µm. (b) SEM images of a line pattern of electrosprayed particles (b-(i)) and magnified images (indicated in (b-(i)) of the nanoparticlenon-deposited (b-(ii)) and nanoparticle-deposited (b-(iii)) areas. Scale bars are 250 µm (b-(i)) and 5 µm (b-(ii and iii)), respectively.

shaped pattern and the university emblem-shaped pattern of PCL nanoparticles were achieved on the flat PDMS substrates (Fig. 5a and b). The ELED was further extended to pattern the electrosprayed PCL nanoparticles on a 3D curved non-conductive PMMA surface due to the flexible formation of electrolyte-solution collector. We successfully achieved a gear-train pattern of PCL nanoparticles on the 3D curved PMMA substrate (Fig. 5c and d). To investigate the patterning performance of electrosprayed nano/microparticles according to the space between the adjacent line patterns of the electrolyte solution, we produced line patterns of electrosprayed PCL nanoparticles with four different widths and spaces of 20, 60, 100, and 200 µm. As shown in Fig. 6a, the line patterns and spaces formed by electrosprayed PCL nanoparticles could be distinguishable even in the 20-µm-width line pattern. However, the magnified images of the space between the adjacent line patterns showed that the number of satellite electrosprayed particles on the space, which were the result of the unwanted particle deposition by the ELED, increased with the decreased space (Fig. 6b). As a measure for quantitative evaluation of the patterning performance of electrosprayed particles by the ELED, we calculated area fractions of the satellite electrosprayed particles on the space for the four different patterns from the binary images of the magnified SEM images in Fig. 6b. The calculated area fraction plotted in Fig. 6c indicated that the decrease in the space between the adjacent line patterns of the electrolyte solution resulted in the retarded patterning performance of

charged electrosprayed particles were deposited as shown in Fig. 4b(iv). Such electrostatic repulsive forces between the next coming and the previously deposited electrosprayed particles have been already reported by the previous work [27]. Thus, the collector system only with a non-conductive substrate and a metal ground would greatly reduce the coating efficiency of electrosprayed particles on the nonconductive surface. Moreover, the patterning of electrosprayed particles on the non-conductive surface was also expected to be limited due to the electrostatic repulsive forces between the electrosprayed particles in the same manner. In contrast to the conventional collector system shown in the model (1), the ELED showed the highly efficient deposition of electrosprayed PCL nanoparticles on the PDMS surface whose region was originally covered by the 0.1 M KCl solution collector. From this result, it was deduced that the electrosprayed particles previously deposited on the electrolyte solution would rarely influence on the next coming electrosprayed particles. We conjectured that this phenomenon was attributed the charge relaxation of the deposited electrosprayed particles on the electrolyte solution during the ELED process. Studying the detail mechanism of the highly efficient deposition of the electrosprayed particles on the electrolyte-solution collector based on the charge relaxation would be an intriguing future work. By virtue of the efficient deposition of the ELED, we could achieve various shaped patterns of PCL nanoparticles on various types of nonconductive substrates, i.e., PDMS and PMMA (Fig. 5). The letter M-

Fig. 4. (a) Numerical simulation of the electric field between the metal capillary and four different collector systems. (b) The magnified images of the numerical simulation of concentrated electric field around collector systems (dashed rectangle in (a)) of a non-conductive substrate on a metal ground (b-(i)), a non-conductive substrate with electrolyte solution (b-(ii)), a nonconductive substrate with patterned electrolyte solution (b-(iii)), and a non-conductive substrate with charged nanoparticles (b-(iv)).

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Fig. 5. Patterning of electrosprayed particles on various substrates with complex shapes of the letter “M” on the PDMS substrate (a), the emblem of POSTECH on the PDMS substrate (b), and gear-train pattern on the 3D-curved PMMA substrate with its magnified image (c and d). Scale bars are 1 mm (a), 10 mm (b, c), and 1 mm (d).

Fig. 7. Cell patterning on the functionalized PDMS substrate with line patterns of PCL nanoparticles. (a) Design of line patterns on a tape mask (white and black areas are blanking and masking region, respectively) and (b) fluorescent image of live NIH/3T3 fibroblasts (green) adhered along the line patterns of PCL nanoparticles. All scale bars are 500 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

electrosprayed PCL nanoparticles. Interestingly, when the space between adjacent line patterns was decreased from 60 to 20 µm, a shape increase in the area fraction of satellite electrosprayed particles was observed, and the satellite electrosprayed particles covered over 10% of the space between adjacent line patterns. Thus, we concluded that the resolution limitation of the ELED process was several tens of micrometers under the present processing condition of flow rate (0.2 ml h−1) and applied voltage (28 kV). Given that the resolution limitation of the ELED process would be affected by the processing conditions and types

Fig. 6. Characterization of the satellite electrosprayed PCL nanoparticles on the space between the line patterns. (a) Magnified SEM images of line patterns formed by the electrosprayed PCL nanoparticles for four different widths of 200 µm (a-(i)), 100 µm (a-(ii)), 60 µm (a-(iii)), and 20 µm (a-(iv)). (b) Binary images of the magnified SEM images of the line patterns clearly indicating the satellite electrosprayed PCL nanoparticles on the space for four different widths of 200 µm (b-(i)), 100 µm (b(ii)), 60 µm (b-(iii)), and 20 µm (b-(iv)) (black and white indicate substrate and satellite electrosprayed PCL nanoparticles, respectively). (c) Calculated area fraction of satellite electrosprayed PCL nanoparticles on the space with respect to the space between the adjacent line patterns. Scale bars are 50 µm (a) and 3 µm (b).

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and spaces of 60, 100, and 200 µm, and then cultured C2C12 cells on the samples to examine the pattern formation of C2C12 cells guided by the PCL nanoparticle line. From Fig. 8a, we could observe that the C2C12 cells were patterned and aligned along the direction of the line pattern of the electrosprayed PCL nanoparticles, and also the smaller width of the line pattern highly promoted the cell alignment. The C2C12 cell alignment was quantified by measuring the angles between the longitudinal direction of the line pattern and the cell elongation axis (Fig. 8b). Around 70% of the C2C12 cells on the 60-µm-width line pattern had angles between 0 and 15°, whereas the C2C12 cells on the 200-µm-width line pattern exhibited a relatively uniform distribution of alignment angles, indicating that the cells were randomly oriented on the PCL nanoparticle pattern (Fig. 8c). Considering that the alignment of C2C12 myoblasts has been known to enhance the myotube formation, a PDMS substrate with 60-µm-width line pattern of the electrosprayed PCL nanoparticles is expected to be an effective substrate to promote skeletal muscle formation. Furthermore, the ELED process enabled to control the diameter of the electrosprayed particles, which allowed to modulate the nanotopography on the substrate such as adherable area and roughness. Our previous work revealed that the decrease in the adherable area of the nanotopography led to the reduction of the focal adhesion formation and subsequently the increase in the migration of the NIH/3T3 cells [30]. Likewise, we considered that the modulation of the diameter of nanoparticles would result in the change in the adherable area of cells and thereby influence cell function and behaviors, such as cell migration. Thus, we believed that the investigation of the influence of the diameter of electrosprayed nanoparticles on cell behaviors would be an interesting future research. To confirm the stable adhesion between the electrosprayed PCL nanoparticles and the PDMS substrate, we examined the remaining electrosprayed PCL nanoparticles on the PDMS substrate after the sequential process of culturing the cells for 3 days. We detached the cells from the PCL nanoparticles by treating trypsin, which was a commonly utilized method to detach the cells from the substrate. The line patterns of electrosprayed PCL nanoparticles were found to be still distinguishable after the trypsin treatment, which confirmed that the patterned

of materials, we expect to achieve a higher resolution limitation of the ELED by fine-tuning of the processing parameters.

3.3. Cell patterning and alignment on PDMS substrate with line patterns of PCL nanoparticles The ELED holds great promises in various fields due to its ability to functionalize the non-conductive surface with the electrosprayed nanoparticles. As one example of such functionalization, selective cell patterning on the PDMS substrate, which was known as a cell-repellent substrate, based on the ELED was demonstrated. Cell micro-patterning is of great importance in the tissue formation or the understanding of cell behaviors. NIH/3T3 fibroblast cells were selectively patterned on the PDMS substrate on which electrosprayed, cell-friendly PCL nanoparticles were deposited by the ELED process. Fig. 7a shows the design of the line-pattern mask, which was replicated as the line patterns of electrosprayed PCL nanoparticles on the PDMS substrate by the ELED. NIH/3T3 fibroblasts were then seeded and cultured for 3 days on the PDMS substrate with the line patterns of PCL nanoparticles, which resulted in the line patterns of NIH/3T3 cells on the PDMS substrate. The fluorescence microscopy image after live/dead staining clearly exhibited that the 3-day cell culture was enough to form the line patterns of NIH/3T3 cells, which were shown in green, on the PCL nanoparticle regions (Fig. 7b). Most of NIH/3T3 cells were adhered and grown on the line patterns of the cell-friendly PCL nanoparticles, whereas only a few numbers of cells were observed on the pristine PDMS surface due to the cell-repellent property of the PDMS. As another example of such functionalization, the enhancement of cell alignment parallel to the line patterns of the electrosprayed PCL nanoparticles was investigated by changing the width of the line patterns. The previous work utilizing a micropatterned substrate demonstrated that the line pattern could enhance the alignment of C2C12 myoblasts, which was considered a critical step for myotube formation [28]. Another previous work reported that the width and space of line pattern could affect the orientation of C2C12 myoblasts [29]. To do so, we fabricated three different cell culture samples with the line patterns of the electrosprayed PCL nanoparticles by the ELED having the widths

Fig. 8. Cell alignment on the functionalized PDMS substrate with line patterns of PCL nanoparticles. (a) Immunofluorescent images of F-actin (red) and nuclei (blue) of C2C12 myoblasts on the different line patterns with different widths of 60 µm (a-(i)), 100 µm (a-(ii)), and 200 µm (a-(iii)). (b) Definition of C2C12 cell orientation angle. (c) Aspect ratio and orientation angle of C2C12 cells cultured on the different line patterns with different widths of 60 µm (c-(i)), 100 µm (c-(ii)), and 200 µm (c-(iii)). Scale bars are 50 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 9

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PCL nanoparticles stably attached to the PDMS substrate during the cell culture (Fig. S2). Unlike the previous methods to fabricate the cell culture platforms to form a micro-patterned cell organization, such as micro-contact printing, photolithography, and femtosecond laser processing [31], the present ELED was able to produce not only a cell-friendly micro-pattern but also generate a nano-scale topography as well as chemical functional groups on the micro-pattern through the electrosprayed nanoparticles. Thus, the cell culture platform generated by the ELED can be expected to be utilized to study complex cell-surface interaction including cell-micro-pattern and nanotopography interactions. In addition to the biomedical application, we believed that the ELED process could be further applied to various research fields. For example, in the triboelectric nanogenerator (TENG) research fields, the nonconductive dielectric substrates are generally utilized as a contact layer, which is one of the most important components to generate the triboelectric energy. Recently, Huang et al. demonstrated that micro/nanostructuring on the contact layer, which was a non-conductive PDMS substrate, could enhance the performance of the TENG [32]. Given that the ELED process directly fabricated nanotopography on a non-conductive substrate, the ELED process could be utilized to produce micro/ nano-structures on the contact layer to boost the electrical output of TENG. Furthermore, the ELED process is expected to be applied to producing a superhydrophobic surface on a non-conductive substrate by developing surface roughness. One of the previous works achieved a superhydrophobic surface on a conductive metal substrate through the conventional electrospraying process [5]. Given that the superhydrophobicity has many advantages including anti-fouling or antiicing, the ELED process would be widely applied to achieve a waterproof plastic case or an anti-icing surface of cables or refrigerators.

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4. Conclusions In this study, we have developed the ELED process, which replaced the metal collector into the electrolyte-solution collector, to overcome the limitation of the conventional electrospray deposition, thereby widening the available substrate materials from conductive to nonconductive. Due to the fluidic nature, the electrolyte-solution collector was readily positioned on the surface of a non-conductive substrate for electrospray deposition and eliminated after the electrospray process, resulting in only the electrosprayed particles remaining on the nonconductive surface. Furthermore, the electrolyte-solution collector generated a concentrated electric field toward it on the non-conductive surface, which enabled the selective patterning of electrosprayed nanoparticles on the non-conductive surface. The experimental and numerical simulation results showed that the ELED dramatically increased the productivity of coating and patterning of electrosprayed particles on the non-conductive surface, which might be attributed to the charge relaxation of the deposited electrosprayed particles through the electrolyte-solution collector. Lastly, we demonstrated the selective functionalization of the non-conductive surface based on the ELED by creating the patterns of NIH/3T3 cells and the alignment of C2C12 cells only on the PCL nanoparticles-patterned PDMS substrate. The ELED has overcome the limitation and expanded the versatility of the existing electrospray deposition, and thus, the ELED is expected to greatly broaden the spectrum of the applications of electrospray deposition. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (Nos. 2017R1A2A1A05001090 and 2017M3A9C6032067). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// 10

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