Poly(3,4-ethylenedioxythiophene)-Based Nanofiber Mats as an

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Poly(3,4-ethylenedioxythiophene)-Based Nanofiber Mats as an Organic Bioelectronic Platform for Programming Multiple Capture/Release Cycles of Circulating Tumor Cells Chia-Cheng Yu, Bo-Cheng Ho, Ruey-Shin Juang, Yu-Sheng Hsiao, R Venkata Ram Naidu, Chiung Wen Kuo, Yun-Wen You, Jing-Jong Shyue, Ji-Tseng Fang, and Peilin Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07042 • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 22, 2017

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ACS Applied Materials & Interfaces

Poly(3,4-ethylenedioxythiophene)-Based Nanofiber Mats as an Organic Bioelectronic Platform for Programming Multiple Capture/Release Cycles of Circulating Tumor Cells Chia-Cheng Yua,+, Bo-Cheng Hoa,+, Ruey-Shin Juangb,c,+, Yu-Sheng Hsiaoa,*, R Venkata Ram Naidud, ChiungWen Kuod, Yun-Wen Youd, Jing-Jong Shyued, Ji-Tseng Fangc,e,* and Peilin Chend,*

a

Department of Materials Engineering, Ming Chi University of Technology, Taishan, New Taipei City 24301, Taiwan. E-mail: [email protected]; Fax: +886-2908-4091 b

Department of Chemical and Materials Engineering, Chang Gung University, Guishan, Taoyuan 33302, Taiwan. c

Division of Nephrology, Department of Internal Medicine, Chang Gung Memorial Hospital, Linkou, Taiwan. E-mail: [email protected]; Fax: +886-3328-2173

d

Research Center for Applied Sciences, Academia Sinica, Taipei 11529, Taiwan. E-mail: [email protected]; Fax: +886-2782-6680 e

College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan.

+

These authors contributed equally to this work.

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Abstract In this investigation, we employed a novel one-step electrospinning process to fabricate poly(ethylene oxide) (PEO)/poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) core/shell nanofiber structures with improved water resistance and good electrochemical properties, and characterized them using scanning electron microscopy, transmission electron microscopy, and time-of-flight secondary ion mass spectrometry imaging. We then integrated a biotinylated poly-(L-lysine–graft–ethylene glycol) (PLL-g-PEG-biotin) coating with three-dimensional (3D) PEDOT-based nanofiber devices for dynamic control over the capture/release performance of rare circulating tumor cells (CTCs) on-chip. The detailed capture/release behavior of the circulating tumor cells was studied using an organic bioelectronic platform comprising PEO/PEDOT:PSS nanofiber mats with 3 wt% of (3-glycidyloxypropyl)trimethoxysilane (GOPS) as an additive. We have demonstrated that these nanofiber mats deposited on five-patterned indium tin oxide (ITO) finger electrodes are excellent candidates for use as functional bioelectronic interfaces for the isolation, detection, sequential collection, and enrichment of rare CTCs through electrical activation of each single electrode. This combination behaved as an ideal model system displaying a high cell-capture yield for antibody-positive cells while resisting the adhesion of antibody-negative cells. Taking advantage of the electrochemical doping/de-doping characteristics of PEDOT:PSS materials, the captured rare cells could be electrically triggered release through the desorption phenomena of PLL-g-PEG-biotin on device surface. More than 90% of the targeted cancer cells were captured on the 3D PEDOT-based nanofiber microfluidic device; over 87% of captured cancer cells were subsequently released for collection; approximately 80% of spiked cancer cells could be collected in a 96-well plate. Therefore, this 3D PEDOT-based nanofiber approach appears to be an economical route for the largescale preparation of systems for enhancing the downstream characterization of rare CTCs. Keywords: Nanofiber; Poly(3,4-ethylenedioxythiopine):Polystyrenesulfonate (PEDOT:PSS); Bioelectronic Interfaces (BEIs); Circulating Tumor Cells (CTCs)

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Introduction Organic bioelectronics have attracted considerable interest in recent years as alternatives to conventional inorganic materials, with several distinct applications; for example, in therapeutics,1–4 monitoring biological activities from electrical signals,5–7 and providing electrically activated responses for cell release in closed-loop feedback control models.8–10 To understand the conditions related to cancer, these systems have been subjected to considerable amounts of materials science to improve the isolation and detection of circulating tumor cells (CTCs). An extremely harmful characteristic of cancer tumors is their affinity to shed cells into blood circulation and later disseminate the cancer by growing metastatic tumors in other parts of the body. Procedures for the isolation and careful examination of cancer cells through liquid biopsies have major advantages over solid tumor biopsies because they can rapidly provide thorough information for the diagnosis and therapeutic treatment of cancer patients.11, 12 There are enormous opportunities to quickly monitor the survival rates and disease conditions of cancer patients depending on the quantity of CTCs in their blood and/or the number of genetic mutations present in the cells.13 Therefore, several novel methods have been constructed for the effective capture and isolation of CTCs from the bloodstream, including physical methods (e.g., microfilters, microposts, centrifugation, dielectrophoresis) based on size, deformability, and electric charge and biological methods based on immunomagnetic separation and microfluidics-enabled immunoseparation.14–16 At present there is only one liquid biopsy test approved by the FDA, which is the CellSearch system used for detecting CTCs in cancer patients. Nevertheless, the CellSearch system has a major limitation in that it is capable of detecting subpopulation of CTCs of interest (especially in lung cancer patients) only because of the miscellaneous behavior of CTCs in the epithelial to mesenchymal transition—that is, it is applicable only to cancers of epithelial origin. Another limitation is that the cell viability is poor after capturing the CTCs through the CellSearch system surface-modified with anti-EpCAM antibodies. These limitations affect the purity and cell capture yield of CTCs and hinder subsequent morphological analyses (e.g., high-resolution fluorescence



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imaging; molecular characterization of circulating tumor cells) that would improve the quality of clinical information given to cancer patients as a form of personalized medicine. In recent years, much effort has been devoted to the development of novel NanoVelcro substrates for capturing CTCs and enhancing their real-time imaging and molecular analysis.18,

19

In 2013, Tseng and

colleagues demonstrated a thermoresponsive NanoVelcro device for the capture and release of CTCs by allowing the system to vary the temperature of the blood.20, 21 Novel organic bioelectronic interfaces (BEIs) integrated with biological systems have several potential applications because they allow low-temperature processing and the ability to alter surface structures (chemical, optical, mechanical, and electrical properties) to facilitate doping/de-doping processes.4,

8, 22–24

Several conducting polymer materials (e.g., polypyrrole;

polyaniline; polythiophene) and their derivatives have been used as organic BEIs, taking advantage of their intrinsic biocompatibility, superior electrochemical and electrical transport properties, and extraordinary manufacturing flexibility.25–30 In earlier studies from our laboratory, we demonstrated that 3D BEIs (e.g., carboxyl-grafted PEDOT nanostructures) can be used as NanoVelcro cell-affinity devices that increase the CTC capture efficiency.8,

31, 32

In this present study, we fabricated 3D PEO/PEDOT:PSS nanofiber mats having

highly water-resistant nanostructures on ITO glass substrates and used them as organic bioelectronic interfaces (OBEIs) for dynamic control over CTC capture and release; we also tested them as electrode mats for multiple rounds of CTC purification. These PEO/PEDOT:PSS nanofiber mats were fabricated through electrospinning and then the devices were subjected to surface functionalization with PLL-g-PEG-biotin, streptavidin, and antiEpCAM-biotin. Cell-capture measurements revealed a cell-capture yield of MCF7 cells of approximately 1900 cells mm–2; electrically triggered release experiments indicated that approximately 90% of the cancer cells were released from devices prepared using various electrospinning deposition times when applying multiple cycles of CV sweeping. Our findings suggest a new approach for using commercial PEDOT:PSS materials as integrated


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organic bioelectronics for dynamic capture and sequential release of rare cancer cells for further clinical applications. Experimental Section PEO/PEDOT:PSS Nanofiber Mats. The detailed materials and reagents for PEDOT:PSS nanofiber mats were described in the Supporting Information. To circumvent the poor wet stability of PEDOT:PSS nanofiber mats, a cross-linking

reaction

[between

PSS

and

poly(ethylene

oxide)

(PEO)]

with

additional

(3-

glycidoxypropyl)trimethoxysilane (GOPS) crosslinker was performed with itself or with PEDOT:PSS, thereby improving the water resistance performance of conductive PEDOT nanofiber mats.29, 33 For the water resistance test, various batches of PEO materials were synthesized as the PEO/PEDOT:PSS blend with 3 wt% of GOPS and 5 wt% of dimethylsulfoxide (DMSO), and later electrospun using a commercial needle-type electrospinning system. Figure 1a presents the experimental setup of the electrospinning system; using a syringe-based method, the PEDOT nanofibers were collected on the cathode electrode by controlling the deposition time (spinning time) with a high voltage supply to a 27-gauge disposable needle to control the diameter of the nanofibers. To prevent of nanofibers from standing up (due to negative charge of the conductive nanofibers) during the electrospinning process, an air blower under different electrospinning parameters was operated at room temperature at a relative humidity of less than 30%. Device Design and Surface Modification on PEDOT-Based Nanofiber Mats. The device configuration of the PEO/PEDOT:PSS nanofiber coated ITO glass was assembled with a polydimethylsiloxane (PDMS) chamber (ca. 10 mm diameter and 5 mm height) for static cell-capture studies or a chaotic mixer of a PDMS microchannel for dynamic cell-capture studies. The microfluidic BEI device configuration of the PEO/PEDOT:PSS nanofiber mats (NF10) coated five-patterned ITO finger electrodes was assembled with a chaotic mixer (approximately 6 mm thick) for a PDMS microchannel with 11 channels (the cross section for each continuous PDMS channel in this device was 1 mm wide, and approximately 22 cm long). The details



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regarding surface modification of PEDOT-based nanofiber device with anti-EpCAM-biotin coatings was described in the Supporting Information and already studied in our earlier work.10 Scanning Electron Microscopy, Transmission Electron Microscopy, and Atomic Force Microscopy. Scanning electron microscopy (SEM) images of the nanodimension PEDOT fibers and the CTCs on the chips were recorded using a Hitachi S-3400N operated at 15 keV. The PEDOT samples for SEM analysis were dehydrated (freeze-dried) and then sputter-coated with gold (

Poly(3,4-ethylenedioxythiophene)-Based Nanofiber Mats as an

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