Thermoresponsive and Active Functional Fiber Mats for Cultured Cell

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Thermo-responsive and Active Functional Fiber Mats for Cultured Cell Recovery Wilaiporn Graisuwan, Songchan Puthong, Hui Zhao, Suda Kiatkamjornwong, Patrick Theato, and Voravee P. Hoven Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00382 • Publication Date (Web): 20 Sep 2017 Downloaded from http://pubs.acs.org on September 25, 2017

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Thermo-responsive and Active Functional Fiber Mats for Cultured Cell Recovery Wilaiporn Graisuwan1, Songchan Puthong2, Hui Zhao3, Suda Kiatkamjornwong4, Patrick Theato3, Voravee P. Hoven5,6* 1

Program in Petrochemistry, Faculty of Science, Chulalongkorn University, Phayathai Road,

Pathumwan, Bangkok 10330, Thailand 2

Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Phayathai

Road, Pathumwan, Bangkok 10330, Thailand 3

Institute for Technical and Macromolecular Chemistry, University of Hamburg, Bundesstrasse

45, D-20146, Hamburg, Germany 4

Faculty of Science, Chulalongkorn University, Phayathai Road, Pathumwan, Bangkok 10330,

Thailand 5

Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road,

Pathumwan, Bangkok 10330, Thailand 6

Center of Excellence in Materials and Bio-interfaces, Chulalongkorn University, Phayathai

Road, Pathumwan Bangkok 10330, Thailand

ACS Paragon Plus Environment

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ABSTRACT: Thermo-responsive and active functional fiber mats were prepared from random copolymer

of

poly(pentafluorophenyl

acrylate-co-N-isopropylacrylamide)

(P(PFPA-co-

NIPAM)), which was synthesized by a controlled radical polymerization process based on reversible addition-fragmentation chain transfer (RAFT). As reactive sites, pentafluorophenyl ester (PFP) groups were incorporated in the copolymer to allow for a multiple postpolymerization modification. UV-crosslinkable moieties were first introduced by partially reacting PFP groups in the copolymer with ortho-nitrobenzyl (ONB)-protected diamine. Electrospinning the resulting ONB-containing P(PFPA-co-NIPAM) followed by UV-induced cross-linking yielded stable cross-linked thermo-responsive PNIPAM-based fiber mats. The remaining PFP active groups on the surface of copolymer fiber mats allowed for further conjugation with an H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS) peptide, a well-known cell adhesive peptide sequence that was selected as a model in order to promote cell growth. At 37oC, fibroblast cells were found to attach, spread and proliferate well on the GRGDS-immobilized cross-linked (CL) fiber mat, as opposed to those on the GRGDS-immobilized uncross-linked (UCL) fiber mat. By decreasing the temperature down to 20oC, i.e. below the lower critical solution temperature (LCST) of thermo-responsive PNIPAM, cultured cells could easily be released from both GRGDS-immobilized CL and UCL fiber mats, whereas no cells were detached from tissue culture polystyrene (TCPS). These results suggest that the thermo-sensitive and active functional fiber mat obtained in this research represent an attractive and versatile platform for cultured cell recovery, which is beneficial for tissue engineering applications.

KEYWORDS:

thermo-responsive,

pentafluorophenyl

acrylate,

post-functionalization,

electrospinning, cell recovery, adhesive peptide


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INTRODUCTION Cell-based regenerative medicine to restore or replace the lost functions of damaged or diseased tissues and organs has recently emerged as a promising medical therapy and has been progressively implemented in clinical trials.1,2,3 Cell harvesting is a key element required whether the treatment is based on direct injection of isolated cells alone, using cells in combination with biodegradable scaffolds, or confluent cell sheets. To recover cells from a cultured surface, conventional approaches rely on enzymatic proteolysis in which trypsin or other proteolytic enzymes are used to digest cells so that the cells adhering to the cultured material can be cleaved off. This process unavoidably destroys cell-to-cell junctions as well as extracellular matrix (ECM), which may affect cell viability and functions.4,5 More importantly, such an invasive process is not feasible for cell recovery in the form of tissue-like cellular monolayers or cell sheets, that can be directly transplanted to host tissues without the requirement for scaffolds, one of the most effective platform for some specific tissue engineering applications. Thermo-responsive polymers have been highly regarded as smart materials for a number of novel biomedical applications covering from bioseparation,6,7 drug delivery carriers8,9 to substrates for cell culture.10 Poly(N-isopropylacrylamide) (PNIPAM), in particular, is an intensively studied thermo-responsive polymer featuring a lower critical solution temperature (LCST) at 32oC in aqueous media. It exhibits a reversible phase transition between a hydrophilic state (below LCST) and a hydrophobic state (above LCST) because of competing intermolecular and intramolecular hydrogen bonding.11 Accordingly, cells can attach and grow on the PNIPAM surface at 37oC (above the LCST) similar to that of normal TCPS dishes because the surface is slightly hydrophobic above LCST. After cells have reached confluency, simply cooling the cell culture medium down to 20oC (below the LCST) renders the surface hydrophilic enough -due to


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the transition of the polymer from collapsed globule to extended, swollen coil to result in the spontaneous release of cells without requiring enzymes.10,12 Employing PNIPAM and its derivatives as such temperature-responsive platforms, Okano's research group has continuously reported their success in developing non-invasive approaches for recovering single cells and cell sheets13 by grafting PNIPAM chains onto tissue culture polystyrene dishes (TCPS).5,14-19 However, cell detachment from PNIPAM-grafted TCPS is a relatively slow process that takes approximately 55 min, which might have negative impact on cell functions.5 Spin coating of a photo-crosslinkable copolymer of PNIPAM on TCPS is an alternative, inexpensive and facile approach to enhance a rapid cell sheet detachment.20,21 Moreover, the use of porous substrates grafted with PNIPAM chains as culture substrates have recently been reported to facilitate a rapid cell sheet detachment.22-26 Because of the porous structure, water molecules can rapidly reach to grafted PIPAM from underneath and peripheral to the attached cells, resulting in a rapid hydration of grafted PIPAM chains and hence in an accelerated detachment of cells.27 Functional polymeric nanofibers produced via electrospinning are very attractive materials for diverse applications. Electrospun nanofiber mats are recognized as one of the most promising and versatile methods for preparing 3D porous platforms with high porosity and high surface area.28 Their ECM-mimicking structure render electrospun fibrous material an excellent candidate to be used in tissue engineering.29-32 However, electrospun nanofibers of PNIPAM are readily soluble in aqueous solution, which limits their application in some bio-related areas, especially in tissue engineering and regenerative medicine.33,34 Copolymerizing NIPAM with comonomers having active functionalities that can undergo cross-linking reactions is a promising strategy to obtain stable nanofibers in aqueous media. Monomers featuring active


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pentafluorophenyl ester groups, such as pentafluorophenyl (meth)acrylate (PFP(M)A), are quite desirable choices for copolymerization with NIPAM because their resulting copolymers are hydrolytically stable in water.35,36 Moreover, these active PFP ester groups are readily available for further chemical modification with bioactive molecules via activated ester-amine chemistry. Designing and producing three-dimensional PFP-containing functional nanofibrous structures via electrospinning have been recently reported in 2010. Gentsch and coworkers37 successfully prepared reactive fiber meshes based on pentafluorophenyl methacrylate (PFPMA) for the first time. The fiber mats proved to be a versatile platform for simple immobilization of sugar molecules via post-polymerization modification. The sugar-functionalized fiber mats enhance the cytokine production of macrophages that triggered specific interaction with biological systems. The group of Theato further employed this approach for the preparation of other functionalized nanoporous fiber mats.38 In the present research project, a random copolymer of P(PFPA-co-NIPAM) is introduced as an electrospinnable and cross-linkable polymeric material that could combine thermoresponsiveness with a reactive character that allows for a further biofunctionalization. The copolymer was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, a controlled radical polymerization that can be used to prepare both random or block copolymers with well-defined molecular weight and molecular weight distribution.39,40 This combination of RAFT polymerization and post-polymerization modification is compatible with a wide range of functional monomers and does not require the use of transition metal catalysts. Further, it has been used successfully for the synthesis of functional PNIPAM copolymers.41,42


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Partial post-polymerization modification of the copolymer was first performed to incorporate UV-crosslinkable moieties of ortho-nitrobenzyl (ONB)-protected diamine into the PFPA part. The ONB group is known to undergo irreversible transformation upon UV irradiation.43 Finally, the ONB-containing copolymers were electrospun into fiber mat. Crosslinked and stable fiber mat was then obtained after UV irradiation. The cross-linked copolymer fiber mat was utilized as functional platform to immobilize GRGDS peptide, a well-known cell adhesive peptide sequence, which could promote cell growth. GRGDS as an extracellular matrix protein contains the RGD moiety that has been shown to mediate cell adhesion through interactions with integrins.44,45

EXPERIMENTAL SECTION Materials. Triethylamine (TEA) (99.5%),

2-nitrobenzyl bromide (98%),

N,N′-

dimethylethylenediamine (85%), 4-cyanopentanoic acid dithiobenzoate (CPADB) (97%), and bovine serum albumin-fluorescein isothiocyanate conjugate (BSA-FITC) were purchased from Sigma-Aldrich (USA) and used as received. N-isopropylacrylamide (NIPAM) (97%, SigmaAldrich, USA) was recrystallized twice from hexane before use. Acrylic acid (AA) (99%, SigmaAldrich, USA) was purified by distillation under reduced pressure prior to use. Pentafluorophenol (99%, Merck, Germany) was used as received. Azobisisobutyronitrile (AIBN) (98%, Fluka, USA) was used as received. Acryloyl chloride (98%, Acros, USA) was used as received. H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS) was purchased from Genscript (USA). Fetal bovine serum (FBS) and RPMI 1640 medium were purchased from InVitromex (USA). Fibroblast (L929) cell line was obtained from the American Type Culture Collection company (ATCC®). All solvents used for reactions are reagent grade and used as received, unless


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otherwise specified. Anhydrous 1,4-dioxane (99.9%) was obtained from Merck, Germany. Anhydrous tetrahydrofuran (THF) (99.9%) and phosphate buffered saline (PBS) pH 7.4 were obtained from Sigma-Aldrich, USA. Dichloromethane was dried over CaH2 under reflux and nitrogen atmosphere. The NMR solvents such as CDCl3 (99.8% D) and DMSO-d6 (99.9%) were obtained from Cambridge Isotope Laboratories, Inc. (USA). ONB-protected diamine was synthesized according to a published procedure46,47 and PFPA was synthesized according to the method of Jochum et al.48 (See supporting information for detailed experimental description and characterization). Methods. 1H NMR spectra were recorded on a Varian, model Mercury-400 nuclear magnetic resonance spectrometer (USA) operating at 400 MHz. Chemical shifts (δ) were reported in part per million (ppm) relative to tetramethylsilane (TMS) signal as a reference. Fourier Transform Infrared (FT-IR) spectra were recorded with a Nicolet Impact 6700 FT-IR spectrometer, Thermo Scientific, USA with 32 scans at a resolution of 4 cm-1 in a frequency range of 400-4000 cm-1. Molecular weight and molecular weight distribution of synthesized polymers were analyzed by gel permeation chromatography (GPC) using Waters 600 controller chromatograph (USA) equipped with HR1 and HR4 columns (Waters, MW resolving range =100-500,000 g/mol) at 35oC and refractive index (RI) detector (Waters 2414). Tetrahydrofuran (THF) was used as an eluent with the flow rate of 1.0 mL/min. Sample injection volume was 80 µL. Five polystyrene standards (996-188,000 g/mol) were used for generating a calibration curve. The surface morphology of electrospun fibers was observed using a JEOL model JSM6610/LV (USA) scanning electron microscope (SEM) and analyzed with an accelerating voltage of 15 kV. A small piece of electrospun fibers on aluminum foil was cut and placed on SEM stub


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using a double-sided adhesive carbon tape. Sample was sputter-coated with gold prior to imaging. The fiber diameters were measured by using a SemAfore 5.21 for 50 fibers per sample. The dynamic advancing (θA) water contact angles were measured using a contact angle goniometer (Ramé-Hart, Inc., USA, model 100-00), equipped with a Gilmont syringe and a 24gauge flat-tipped needle. A droplet of Milli-Q water was placed on the tested surface by bringing the surface into contact with a droplet suspended from a needle on the syringe. Measurements were carried out at 25 and 37oC. The samples were placed in a controlled temperature attachment mounted on a tilt stage. A thermocouple was attached close to the sample. For advancing contact angle (θA) measurement, the needle was inserted into the center of the drop from above. The drop’s volume was slowly increased by pumping liquid into the drop. The reported angle is an average of 5 measurements on different areas of each sample, after which they were expressed as the arithmetic mean ± standard deviation (SD). Synthesis of P(PFPA-co-NIPAM) by RAFT polymerization. PFPA (2.38 g, 10 mmol), NIPAM (1.13 g, 10 mmol), CPADB (2.79 mg, 0.01 mmol), and AIBN (0.21 mg, 1.28x10-3 mmol), were added to a vial followed by 10 mL of dry 1,4-dioxane. The vial was sealed with a rubber septum and the solution was purged with nitrogen gas for 30 min under stirring. Polymerization was conducted under nitrogen atmosphere at 70oC for 24 h. The polymer solution was precipitated in diethyl ether, centrifuged, and finally dried under vacuum at room temperature. The dried copolymer was then dissolved in THF and precipitated again in diethyl ether. This procedure was repeated two times. The product was obtained as pink powder in 80% yield (2.81 g, 8 mmol). 1H NMR (400 MHz, CDCl3): δ/ppm: 4.10 (br,–NHCH(CH3)2), 3.10 (br, CH2CH), 2.51-1.50 (m, protons in backbone), 1.13 (br, –NHCH(CH3)2). FT-IR (ATR-mode):


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1782 cm-1 (C=O reactive ester band), 1645 cm-1 (C=O of amide), 1515 cm-1 (C=C aromatic band), 1090 cm-1 (C-O ester band). Post-polymerization modification of P(PFPA-co-NIPAM) with ONB-protected diamine. UV-crosslinkable moieties in the form of an ONB-protected diamine were introduced to the PFPA part of P(PFPA-co-NIPAM) via post-polymerization modification.47 P(PFPA-coNIPAM) (1.0 g, 1 equiv. of PFPA unit) was dissolved in 10 mL of dry THF under nitrogen atmosphere for 30 min. Separately, ONB-protected diamine (0.5 equiv.) and TEA (0.1 equiv) were dissolved in 1 mL of dry THF. The ONB-protected diamine solution was quickly added to the polymer solution and continued purging with nitrogen gas for 20 min. The solution was stirred in the dark at room temperature for 24 h. The resulting copolymer was purified by precipitation in diethyl ether, centrifuged, and vacuum dried at room temperature overnight. The product was obtained as pale-yellow powder in 71% yield (1.16 g, 2.02 mmol). 1H NMR (400 MHz, CDCl3): δ/ppm: 7.52-8.09 (br, protons in o-nitrobenzene), 4.10 (br, –NHCH(CH3)2), 3.21.5 (m, protons in backbone and linker of ONB), 1.13 (br, –NHCH(CH3)2). FT-IR (ATR-mode): 1782 cm-1 (C=O reactive ester band), 1645 cm-1 (C=O of amide I band), 1515 cm-1 (C=C aromatic band), 1090 cm-1 (C-O ester band). Electrospinning of P(PFPA-co-NIPAM) and ONB-containing P(PFPA-co-NIPAM). Electrospinning technique was employed to fabricate the P(PFPA-co-NIPAM) and ONBcontaining P(PFPA-co-NIPAM) fiber mats. The electrospinning apparatus consisted of a variable syringe pump (ProSense B.V. Laboratory & process equipment model NE1000, Netherlands) and a high voltage power supply (Gamma High Voltage Research, model ES30P, USA). The copolymers of P(PFPA-co-NIPAM) and ONB-containing P(PFPA-co-NIPAM) with different concentrations (10-30% w/v) were dissolved in a mixture of THF/DMF (3:1, v/v). The


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polymer solutions were then stirred at room temperature for 24 h. The polymer solution was loaded in a 5 mL plastic syringe equipped with a metallic needle of 0.7 mm inner diameter. The syringe was fixed horizontally on the syringe pump and an electrode of high power supply was clamped to the metal needle tip. A grounded stationary rectangular metal collector covered with a piece of clean aluminum foil was used for the fiber collection. The distance between the needle tip and the collector was set to 20 cm. The flow rate of polymer solution was fixed at 3 mL/h, and the applied voltage was set at 20 kV. The electrospun fibers were collected on a flat aluminum foil and then vacuum dried at room temperature overnight prior to further studies. For preparation of cross-linked P(PFPA-co-NIPAM) fiber mat, the electrospun fiber mat of ONB-containing P(PFPA-co-NIPAM) fiber mat was exposed to UV light (365 nm) for 2 h to generate cross-linked networks. Immobilization of GRGDS peptide on the electrospun fiber mat. GRGDS peptide (0.05 M) was dissolved in PBS buffer pH 7.4. The electrospun fiber mats of P(PFPA-co-NIPAM) and cross-linked P(PFPA-co-NIPAM) were cut into small circle pieces with diameter of 15 mm and placed into the bottom of 24-well tissue culture polystyrene (TCPS) plates. The GRGDS peptide solution was then pipetted into each well of the electrospun fiber mats. After immobilization for 24 h, the samples were rinsed twice with PBS and DI water, respectively. The electrospun fiber mat were then air dried overnight. GRGDS-immobilized uncross-linked P(PFPA-co-NIPAM) and cross-linked P(PFPA-co-NIPAM) fiber mats are abbreviated as GRGDS-UCL and GRGDSCL fiber mats, respectively. Additionally, bovine serum albumin-fluorescein isothiocyanate conjugate (BSA-FITC) was used as a fluorescent-labeled biomolecule model to prove whether or not amino-containing molecules can react with the active ester groups of PFPA units in the copolymer. The


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immobilization process was performed using the same procedure as that for the immobilization of GRGDS peptide as mentioned above. The stability of UCL and CL P(PFPA-co-NIPAM). fiber mats. The stability of UCL and CL fiber mats of P(PFPA-co-NIPAM) were evaluated in phosphate-buffered saline (10 mM PBS, pH 7.4) and BSA-FITC solution. Samples were immersed in the solutions at room temperature for 24 h and then washed with distilled water twice, followed by drying overnight under ambient condition and then vacuum dried for another 24 h. The morphological changes of the samples were observed by SEM. The fiber diameters were measured and averaged from at least 50 samples. Cell viability test. Fibroblast (L929) cell line was used to study cell adhesion and proliferation of the electrospun fiber mat. The L929 cells were cultured in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS), penicillin (100,000 U/L) and streptomycin (100 mg/mL). They were incubated at 37°C in atmosphere containing 5% CO2 where the culture medium was changed every 3 days. For cell culture, the electrospun fiber mats in 24-well TCPS plates were sterilized with UV light overnight prior to use. Eight replicated samples were used for each condition. Approximately 2x104 of the L929 cells in 200 µL culture medium were pipetted into each well containing the substrates as well as into the bottom of TCPS plates as a control and then incubated under 5% CO2 at 37°C. 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was used to investigate cell adhesion and proliferation. After cells were cultured for a set of incubation time, the culture medium was removed to discard the unattached cells and the 200 µL of fresh culture medium was pipetted into each well followed by 10 µL of 0.5 mg/mL of MTT in normal saline solution. After incubation for 1 h, the supernatant solution was removed and 150 µL of DMSO was pipetted into


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each well to dissolve the purple crystals of formazan. Next, 25 µL of 0.1 M glycine (pH 10.5) was added. The optical density of sample was measured using a microplate reader at the wavelength at 540 nm. For the cell adhesion studies, the cells were allowed to attach on the surfaces for 6 h. For the cell proliferation studies, the cells after having been allowed to attach on the surfaces for 6 h, were cultured for either 1, 3, and 5 days. The viability of the attached and proliferated cells was quantified by MTT assay. Recovery of cultured cells. Detachment of attached cells was achieved by lowering the temperature after incubation for 5 days. For lowering temperature, spread cells on each surface were transferred to a controlled temperature incubator fixed at 20oC. The morphology and detachment rate were observed with SEM and MTT assay as a function of lowering temperature treatment time. Statistical Analysis. For the cell adhesion and proliferation tests, the data are expressed as the mean ±standard deviation (S.D.) Statistical analysis to test for significant differences between means was performed using the Statistical Package for the Social Science (SPSS) version 17.0 software, using One-Way Analysis of Variance (ANOVA) with the Least Square Difference (LSD) tests were used for post hoc evaluations of differences between groups. The threshold level for accepting statistical significance was set at p < 0.05.

RESULTS AND DISCUSSION Preparation of P(PFPA-co-NIPAM) by RAFT polymerization. RAFT polymerization was particularly chosen as a controlled polymerization process for the synthesis of P(PFPA-coNIPAM) using 4-cyanopentanoic acid dithiobenzoate (CPADB) and azobisisobutyronitrile (AIBN) as chain transfer agent (CTA) and initiator, respectively in 1,4-dioxane at 70oC. It has


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been previously shown that RAFT polymerization can yield copolymers with well-defined molecular weight and comonomer composition.47 Further, in order to minimize the effect of molecular weight on the thermo-responsive behavior, RAFT polymerization was chosen. All synthesized copolymers were characterized by 1H NMR and FT-IR analyses and the results are shown in Figure 1 and 2, respectively. The characteristic proton signal of the NIPAM repeating unit appeared at 4.10 ppm (signal k), which can be assigned to the methine proton of NIPAM part. The characteristic proton signal at 3.10 ppm (signal b) is assignable to the CH group of the PFPA polymer backbone. The signals around δ = 2.51-1.13 ppm (signals a,c,d,e,f,g,i, and j) can be attributed to the protons of polymer backbone from both of PFPA and NIPAM units. In addition, FT-IR spectra also showed the characteristic peaks of both of PFPA (C=O of ester at 1782 cm-1) and NIPAM (C=O of amide I at a 1645 cm-1) as shown in Figure 2(a). The comonomer composition (mol%) of PFPA to NIPAM in the copolymer was determined from 1H NMR data based on relative integration of the peak at 4.10 ppm (for methine proton of NIPAM) to that of the peak at 3.10 ppm of the PFPA. The comonomer ratios of PFPA to NIPAM were found to be 28:72 and 25:75 for the Mn of 47.000 g/mol and 60.000 g/mol as shown in Table S1 (Supporting Information). It should be emphasized that high molecular weights of the copolymer were targeted, because this is mandatorily required to provide enough chain entanglement for the copolymer to be electrospinnable. Relatively high dispersities (Ð > 1.5) are the result of RAFT process being less controllable at such high molecular weight. The change in molecular weight and Ð value of the copolymer as a function of polymerization time (Table S2, Supporting Information) can also be used as a supporting evidence. This may be attributed to an increase of termination reactions as the monomers become increasingly consumed and the


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viscosity increase of the medium would limit termination reactions of growing polymer chains especially at higher conversions.49,50

Figure 1. 1H NMR spectra in CDCl3 of (a) P(PFPA-co-NIPAM), (b) ONB-containing P(PFPAco-NIPAM).


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Figure 2. FT-IR spectra of (a) P(PFPA-co-NIPAM), ONB-containing P(PFPA-co-NIPAM) (b) before and (c) after cross-linking by UV irradiation, and (d) cross-linked P(PFPA-co-NIPAM) fiber mat after GRGDS immobilization.

Post-polymerization modification of P(PFPA-co-NIPAM). UV-crosslinkable ONBcontaining P(PFPA-co-NIPAM) were prepared via post-polymerization modification of PFPA units in the copolymer with a mono ONB-protected diamine. Upon UV irradiation at 365 nm, the ONB-protected amine group can be released, which can subsequently induce cross-linking via activated ester-amine chemistry resulting in a network formation as shown in Scheme 1. A successful attachment of ONB-protected diamine was confirmed by 1H NMR and FT-IR analyses. Figure 1 (b) represents the 1H NMR spectrum of ONB-containing P(PFPA-coNIPAM). The signals of the four aromatic protons of the ONB moieties in a range of δ = 7.528.09 ppm clearly confirmed the attachment of the ONB groups to the P(PFPA-co-NIPAM). The amount of incorporated ONB-protected amine in the copolymer was calculated by 1H NMR from the relative integral ratio between the peak of the four aromatic protons (δ = 7.52-8.09 ppm) and the peak of one proton at 3.10 ppm of the PPFPA polymer backbone. The percentage of ONB incorporation (%ONB) was found to be 20 mol% of active ester groups of the PPFPA. Herein, the %ONB in the copolymer of 20 mol% were chosen to assure that only a minor fraction of PFPA units were converted with ONB-protected diamine allowing for cross-linking reaction later via the incorporated ONB-protected diamine. The majority of the unreacted PFPA was hence available for subsequent post-functionalization with GRGDS peptides. Additionally, the successful conversion of the parent PFPA units in the copolymer to the corresponding ONBprotected group was also confirmed by FT-IR analysis from the decrement of C=O peak at 1782


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cm-1 of the PFP active ester group and the increment of the amide carbonyl group at 1645 cm-1 (Figure 2 (b)). After UV irradiation, a new peak of C=O stretching of nitrosobenzaldehyde appeared at 1725 cm-1 confirming the formation of the cleaved by-product as shown in Figure 2 (c). It was our intention in this specific experiment not to wash the fibers in order to confirm that the cross-linking took place and there was the release of by-product. In further experiments, the cross-linked fibers were thoroughly rinsed to assure that nitrosobenzaldehyde was entirely removed. The signal from C=O stretching of nitrosobenzaldehyde diminished upon subsequent peptide conjugation as can be realized from Figure 2(d). The active ester peak at 1782 cm−1 almost disappeared after GRGDS immobilization, indicating that most of the active ester groups in the precursor copolymer were consumed after this second post-functionalization step as shown in Figure 2(d). A trace amount of PFP activated ester (C=O signal at 1782 cm−1) still remained, which may be ascribable to the high hydrophobicity and surface area to volume ratio of the fibers that limited the accessibility of peptide.

Scheme 1. Synthetic pathway for the preparation of ONB-containing P(PFPA-co-NIPAM) and GRGDS immobilized cross-linked P(PFPA-co-NIPAM) via post-polymerization modification and cross-linked reaction induced by UV irradiation.


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Fiber fabrication by electrospinning process. The P(PFPA-co-NIPAM) was fabricated into fiber mat by electrospinning technique. The effects of solvent, molecular weight and polymer solution concentration were investigated in order to develop optimal conditions that yield fibers with uniform size distribution. Effect of solvent. P(PFPA-co-NIPAM) having Mn of 47 kg/mol was electrospun from THF or binary mixtures of THF/DMF (3:1 v/v) to study the effect of solvent on the fiber formation (Figure 3). For each solvent system, the polymer solution concentration was varied from 10 to 30% (w/v). The fibers electrospun from THF showed only beads and beaded fibers on the collector at polymer concentrations of 10 and 20% w/v, respectively, while homogenous fibers with an average diameter of 2.02 ± 0.84 µm were obtained when electrospun from THF at 30% (w/v). Slightly beaded fibers were obtained when electrospun from a mixture of THF/DMF (3:1 v/v) at a polymer concentration of 30% (w/v). However, the average diameter of fibers electrospun from the THF/DMF was smaller than that of the fibers electrospun from THF. DMF has a relatively high dielectric constant (36.4) as compared with that of THF (7.6) and hence can increase the elongational force within the solution jet, resulting in fibers with a smaller diameter when electrospun from THF/DMF.51 Therefore, the binary mixture of THF/DMF (3:1 v/v) was selected and used for all further investigations.


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Figure 3. SEM micrographs of P(PFPA-co-NIPAM) fibers electrospun from two molecular weights (47 and 60 kg/mol) in THF and binary mixture of THF/DMF (3:1 (v/v)) with different polymer concentrations from 10 to 30% w/v at a constant voltage of 20 kV, a flow rate of 3 mL/h. Number appearing below each micrograph is an average diameter of the electrospun fibers (scale bar 10 µm).

Effect of molecular weight and polymer solution concentration. The molecular weight and polymer solution concentration are important parameters that have a strong impact on the fiber formation. Figure 3 shows SEM micrographs of the P(PFPA-co-NIPAM) electrospun fiber mats fabricated from two different molecular weights (47 and 60 kg/mol) with different polymer concentrations (10-30% (w/v)) at a constant voltage of 20 kV and a flow rate of 3 mL/h. For the copolymer with Mn of 47 kg/mol, the concentration of 10% (w/v) was too low to provide a


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reasonable chain entanglement and viscosity so that only beads were formed. For the same concentration, beads with some fibers were observed for the copolymer with Mn of 60 kg/mol. At 20% (w/v), beaded fibers with diameter of 0.18 ± 0.07 µm were formed for the copolymer with Mn of 47 kg/mol. In contrast, the copolymer with Mn of 60 kg/mol yielded fibers with fewer beads. As the concentration of polymer in solution was increased to 30% (w/v), chain entanglement and viscosity were high enough to yield well-defined fibers without any beads especially in the case of copolymer with high molecular weight. Moreover, the corresponding fiber diameter of P(PFPA-co-NIPAM) increased as a function of polymer concentration. Therefore, an optimal molecular weight and polymer concentration of the P(PFPA-co-NIPAM) in the present study were identified as 60 kg/mol and 30% (w/v), respectively. Following the optimal conditions formerly identified for the electrospinning of P(PFPAco-NIPAM) fibers, ONB-containing P(PFPA-co-NIPAM) fiber mat was fabricated using a polymer solution concentration of 30% w/v, a constant voltage of 20 kV, and a flow rate was 3 mL/h. The average fiber diameter of ONB-containing P(PFPA-co-NIPAM) fiber mat was 0.78 ± 0.21 µm. After UV irradiation, the average fiber diameter of cross-linked (CL) fiber mat was found to be slightly smaller than those of uncross-linked (UCL) fiber mat, implying that the covalently cross-linked network of the ONB-protected diamine with PFPA esters were formed as shown in Figure S1 in the Supporting Information. Stability of of P(NIPAM-co-PFPA) fiber mat. The morphological changes and the diameters of the electrospun fiber mat before and after treatment with the solutions of phosphatebuffered saline (10 mM PBS, pH 7.4) and BSA-FITC were investigated and the results are shown in Figure S2 in the Supporting Information. Before immersion, all the samples were of similar diameters (around 600-700 nm). After immersion in the solutions for 24 h, both of the


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UCL and CL electrospun fiber mats maintained their fibrous structure, although the diameters of both fiber mats slightly increased as a result of PNIPAM swelling. We describe this rather unexpected stability of the UCL electrospun fiber mat as a result of their inherit hydrophobicity, which is also verified by contact angle measurements, vide infra. It is believed that the extreme hydrophobic nature renders the UCL electrospun fiber mat as an inferior substrate for cell attachment/proliferation although the fibers were conjugated with GRGDS peptide (See data in Figure 4 and 5). It should also be emphasized that this experiment was performed only for 24 h, hence, this information is only preliminary and does not account for their long-term stability. In this respect, the cross-linking was performed not only to guarantee long-time stability of the fibers, but also helped tuning the hydrophobicity of their surface to the right level so that cell attachment/proliferation as well as subsequent cell detachment are desirable. Immobilization of GRGDS peptide on the P(PFPA-co-NIPAM) fiber mat. Prior to immobilization of GRGDS peptide on both UCL and CL fiber mats, bovine serum albuminfluorescein isothiocyanate conjugate (BSA-FITC) was used as a fluorescent-labeled biomolecule model to prove whether or not amino-containing molecules can react with the active ester groups of PFPA units in the copolymer. Figure 4 shows fluorescence images of uncross-linked and cross-linked P(PFPA-co-NIPAM) fiber mats both before and after conjugation. The green fluorescence signal of both UCL and CL fiber mats on the right column indicated the successful immobilization of the BSA-FITC onto the fiber mats. The even dye distribution along the fibers also implies a homogeneous distribution of PPFPA moieties on the fiber surface. Changes in wettability of the fiber mat surfaces were monitored by dynamic water contact angle measurements. However, in Table 1 only the advancing water contact angles are shown, because the presence of hydrophilic PNIPAM entities within the fibers together with the roughness


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account for the water drop pinning during receding angle measurements so that reducing water contact angle became un-measurable, i.e. its “stick/slip behavior”.52 Measurements were carried out at 25 and 37oC. The UCL fiber mat exhibited much higher water contact angle than the CL fiber mat at both temperatures, which is a strong indication of an increased amount of hydrophobic PFP groups at the fiber mat surface before cross-linking. The water contact angle significantly decreased after reacting with the GRGDS peptide, suggesting that some of hydrophobic moieties of PFPA units have been replaced by the more hydrophilic GRGDS peptide. The fact that the water contact angles measured at 37oC, i.e at a temperature above LCST of PNIPAM, were greater than those measured at 25oC verifies the thermo-responsiveness of the developed PNIPAM-based fiber mats. Hence, their surface would become more hydrophobic upon temperature increase above LCST as a consequence of collapsed polymer chains. These results presumably suggested that the trend in wettability seen at ambient temperature should not be altered once the fiber mats were put in contact with cells at 37 oC.


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Figure 4. Fluorescence micrographs of UCL and CL P(PFPA-co-NIPAM) fiber mats before and after immobilization with BSA-FITC (scale bar 200 µm).

Table 1. Advancing water contact angles of P(PFPA-co-NIPAM) fiber mats before and after immobilization of GRGDS peptide measured at 25 and 37oC. Advancing water contact angle (degree) Sample measured at 25oC

measured at 37oC

UCL fiber mat

134 ± 4

141 ± 3

CL fiber mat

120 ± 3

131 ± 2

GRGDS-UCL fiber mat

91 ± 3

105 ± 2

GRGDS-CL fiber mat

77 ± 1

86 ± 1

Cytocompatibility test. In this research, in vitro cytocompatibility of both uncross-linked and cross-linked P(PFPA-co-NIPAM) fiber mats after GRGDS immobilization, which are


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designated as GRGDS-UCL and GRGDS-CL fiber mats, respectively, was carried out against a mice L929 fibroblast cell line. To investigate the mitochondrial functions of the cultured L929, reduction of MTT reagent was used as an assay of mitochondrial redox activity. MTT reagent is a pale yellow substance that is reduced to a dark blue formazan product when incubating with viable cells by mitochondrial succinate dehydrogenase in complex II, which plays a critical role in both oxidative phosphorylation and tricarboxylic acid cycle. Therefore, the production of formazan can reflect the level of cell viability. The cell adhesion was tested after 6 h and cell proliferation was evaluated after 1 day, 3 days and 5 days of cell culturing. The results are reported in terms of the cell adhesion and proliferation ratio (% relative to TCPS), which is directly correlated to the number of viable cells. After 6 h of cell culture, the cell adhesion ratios (% relative to TCPS) on UCL and CL fibers were 35 and 49%, respectively, whereas much higher cell adhesion ratios of 60% and 109 % were found on GRGDS-UCL and GRGDS-CL fiber mats, respectively. This outcome strongly suggested that the immobilized GRGDS peptide can efficiently support cell adhesion at 6 h (Figure 5 (a)). Being slightly more hydrophobic with a higher θA than TCPS (65 ± 1o) together with its conjugated adhesive peptide, the GRGDS-CL fiber mat exhibited an enhanced cell adhesion ratio to TCPS. We explain the inferior cellular adhesion of both the UCL and CL fibers before peptide immobilization as a consequence of the fiber mats being too hydrophobic (with advancing water contact angles θA > 100o at 37oC) as opposed to the TCPS and the fibers after peptide immobilization. Apparently, the degree of cell adhesion corresponded quite well with the hydrophobicity of the fiber mat (see water contact angle data in Table 1). Cell proliferation was observed after 1 day, 3 days, and 5 days of cell culturing. The data of cell proliferation ratio (% relative to TCPS) are displayed in Figure 5 (b). The percentage of


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live cells increased with an increase in cell culture time. The percentage of live cells on GRGDSCL fiber mat was twice as high as those found on GRGDS-UCL fiber mat. This outcome suggested that not only the immobilized peptide but also the hydrophilicity of the surface are important parameters that dictate cell growth. The performance of the GRGDS-CL fiber mat in supporting cell attachment and proliferation is apparently comparable to TCPS, the conventional substrate.


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Figure 5. Responses of L929 cells (% relative to TCPS) with a seeding density of 2.0x104 cells/well on uncross-linked (UCL), cross-linked (CL), GRGDS-immobilized uncross-linked (GRGDS-UCL) and cross-linked (GRGDS-CL) fiber mats and TCPS in terms of (A) the cell adhesion ratios at 6 h and (B) the cell proliferation ratios at 1, 3 and 5 days. Statistical significance with p < 0.05 is compared with the control (TCPS).


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For the cell detachment, substrates having spread cells were transferred to an incubator equipped with a cooling unit fixed at 20oC. The morphology of both attached (37oC) and detached cells (20oC) were observed by SEM as shown in Figure 6. At 37oC, cells can attach and proliferate very well on GRGDS-CL fiber mat. The cells spread out and almost covered the complete substrate after 3 days of incubation. GRGDS-UCL fiber mat, on the other hand, seem to be an unfavorable substrate for cell attachment and proliferation as evidenced from less number of attached cells and some of them were rounded. The information from the cell morphology is in good agreement with the quantitative data displayed in Figure 5. When the culture temperature was reduced to 20oC after incubation at 37oC for 3 and 5 days, the spread cells became rounded and detached from both GRGDS-UCL and GRGDS-CL fiber mats due to their thermo-responsiveness. In addition, Ebara et al.,53 reported that at temperatures below the grafted polymer’s LCST, integrin-RGDS association decreases due to loss of cell tension and surface anchoring, prompting cells to round and then detach. This surface property changes obviously weakened cellular adhesion, resulting in spontaneous cell detachment.


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Figure 6. SEM micrographs of fibroblasts on the GRGDS-UCL fiber mats (left column) and GRGDS-CL fiber mats (right column): cell attachment at 37oC for a set culture time and cell detachment at 20oC for 30 min (scale bar 50 µm).

As demonstrated in Figure 7, the percentage of attached cells on the surfaces after temperature reduction from 37 to 20oC decreased rapidly on both GRGDS-UCL and GRGDS-CL fiber mats. After 30 min of temperature decrease, there were approximately 55 % cells detached from both GRGDS-UCL and GRGDS-CL fiber mats whereas no cells detached from TCPS in the same time, due to the lack of thermo-responsiveness. The cell detachment was almost complete within 90 min of incubation in the case of GRGDS-CL fiber mats (95% cell detachment). Once again, it should be highlighted that the initial number of attached cells were higher on the GRGDS-CL fiber mats than the GRGDS-UCL fiber mats. This presumably suggests that a greater number of cells are recovered from the GRGDS-CL fiber mats than from


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the GRGDS-UCL one. The cell detachment from both GRGDS-UCL and GRGDS-CL fiber mats may be explained as a result of water molecules being capable of efficiently reaching PNIPAM chains within the fiber through pores from both underneath and side. Therefore, the hydration of PNIPAM chains can take place instantaneously and thus promoted detachment of the attached cells. These results are in good agreement with that reported by Kwon et al. in that the percentage of remaining cells after low temperature treatment was found to decrease rapidly around 50% within 30 min. on the PIPAM-grafted porous membrane.26 From practical perspective, it is quite important to determine the viability of the detached cells. We have therefore performed an additional test by reseeding the detached cells at 37oC. As evaluated by MTT assay, it was found that cell proliferation ratio reached approximately 82% relative to TCPS after 5 days of incubation suggesting that the cells remained viable and can still proliferate. After the attached cells in the first round of test were detached upon temperature reduction, the GRGDS-CL fiber mat was washed and re-sterilized before subjected to the second round of cell incubation for another 5 days. Impressively, the cell proliferation ratio remained as high (88% relative to TCPS) as its original value found in the first round of the test suggesting that the GRGDS-CL fiber mat can be repeatedly used. This satisfactory feature has also been previously highlighted on solvent-cast PNIPAM film used as thermo-responsive platform for recovery of human mesenchymal stem cells.54 The ability of the GRGDS-CL fiber mat to yield detached cells with cell-to-cell junctions maintained can be observed by optical microscope. A representative brightfield image of a rolling cell sheet is shown in Figure S3, Supporting Information. Refinement on experimental parameters (i.e. cell type, initial cell seeding density, incubation time) is necessary in order to construct good-quality cell sheet and to be able to compare its effectiveness in cell recovery with


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other methods. Nevertheless, this comparable cell detachment efficiency together with its equivalent performance in supporting for cell attachment and proliferation to TCPS, recovered cell viability and its re-usability, renders the GRGDS-CL fiber mat as a promising alternative biomaterial platform to recover intact cultured cells for applications in the area of regenerative medicine and tissue engineering.

Figure 7. The percentage of remaining cells on GRGDS-immobilized uncross-linked (GRGDSUCL) and cross-linked (GRGDS-CL) fiber mats and TCPS as a function of incubation time at 20oC.

CONCLUSIONS As successfully synthesized by RAFT polymerization, random copolymers of P(PFPA-coNIPAM) with molecular weights high enough (up to 60 kg/mol) allowed for the fabrication of non-woven fiber mats with well-defined morphology with sub-micrometer diameters by electrospinning. The presence of active PFP groups provides a build-in feature for the postpolymerization modification of the copolymer with UV-crosslinkable ONB-protected diamine.


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Upon UV irradiation, in situ cross-linking of the electrospun ONB-containing copolymer can be spontaneously induced and yielded cross-linked (CL) fiber mats. Crosslinking not only can ensure the stability of the fiber mat but also provide an appropriate level of hydrophilicity to the fiber mats so that it became favorable substrate in supporting cell adhesion and proliferation after subsequently being immobilized

with adhesive GRGDS

peptide via second

post-

functionalization of the remaining PFP moieties within the fiber. The ability to release intact cultured cell sheets from its surface after temperature has been reduced below the LCST of PNIPAM promptly suggests that the GRGDS-immobilized CL fiber mat could serve as an alternative promising material for regenerative medicine and tissue engineering applications. The fact that the surface character of the fiber mat can be specifically tuned by post-polymerization modification of active PFP groups opens up a broad opportunity to be further developed into other biomaterials that can benefit from thermo-responsiveness.

ASSOCIATED CONTENT Supporting Information Synthesis of ortho-nitrobenzyl (ONB) protected diamine, Synthesis of pentafluorophenylacrylate (PFPA), Molecular weight information of P(PFPA-co-NIPAM), SEM micrographs of ONBcontaining P(PFPA-co-NIPAM) fibers, SEM micrographs of uncross-linked and cross-linked P(PFPA-co-NIPAM) fibers before and after immersion in PBS and BSA-FITC, A brightfield image of rolling cell sheet recovered from the GRGDS-CL fibers having attached fibroblasts. This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author *Tel.: +66 22187627. Fax: +66 22187598. E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENTS This research was financially supported by Thailand Research Fund (DBG5580003), the Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University (RES560530126-AM), and the Thai Government Stimulus Package 2 (TKK2555), under the Project for Establishment of Comprehensive Center for Innovative Food, Health Products and Agriculture. WG gratefully acknowledges the Commission on Higher Education, Thailand for the program Strategic Scholarships for Frontier Research Network for the Joint Ph.D. Program Thai Doctoral degree.

ABBREVIATIONS ECM,

extracellular

matrix;

TCPS,

tissue

culture

polystyrene;

PNIPAM,

poly(N-

isopropylacrylamide); LCST, lower critical solution temperature; PFP(M)A, pentafluorophenyl (meth)acrylate; ONB, ortho-nitrobenzyl; GRGDS-UCL, GRGDS-immobilized uncross-linked P(PFPA-co-NIPAM) fibers; GRGDS-CL, cross-linked P(PFPA-co-NIPAM) fibers; BSA-FITC, bovine serum albumin-fluorescein isothiocyanate conjugate.


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Thermoresponsive and Active Functional Fiber Mats for Cultured Cell

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