Ultrathin Poly(glycidyl ether) Coatings on Polystyrene for Temperature

Subscriber access provided by Georgetown University | Lauinger and Blommer Libraries

Article

Ultrathin Poly(glycidyl ether) Coatings on Polystyrene for Temperature-Triggered Human Dermal Fibroblast Sheet Fabrication Daniel David Stöbener, Melanie Uckert, José Luis Cuellar Camacho, Anke Hoppensack, and Marie Weinhart ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00270 • Publication Date (Web): 18 Jul 2017 Downloaded from http://pubs.acs.org on July 24, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Biomaterials Science & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Ultrathin Polystyrene

Poly(glycidyl for

ether)

Coatings

Temperature-Triggered

on

Human

Dermal Fibroblast Sheet Fabrication Daniel David Stöbener, Melanie Uckert, José Luis Cuellar-Camacho, Anke Hoppensack, Marie Weinhart* Institute of Chemistry and Biochemistry, Freie Universitaet Berlin, Takustrasse 3, D-14195 Berlin, Germany *E-mail: [email protected]

ABSTRACT

The fabrication of cell sheets is a major requirement for bottom-up tissue engineering purposes (e.g. cell sheet engineering) and regenerative medicine. Employing thermoresponsive polymer coatings as tissue culture substrates allows for the mild, temperature-triggered detachment of intact cell sheets along with their extracellular matrix (ECM). It has been shown before that biocompatible, thermoresponsive poly(glycidyl ether) monolayers on gold substrates can be utilized to harvest confluent cell sheets by simply reducing the temperature to 20 °C. Herein, we report on the first poly(glycidyl ether)-based coating on an application-relevant tissue culture plastic substrate. We devised a simple, substrate geometry-independent method to functionalize

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 34

polystyrene (PS) surfaces from dilute ethanolic solution via the physical adsorption process of a thermoresponsive poly(glycidyl ether) block-copolymer (PGE) bearing a short, hydrophobic, and photoreactive benzophenone (BP) anchor block. Subsequently, the PGE-coated PS is UV irradiated for covalent photo-immobilization of the polymer on the PS substrate. Online monitoring of the adsorption process via QCM-D measurements and detailed characterization of the resulting coatings via AFM, ellipsometry, and water contact angle (CA) measurements revealed the formation of an ultrathin PGE layer with an average dry thickness of 0.7 ± 0.1 nm. Adhesion and proliferation of human dermal fibroblasts on PGE-coated PS and TCPS were comparable. For temperature-triggered detachment, fibroblasts were cultured in PGE-coated PS culture dishes at 37 °C for 24 h until they reached confluency. Intact cell sheets could be harvested from the thermoresponsive substrates within 51 ± 17 min upon cooling to 20 °C, whereas sheets could not be harvested from uncoated PS and tissue culture PS (TCPS) control dishes. Live/dead staining and flow cytometry affirmed a high viability of the fibroblasts within the cell sheets. Hence, ultrathin layers of thermoresponsive poly(glycidyl ether)s on hydrophobic PS substrates are functional coatings for cell sheet fabrication. KEYWORDS thermoresponsive polymer coating, cell sheet detachment, photo-immobilization, physical adsorption, C, H-insertion INTRODUCTION Thermoresponsive polymer coatings on cell culture substrates enable the fabrication of confluent cell sheets with their intact ECM for potential use in tissue engineering and regenerative medicine via a mild and enzyme-free harvesting method.1-4 On surfaces modified with thermoresponsive polymers, which have been optimized for cell culture applications, cell


2

Page 3 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


adhesion ideally occurs at 37 °C and is mediated by protein adsorption on the thermally collapsed and dehydrated polymer chains.5-9 During cell detachment, the reduction of the temperature to 20 °C induces a volume phase transition of the tethered polymer chains and thereby facilitates the release of single cells or confluent cell sheets.10-11 In contrast to conventional cell harvesting methods, such as trypsin treatment, intact confluent cell sheets can be harvested from thermoresponsive surfaces maintaining cell-cell junctions, cell surface proteins, cellular polarization, and secreted ECM. The cell sheets can be used as single cell sheets or can be layered to form 3D scaffold-free tissues.4 Cell sheets have been shown to be effective in regenerative medicine applications, such as cornea reconstruction12-13, prevention of esophageal stricture after tumor removal14, and recovery of cardiac function.15 It has also been demonstrated that cell sheet transplantation leads to a higher efficiency of cell delivery, a higher cell survival rate, and tissue functionality compared to injection of a single cell suspension.16-17 Aside from poly(N-isopropyl acrylamide) (PNIPAM) only a few thermoresponsive polymer coatings, such as poly(2-oxazoline)s18 and poly[tri(ethylene glycol) ethyl ether methacrylate]s19, do not require additional modification with cell-adhesive peptides or ligands to support cell adhesion.20-22 Thermoresponsive and biocompatible poly(glycidyl ether) monolayers on gold as a model substrate have demonstrated to be suitable coatings for cell culture applications.23-25 These coatings exhibit intrinsic cell-adhesive properties at 37 °C without further modification and allow cell sheet detachment at 20 °C. However, for a widespread application of functional poly(glycidyl ether) coatings based on glycidyl methyl ether (GME) and ethyl glycidyl ether (EGE), common cell culture substrates such as PS need to be addressed rather than gold surfaces. Diverse coating strategies have been applied for PNIPAM as the most prominent thermoresponsive polymer coating.4,

11, 26

Furthermore, extensive structural and mechanistic


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 34

studies have been performed with these coatings in order to generate an optimized cell response.10,

27-33

General design guidelines for thermoresponsive surface coatings with

application in cell culture are yet rare. Commercial thermoresponsive cell culture substrates are for example manufactured via electron beam (EB) polymerization of the monomer N-isopropyl acrylamide (NIPAM) onto TCPS substrates.10,

31-33

This yields structurally undefined coatings

due to a simultaneous, uncontrolled crosslinking process.32,

34

Further, a dry layer thickness

between 15 and 30 nm is required on such surfaces for effective cell adhesion and efficient cell sheet detachment.26, 32 In addition, EB polymerization requires expensive machinery and is prone to experimental variations.26 Alternative grafting-from techniques of NIPAM on plastic cell culture substrates include plasma polymerization and radical photo-grafting.35-37 The detriments of EB polymerization similarly apply to plasma polymerization.26,

35-37

In order to overcome

these limitations, physical adsorption processes of thermoresponsive polymers on surfaces have shown to be a potent alternative to conventional covalent coating methods.4,

11

Aside from

electrostatic coatings which are tethered to surfaces via coulombic forces38-39, adsorption techniques based on hydrophobic interactions offer a straight-forward, cost- and materialefficient, as well as substrate geometry-independent method for the functionalization of common cell culture substrates. For example, Ito and co-workers40-41 developed PNIPAM-block-poly[(R)3-hydroxybutyrate]-block-PNIPAM triblock-copolymers to coat polyethylene terephthalate (PET) based Thermanox™ coverslips. Through physical adsorption of the rather hydrophobic poly[(R)-3-hydroxybutyrate]-block onto the PET substrates, they obtained stable, homogeneous coatings. Polymer layers with dry thicknesses in the range of 4-8 nm allowed to culture and harvest both mouse embryonic stem cells and human mesenchymal stem cells. Nakayama et al.42 synthesized diblock-copolymers comprising a thermoresponsive PNIPAM block and a


4

Page 5 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


poly(butyl methacrylate) (PBMA) block using the controlled reversible addition-fragmentation chain-transfer polymerization. They spin-coated TCPS substrates with the diblock-copolymers and obtained stable, smooth layers with phase-separated PBMA and PNIPAM domains and dry thicknesses of 15 nm from which they detached bovine carotid endothelial cell (BAEC) sheets. Sakuma et al.43-44 coated hydrophobically modified glass substrates with Langmuir-Schaefer films comprising dodecyl-terminated PNIPAM or PS-PNIPAM block-copolymers and obtained stable coatings from which they also detached confluent BAEC sheets. In more recent studies, Kakimoto and co-workers45-46 used hyperbranched PS grafted with PNIPAM to coat commercial PS dishes via a physical adsorption process. They cultured mouse 3T3 fibroblasts on coated substrates and were able to detach intact cell sheets upon reducing the temperature. In a slightly different approach, Healy et al.47-48 used the NIPAM monomer together with the hydrophobic Ntert-butyl acrylamide (NtBAM) and the photoreactive acrylamide BP (AcBzPh) and synthesized statistical poly(NIPAM-co-NtBAM), poly(NIPAM-co-AcBzPh), and poly(NIPAM-co-NtBAMco-AcBzPh) copolymers via free radical polymerization. All polymers were adsorbed onto TCPS substrates to obtain NtBAM- and BP-stabilized multilayers. Only the BP-containing polymer layers were further covalently immobilized and crosslinked by irradiation with UV light to form gel layers with a dry thickness of about 20 nm. Both types of coatings were used to culture and detach confluent sheets of human pulmonary microvascular endothelial cells. Herein, we report on the synthesis of a novel BP-bearing glycidyl ether monomer which was copolymerized as a short, photoreactive anchor block on a thermoresponsive, random copolymer based on GME and EGE via the monomer-activated anionic ring-opening polymerization (AROP).49-51 The resulting thermoresponsive copolymer with a hydrophobic and photoreactive anchor block readily adsorbs onto PS substrates as an ultrathin layer (0.7 ± 0.1 nm) which can be


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 34

covalently immobilized by irradiation with UV light. The coated PS dishes were applied in cell culture in order to fabricate confluent sheets composed of human dermal fibroblasts with high viability within the harvested cell sheets. EXPERIMENTAL SECTION A detailed description of all materials applied and analytical methods used is given in the Supporting Information (SI). Synthesis of 4-[2-(2,3-epoxypropoxy)ethoxy]benzophenone (EEBP). 4-(2-hydroxyethoxy)benzophenone (HEBP) was synthesized from 4-hydroxybenzophenone and 2-bromoethanol according to literature with 47% yield.52 EEBP was synthesized based on a procedure reported by Jabeen et al. with slight modification.53 A solution of HEBP (7.8 g, 32.2 mmol) in epichlorohydrin (ECH, 50 mL, 644.4 mmol) was treated with solid NaOH (3.2 g, 80.5 mmol) at room temperature. The suspension was refluxed for 5 h and stirred at room temperature overnight. The residue was filtered off and washed with Et2O (20 mL). The solvent was evaporated and the remaining opalescent oil was dissolved in DCM (100 mL). The organic solution was washed with water (3 x 100 mL). The separated organic phase was dried over anhydrous Na2SO4, filtered and the solvent was removed under reduced pressure. The product was purified by silica column chromatography using a mixture of ethyl acetate and hexane as eluents (1:1 (v/v)). After solvent evaporation, a slightly yellow opalescent oil was obtained (yield: 7.1 g, 80%). Detailed characterization is given in the Supporting Information (SI). Synthesis of poly(GME-ran-EGE)-block-EEBP (PGE). Block-copolymers were synthesized by sequential monomer-activated anionic ring-opening polymerization according to literature using N(Oct)4Br as initiator and (i-Bu)3Al as activator.51 In brief, N(Oct)4Br (49.6 mg, 0.091


6

Page 7 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mmol) was melted and dried at 110 °C under high vacuum in a Schlenk-flask, subsequently flushed with argon and dissolved in freshly distilled, dry toluene (15 mL). The solution was cooled down to 0 °C using an ice bath and both monomers, GME (0.5 g, 5.7 mmol) and EGE (1.74 g, 17.0 mmol) for the random, thermoresponsive block, were added. The polymerization was started by fast addition of (i-Bu)3Al (1.1 M in toluene) (0.33 mL, 0.364 mmol) to the reaction mixture at 0 °C. After 10 min at 0 °C, a solution of EEBP in dry toluene (0.1 M, 4.6 mL, 0.46 mmol) was added to form the anchor block and the reaction was allowed to reach room temperature while stirring overnight. The reaction was quenched by addition of water (MilliQ, 2 mL). The mixture was vigorously stirred for 1 h and dried over Na2SO4. After filtration and concentration of the filtrate, the raw product was dissolved in Et2O (15 mL) in order to precipitate residual tetraoctylammonium salts. Therefore, the mixture was stored in the fridge overnight, centrifuged at 0 °C at 10000 rpm for 20 min with a Rotina 380 R centrifuge (Hettich GmbH & Co. KG, Tuttlingen, Germany), the supernatant was decanted and concentrated. For further purification, the concentrate was dialyzed in methanol (MWCO = 3.5 kDa) to remove remaining impurities. After concentrating under reduced pressure, a clear viscous oil was obtained (yield: 2.32 g, 94%). Detailed characterization is given in the Supporting Information (SI). Below, the thermoresponsive block-copolymer is referred to as PGE. Surface Preparation. In order to characterize the thermoresponsive coatings, PS-coated silicon wafers were used as a model substrate. Silicon wafers (11 x 11 mm) were rinsed with ethanol and dried under a stream of N2. The samples were spin-coated at 3000 rpm for 60 s on a WS650-23 spin-coater from Laurell Technologies Corporation (North Wales, PA, USA) applying 30 µL PS solution in toluene (0.5% (w/w)). Spin-coated samples were dried at ambient conditions overnight. The thickness and the contact angle (CA) of the PS layer were determined by


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 34

ellipsometry and static water CA measurements, respectively. The samples were kept in PS culture dishes (diameter 3.5 cm) and immersed in a solution of PGE in ethanol (0.01 mM, 2 mL) for 30 min. Subsequently, the surfaces were thoroughly washed with ethanol, dried under a stream of N2, and irradiated by UV light using a UV-KUB 2 from KLOÉ (Montpellier, France) with a wavelength of 365 nm and a radiant exposure of 4.0 J cm-2 for 160 s using an irradiance of 25 mW cm-2 (100%) in order to covalently immobilize the PGE layer on PS. After thoroughly washing with water (MilliQ) and ethanol, the coatings on silicon wafers were characterized by ellipsometry, static water CA, and atomic force microscopy (AFM) measurements. Bare PS surfaces treated with UV light and PGE-coated PS surfaces, which have not been treated with UV light, were used as control surfaces and characterized by ellipsometry, static water CA, and AFM measurements. PS and TCPS culture dishes (diameter 35 mm) and 24-well plates (PS) were coated with PGE as described above for PS-coated silicon wafers. After thoroughly washing with water (MilliQ) and ethanol, the dishes were used for culturing human dermal fibroblasts and the temperature-triggered detachment of the respective cell sheets. Cell isolation and culture. Dermal fibroblasts were isolated from human foreskin biopsies after ethical approval and informed parental consent. Connective tissue was mechanically removed and remaining skin tissue was dissected into approximately 1 x 10 mm stripes. They were incubated in dispase II (2 U mL-1 in PBS) for 16 h at 4 °C. Epidermis and dermis were mechanically separated followed by mincing of the dermis. Minced tissue was digested with collagenase NB4 (0.5 U mL-1 in PBS with Ca2+ and Mg2+) for 45 min at 37 °C. After centrifugation (5 min at 200 x g), the pellet was washed with culture medium consisting of DMEM supplemented with 10% FBS, 100 U mL-1 penicillin, and 100 µg mL-1 streptomycin. The tissue fragments were resuspended in 2 mL medium and seeded into a 75 cm² tissue culture


8

Page 9 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


flask. Additional 6 mL medium were added to the outgrowing cells after 24 h. Cells were cultured in a humidified atmosphere at 37 °C and 5% CO2 and used in passages 3 to 7. Adhesion and proliferation study. In adhesion and proliferation studies, 5x103 cells/cm2 were seeded on PGE-coated PS dishes in culture medium (DMEM with 10% FBS) for microscopical observations and 24-well plates for a resazurin-based PrestoBlue® viability assay. TCPS surfaces served as a control. PGE-coated surfaces were sterilized with 70% ethanol for 10 minutes and washed twice with PBS before cell seeding. Cells were observed microscopically every 24 h until confluency was reached. PrestoBlue® viability assay was performed according to the manufacturer’s instructions. Briefly, PrestoBlue® solution was diluted 1:10 with culture medium. Cells cultured on different substrates were incubated with 500 µL of the solution at 37 °C for 2 h. Control wells without cells containing only the diluted PrestoBlue® solution were included on each plate as a blank. Afterwards, the supernatant of each well was transferred in triplicates of 100 µL to a 96-well plate for absorbance measurements at 570 nm and 600 nm using an Infinite® M200 plate reader (Tecan, Maennedorf, Switzerland). Remaining solution was removed from the cells and fresh culture medium was added for further culture. For each well, the absorbance at 600 nm (reference wavelength) was subtracted from the absorbance at 570 nm. Afterwards, the average blank value was subtracted from each experimental well to obtain background-corrected data. Cell sheet fabrication. Experiments were performed with PGE-coated Falcon® PS culture dishes and bare Falcon® PS culture dishes. PGE-coated Corning® TCPS and bare Corning® TCPS dishes were used as controls. The dishes were sterilized with 70% ethanol for 10 minutes and washed twice with PBS before cell seeding. 1.6 x 105 cells cm-2 in 2 mL DMEM with 10% FBS, 100 U mL-1 penicillin, and 100 µg mL-1 streptomycin were seeded per dish (35 mm diameter),


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 34

followed by culture at 37 °C and 5% CO2 for 24 h. Cells were analyzed after 24 h via phase contrast microscopy. For temperature-triggered detachment, confluent cell cultures were incubated in PBS at 20 °C for 10 min followed by incubation in fresh PBS (37 °C) for 5 min at 37 °C. Afterwards, cultures were kept at room temperature (20 °C) until the cell sheets detached from the surfaces. Cell sheet detachment was observed and documented microscopically and visually. Viability assays. For live/dead staining of adherent cultures, cells were washed with PBS followed by incubation with 50 µM propidium iodide (PI) and 10 µM fluorescein diacetate in DMEM for 5 min at 37 °C. Samples were washed with PBS and imaged on a Zeiss Observer Z1 microscope in fluorescent mode with appropriate filter sets. For viability testing of detached cell sheets, they were trypsinized, centrifuged and resuspended in PBS. For staining of dead cells, 7.5 µM PI was added and incubated for 5 min. The samples were analyzed using a BD Accuri™ C6 Flow Cytometer (BD, Heidelberg, Germany). 10,000 events were measured per sample with a 488-nm laser and 585/40 emission filter. Statistical analysis. Statistical comparison of water CAs (Figure 1c) was performed using the unpaired t-test for two independent sample sets following a normal distribution (*: p < 0.05, **: p < 0.01, ***: p < 0.005). Adhesion forces determined by AFM nanoindentation (Figure 2b) were statistically compared using the nonparametric Mann-Whitney U test for two independent sample sets not following a normal distribution (*: p < 0.01, **: p < 0.005). Statistical significance of PrestoBlue® assay values was also determined using the Mann-Whitney U test. Differences with p < 0.05 were considered to be significant.


10

Page 11 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RESULTS Polymer Synthesis. Thermoresponsive block-copolymers based on glycidyl ethers were synthesized via the monomer-activated AROP. It was previously shown that, via this method, GME and EGE copolymerize in a truly random fashion to yield thermoresponsive polymers with adjustable molecular weights and narrow polydispersity indices (PDIs).51 Furthermore, the polymerization proceeds fast, thus after roughly 10 min at 0 °C and an activator to initiator ratio of 4, a copolymer of 20 kDa was obtained. By means of monolayers on gold, it was further demonstrated that surface-anchored thermoresponsive random copolymers with a monomer composition of 1:3 (GME/EGE) facilitate the fabrication of cell sheets.23-25 In order to covalently immobilize thermoresponsive glycidyl ether-based copolymers on more cell culture-relevant PS substrates, we synthesized a novel, photoreactive EEBP monomer (Scheme 1a). The latter was block-copolymerized with a thermoresponsive, random GME/EGE block via sequential addition of the EEBP monomer 10 min after the random GME/EGE copolymerization was initiated (Scheme 1b). After the addition of EEBP, the initially colorless solution turned to a bright yellow color, indicating complex formation between the activator (i-Bu)3Al and the carbonyl moiety of the EEBP monomer.54 The color slowly faded while stirring overnight and completely disappeared after quenching of the reaction with water. Unchanged chemical shifts of the EEBP signals with line-broadening in 1H- and

13

C-NMR spectra of the PGE block-copolymer clearly

indicate the successful grafting of the EEBP block onto the thermoresponsive copolymer block (Figure S1 and S2) without the occurrence of traceable side reactions.54 Based on 1H NMR and GPC measurements, a PGE consisting of a 25.8 kDa thermoresponsive block with a GME/EGE ratio of 1:2.8 and a 1 kDa EEBP-based anchor block was obtained. The targeted (Mn, theor. (GME/EGE) = 30 kDa and Mn, theor. (EEBP) = 1.5 kDa) and the experimentally obtained


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 34

(Mn, exp. = 26.8 kDa) molecular weight values of the synthesized PGE matched reasonably well and a narrow molecular weight distribution (PDI = 1.05) was obtained. The presence of the hydrophobic BP-anchor block does not induce aggregation of the PGE in ethanolic solution at 20 mg mL-1 as confirmed by DLS measurements at 20 °C (Figure S3).

Scheme 1. (a) Synthesis of a photoreactive, polymerizable glycidyl ether monomer 4-[2-(2,3-epoxypropoxy) ethoxy] benzophenone (EEBP); (b) Monomer-activated, sequential AROP of a block-copolymer (PGE) consisting of a thermoresponsive, random GME and EGE block and a short, photoreactive EEBP anchor block. Surface Functionalization. Untreated PS substrates were chosen over TCPS as a substrate for surface functionalization due to their hydrophobicity (CA~90°) and the fact that cell adhesion is not supported on these surfaces.55-57 Physical adsorption of PGE on hydrophobic surfaces is expected to be increased, owing to an enhanced hydrophobic interaction between the PGE and the substrates. In order to investigate the coating process, we monitored PGE adsorption over 30 min in real-time on PS-coated gold chips via QCM-D measurements. PGE was adsorbed from


12

Page 13 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dilute ethanolic solution (0.01 mM) under static conditions and the stability of the adsorbed polymer layer was determined by flushing with ethanol, which is an adequate solvent for PGE. Representative frequency and dissipation curves of the adsorption process are shown in Figure 1a. The average areal mass of adsorbed PGE was calculated via the Sauerbrey equation using the decline in frequency ∆f (green curve, Figure 1a) and was determined to be 136 ± 8 ng cm-2 (n = 3). The minor increase in dissipation ∆D (grey curve, Figure 1a) indicates the formation of a rigid PGE layer with negligible viscoelastic properties58, which was stable against flushing with ethanol even at maximum flow rates of 0.975 mL min-1 (data not shown). We further calculated the wet thickness of the adsorbed PGE layers to be 1.36 ± 0.08 nm (n = 3) from the adsorbed areal mass and the density of the coating. The latter was assumed as 1 g mL-1 corresponding to a 50% solvation of the adsorbed polymer with ethanol. The dry thickness and the CAs of the coatings were determined before and after UV irradiation on PS-coated silicon wafers and revealed no significant difference (Figure S4). Furthermore, neither the layer thickness nor the CA of bare PS substrates was changed when irradiated by UV light (Figure S4). We obtained a reproducible dry thickness of 0.7 ± 0.1 nm (n = 12) after UV irradiation of PGE-coated PS, which was in good agreement with QCM-D results and justified our assumption of a PGE layer solvation of 50% and a swelling ratio of 2 (Figure 1b). In addition, static water CA measurements showed a significant decrease in CA from 90 ± 2° (n = 12) for the bare PS to 70 ± 3° (n = 12) for the PGE-coated PS surfaces (Figure 1c).


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 34

Figure 1. (a) Representative frequency and dissipation curves of the PGE adsorption on spincoated gold chips measured by QCM-D (areal mass: 136 ± 8 ng cm-2, n = 3); (b) Dry PGE layer thickness measured by ellipsometry on PS-coated silicon wafers (n = 12, error bars indicate SEM) and wet PGE layer thickness measured by QCM-D (n = 3, error bars indicate SD); (c) Static water CA of PS-coated silicon wafers before and after adsorption and photoimmobilization of PGE (n = 12, error bars indicate SEM; t-test: ***: p < 0.005). In order to investigate the adsorption behavior of PGE on TCPS-like surfaces, adsorption experiments were performed on UV/ozone-treated substrates with static water CA of 60°. QCM-D measurements revealed significantly lower amounts of adsorbed PGE on TCPS-like substrates compared to hydrophobic PS substrates with an average areal mass of 66 ± 13 ng cm-2 (n = 3) and a solvated layer thickness of 0.66 ± 0.13 nm (n = 3) (Figure S5a). The dry layer thickness and the water CA of photo-immobilized PGE coatings on TCPS-like surfaces were determined to be 0.56 ± 0.46 nm (n = 3) and 62 ± 1° (n = 3), respectively. The high relative standard deviation for the measured mean layer thickness might be attributed to the roughness of the underlying ozone treated substrate.


14

Page 15 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


AFM. Three different AFM methods were applied to verify the functionalization of PS substrates with PGE coatings. First, the PeakForce QNM imaging technique, which is schematically described in Figure S6, was used to record images of the surface topography in MilliQ water. The morphology and roughness of the bare and PGE-coated PS surfaces were compared at 37 and 20 °C. As illustrated by the AFM topography images in Figure 2a, the roughness of the PGE-coated surface at 20 °C is slightly higher compared to the bare PS surface, supporting a successful functionalization of the PS substrates with a thin PGE layer. The morphology of the coated surfaces was also investigated at 37 °C (Figure S7) and revealed a similar roughness at this temperature. The root mean squared (Rq), average (Ra) and maximum peak to valley (Rmax) roughness of the bare PS at 20 °C and the PGE-coated PS at 37 and 20 °C in MilliQ water are summarized in Table S2. In the second approach, surface interaction forces between a blunt AFM tip and the PGE-coated and bare PS surfaces were investigated by nanoindentation measurements using approachretraction cycles at 37 and 20 °C in MilliQ water. Analysis of the obtained force-distance curves allowed to monitor the adhesion forces between the AFM tip and the surfaces. Through consecutive approach-retraction cycles, force-distance curves were obtained with which attractive or repulsive long- and short-range interactions can be directly discriminated. Measurements revealed significantly higher adhesion forces between the silicon nitride tips and the bare PS surface compared to the adhesion forces between the tip and the PGE-coated PS surface (Figure 2b) at both 37 and 20 °C. Representative force-distance curves recorded at 37 and 20 °C are shown in Figure 2c and 2d, respectively. As illustrated, higher adhesion forces were measured on bare PS substrates (blue curves, Figure 2c-d), whereas they were drastically reduced on PGE-coated surfaces (green curves, Figure 2c-d). Further, at both temperatures long


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 34

range repulsive forces of about 200 nm in length were observed in the approach curves before the tip contacted the PS surfaces (dark grey curves, Figure 2c-d). In contrast, the repulsive forces in the approach curves are markedly reduced on PGE-coated PS substrates (light grey curves, Figure 2c-d), which indicates the screening of the repulsive force by the PGE coating. In addition, measurements in PBS at both temperatures showed that the long range repulsive interactions upon the approaching AFM tip can be effectively screened by the addition of salt, whereas adhesion forces remained unaffected (data not shown).

Figure 2. AFM measurements on PS and PGE-coated, photo-immobilized PS (a) 3D images in MilliQ water at 20 °C; (b) Adhesion forces of a silicon nitride tip with the surfaces in MilliQ


16

Page 17 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


water at 37 and 20 °C (n = 3, error bars indicate SEM; Mann-Whitney U test: *: p < 0.01, **: p < 0.005); (c-d) Representative force-distance curves at 37 (c) and 20 °C (d). Finally, the material properties of the surfaces in the dry state were investigated at large indentation forces in order to measure the surface morphology, elastic (Young’s) modulus, deformation and adhesion via PeakForce QNM. Maps for surface height (Figure S7a,b), elastic modulus (Figure S7d,e), induced deformation (Figure S7g,h), and adhesion force (Figure S7j,k) were obtained and compared between bare and PGE-coated PS substrates. The distribution values obtained from the analysis of the parameter maps via the depth tool all show measurable differences between the PGE-coated and the bare PS substrates (Figure S7c,f,i,l). A detailed description of the material properties of PGE-coated and bare PS is given in the Supporting Information (SI). Non-specific Protein Adsorption. Since cell adhesion is generally mediated through the adsorption of cell-adhesive proteins, such as fibronectin and vitronectin, we investigated the nonspecific protein adsorption on bare PS and photo-immobilized, PGE-coated PS surfaces simulating cell culture conditions. Therefore, protein adsorption from DMEM (10% FBS, high glucose) at 37 °C was determined via QCM-D measurements on bare PS, PGE-coated PS, and PGE-coated TCPS-like substrates. Representative frequency curves revealed the adsorption of similar amounts of proteins on both, bare PS (blue curve, Figure 3a) and PGE-coated PS (green curve, Figure 3b) substrates. However, the steady decrease in frequency during protein adsorption on bare PS surfaces showed a constantly increasing amount of adsorbing proteins on the surface, which does not reach an equilibrium state within 20 minutes. This indicates either multilayer formation or protein exchange and rearrangement on the surface, which is known as the Vroman effect. In contrast, on PGE-coated PS substrates, an equilibrium state in protein


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 34

adsorption was quickly reached after exposure to DMEM containing FBS within 10 min. Furthermore, the stronger decrease in dissipation during flushing of the PGE-coated surface after protein exposure with PBS indicated the formation of a less elastic, more densely packed protein layer on the PGE coatings (grey curve, Figure 3b) compared to the bare PS substrates (grey curve, Figure 3a). Interestingly, protein adsorption on PGE-coated TCPS-like surfaces was significantly higher (728 ± 49 ng cm-2, n = 3) than on bare and PGE-coated PS substrates (Figure S5b).

Figure 3. Protein adsorption from high glucose DMEM containing 10% FBS at 37 °C (a) on bare PS (areal mass: 359 ± 25 ng cm-2, n = 3) and (b) on PGE-coated PS (areal mass: 432 ± 96 ng cm-2, n = 3). Cell Adhesion and Proliferation. Human dermal fibroblast adhesion and proliferation on PGEcoated PS was evaluated and compared to the TCPS control. Therefore, 5 x 10³ cells cm-² were seeded on these surfaces. Cultures were investigated microscopically and by a PrestoBlue® viability assay. Microscopical observations showed comparable adhesion and growth on PGEcoated PS and TCPS control. Cells reached confluence within 4 days (Figure 4a). Accordingly,


18

Page 19 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the PrestoBlue® assay also showed comparable metabolic activity of cells grown on both surfaces without significant differences (Figure 4b).

Figure 4. Adhesion and proliferation of human dermal fibroblasts on PGE-coated PS and on TCPS control. (a) Microscopic phase contrast images (scale bar = 100 µm) and (b) PrestoBlue® Assay. Values are displayed as mean ± SEM (n = 3). No significant differences between cultures grown on PGE-coated PS and TCPS were observed. Cell Sheet Fabrication and Viability Assessment. Human dermal fibroblasts were cultured on bare PS and PGE-coated PS culture dishes. Bare TCPS and PGE-coated TCPS dishes were used


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 34

as controls. To obtain confluent cell sheets within 24 h, human dermal fibroblasts were seeded at a high density of 1.6 x 105 cells cm-2. Accordingly, cells reached confluency on the PGE-coated PS and both, PGE-coated and non-coated TCPS controls after 24 h in culture at 37 °C (Figure 5a, c, d). In contrast, fibroblasts hardly attached and thus did not form confluent cell layers on the bare PS dishes (Figure 5b). After reducing the temperature from 37 to 20 °C, intact cell sheets were only harvested from the PGE-coated PS surfaces within 51 ± 17 min (n = 7, Figure 5a). No cell sheet detachment was observed on both TCPS controls (Figure 5c, d).

Figure 5. Representative phase contrast images of human dermal fibroblasts 24 h after seeding on (a) PS culture dishes coated with PGE, (b) bare PS dishes, (c) TCPS dishes coated with PGE, and (d) bare TCPS. Underneath the microscopy images, representative macroscopic photographs of the respective culture dishes are shown after temperature reduction to 20 °C. Only PGEcoated PS dishes released cell sheets when triggered thermally (n = 7). In order to assess the cell viability of fibroblasts grown on PGE-coated PS dishes, the obtained confluent cell layers after 24 h culture were subjected to a fluorescent live/dead staining without


20

Page 21 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


detaching them from the substrate. As illustrated in Figure 6, a confluent layer of living cells was obtained on the PGE-coated PS surfaces. Comparable results were obtained from confluent cell layers cultured on the TCPS control.

Figure 6. Representative fluorescent images of live/dead stained human dermal fibroblasts cultured for 24 h on PGE-coated PS and a TCPS control (n = 3). A confluent layer of living cells was obtained on both surfaces. Adherent cells were stained with fluorescein diacetate (green) and propidium iodide (PI, red) (scale bar = 50 µm). The viability of fibroblasts after thermal detachment from the surface was further quantified using flow cytometry. Therefore, cell sheets harvested from thermoresponsive PGE-coated substrates via the temperature-trigger were singularized via conventional trypsinization and compared to cells grown and conventionally trypsinized from TCPS controls. Flow cytometry analysis after PI staining revealed similar viabilities with both harvesting methods (Figure 7).


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 34

Figure 7. Quantitative viability assessment of thermally detached cells from PGE coatings (a) and conventionally harvested cells from TCPS (b), respectively. Dead cells were discriminated from living cells by staining with PI and counted by flow cytometry. Displayed are the mean percentages of PI-positive dead cells (n = 4, error is given as SD).

DISCUSSION The sequential copolymerization of a mixture of GME and EGE with the novel glycidyl ether monomer

EEBP

yielded

a

block-copolymer

comprising

a

long-chained,

random,

thermoresponsive and a short-chained, hydrophobic, and photoreactive anchor block (Scheme 1). Due to the living nature of the polymerization, the molecular weight is adjustable by the monomer to initiator ratio (Mn, exp. = 26.8 kDa; Mn,

theor.

= 31.5 kDa) and the obtained

polydispersity is low (PDI = 1.05). Combined NMR and GPC analysis of the PGE blockcopolymer revealed a GME to EGE ratio of 1:2.8 with overall 270 repeating units in the thermoresponsive block and 3.3 instead of the intended 5 EEBP repeating units in the anchor block. We attributed this deviation to the sterically demanding EEBP side group, which


22

Page 23 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


potentially prevents further growth of the polymer chain. Transalkylation as a common side reaction of the carbonyl group of EEBP with the activator triisobutyl aluminum has not been observed under the applied block-copolymerization conditions.54 However, it has to be noted that increasing the relative amount of activator in this polymerization leads to a partial transalkylation of the BP moiety, which renders the EEBP block unreactive towards C, H -insertion upon UV irradiation. Spontaneous adsorption of PGE from dilute ethanolic solution onto PS substrates via hydrophobic interactions is material-efficient and independent on the substrates’ geometry in comparison to, e.g., spin-coating. The formed polymeric layer is tightly bound to the PS substrates since ethanol or PBS washing under sheer stress cannot remove the physisorbed polymer. Monitoring the adsorption process in real-time by QCM-D measurements indicated the formation of an ultrathin PGE layer (Figure 2a). A highly reproducible average dry thickness of 0.7 ± 0.1 nm (Figure 2b) and a significant reduction in water CA from 90 ± 2° to 70 ± 3° (Figure 2c) substantiated these findings. The PGE coatings were covalently immobilized onto the substrate via a radical C, H-insertion mechanism initiated by short exposure to UV light of 365 nm which is in the absorption range of BP.59-62 Although we cannot prove experimentally that a covalent bond is formed between PGE and PS, which is due to the very low surface concentration of BP groups in the ultrathin PGE layers, numerous reports in literature have shown analogous reactions of BP derivatives with PS.60, 62-65 DLS measurements of the blockcopolymer in ethanol indicated no aggregation due to the presence of the hydrophobic BP block (Table S1, Figure S3). The measured solvated diameter (Z-average: 10 ± 0.4 nm) is located in a similar size range of a comparable thermoresponsive poly(GME-ran-EGE) copolymer (Zaverage: 9 ± 0.4 nm) without a BP anchor block.51 Adsorption experiments of PGE on a TCPS-


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 34

like PS substrate, which was treated with UV/ozone prior to coating, revealed lower amounts of adsorbed polymer on the surface (Figure S5a), which demonstrates the hydrophobically-driven nature of the adsorption process. AFM PeakForce QNM imaging in MilliQ water at 37 and 20 °C confirmed the presence of a homogeneous PGE layer due to a higher surface roughness of PGE-coated compared to the bare PS substrates (Table S2, Figure S7). Furthermore, nanoindentation measurements in MilliQ water revealed substantially lower adhesion forces between AFM tips and PGE-coated surfaces compared to bare PS surfaces, indicating a homogeneous coverage of the PS substrates by PGE (Figure 2b-d). The hydrophobic nature of PS and its adhesion strength with AFM tips has already been reported in literature and is consistent with the results obtained in our study.66 Compared to the bare PS substrates, the lower adhesion and long range repulsive forces between the AFM tips and the PGE layers can be explained by decreasing hydrophobic interactions between the PGEcoated surfaces and the AFM tip66 compared to the bare PS and the shielding of the characteristic electrostatic double layer at the PS-water interface by the ultrathin PGE coating67, respectively (Figure 2b-c). Together with slightly different surface properties of PGE-coated PS compared to bare PS substrates in the dry state (Figure S8-S9), these findings demonstrate the effectiveness of the employed functionalization method. The non-specific FBS protein adsorption from cell culture medium on bare PS and PGE-coated substrates was investigated by QCM-D measurements and revealed similar amounts of adsorbed proteins of 359 ± 25 ng cm-2 and 432 ± 96 ng cm-2, respectively (Figure 3). However, the adsorption kinetics indicate a faster protein adsorption on PGE-coated substrates, which reaches equilibrium within 10 min, as opposed to the steadily increasing protein adsorption on bare PS substrates which indicates multilayer formation or protein exchange. In contrast, FBS protein


24

Page 25 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


adsorption on PGE-coated TCPS-like substrates (Figure S5b) was noticeably higher (areal mass: 728 ± 49 ng cm-2) than on PGE-coated and bare PS substrates. This might be a consequence of insufficient coverage of the TCPS-like substrate with adsorbed PGE, which is indicated by a significantly lower PGE layer thickness (~0.5 nm) and is also reflected by the unchanged water CAs of TCPS before and after PGE coating (60 ± 1 and 62 ± 1°, respectively). Insufficient surface coverage with PGE might permit additional protein adsorption directly onto the underlying TCPS-like substrate. Despite the differences in the amount and the kinetics of protein adsorption between PGE-coated PS and bare TCPS, human dermal fibroblast adhesion and proliferation are comparable according to a PrestoBlue® viability assay (Figure 4). For cell sheet fabrication on PGE-coated and bare PS substrates a high seeding density of 1.6x105 cells cm-² and short culture time of 24 hours were chosen because this turned out to result in effective cell sheet detachment in preliminary experiments with human dermal fibroblasts. We hypothesize that this is caused by the fact that cell-substrate contacts are still weak which facilitates the temperature-triggered detachment from the thermoresponsive surfaces. Moreover, the short culture time enables a fast generation of cell sheets for further use. In order to demonstrate the functionality of the thermoresponsive surfaces, the temperaturetriggered cell sheet detachment was investigated by replacing medium against PBS at room temperature. In addition to PGE-coated PS and bare TCPS, bare PS and PGE-coated TCPS were included in these experiments. In contrast to bare PS on which cells did not grow, cells adhered on both, PGE-coated PS and TCPS surfaces, in a similar manner as on conventional bare TCPS surfaces. The weak cell-adhesiveness of bare PS might be attributed to the adsorption of cellrepellent BSA, the major component of FBS, which was shown in literature to primarily adsorb


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 34

on hydrophobic surfaces.68 In contrast, cell adhesion-promoting proteins such as fibronectin rather adsorb on more hydrophilic substrates68, such as our PGE-coated PS or TCPS. Intact cell sheets with viabilities > 97% were reproducibly harvested from PGE-coated PS surfaces within 51 ± 17 min, whereas no cell sheet detachment was observed on conventional, non-coated or PGE-coated TCPS (Figure 5e-h). The failure to detach cells from PGE-coated TCPS is presumably caused by the cell-adhesiveness of the exposed hydrophilic background of TCPS as well as the lower amounts of adsorbed PGE. Both hamper the temperature-triggered detachment of the cell sheets.69 Reversely, the rather cell repellant properties of the bare PS substrates are likely to assist the detachment of confluent cell sheets from PS substrates comprising ultrathin PGE layers. This displays the importance of the interplay between thermoresponsive coatings and the substrate properties, such as hydrophobicity, elasticity, and porosity, with respect to cell sheet fabrication. Fluorescent live/dead staining of adherent and confluent human dermal fibroblasts, which were cultured for 24 h on PGE-coated PS and TCPS controls, did not reveal any toxicity of the PGE coating as comparable viabilities of the cells on both dishes were detected (Figure 6). Additionally, flow cytometry analysis of thermally harvested and subsequently singularized fibroblasts and conventionally harvested fibroblasts indicated no difference in cell viability supporting the cytocompatibility of the thermal harvesting method for human dermal fibroblasts (Figure 7).


26

Page 27 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CONCLUSION For the first time, the modification of a conventional plastic surface with a thermoresponsive poly(glycidyl ether) was reported for application in cell sheet fabrication. PGE block-copolymers bearing a short photoreactive BP anchor block were physically adsorbed and covalently immobilized onto PS dishes via a straight forward, material-efficient method, which is independent of the geometry of the substrate. This is particularly useful for many plastic cell culture vessels such as culture flasks, multiwell plates, and dishes. Without the need of cell adhesion-promoting ligands, the ultrathin PGE coatings support the attachment and growth of human dermal fibroblasts under standard cell culture conditions at 37 °C similar to TCPS surfaces. Further, the coatings allow the temperature-triggered detachment of confluent cell sheets at 20 °C with similar viability to cells harvested via conventional enzymatic treatment from TCPS surfaces. The apparent cooperative interplay between the ultrathin, thermoresponsive PGE coatings and the hydrophobic PS substrates during cell sheet detachment might help to elucidate the underlying mechanisms involved in the detachment of cell sheets and advance the discussion on general material design guidelines for thermoresponsive polymer coatings. In order to further improve the performance of poly(glycidyl ether) based coatings on plastic surfaces in terms of a faster cell sheet detachment and a broadened applicability to various other cell types, further structural optimization of poly(glycidyl ether) coatings on cell culture-relevant substrates are currently under investigation.


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 34

SUPPORTING INFORMATION Detailed description of materials and methods used, 1H and 13C NMR spectra of EEBP and PGE, DLS measurements, QCM-D measurements of PGE adsorption on TCPS-like substrates and protein adsorption from cell culture medium at 37 °C, investigation of surface morphology and material properties by AFM in PeakForce QNM mode.

ACKNOWLEDGEMENTS The authors are grateful to Prof. Dr. Sarah Hedtrich from the Institute of Pharmacy (FU Berlin) for cooperation. J. Scholz is kindly acknowledged for cell viability assessment. M.W. is grateful to financial support from the Federal Ministry of Education and Research through grant FKZ: 13N13523. REFERENCES (1) Elloumi-Hannachi, I.; Yamato, M.; Okano, T., Cell Sheet Engineering: A Unique Nanotechnology for Scaffold-Free Tissue Reconstruction with Clinical Applications in Regenerative Medicine. J. Intern. Med. 2009, 267, 54-70, DOI: 10.1111/j.13652796.2009.02185.x (2) Yamato, M.; Okano, T., Cell Sheet Engineering. Mater. Today 2004, 7, 42-47, DOI: 10.1016/S1369-7021(04)00234-2 (3) Matsuda, N.; Shimizu, T.; Yamato, M.; Okano, T., Tissue Engineering Based on Cell Sheet Technology. Adv. Mater. 2007, 19, 3089-3099, DOI: 10.1002/adma.200701978 (4) Tang, Z.; Okano, T., Recent Development of Temperature-Responsive Surfaces and Their Application for Cell Sheet Engineering. Regener. Biomater. 2014, 1, 91-102, DOI: 10.1093/rb/rbu011 (5) Yim, H.; Kent, M. S.; Satija, S.; Mendez, S.; Balamurugan, S. S.; Balamurugan, S.; Lopez, G. P., Study of the Conformational Change of Poly(N-Isopropyl-Acrylamide)-Grafted Chains in Water with Neutron Reflection: Molecular Weight Dependence at High Grafting Density. J. Polym. Sci., Part B: Polym. Phys. 2004, 42, 3302-3310, DOI: 10.1002/polb.20169 (6) Choi, S.; Choi, B.-C.; Xue, C.; Leckband, D. E., Protein Adsorption Mechanisms Determine the Efficiency of Thermally Controlled Cell Adhesion on Poly(N‑Isopropyl Acrylamide) Brushes. Biomacromolecules 2013, 14, 92-100, DOI: 10.1021/bm301390q


28

Page 29 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(7) Plunkett, K. N.; Zhu, X.; Moore, J. S.; Leckband, D. E., PNIPAM Chain Collapse Depends on the Molecular Weight and Grafting Density. Langmuir 2006, 22, 4259-4266, DOI: 10.1021/la0531502 (8) Xue, C.; Choi, B.-C.; Choi, S.; Braun, P. V.; Leckband, D. E., Protein Adsorption Modes Determine Reversible Cell Attachment on Poly( N-Isopropyl Acrylamide) Brushes. Adv. Funct. Mater. 2012, 22, 2394-2401, DOI: 10.1002/adfm.201103056 (9) Xue, C.; Yonet-Tanyeri, N.; Brouette, N.; Sferrazza, M.; Braun, P. V.; Leckband, D. E., Protein Adsorption on Poly(N-Isopropylacrylamide) Brushes: Dependence on Grafting Density and Chain Collapse. Langmuir 2011, 27, 8810-8818, DOI: 10.1021/la2001909 (10) Yamada, N.; Okano, T.; Sakai, H.; Karikusa, F.; Sawasaki, Y.; Sakurai, Y., ThermoResponsive Polymeric Surfaces; Control of Attachment and Detachment of Cultured Cells. Marcomol. Chem., Rapid Commun. 1990, 11, 571-576, DOI: 10.1002/marc.1990.030111109 (11) Tang, Z.; Akiyama, Y.; Okano, T., Recent Development of Temperature-Responsive Cell Culture Surface Using Poly(N-Isopropylacrylamide). J. Polym. Sci., Part B: Polym. Phys. 2014, 52, 917-926, DOI: 10.1002/polb.23512 (12) Burillon, C.; Huot, L.; Justin, V.; Nataf, S.; Chapuis, F.; Decullier, E.; Damour, O., Cultured Autologous Oral Mucosal Epithelial Cell Sheet (Caomecs) Transplantation for the Treatment of Corneal Limbal Epithelial Stem Cell Deficiency. Invest. Ophthalmol. Visual Sci. 2012, 53, 1325-1331, DOI:10.1167/iovs.11-7744 (13) Nishida, K.; Yamato, M.; Hayashida, Y.; Watanabe, K.; Yamamoto, K.; Adachi, E.; Nagai, S.; Kikuchi, A.; Maeda, N.; Watanabe, H.; Okano, T.; Tano, Y., Corneal Reconstruction with Tissue-Engineered Cell Sheets Composed of Autologous Oral Mucosal Epithelium. N. Engl. J. Med. 2004, 351, 1187-1196, DOI: 10.1056/NEJMoa040455 (14) Ohki, T.; Yamato, M.; Ota, M.; Takagi, R.; Murakami, D.; Kondo, M.; Sasaki, R.; Namiki, H.; Okano, T.; Yamamoto, M., Prevention of Esophageal Stricture after Endoscopic Submucosal Dissection Using Tissue-Engineered Cell Sheets. Gastroenterology 2012, 143, 582588, DOI: 10.1053/j.gastro.2012.04.050 (15) Sawa, Y.; Miyagawa, S.; Sakaguchi, T.; Fujita, T.; Matsuyama, A.; Saito, A.; Shimizu, T.; Okano, T., Tissue Engineered Myoblast Sheets Improved Cardiac Function Sufficiently to Discontinue Lvas in a Patient with Dcm: Report of a Case. Surg. Today 2012, 42, 181-184, DOI: 10.1007/s00595-011-0106-4 (16) Memon, I. A.; Sawa, Y.; Fukushima, N.; Matsumiya, G.; Miyagawa, S.; Taketani, S.; Sakakida, S. K.; Kondoh, H.; Aleshin, A. N.; Shimizu, T.; Okano, T.; Matsuda, H., Repair of Impaired Myocardium by Means of Implantation of Engineered Autologous Myoblast Sheets. J. Thorac. Cardiovasc. Surg. 2005, 130, 1333-1341, DOI: 10.1016/j.jtcvs.2005.07.023 (17) Takeuchi, R.; Kuruma, Y.; Sekine, H.; Dobashi, I.; Yamato, M.; Umezu, M.; Shimizu, T.; Okano, T., In Vivo Vascularization of Cell Sheets Provided Better Long-Term Tissue Survival Than Injection of Cell Suspension. J. Tissue Eng. Regener. Med. 2016, 10, 700-710, DOI: 10.1002/term.1854 (18) Dworak, A.; Utrata-Wesołek, A.; Oleszko, N.; Wałach, W.; Trzebicka, B.; Anioł, J.; Sieroń, A. L.; Klama-Baryła, A.; M, K., Poly(2-Substituted-2-Oxazoline) Surfaces for Dermal Fibroblasts Adhesion and Detachment. J. Mater. Sci. Mater. Med. 2014, 25, 1149-1163, DOI: 10.1007/s10856-013-5135-7 (19) Dworak, A.; Utrata-Wesołek, A.; Szweda, D.; Kowalczuk, A.; Trzebicka, B.; Anioł, J.; Sieroń, A. L.; Klama-Baryła, A.; Kawecki, M., Poly[Tri(Ethylene Glycol) Ethyl Ether


29


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 34

Methacrylate]-Coated Surfaces for Controlled Fibroblasts Culturing. ACS Appl. Mater. Interfaces 2013, 5, 2197-2207, DOI: 10.1021/am3031882 (20) Teichmann, J.; Nitschke, M.; Pette, D.; Valtink, M.; Gramm, S.; Härtel, F. V.; Noll, T.; Funk, R. H. W.; Engelmann, K.; Werner, C., Thermo-Responsive Cell Culture Carriers Based on Poly(Vinyl Methyl Ether)—the Effect of Biomolecular Ligands to Balance Cell Adhesion and Stimulated Detachment. Sci. Technol. Adv. Mater. 2015, 16, 045003 (045013pp), DOI: 10.1088/1468-6996/16/4/045003 (21) Teichmann, J.; Valtink, M.; Gramm, S.; Nitschke, M.; Werner, C.; Funk, R. H.; Engelmann, K., Human Corneal Endothelial Cell Sheets for Transplantation: ThermoResponsive Cell Culture Carriers to Meet Cell-Specific Requirements. Acta Biomater. 2013, 9, 5031-5039, DOI: 10.1016/j.actbio.2012.10.023 (22) Uhlig, K.; Boerner, H. G.; Wischerhoff, E.; Lutz, J.-F.; Jaeger, M. S.; Laschewsky, A.; Duschl, C., On the Interaction of Adherent Cells with Thermoresponsive Polymer Coatings. Polymers 2014, 6, 1164-1177, DOI: 10.3390/polym6041164 (23) Weinhart, M.; Becherer, T.; Haag, R., Switchable, Biocompatible Surfaces Based on Glycerol Copolymers. Chem. Commun. 2011, 47, 1553-1555, DOI: 10.1039/c0cc04002a (24) Becherer, T.; Heinen, S.; Wei, Q.; Haag, R.; Weinhart, M., In-Depth Analysis of Switchable Glycerol Based Polymeric Coatings for Cell Sheet Engineering. Acta Biomater. 2015, 25, 43-55, DOI: 10.1016/j.actbio.2015.06.036 (25) Heinen, S.; Cuellar-Camacho, J. L.; Weinhart, M., Thermoresponsive Poly(Glycidyl Ether) Brushes on Gold: Surface Engineering Parameters and Their Implication for Cell Sheet Fabrication. Acta Biomater. 2017, in press, DOI: 10.1016/j.actbio.2017.06.029 (26) Nash, M. E.; Healy, D.; Carroll, W. M.; Elviraa, C.; Rochev, Y. A., Cell and Cell Sheet Recovery from Pnipam Coatings; Motivation and History to Present Day Approaches. J. Mater. Chem. 2012, 22, 19376-19389, DOI: 10.1039/c2jm31748f (27) Kobayashi, J.; Okano, T., Thermoresponsive Thin Hydrogel-Grafted Surfaces for Biomedical Applications. React. Funct. Polym. 2013, 73, 939-944, DOI: 10.1016/j.reactfunctpolym.2013.01.017 (28) Yamato, M.; Konno, C.; Kushida, A.; Hirose, M.; Utsumi, M.; Kikuchi, A.; Okano, T., Release of Adsorbed Fibronectin from Temperature-Responsive Culture Surfaces Requires Cellular Activity. Biomaterials 2000, 21, 981-986, DOI: 10.1016/S0142-9612(99)00239-2 (29) Yamato, M.; Okuhara, M.; Karikusa, F.; Kikuchi, A.; Sakurai, Y.; Okano, T., Signal Transduction and Cytoskeletal Reorganization Are Required for Cell Detachment from Cell Culture Surfaces Grafted with a Temperature-Responsive Polymer. J. Biomed. Mater. Res. 1999, 44, 44-52, DOI: 10.1002/(SICI)1097-4636(199901)44:13.0.CO;2-X (30) Okano, T.; Yamada, N.; Okuhara, M.; Sakai, H.; Sakurai, Y., Mechanism of Cell Detachment from Temperature-Modulated, Hydrophilic-Hydrophobic Polymer Surfaces. Biomaterials 1995, 16, 297-303, DOI: 10.1016/0142-9612(95)93257-E (31) Fukumori, K.; Akiyama, Y.; Yamato, M.; Kobayashi, J.; Sakai, K.; Okano, T., Temperature-Responsive Glass Coverslips with an Ultrathin Poly(N-Isopropylacrylamide) Layer. Acta Biomater. 2009, 5, 470-476, DOI: 10.1016/j.actbio.2008.06.018 (32) Fukumori, K.; Akiyama, Y.; Kumashiro, Y.; Kobayashi, J.; Yamato, M.; Sakai, K.; Okano, T., Characterization of Ultra-Thin Temperature-Responsive Polymer Layer and Its Polymer Thickness Dependency on Cell Attachment/Detachment Properties. Macromol. Biosci. 2010, 10, 1117-1129, DOI: 10.1002/mabi.201000043


30

Page 31 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(33) Akiyama, Y.; Kikuchi, A.; Yamato, M.; Okano, T., Ultrathin Poly(NIsopropylacrylamide) Grafted Layer on Polystyrene Surfaces for Cell Adhesion/Detachment Control. Langmuir 2004, 20, 5506-5511, DOI: 10.1021/la036139f (34) Akiyama, Y.; Kikuchi, A.; Yamato, M.; Okano, T., Accelerated Cell-Sheet Recovery from a Surface Successively Grafted with Polyacrylamide and Poly(N-Isopropylacrylamide). Acta Biomater. 2014, 10, 3398-3408, DOI: 10.1016/j.actbio.2014.03.024 (35) Canavan, H. E.; Graham, D. J.; Cheng, X.; Ratner, B. D.; Castner, D. G., Comparison of Native Extracellular Matrix with Adsorbed Protein Films Using Secondary Ion Mass Spectrometry. Langmuir 2007, 23, 50-56, DOI: 10.1021/la062330o (36) Canavan, H. E.; Cheng, X.; Graham, D. J.; Ratner, B. D.; Castner, D. G., Surface Characterization of the Extracellular Matrix Remaining after Cell Detachment from a Thermoresponsive Polymer. Langmuir 2005, 21, 1949-1955, DOI: 10.1021/la048546c (37) Pan, Y. V.; Wesley, R. A.; Luginbuhl, R.; Denton, D. D.; Ratner, B. D., Plasma Polymerized N-Isopropylacrylamide: Synthesis and Characterization of a Smart Thermally Responsive Coating. Biomacromolecules 2001, 2, 32-36, DOI: 10.1021/bm0000642 (38) Liao, T.; Moussallem, M. D.; Kim, J.; Schlenoff, J. B.; Ma, T., N-IsopropylacrylamideBased Thermoresponsive Polyelectrolyte Multilayer Films for Human Mesenchymal Stem Cell Expansion. Biotechnol. Prog. 2010, 26, 1705-1713, DOI: 10.1002/btpr.471 (39) Schmidt, S.; Zeiser, M.; Hellweg, T.; Duschl, C.; Fery, A.; Möhwald, H., Adhesion and Mechanical Properties of PNIPAM Microgel Films and Their Potential Use as Switchable Cell Culture Substrates. Adv. Funct. Mater. 2010, 20, 3235-3243, DOI: 10.1002/adfm.201000730 (40) Loh, X. J.; Cheong, W. C. D.; Li, J.; Ito, Y., Novel Poly(N-Isopropylacrylamide)Poly[(R)-3-Hydroxybutyrate]-Poly(N-Isopropylacrylamide) Triblock Copolymer Surface as a Culture Substrate for Human Mesenchymal Stem Cells. Soft Matter 2009, 5, 2937-2946, DOI: 10.1039/b904171k (41) Loh, X. J.; Gong, J.; Sakuragi, M.; Kitajima, T.; Liu, M.; Li, J.; Ito, Y., Surface Coating with a Thermoresponsive Copolymer for the Culture and Non-Enzymatic Recovery of Mouse Embryonic Stem Cells. Macromol. Biosci. 2009, 9, 1069-1079, DOI: 10.1002/mabi.200900081 (42) Nakayama, M.; Yamada, N.; Kumashiro, Y.; Kanazawa, H.; Yamato, M.; Okano, T., Thermoresponsive Poly(N-Isopropylacrylamide)-Based Block Copolymer Coating for Optimizing Cell Sheet Fabrication. Macromol. Biosci. 2012, 12, 751-760, DOI: 10.1002/mabi.201200018 (43) Sakuma, M.; Kumashiro, Y.; Nakayama, M.; Tanaka, N.; Umemura, K.; Yamato, M.; Okano, T., Control of Cell Adhesion and Detachment on Langmuir-Schaefer Surface Composed of Dodecyl-Terminated Thermo-Responsive Polymers. J. Biomater. Sci. Polym. Ed. 2014, 25, 431-443, DOI: 10.1080/09205063.2013.866761 (44) Sakuma, M.; Kumashiro, Y.; Nakayama, M.; Tanaka, N.; Umemura, K.; Yamato, M.; Okano, T., Thermoresponsive Nanostructured Surfaces Generated by the Langmuir−Schaefer Method Are Suitable for Cell Sheet Fabrication. Biomacromolecules 2014, 15, 4160-4167, DOI: 10.1021/bm501187a (45) Sudo, Y.; Sakai, H.; Nabae, Y.; Hayakawa, T.; Kakimoto, M., Preparation of Hyperbranched Polystyrene-G-Poly(N-Isopropylacrylamide) Copolymers and Its Application to Novel Thermo-Responsive Cell Culture Dishes. Polymer 2015, 70, 307-314, DOI: 10.1016/j.polymer.2015.06.035 (46) Sudo, Y.; Sakai, H.; Nabae, Y.; Hayakawa, T.; Kakimoto, M., Role of Hyperbranched Polystyrene on Thermo-Responsive Cell Culture Dishes Prepared by Hyperbranched


31


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 34

Polystyrene-G-Poly(N-Isopropylacrylamide). Polymer 2016, 100, 77-85, DOI: 10.1016/j.polymer.2016.08.025 (47) Healy, D.; Nash, M. E.; Gorelov, A.; Thompson, K.; Dockery, P.; Belochapkine, S.; Madden, J.; Rochev, Y. A., Nanometer-Scale Physically Adsorbed Thermoresponsive Films for Cell Culture. Int. J. Polym. Mater. Po. 2017, 66, 221-234, DOI: 10.1080/00914037.2016.1201765 (48) Healy, D.; Nash, M. E.; Gorelov, A.; Thompson, K.; Dockery, P.; Beloshapkin, S.; Rochev, Y. A., Fabrication and Application of Photocrosslinked, Nanometer-Scale, Physically Adsorbed Films for Tissue Culture Regeneration. Macromol. Biosci. 2016, 17, 1600175, DOI: 10.1002/mabi.201600175 (49) Labbé, A.; Carlotti, S.; Deffieux, A.; Hirao, A., Controlled Polymerization of Glycidyl Methyl Ether Initiated by Onium Salt/Triisobutylaluminum and Investigation of the Polymer LCST. Macromol. Symp. 2007, 249-250, 392-397, DOI: 10.1002/masy.200750409 (50) Gervais, M.; Brocas, A.-L.; Cendejas, G.; Deffieux, A.; Carlotti, S., Synthesis of Linear High Molar Mass Glycidol-Based Polymers by Monomer-Activated Anionic Polymerization. Macromolecules 2010, 43, 1778-1784, DOI: 10.1021/ma902286a (51) Heinen, S.; Rackow, S.; Schäfer, A.; Weinhart, M., A Perfect Match: Fast and Truly Random Copolymerization of Glycidyl Ether Monomers to Thermoresponsive Copolymers. Macromolecules 2017, 50, 44-53, DOI: 10.1021/acs.macromol.6b01904 (52) Lehaf, A. M.; Moussallem, M. D.; Schlenof, J. B., Correlating the Compliance and Permeability of Photo-Cross-Linked Polyelectrolyte Multilayers. Langmuir 2011, 27, 4756-4763, DOI: 10.1021/la200229h (53) Jabeen, I.; Pleban, K.; Rinner, U.; Chiba, P.; Ecker, G. F., Structure−Activity Relationships, Ligand Efficiency, and Lipophilic Efficiency Profiles of Benzophenone-Type Inhibitors of the Multidrug Transporter P-Glycoprotein. J. Med. Chem. 2012, 55, 3261-3273, DOI: 10.1021/jm201705f (54) Radhakrishnan, K.; Cramail, H.; Deffieux, A.; Francois, P.; Momtaz, A., Ethylene Polymerization Studies with an MAO Synthesized by a Non-Hydrolytic Synthetic Route. Macromol. Rapid Commun. 2002, 23, 829-833, DOI: 10.1002/15213927(20021001)23:143.0.CO;2-5 (55) Lee, J. H.; Khang, G.; Lee, J. W.; Lee, H. B., Interaction of Different Types of Cells on Polymer Surfaces with Wettability Gradient. J. Colloid Interface Sci. 1998, 205, 323-330, DOI: 10.1006/jcis.1998.5688 (56) Wei, J.; Igarashi, T.; Okumori, N.; Igarashi, T.; Maetani, T.; Liu, B.; Yoshinari, M., Influence of Surface Wettability on Competitive Protein Adsorption and Initial Attachment of Osteoblasts. Biomed. Mater. 2009, 4, 045002 (045007pp), DOI: 10.1088/1748-6041/4/4/045002 (57) Hickman, G. J.; Boocock, D. J.; Pockley, A. G.; Perry, C. C., The Importance and Clinical Relevance of Surfaces in Tissue Culture. ACS Biomater. Sci. Eng. 2016, 2, 152-164, DOI: 10.1021/acsbiomaterials.5b00403 (58) Höök, F.; Kasemo, B.; Nylander, T.; Fant, C.; Sott, K.; Elwig, H., Variations in Coupled Water, Viscoelastic Properties, and Film Thickness of a Mefp-1 Protein Film During Adsorption and Cross-Linking: A Quartz Crystal Microbalance with Dissipation Monitoring, Ellipsometry, and Surface Plasmon Resonance Study. Anal. Chem. 2001, 73, 5796-5804, DOI: 10.1021/ac0106501 (59) Dormán, G.; Prestwich, G. D., Benzophenone Photophores in Biochemistry. Biochemistry 1994, 33, 5661-5673, DOI: 10.1021/bi00185a001


32

Page 33 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(60) Prucker, O.; Naumann, C. A.; Rühe, J.; Knoll, W.; Frank, C. W., Photochemical Attachment of Polymer Films to Solid Surfaces Via Monolayers of Benzophenone Derivatives. J. Am. Chem. Soc. 1999, 121, 8766-8770, DOI: 10.1021/ja990962+ (61) Toomey, R.; Freidank, D.; Rühe, J., Swelling Behavior of Thin, Surface-Attached Polymer Networks. Macromolecules 2004, 37, 882-887, DOI: 10.1021/ma034737v (62) Körner, M.; Prucker, O.; Rühe, J., Kinetics of the Generation of Surface-Attached Polymer Networks through C, H‑Insertion Reactions. Macromolecules 2016, 49, 2438-2447, DOI: 10.1021/acs.macromol.5b02734 (63) Carbone, N. D.; Ene, M.; Lancaster, J. R.; Koberstein, J. T., Kinetics and Mechanisms of Radical-Based Branching/Cross-Linking Reactions in Preformed Polymers Induced by Benzophenone and Bis-Benzophenone Photoinitiators. Macromolecules 2013, 46, 5434-5444, DOI: 10.1021/ma4007347 (64) Zillessen, A.; Bartsch, E., Synthesis of Photo-Cross-Linkable Microgel Colloids for Cluster Formation Studies. Langmuir 2010, 26, 89-96, DOI: 10.1021/la9021454 (65) Millan, M. D.; Locklin, J.; Fulghum, T.; Baba, A.; Advincula, R. C., Polymer Thin Film Photodegradation and Photochemical Crosslinking: FT-IR Imaging, Evanescent Waveguide Spectroscopy, and QCM Investigations. Polymer 2005, 46, 5556-5568, DOI: 10.1016/j.polymer.2005.05.050 (66) Ghorbal, A.; Brahim, A. B., Evaluation of Nanotribological Behavior of Amorphous Polystyrene: The Macromolecular Weight Effect. Polym. Test. 2013, 32, 1174-1180, DOI: 10.1016/j.polymertesting.2013.07.012 (67) Vinogradova, O. I.; Yakubov, G. E., Forces between Polystyrene Surfaces in Water– Electrolyte Solutions: Long-Range Attraction of Two Types? J. Chem. Phys. 2001, 114, 81248131, DOI: 10.1063/1.1365954 (68) Zelzer, M.; Albutt, D.; Alexander, M. R.; Russell, N. A., The Role of Albumin and Fibronectin in the Adhesion of Fibroblasts to Plasma Polymer Surfaces. Plasma Process. Polym. 2012, 9, 149-156, DOI: 10.1002/ppap.201100054 (69) Matsuzaka, N.; Takahashi, H.; Nakayama, M.; Kikuchi, A.; Okano, T., Effect of the Hydrophobic Basal Layer of Thermoresponsive Block Co-Polymer Brushes on ThermallyInduced Cell Sheet Harvest. J. Biomater. Sci. Polym. Ed. 2012, 23, 1301-1314, DOI: 10.1163/092050611X580454


33


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 34

TABLE OF CONTENTS GRAPHIC Manuscript Title: Ultrathin Poly(glycidyl ether) Coatings on Polystyrene for Temperature-Triggered Human Dermal Fibroblast Sheet Fabrication Author Names: Daniel David Stöbener, Melanie Uckert, José Luis Cuellar-Camacho, Anke Hoppensack, Marie Weinhart*


34

Ultrathin Poly(glycidyl ether) Coatings on Polystyrene for Temperature

Recommend Documents