Acrylate End-Capped Poly(ester-carbonate) and Poly(ether-ester)s for

Biomacromolecules 2008, 9, 867–878

867

Acrylate End-Capped Poly(ester-carbonate) and Poly(ether-ester)s for Polymer-on-Multielectrode Array Devices: Synthesis, Photocuring, and Biocompatibility Gaëtan R. P. Henry,† Andreas Heise,†,‡ Daniele Bottai,§ Alessandro Formenti,4 Alfredo Gorio,§ Anna Maria Di Giulio,§ and Cor E. Koning*,† Laboratory of Polymer Chemistry, Eindhoven University of Technology Technology (TU/e), P.O. Box 513, 5600 MB Eindhoven, The Netherlands, DSM Research, P.O. Box 18, 6160 MD Geleen, The Netherlands, Dipartimento di Medicina, Chirurgia e Odontoiatria Ospedale S. Paolo, Università di Milano, Via Di Rudinì 8, Milan I-20142, Italy, and Instituto di Fisiologia Umana II, Università di Milano, Via Mangiagalli 32, Milan I-20133, Italy Received October 29, 2007; Revised Manuscript Received December 19, 2007

Polymeric materials based on -caprolactone (CL), 1,5-dioxepan-2-one (DXO), and trimethylene carbonate (TMC) were prepared and evaluated as possible candidates for polymer-on-multielectrode (PoM) applications. CL was copolymerized with either DXO or TMC in the presence of the diol initiator 1,4-benzenedimethanol (BDM). The ring-opening polymerization experiments, carried out in bulk and using tin(II) catalysis, yielded the desired low molecular weight random copolymer diols, as evidenced by NMR, IR, MALDI-ToF MS, and DSC techniques. Upon reaction with acryloyl chloride, the corresponding diacrylate end-capped copolymers were obtained. The latter were characterized by NMR and IR spectroscopy, and their photocross-linking (in the presence of a UV initiator) was followed by ATR-FTIR spectroscopy. Transparent and soft thin films of the copoly(ether-ester) and copoly(ester-carbonate) diacrylates were prepared and cured under UV irradiation. The resulting polymeric films showed good biocompatibility properties as far as in vitro neural stem cells proliferation and differentiation to neurons and astrocytes are concerned. Noteworthy are the beneficial effects obtained upon preconditioning the copolymers by means of the cell-culture medium and the excellent properties shown particularly by the CL-TMC copolymer. Moreover, preliminary results show that microchannel formation by photocuring is possible with the synthesized polymers.

Introduction Polymer-on-multielectrode (PoM) devices combine polymeric microfluidic microstructures and solid substrates with embedded multielectrode arrays (MEA). The MEA technology proved to be a very useful tool for the extracellular electrical recording and stimulation of cultured patterned neurons.1,2 In typical PoM devices, the polymeric layer is deposited on a glass support with embedded indium-tin oxide (ITO) electrodes; the former comprises networks of microwells and buried microchannels, allowing the seeding of neuron cells and the subsequent formation of neuronal networks upon connection of the growing neurites through the channels. Various strategies have been developed in the past decade for the fabrication of these polymeric microstructures such as the patterning of poly(dimethylsiloxane) (PDMS) films using masters and microhole punchers,1,2 a multilevel resist technique combining thick liquid and laminated photopolymers,3 a two-step photolithography method using the polyester resist SU-8,4 or the photothermal etching of agarose layers.5,6 The aim of this project is to develop new fabrication processes and therefore new materials in order to reduce the production * To whom correspondence should be addressed. E-mail: c.e.koning@ tue.nl. Telephone: (+) 31 40 2475353. Fax: (+) 31 40 2463966. † Laboratory of Polymer Chemistry, Eindhoven University of Technology Technology (TU/e). ‡ DSM Research. § Dipartimento di Medicina, Chirurgia e Odontoiatria Ospedale S. Paolo, Università di Milano. 4 Instituto di Fisiologia Umana II, Università di Milano.

time and cost of custom-designed laboratory-on-a-chip (PoM) devices.7 Specifically, we are aiming for a one-step process in which the device topology (comprising buried microchannels) would be directly laser-written into a photosensitive material, thereby avoiding preparation of masters or stamping. This paper reports on the material aspects, i.e., the design of polymers suitable for application in the field of such PoM devices. The latter require the use of materials that are, among others, photopatternable (for instance UV-curable), compatible with the culture and differentiation of stem cells, transparent and physically and chemically stable in a cell culture medium. Moreover, they preferably exhibit low glass transition temperatures (Tgs) and good adhesion to the solid substrate, which usually consist of a glass support with embedded indium-tin oxide (ITO) electrodes. We anticipated that polymeric materials based on lactones or cyclic carbonates such as -caprolactone (CL), 1,5-dioxepan-2-one (DXO), or 1,3-dioxan-2-one (trimethylene carbonate, TMC) would be ideal candidates for our purpose, as they are known for their usually good biocompatibility8–11 and slow hydrolytic degradation behavior.12–14 Moreover, while CL homopolymers are crystalline opaque materials, incorporation of randomly distributed DXO or TMC noncrystallizable units leads to transparent amorphous copolymers with Tgs below 0 °C.15,16 Because of their biocompatibility, biodegradability, and tunable mechanical properties, CL-DXO and CL-TMC copolymers have been extensively studied as candidates for tissue engineering,11,17–20 drug delivery,8 and surgical21 applications. However, as explained above, we are interested in the use of these

10.1021/bm701191e CCC: $40.75  2008 American Chemical Society Published on Web 02/08/2008

868 Biomacromolecules, Vol. 9, No. 3, 2008

copolymers in the presence of cultured neuron and stem cells. Although recent publications report on the combination of various types of stem cells with CL-based polymers for applications in tissue engineering22,23 (including photopatternable polymers24), no example was found in the literature regarding the compatibility of amorphous and photopatternable CL-DXO and CL-TMC copolymers with cultured stem cells or laboratory-on-a-chip applications. The synthesis of statistical and/or amorphous copolymers of CL with either DXO or TMC can be achieved easily by ring-opening polymerization (ROP) in the presence of organometallic15,16,19,20,25–29 or enzymatic30,31 catalysts. Depending on the conditions, i.e., type of catalyst or cocatalyst system, type of initiator, temperature, reaction in bulk or in a solvent, etc., various polymeric structures can be obtained. This concerns the nature and number of the end groups, the distribution of the comonomers along the chain, the average molecular weight and molecular weight distribution, as well as the presence of degradation or side-products. It is thus important to control those parameters because they determine the physical, thermal, and microstructural properties of the final product. Another requirement for the targeted application is the ability to form cross-linked films. In the literature, cross-linked materials based on lactones and carbonates were directly obtained from polymerization using cross-linking agents as comonomers19,32,33 or from reaction of the polymers with polyfunctional cross-linkers.34 However, the design of photopatternable polymers based on lactone and aliphatic carbonate monomers requires their functionalization with reactive moieties such as coumarins35,36 or acrylates.37–42 The more frequently adopted acrylates can be introduced as end groups to hydroxy end-capped (co)polymers by lipase catalysis43,44 or by reaction with acryloyl chloride.37,38,40,41 The resulting acrylated (co)polymers are able to cross-link efficiently under visible or UV irradiation in the presence of a suitable photoinitiator. In this work, we report on the preparation and characterization of diol end-capped low molecular weight copolymers of CL with either DXO or TMC, synthesized by tin(II)-catalyzed ROP. The resulting amorphous copoly(ether-ester)s and copoly(estercarbonate) were functionalized with acrylate end groups, and curing experiments were carried out in the presence of a UV initiator. Special emphasis was given to a thorough characterization of the materials, as impurities could significantly influence the biocompatibility. Biocompatibility tests have been carried out to assess the effects of the resulting cured films on neural stem cell (NSC) proliferation as neurospheres in suspension in the culture medium. The cell adhesion on the polymer surface and the possibility for NSCs to differentiate to nervous cells, neurons, and astrocytes were also evaluated.

Experimental Section Materials. Unless otherwise specified, all solvents, reagents and salts were used as received. Ethyl acetate, dichloromethane, diethyl ether, and methanol were purchased from Biosolve. Tetrahydro-4H-pyran4-one, m-chloroperbenzoic acid (mCPBA, 70–75%), 1,4-benzenedimethanol (BDM), Celite 545, 2-hydroxy-4′-(2-hydroxyethoxy)-2methylpropiophenone (IRGACURE 2959), Na2CO3, and MgSO4 were all purchased from Acros. tert-Butyl methyl ether was purchased from Aldrich, NaCl and P2O5 from Fluka, tin(II) 2-ethylhexanoate (95%) from Sigma, and Na2S2O3 (97%) from Merck. Triethylamine (Merck) was dried and purified by distillation over CaH2 (Acros, 93% purity 10–100 mm pieces that were smashed just before use). CL (g99%, Aldrich) and DXO (synthesized in the laboratory) were distillated under

Henry et al. reduced pressure over CaH2, and TMC was a gift from Boehringer Ingelheim, Germany. Before use, the monomers (CL, DXO, and TMC) and the diol initiator (BDM) were dried over P2O5 overnight under vacuum. Synthesis of DXO. DXO was synthesized by Baeyer–Villiger oxidation of tetrahydro-4H-pyran-4-one, as reported by Mathisen et al.45 according to the following modified procedure. Tetrahydro-4Hpyran-4-one, 14.9 g (0.15 mol), was added to a solution of 44.3 g (∼0.18 mol) of mCPBA in dichloromethane (180 mL) at 0 °C under stirring. The reaction mixture was maintained at 0 °C for 1 h and then refluxed for 17 h. The resulting suspension was cooled down to 0 °C and filtered over Celite, and the solids were washed three times with dichloromethane. The filtrate was washed with 2 × 180 mL of a 10% solution of Na2S2O3, with 2 × 150 mL of a saturated solution of Na2CO3, and finally with 150 mL of brine. After drying over MgSO4, filtration, and evaporation of the solvent under reduced pressure, a slightly yellow oil was obtained. The crude product was recrystallized from diethyl ether and then distillated under reduced pressure (46 °C, 10-2 mbar), yielding 11.84 g of clean DXO (mp ) 35 °C) (yield ) 68%). Before use, DXO was distillated under reduced pressure over CaH2 and dried over P2O5 under vacuum overnight. 1H NMR (CDCl3, 400 MHz), δ (ppm): 2.90 (m, 2H, C-3 protons), 3.83 (m, 2H, C-4 protons), 3.90 (m, 2H, C-6 protons), 4.30 (m, 2H, C-7 protons). 13C NMR (CDCl3), δ (ppm): 39.0 (C-7), 64.4, 70.1, 70.5, 174.0 (C-1). Synthesis of the Copolymer Diols. In a typical polymerization, a dried three-necked flask equipped with a magnetic stirrer, a gas inlet, and a septum was filled with the desired amount of dried monomers (CL with either DXO or TMC) and diol initiator (BDM) under a nitrogen atmosphere. The mixture was stirred (and slightly heated) for a short time until a homogeneous melt was obtained. The polymerization was started by adding tin(II) 2-ethylhexanoate (monomer to catalyst ratio about 450) via a syringe through the septum and immersing the flask into an oil bath preheated at the required temperature. The system was allowed to stir until the desired conversion or time was reached: from time to time, samples were withdrawn via a syringe through the septum (or with a pipet under nitrogen flow when the reaction mixture was too viscous) and analyzed by 1H NMR and SEC. Once the desired conversion was reached, the reaction mixture was cooled down to room temperature and solubilized in a small amount of dichloromethane (typically 1.5 to 3 mL per g of polymer) and then poured into cold methanol. The resulting suspension was cooled down in a freezer for 10 min and then centrifuged at 2000 rpm for 10 min. The bottom layer, containing the polymer, was isolated and dried under vacuum for several days (at a temperature below 40 °C to avoid methanolysis of the ester and carbonate bonds in the polymer). The purified polymers were transparent, viscous, and tacky oils in the case of P(CL-co-DXO)-diols, or a transparent tacky paste in the case of P(CL-co-TMC)-diol. Preparation of the Copolymer Diacrylates. The preparation of the P(CL-co-TMC)-diacrylate (assigned “CT-1-DA” in the Results and Discussion Section) is given here as a representative procedure for acrylation reactions of all copolymers. Then 7.00 g (∼2.3 mmol of hydroxy end groups) of the starting P(CL-co-TMC)-diol were introduced in a three-necked flask equipped with a magnetic stirrer, a gas inlet, and a septum. The flask containing the copolymer was dried overnight under vacuum over P2O5, and then equipped with a dried addition funnel. Under nitrogen atmosphere, 30 mL of anhydrous dichloromethane were added to solubilize the polymer; a solution of 0.56 mL of acryloyl chloride (6.9 mmol, 3.0 equiv) in the same volume of dichloromethane was introduced in the addition funnel. The solution of the polymer was cooled down below 0 °C using an ice/NaCl bath, and 0.99 mL of dry triethylamine (7.1 mmol, 3.1 equiv) was added via the septum. The acryloyl chloride solution was then added dropwise and under vigorous stirring over 1.5 h. Upon complete addition of the acid chloride, the reaction mixture was allowed to stir for 2 h at 0 °C and for another 22 h at room temperature. The resulting slightly yellow solution was then poured into 25 mL of a 5% NaOH solution and stirred for 10 min (process repeated twice); the combined aqueous layers were

End-Capped Poly(ester-carbonate) and Poly(ether-ester)s reextracted by 15 mL of dichloromethane. The combined organic layers were washed respectively with 25 mL of a 5% HCl solution, 25 mL of water, and 2 × 25 mL of brine. The organic phase was finally dried over MgSO4, filtrated, and concentrated by rotoevaporation. The resulting slightly yellow oil was dried under vacuum at 40 °C for several days until no solvent was detected anymore in 1H NMR. Cross-Linking of the Copolymer Diacrylates into Films. The UV curing of the copolymer diacrylates was carried out in the presence of the UV initiator IRGACURE 2959. Given their high viscosity, the polymers were solubilized in a 1.7 mM solution of IRGACURE 2959 in ethyl acetate to ensure a homogeneous dispersion of the initiator in the polymer. The volume of added initiator solution was chosen in order to obtain a concentration of 0.15 wt % of IRGACURE 2959 in the polymer, corresponding to about 1 initiator molecule per 48((4) acrylate end groups. The solvent was removed from the formulation by applying vacuum overnight. The films designed for cytocompatibility testing were prepared on glass plates that were previously cleaned by sonication in 2-propanol and dried. The formulations (copolymer diacrylate containing UV initiator) were applied as 120 µm thick films on the glass plates using a rod and placed in a chamber under nitrogen flow under a Hönle H2-filter (cutting off wavelengths below 295 nm) inside a Hönle UVACUBE equipped with a F-lamp. The UV intensity measured at the same position was 33 mW · cm-2 in UV-A and 22 mW · cm-2 in UV–vis. Patterned films were prepared in the same way. They were irradiated through a mask with lines (50 µm wide) and squares (1 mm × 1 mm) for 180 s at 35.1 mW/cm2 (UV-A) (power intensity measured at sample location). The obtained film was then developed in acetone/tert-butyl methyl ether 9:1 v/v for 6 h. Water uptake and hydrolytic degradation of the cured films were estimated as follow. Photocross-linked films (120 µm thick) were peeled off from their glass support, weighed (initial mass, m0), and then immersed in a pH 7.4 phosphate buffer solution (PBS) maintained at 37 °C under static conditions (the PBS was prepared by mixing 174.16 g Na2HPO4, 37.36 g NaH2PO4 · H2O, 0.71 g NaCl, 7.63 g NaN3, and 15 L of Milli-Q water). After 4 weeks, the films were removed from the buffer solution and washed with distilled water. The films were weighed after wiping their surface with paper (swollen mass, mS) and after drying for 5 days under vacuum (dried mass, mD). The weight loss (WL) and water absorption (WA) were then calculated from: WL (%) ) 100 × ([m0 - mD]/m0); WA (%) ) 100 × ([mS - mD]/mD). The gel content of the cured films was determined by extraction at room temperature in deuterated chloroform for 24 h and drying under vacuum until constant weight. Using the initial mass of the film (m0) and its mass after exctraction and drying (mE), the gel content is given by: gel content (%) ) 100 × (mE/m0). The CDCl3-soluble fraction was analyzed by 1H NMR, dried, redissolved in THF, and then analyzed in MALDI-ToF MS. Neural Stem Cells (NSCs): Preparation and Validation. Primary cultures and cultivation of the NSCs from adult mice, their differentiation, and their immunostaining were performed as described elsewhere.47,48 Briefly, two- to three-month-old CD-1 albino mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.1 mL/10 g body weight) and killed by decapitation. The brains were removed from adult mice, and tissues containing the subventricular zone (SVZ) were dissected out. Tissues derived from a single mouse were used to generate each culture. The dissected tissue was transferred to a phosphate buffer solution containing penicillin and streptomycin 100 U/mL each (Invitrogen, San Diego, CA) and glucose (0.6%) at 4 °C until the end of the dissection. We carried out an enzymatic dissociation transferring the tissue a Earl’s Balanced Salt Solution (EBSS) (SigmaAldrich, St. Louis, MO) containing 1 mg/mL papain (27 U/mg; Worthington DBA, Lakewood, NJ), 0.2 mg/mL cysteine (SigmaAldrich, St. Louis, MO), and 0.2 mg/mL EDTA (Sigma-Aldrich, St. Louis, MO) and incubated for 45 min at 37 °C on a rocking platform. Tissues were then centrifuged at 123g, and the supernatant was discarded. The pellet was resuspended in a 1 mL of EBSS and

Biomacromolecules, Vol. 9, No. 3, 2008 869 mechanically dissociated using an aerosol-resistant tip (1000 µm Gilson Pipette). Cells were resuspended in 10 mL of EBSS and centrifuged at 123g for 10 min. The supernatant was discarded, and the pellet resuspended in 200 µL of EBSS. The pellet was again dissociated mechanically using an aerosol-resistant tip (200 µm Gilson Pipette). Cells were resuspended in 10 mL of EBSS and centrifuged at 123g for 10 min. The supernatant was discarded and the pellet resuspended in 1 mL of chemically defined DMEM-F-12 containing FGF2 (human recombinant, 10 ng/mL; Peprotech, Rocky Hill, NJ, or Upstate Biotechnology, Lake Placid, NY) and EGF (human recombinant, 20 ng/mL; Peprotech). The cells were counted and plated at 3500 cell · cm-2, the spheres formed after 5–7 days were harvested, collected by centrifugation (10 min at 123 gs), mechanically dissociated to a single-cell suspension, and replated in medium containing the appropriate growth factors, (GF(s)).47,48 This procedure can be repeated every 3–5 days in vitro (DIV) for up to 12 months. The total number of viable cells was assessed at each passage by trypan blue exclusion. Stem cells used in these experiments were between the 5-15th passage in culture. Proliferation of NSCs in Presence of the Copolymers. The first biocompatibility test evaluated the proliferation capacity of the NSC (utilizing growth factors) in the presence of CL-TMC or CL-DXO copolymers in comparison to control cultures. Round polymer samples, 4.3 mm in diameter, were obtained by punching the 120 µm thick copolymer films prepared on microscope glasses under sterile conditions. Part of the samples was utilized without further treatments, whereas another part was preconditioned for 6 days either in distilled water or in culture medium. Then 10000 cells obtained from the dissociation of the neurospheres were plated per well (10000 cell · cm-2) in standard polystyrene 48-well plates (CELLSTAR, Greiner Bio-One, Germany). The only difference between control and test cultures was the presence in the latter of the sample polymer. The cells were cultured for 6 days at 37 °C in 5% CO2 atmosphere in a tissue culture incubator. The cells of each well were harvested in an Eppendorf tube, spun down at 150g for 10 min; 200 µL of supernatant was left in the tube and the cells were dissociated using a 200 µL Gilson pipet. A fraction of the cells was counted in an emocitometer, and the total amount of cells per well was calculated. The number of cells in the control sample was averaged, and the number of cell counted in each well was normalized to this average. The whole experiment was performed twice. The normalized results were analyzed for statistical differences using the one-way Anova method followed by the Bonferroni post test. Differentiation of NSCs. The differentiation of the NSC was performed as already described by Gritti and co-workers47,48 plating the dissociated neurospheres in a medium containing serum, adhesion molecules (Matrigel), and without growth factors. The differentiation was achieved 7 days after these treatments. Immunofluorescence tests were carried out as previously described.47,48 Briefly, cultures were fixed for 10 min in PBS containing 4% paraformaldehyde then rinsed with PBS and incubated for 2 h at 37 °C in PBS containing 10% normal goat serum (NGS), 0.1% Triton X-100, and appropriate primary antibodies or antisera (primary antibodies for β-tubulin III (1:1000) and GFAP (1:1000), Covance) and then washed three times in PBS for 10 min. The samples were incubated for 45 min at room temperature with Alexa 488 and Alexa 546 (1:1000) antimouse or antirabbit (Invitrogen). Samples were then rinsed three times with PBS for 10 min and counterstained with DAPI (4,6-diamidine-2-phenylindole dihydrochloride) for 10 min at room temperature. As final step, the samples were washed once in PBS and once with distilled water and mounted with Fluorsave (Calbiochem, La Jolla, CA). As negative control, we run the staining on the samples omitting the primary antibodies; these samples were used as references in the confocal analysis. Nuclear Magnetic Resonance (NMR). For NMR spectroscopy experiments, CDCl3 (Cambridge Isotope Laboratories) was used as solvent as well as internal standard: δ[CHCl3] ) 7.26 ppm (1H NMR) and δ[CDCl3] ) 77.16 ppm (13C NMR). NMR spectra were recorded at room temperature on a Varian Mercury Vx spectrometer operating


Henry et al.

Scheme 1. General Reaction Scheme for the Synthesis of the Copolymer Diols and their Subsequent Acrylation and Cross-Linking (x ) molar fraction in the copolymer)

at 400.16 MHz (1H) and 100.63 MHz (13C) and processed with VNMR software. For 13C NMR spectra (under proton decoupling), a minimum number of 4000 transients and a relaxation time of 12 s were used in order to obtain semiquantitative spectra with good signal-to-noise ratios, suitable for integration of the carbonyl peaks. Infrared (IR) and Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) Spectroscopy. The copolymers were analyzed by infrared (IR) spectroscopy using a Biorad Excalibur FTIR spectrometer, piloted from a PC equipped with Varian Resolution software. Each spectrum was recorded with a resolution of 2 cm-1, coadding 200 scans. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used in combination with UV irradiation in order to follow the kinetics of the cross-linking reaction for the acrylated polymers (in the presence of IRGACURE 2959). These experiments were carried out using a Biorad Excalibur FTIR spectrometer using a MCT detector to collect the data. The spectrometer was piloted from a PC equipped with Varian Resolution Pro software. The spectrometer was equipped with a Golden Gate ATR unit (Specac) and a light guide connected to a UV irradiating device (Oriel Instruments, Spectral Luminator). A thin film of the formulation (acrylated polymer with 0.15 wt % IRGACURE 2959) was spread over the ATR cell, and a nitrogen flow was applied for 5 min. UV irradiation (λ ) 315 ( 10 nm, 0.45 mW/cm2) was applied for 5 min and IR spectra were recorded every 5 s with a resolution of 4 cm-1. Size Exclusion Chromatography (SEC). SEC analysis was carried out using a WATERS 2695 separation module, model 486 UV detector (254 nm), and model 2414 refractive index detector (at 40 °C). The samples were dissolved in THF, filtered through 0.2 µm PTFE filters

(13 mm, PP housing, Alltech), and 50 µL of the solution was injected. Tetrahydrofuran (Biosolve, stabilized with 2,6-dimethyl-4-tert-butylphenol) was used as eluent at a flow rate of 1.0 mL min-1. The column set consisted of a Polymer Laboratories PLgel guard column (5 µm particles, 50 mm × 7.5 mm), followed by two PLgel mixed-C columns in series (5 µm particles 300 mm × 7.5 mm) at 40 °C. Calibration was done using polystyrene standards (Polymer Laboratories, M ) 580 up to M ) 7.1 × 106 g/mol). Data acquisition and processing were performed using WATERS Empower 2 software. Differential Scanning Calorimetry (DSC). DSC was performed using a TA Instruments Q100 DSC, equipped with a refrigerated cooling system (RCS) and an autosampler, and piloted from a PC equipped with a TA Qseries Advantage software. The DSC cell was purged with a nitrogen gas flow of 50 mL min-1. Experiments were performed in aluminum hermetic pans using a heating and cooling rate of 10 °C min-1. Melting (Tm) and glass transition (Tg) temperatures, as well as the heat of fusion (∆H) were extracted from the second heating curve. The Tgs were determined by applying the half-extrapolated tangent method. The ∆H was converted to the mass fraction of crystallinity wc according to the expression: wc ) ∆H/∆H0, where ∆H0 is the heat of fusion of 100% crystalline PCL, reported to be 139.5 J/g.46 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-ToF-MS). MALDI-ToF-MS analysis was performed on a Voyager DE-STR (Applied Biosystems, Framingham, MA) equipped with a 337 nm nitrogen laser. An accelerating voltage of 25 kV was applied. Mass spectra of 1000 shots were accumulated. The polymer samples were dissolved in THF at a concentration of 1 mg mL-1. The cationization agents used were potassium trifluoroacetate (Fluka, >99%) dissolved in THF at a

End-Capped Poly(ester-carbonate) and Poly(ether-ester)s

Biomacromolecules, Vol. 9, No. 3, 2008 871

Table 1. SnOct2/BDM Initiated Bulk Copolymerizations of CL and DXO or TMC at 110 °C Mnc CL:DXO: TMC (feed)a CD-1 CD-2 CT-1 a

23:23:0 11:34:0 23:0:23

T (h)

yield

b

22 3 20

(wt %)

CL:DXO:TMC (found)a,b,c

89 85 84

Molar equivalents per mole of BDM initiator.

b

30:28:0 13:39:0 24:0:24

After precipitation. c From 1H NMR.

Mwd

(103 g · mol-1) 6.8 6.2 6.0

d

Mnd 7.5 8.0 10.1

From SEC.

e

Mw/Mnd

11.3 10.3 15.9

From

1.51 1.29 1.58

Lj CL

Lj DXOe

e

2.3 1.4e 2.17c

Lj TMCe

2.0 4.6 2.1

13

C NMR.

Table 2. Thermal Properties of the Homo and Copolymer Diols Mn (g · mol-1) d

PCL PDXOd PTMCe CD-1 CD-2 CT-1

a

6200 6700 4200 6800 6200 6000

Tm (°C)b

wc (%)b, c

Tg (°C)b

Tg (°C) from Fox equation

52.7

50.9

17.6

8.2 0 0

-57.6 -43.4 -25.2 -54.6 -46.8 -48.4

-50.9 -47.1 -43.4

a From 1H NMR. b From DSC. c wt % crystallinity. d From Novozym 435 catalyzed, BDM-initiated ROP followed by precipitation. e From SnOct2 catalyzed, BDM-initiated ROP followed by precipitation.

concentration of 5 mg mL-1. The matrix used was trans-2-[3-(4-tertbutylphenyl)-2-methyl-2-propenylidene]malononitrile (Fluka) and was dissolved in THF at a concentration of 40 mg mL-1. Solutions of matrix, salt, and polymer were mixed in a volume ratio of 4:1:4, respectively. The mixed solution was hand-spotted on a stainless steel MALDI target and left to dry. The spectra were recorded in the reflectron mode. Baseline corrections and data analyses were performed using Data Explorer version 4.0 from Applied Biosystems.

Results and Discussion Aiming for diol end-capped low molecular weight amorphous copolymers, CL was copolymerized with DXO and with TMC, in the presence of a diol initiator (1,4-benzenedimethanol, BDM). As depicted in Scheme 1, the resulting copolymer diols were subsequently functionalized by acrylation, and crosslinking experiments were carried out in the presence of a UV initiator (IRGACURE 2959). In the following sections, the short forms “CD” and “CT” will refer to CL-DXO and CL-TMC copolymers, respectively, and the abbreviations “DA” and “XL” will stand for “diacrylate” and “cross-linked”, respectively. Synthesis and Characterization of the Copolymer Diols. For both CL-DXO and CL-TMC copolymers, the polymerizations were carried out in the bulk and in dry conditions in order to prevent water initiation. For the synthesis of P(CL-co-DXO)-diols, tin(II) 2-ethylhexanoate (SnOct2) was selected as catalyst and the polymerization temperature was set to 110 °C, as thermal degradation is known to occur above 130 °C for the polymerization of DXO.13 The reactivity ratios of both CL and DXO were reported to be close to unity for this catalyst at 110 °C, leading to random copolymers.15 A proportion of at least 50 mol % of DXO in the feed ratios was selected because it was reported that poly(CL-co-DXO)s are amorphous transparent copolymers when the content of noncrystallizable DXO is above 40 to 50 mol %.15,20 The formation of diol end-capped copolymers with controlled molecular weights was ensured by the use of the difunctional initiator BDM. The same catalyst/initiator system (SnOct2/BDM) was used for the preparation of the poly(CL-co-TMC)-diol. It is known that the use of SnOct2 together with a diol enables fast copolymerization of CL and TMC, together with high conversions and control over the average molecular weight.29,49 Moreover, although CL exhibits a slightly higher reactivity than TMC in tin(II)-catalyzed polymerizations,16 longer reaction times and higher temperatures lead to more randomized microstructures due to extensive intermolecular transesterification that takes place during and after polymerization

when carbonate bonds are present along the polymer chain.29,50 A polymerization temperature of 110 °C was chosen in order to favor randomization by transesterification while avoiding thermal degradation, which is known to occur at higher temperatures for polymers based on TMC.16,51 Besides the control BDM provides over the final microstructure and molecular weight of the copolymers, this initiator should allow a relatively precise estimation of the average molecular weights by NMR analysis. Indeed, both aromatic and methylenic protons of BDM appear at lower field in the 1H NMR spectra (between 4.6 and 7.4 ppm) compared to the signals of the aliphatic esters or carbonates used in this study (all below 4.4 ppm). This is especially useful in the case of the P(CL-coDXO)-diols, for which the caprolactone CH2OH end group signal overlaps with peaks of the polymer backbone, preventing the calculation of the molecular weight from end group analysis. The conditions and results regarding the synthesis and characterization of the copolymer diols are given in Tables 1 and 2 and commented on below. P(CL-co-DXO)-diols. The first polymerization experiment (CD-1) was carried out with a 50:50 mixture of CL and DXO. Although the conversion was higher than 95% for both monomers, after about 5 h, the reaction time was extended to 22 h in order to increase the proportion of transesterification reactions and to obtain an even more randomized microstructure. In the second experiment (CD-2), a CL:DXO feed ratio of 25:75 was used and the reaction was stopped as soon as a conversion of 95% was reached for both monomers. As mentioned before, the peaks of the caprolactone-related CH2-OH end groups (about 3.62 ppm) are overlapping with other peaks of the polymer in the 1H NMR spectra (see CD-2 in Figure 1). Hence, the signals corresponding to the initiator (BDM) were used to calculate the average molecular weight Mn according to eq 1, assuming one BDM moiety per chain:

Mn )

2.3 2.6 2 × (ICL × MCL + IDXO × MDXO) 7.3 IBDM

+ MBDM

(1)

2.3 where ICL is the integration of the CL multiplet at 2.3 ppm, 2.6 7.3 IDXO the integration of the DXO multiplet at 2.6 ppm, IBDM the integration of the BDM singlet at 7.3 ppm, MCL, MDXO, and MBDM the molar masses of CL, DXO, and BDM, respectively. This method may slightly overestimate the molecular weight, as it does not take into account the water initiated and cyclic species. This possible deviation is however minor


Henry et al.

Figure 1. Low field region of the 1H NMR spectra (CDCl3, 400 MHz) of the copolymer diols CT-1 and CD-2 and diacrylates CT-1-DA and CD-2-DA, with assignment of initiator and end group signals; the singlet at 7.26 ppm is CHCl3.

compared to the overestimation given by SEC analysis when polystyrene standards are used for calibration.27,52 The Mn values obtained by NMR analysis are 7.5 and 8.0 × 103 g · mol-1 for CD-1 and CD-2, respectively, versus 11.3 and 10.3 × 103 g · mol-1 as measured by SEC. As can be seen from the results listed in Table 1, the ratio CL:DXO:BDM measured by NMR for CD-2 after precipitation (24:24:1) fits almost perfectly with the feed ratio (23:23:1), while a higher proportion of monomers is observed for CD-1 (30:28:1 from NMR, for a feed ratio of 23:23:1). The latter observation can be explained by the higher extent of transesterification that occurred in the case of CD-1, leading to a broader molecular weight distribution (MW/Mn ) 1.51 for CD-1 and 1.29 for CD2). The fraction of CD-1 solubilized in methanol during precipitation thus contained a higher proportion of chains having very low monomer-to-initiator ratios, in comparison to CD-2. A clear difference between CD-1 and CD-2 is observed in the MALDI-ToF analyses (Figure 2). While both spectra show only one main series of peaks, which corresponds to the polymer diol initiated by BDM, the distribution of the peaks is different. CD-2 shows a molecular weight distribution typical for polymers obtained from a controlled polymerization. The peak distribution of CD-1, on the other hand, is more representative of a polymer obtained by a polycondensation. This can be explained by the longer reaction time of CD-1, which favors transesterification reactions and thus not only randomization of the monomer units but also of the molecular weight distribution. Moreover, cyclic species were identified in the MALDI-ToF mass spectra of both

copolymers at low m/z values (Figure 2). These cyclics could not be quantified because MALDI-ToF is not a quantitative technique because of possible mass-discrimination effects. It has to be noted that the difference in mass between CL and DXO is only 2 mass units, leading to very narrow groups of peaks in the mass spectra, as can be seen in the expanded regions of Figure 2. The average CL and DXO block lengths (LCL and LDXO, respectively) were calculated from the 13C NMR spectra (Figure 3) according to the following equations:53

LCL )

ICC +1 ICD

(2)

IDD +1 IDC

(3)

LDXO )

where ICC and ICD are the integrations of the CL carbonyl peaks at 173.6 and 173.5 ppm, corresponding to the CL-CL and CL-DXO dyads, respectively, and IDD and IDC are the integrations of the DXO carbonyl peaks at 171.4 and 171.5 ppm, corresponding to the DXO-DXO and DXO-CL dyads, respectively. For both copolymers, the average block lengths calculated from the 13C NMR spectra (summarized in Table 1) fit with random microstructures. There are however CL blocks long enough to form crystallites in the case of CD-1, which exhibits 8.2% crystallinity in DSC (Table 2). The random microstructures



w1 w2 1 ) + Tg Tg1 Tg2

(4)

where Tg is the glass transition temperature of the random copolymer, and Tg1 and Tg2 those of the corresponding homopolymers, determined by DSC for low molecular weight PCL (-57.6 °C) and PDXO (-43.4 °C), w1 and w2 are the weight fractions of the two monomers in the copolymer after purification. The Tgs measured by DSC for CD-1 and CD-2 (-54.6 and -46.8 °C, respectively) fit well with the values calculated from the Fox equation (-50.9 and -47.1 °C, respectively).55 P(CL-co-TMC)-diol. Although the conversion was g99% in this polymerization for both monomers, after about 5 h, the reaction time was extended to 20 h, resulting in an increased proportion of transesterification reactions as indicated by the high polydispersity index (MW/Mn ) 1.58). For this copolymer, the average molecular weight can be calculated from the 1H NMR spectrum, either by integration of the BDM signals or on the basis of the hydroxy end groups, as the latter are not overlapping with other peaks (see CT-1 in Figure 1). Because transesterification reactions involving TMC-based polymers can lead to polymer species containing zero, one, or several initiator moieties,50 the Mn is preferably estimated from the integration of the CH2-OH end groups (triplets at 3.6–3.7 ppm). Assuming two hydroxy chain ends per macromolecule, Mn is calculated from the NMR spectrum according to the expression:

Mn )

Figure 2. MALDI-ToF mass spectra of CD-1 (upper trace) and CD-2 (bottom trace). For clarity reasons, only the composition of the central peak of every distribution is reported in the zoomed regions.

2.3 4.2 (2 × ICL × MCL + ITMC × MTMC) + MBDM IOH

2.3 where ICL is the integration of the CL multiplet at 2.3 ppm, 4.2 ITMC is the integration of the TMC multiplets around 4.2 ppm (after subtraction of the CL signal that overlaps), IOH is the integration of the CH2-OH end group triplets between 3.6 and 3.75 ppm, MCL, MTMC, and MBDM are the molar masses of CL, TMC, and BDM, respectively. Applying this method, a molecular weight of 6.0 × 103 g · mol-1 was determined for CT-1. As for the P(CL-co-DXO)-diols, the Mn calculated from NMR is more precise than the one obtained from SEC analysis using calibration with polystyrene standards (10.1 × 103 g · mol-1), as the latter technique is reported to overestimate the molecular weight.16 The average CL block length (LCL) was calculated from the dyad splitting of the caprolactones CH2-O signals in the 1H NMR spectrum27 (signals at 4.05 and 4.12 ppm in Figure 4). The signal at 4.05 ppm can be directly integrated, and the intensity of the signal at 4.12 ppm (overlapping with TMC 2.3 4.05 4.12 peaks) is easily obtained from ICL - ICL ) ICL (see Figure 4). The average TMC block length (LTMC) was calculated from the 13C NMR spectrum (Figure 3). From the above result the following expressions:

LCL )

ICC +1 ICT

(6)

ITT +1 ITC

(7)

LTMC ) Figure 3. Carbonyl region of 13C NMR spectra of the copolymer diols (see text for assignment of dyads).

are further validated by the observation of only one glass transition (Table 2). These Tgs are comprised between those of the corresponding homopolymers, in accordance with the Fox equation:54

(5)

where ICC and ICT are the intensities of the caprolactone’s CH2-O signals at 4.05 and 4.12 ppm (1H NMR), corresponding to the CL-CL and CL-TMC dyads, respectively, IDD and IDC are the integrations of the TMC carbonyl peaks at 155.0 and 155.2 ppm (13C NMR), corresponding to the TMC-TMC and TMC-CL dyads, respectively. Using eqs 6 and 7, average CL and TMC block lengths were found to be 2.17 and 2.1, respectively, which is in accordance with a random microstructure.


Henry et al.

Figure 4. Expanded regions of the CT-1 1H NMR spectrum. “CT” and “CC” are CL signals assigned to CL-TMC and CL-CL dyads, respectively.

As mentioned before, the long reaction time used for this experiment allowed a high number of transesterification reactions to occur. This is illustrated by the evolution of LCL, which decreased from 2.37 (sample withdrawn at t ) 5 h) to 2.12 (t ) 20 h) after complete conversion of the monomers (the value LCL ) 2.17 reported in Table 1 was measured after purification). The chemical composition of CT-1 fits well with the feed ratio, but this does not reflect the microstructure of all the species present in the polymer. Indeed, the extensive transesterification led to several types of polymer species, containing zero, one, or several BDM initiator moieties.50 This is illustrated by the MALDI-ToF mass spectrum of CT-1, rendered complicated by the superposition of several distributions. In Figure 5, only the two major distributions are assigned. They consist of the linear species having either BDM or propane-1,3-diol (PD) as initiator, which forms in situ from TMC. Other species are probably present such as cyclics, or linear chains containing 2 BDM moieties, but the low intensity of their signals and the complexity of the spectrum do not allow a complete analysis. Noticeably, the integration of the BDM signal in 1H NMR is slightly higher than the integration of the hydroxy end groups (ratio initiator-to-end groups of about 1.1 after precipitation). This suggests the existence of either linear species containing more than one BDM moiety per chain or cyclic species containing BDM. The random microstructure is evidenced by the low LCL and LTMC, obtained by the absence of crystallinity and by the fact that only one glass transition at -48.4 °C is observed between the Tgs of the corresponding homopolymers. Once again, this value is close to the Tg calculated from eq 4 (-43.4 °C). Acrylate Functionalization of the Copolymers. The copolymer diols were functionalized by reacting them with acryloyl chloride in the presence of triethylamine (Et3N) under anhydrous conditions. To decrease the probability of side reactions, a low excess of reagents was used and both the solvent and triethylamine were distilled and anhydrous. Using a small excess of acryloyl chloride and amine also facilititated the purification, which was carried out by washing the organic phase containing the crude product with acidic and basic aqueous solutions. This procedure yielded clean P(CL-co-TMC)-diacrylate (assigned CT-1-DA) in very good yield (94% in weight). The workup was more difficult for the P(CL-co-DXO)-diacrylates (CT-1-DA and CT-2-DA), mainly because these products tend to form an emulsion in the presence of water. Moreover, nonidentified fine particles were sometimes present in acrylated

Figure 5. MALDI-ToF mass spectrum of CT-1. For clarity reasons, only the composition of the central peak of every distribution is reported in the expanded region: BDM-initiated (3) and (pseudo-)PDinitiated (b) species.

Figure 6. Evolution of acrylate absorption band at 1637 cm-1 upon UV curing, measured by ATR-FTIR for CT-1-DA and CD-1-DA.

CL-DXO copolymers after workup and drying over MgSO4, making the last filtration step difficult (results not shown). In these cases, filtration through glass microfiber filters was necessary. Clean P(CL-co-DXO)-diacrylates were, however, obtained in satisfactory yields of about 60% and molecular weights in agreement with the diol precursors. The removal of acrylic acid and other impurities upon purification was confirmed in 1H NMR by the disappearance of small signals at 3.05, 3.45, and in the range 6.45–6.60 ppm, which were visible in the spectra of the crude products. The conversion of the hydroxy end groups into acrylates was assessed by 1H NMR analysis, as illustrated for CD-2-DA and CT-1-DA in Figure 1: no more CH2-OH signal is observed, and three sets of olefinic protons appear around 5.8, 6.1, and 6.4 ppm. Preparation of the Copolymers Networks. Many parameters can affect the compatibility of the cross-linked material



Table 3. Results from Thermal Analysis, Extraction, and Hydrolysis of the Cured Films

CD-1-XL CD-2-XL CT-1-XL a

From DSC.

b

Tg (°C)a

Tm (°C)a

wC (%)a

gel content (%)

WA (%)b,c

WL (%)b,d

-50.2 -42.0 -42.7

22.2

0.9

94 98 98

16.3 49.8 1.8

5.9 10.3 0.4

After 4 weeks in phosphate buffer solution (PBS) at pH 7.4 and 37 °C. c Water absorption.

with cultured cells such as the purity of the prepolymer used, the type and concentration of UV initiator, and the curing conditions. 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (IRGACURE 2959) was selected as the UV initiator for the curing experiments because it was shown that this compound is less toxic to cultured cells than other common initiators.56 The initiator was homogeneously dispersed into the copolymer diacrylates as described in the Experimental Section. Infrared (IR) spectroscopy was chosen to evidence the cross-linking reaction of the acrylated copolymers. The IR spectra of the copolymer diacrylates are almost identical to those of the copolymer diols, except for the appearance of the characteristic acrylates absorption bands at 1637 and 811 cm-1 (see Supporting Information). The first photocuring experiments (for CD1-DA and CT-1-DA) were therefore followed by ATR-FTIR in order to assess the conversion of the acrylate end groups into a network upon UV irradiation. For each experiment, a thin film of the copolymer was spread over the ATR cell and a nitrogen flow was applied. The samples were irradiated for 5 min at a wavelength of 315 ( 10 nm (0.45 mW/cm2) and spectra were recorded every 5 s. The surfaces under the acrylate absorption bands centered at 1637 and 811 cm-1 were integrated and plotted versus the time of irradiation (Figure 6). The irradiation was actually started after the five first scans so that the cross-linking reaction started at t ) 25 s in Figure 6. It was observed that the cross-linking process was slightly faster for CT-1-DA than for CD-1-DA. This can be explained by the fact that the ratio acrylate-to-initiator is slightly lower for CT-1-DA than for CD-1-DA (44:1 versus 52:1, respectively), leading to a faster consumption of the CdC bonds. While the acrylate absorption band at 811 cm-1 is overlapping with signals of the polymer backbone (especially in the case of CT-1-DA, where it appears as a shoulder), the bands around 1637 cm-1 are not observed anymore after 4 min of UV curing. The same formulations of polymers and initiator were then used for the preparation of films designed for cytocompatibility testing. Films 120 µm thick of the copolymer diacrylates CD1-DA, CD-2-DA, and CT-1-DA were applied on glass plates and cured for 120 s under nitrogen, as described in the Experimental Section. The obtained films were transparent, solid, and nontacky and adhered well to the glass support (films cured under air were also solid but slightly tacky, and UV curing experiments performed in the same conditions but without added UV initiator did not lead to cross-linked films). Gel content, water absorption (WA), and weight loss (WL) upon hydrolysis were measured for the three copolymer networks, which were also characterized by thermal analysis. The results of these experiments are reported in Table 3. As expected, the Tgs of the copolymer networks are slightly higher (between 4.4 and 5.7 °C higher) than those of the starting linear copolymer diols (see Table 2). The gel content of the cured films, determined by extracting the networks with CDCl3 at room temperature for 24 h, was found to be 94% for CD1-XL and 98% for the two other networks. 1H NMR and MALDI-ToF MS analyses of the extractables (spectra not shown here) revealed that the latter consist mainly of hydroxy endcapped and cyclic species (both of low molecular weight) in

d

Weight loss.

Figure 7. Proliferation of the neural stem cells in presence of the copolymers CT-1-XL and CD-2-XL and following polymer conditioning in distilled water or culture medium (see text). Each bar represent the normalized mean of the number of cells present in n culture wells and compared to the mean number of the cells in the control wells, fixed to 1. (Number of control wells: n ) 52; CT-1-XL wells: n ) 32; CT-1-XL + H2O wells: n ) 12; CT-1-XL + culture medium wells: n ) 33; CD-2-XL wells: n ) 34; CD-2-XL + H2O wells: n ) 16; CD-2-XL + culture medium wells: n ) 34). Asterisks indicate the bars that are significantly different with respect to control. *** ) P < 0.001.

the case of CT-1, and of acrylate end-capped and low molecular weight cyclic species in the case of CD-1 and CD-2. The lower gel content of CD-1 is probably due to a higher content in cyclic species compared to CD-2 and CT-1, as a longer polymerization time was used for the preparation of the starting copolymer diol CD-1 (22 h), which led to a higher proportion of backbiting reactions. The hydroxy and acrylate end-capped species mentioned above are barely detected in NMR and result from noncomplete acrylation and cross-linking reactions, respectively. It is known, for polymers containing CL and in contact with aqueous media, that hydrolytic degradation is faster in the amorphous phase than in the crystalline one.57 As our polymers are amorphous, the cured films were peeled off from their support and immersed in a phosphate buffer solution (PBS) at pH 7.4 and 37 °C, and their water absorption and weight loss were measured after 4 weeks (typical lifetime for a PoM device). As can be seen in Table 3, CT-1 gave the best results, with very low water absorption (1.8%) and weight loss (0.4%). The copolymers containing DXO (CD-1 and CD-2) are more hydrophilic and therefore exhibit higher water absorptions and weight losses upon hydrolysis, especially CD-2 (WA ) 50% and WL ) 10%), which contains the higher DXO:CL ratio (75:25). Although the integrity of the DXO-containing films was not affected by the degradation process, the relatively high water uptake resulted in a decrease of transparency, in contrast to CT-1, which remained perfectly transparent. The water uptake, weight loss, and partial loss of transparency observed for CD-1 and CD-2 are however expected to be less important for the DXO-containing copolymers when used in the aimed application (PoM device) because only one side of the film would be exposed to the aqueous medium.


Henry et al.

Figure 8. Neural stem cells differentiated to neurons and astrocytes on the surface of copolymers. Confocal microscopy images of cells cultured in control conditions (A, B, C, and D) and on the surface of the polymer samples CT-1-XL (E, F, G, and H) and CD-2-XL (I, J, K, and L). The neurons were stained for β-tubulin in column A, E, and I (as secondary antibody it was used Alexa 546, red); the astrocytes were stained for GFAP in column B, F, and J (as secondary antibody it was used Alexa 488, green), and cell nuclei were stained by means of DAPI in C, G, and K (blue). D, H, and L represent the merge of the previous column images. Images were taken using a Leica SP2 microscope with He/Kr and Ar lasers. Bar is 50 µm and it is representative for all the pictures.

Figure 9. SEM picture of a channel obtained by photocuring of a 120 µm film of CT-1-DA irradiated through a mask to produce microchannels of 50 µm width. Actual width of the microchannel is around 20 µm.

Cytocompatibility Testing. As the toxicity of the compounds involved in the cross-linking may be an important issue, it is worth to mention that the cured films were only washed with ethanol (nonsolvent for these polymers) and not extracted prior to the cytocompatibility tests. Cell Proliferation Assay. The first biocompatibility test was on the effects of the copolymers on the rate of proliferation of NSCs. Both CT-1-XL and CD-2-XL copolymer samples added to the cell culture wells induced a significant reduction in NSC replication in comparison to control cultures (Figure 7). However, utilizing a quite common practice in cell culturing to improve the cell vitality after plating, a treatment of the samples consisting in maintaining them in the culture medium for six days was carried out. This preconditioning significantly reduced or abolished the inhibitory effects of copolymers on NSC

reproduction. In fact, the number of cells after 6 days in culture in the presence of conditioned CT-1-XL samples was comparable to that in the control, whereas the conditioned CD-2-XL copolymers inhibited stem cell proliferation to a significantly (P < 0.001) lower extent. To understand if the beneficial effect was due to a simple wash out of undesirable molecules or if it was produced by a more complex functionalization of the polymer surface, a test with distilled water was performed. As shown in Figure 7, even the 6 days immersion in distilled water was beneficial, but significantly (CT-1-XL: P < 0.01; CD-2XL: P < 0.05) less than that in culture medium. These results suggest that although a wash out of undesirable substances seems to take place, improving cell proliferation, the conditioning of copolymers in culture medium displays a more complex effect, either a better extraction process of undesirable substances or a beneficial functionalization of the copolymer surface. Neural Stem Cell Differentiation on the Surface of CT-1-XL and CD-2-XL Copolymers. NSCs attachment and differentiation to nervous cell at the surface of the sample polymers were evaluated: 40,000 NSCs were plated in the wells on a 48-multiwell plate where a sample disk was previously placed on the bottom, and the differentiation induction protocol was followed. The cells attached at the surfaces of the samples after 7 days from the onset of the differentiation protocol were immunostained for β-tubulin III and GFAP to identify neurons and astrocytes, respectively, and observed by means of the confocal microscopy. Many cells were found positive either to β-tubulin III or to GFAP, indicating that at least part of neural stem cells that were attached to the polymeric surface differentiated to neurons and astrocytes, as it is shown in Figure 8. Preparation of Patterned Films. Preliminary experiments were conducted to investigate whether the synthesized materials can be processed into patterned films by photocuring. Figure 9 shows the SEM images of a 120 µm film of CT-1-DA irradiated through a mask in order to produce microchannels of 50 µm width. From the figure, it can be observed that the channel is


only about 20 µm wide, which might be due to material mobility during the curing process. While this shows the applicability of the synthesized materials for the fabrication of patterned PoMs, further optimization of the irradiation (irradiation time and power) and/or of the development process (type of solvent, development time and temperature) is necessary.

Conclusions Low molecular weight random copolymer diols were successfully synthesized by tin(II)-catalyzed ring-opening copolymerization of CL with either DXO or TMC (in the bulk), initiated by 1,4-benzenedimethanol (BDM). The resulting copoly(ether-ester)s and copoly(ester-carbonate) were characterized by NMR and IR spectroscopy, MALDI-ToF MS, SEC, and DSC. The random nature was confirmed by the relatively short average monomer block lengths calculated from the NMR spectra and by thermal analyses, which showed only one glass transition for every copolymer as well as a lack of crystallinity. According to NMR and MALDI-ToF MS techniques, the polymeric chains were found to be almost exclusively diol endcapped. Clean diacrylated copolymers were successfully obtained by reacting the hydroxy end groups with acryloyl chloride, as demonstrated by NMR and IR spectroscopies. A low concentration of a UV initiator (0.15 wt % IRGACURE 2959) was introduced into the functionalized copolymers, and the latter were photocross-linked under UV irradiation as thin films (120 µm thick). The resulting transparent films are characterized by low Tgs and a good cytocompatibility. The biocompatibility tests confirm the good properties of these copolymers as far as in vitro NSCs proliferation and differentiation to neurons and astrocytes are concerned. Noteworthy are the beneficial effects obtained upon preconditioning the copolymers by means of the cell-culture medium, and the excellent properties shown particularly by the CL-TMC that make this polymer a good candidate for the aimed application (“polymeron-multielectrode arrays” devices). Moreover, preliminary results show that microchannel formation by photocuring is possible with the synthesized polymers. Acknowledgment. This work was financed by the European Union (European Regional Development Fund) within the INTERREG IIIC Programme. We thank Francesca Scaltro and Otto van Asselen for their help with the curing experiments and the ATR-FTIR spectroscopy, respectively, and Drs. Raffaella Adami for the confocal images and Daniela Cigognini for the help with neural stem cells culturing. Furthermore, Casper L. van Oosten and An M. Prenen are thanked for their help in the preparation of the patterned films and the SEM pictures. Supporting Information Available. 1H, 13C NMR and IR spectra of the copolymer diols and diacrylates. This material is available free of charge via the Internet at http://pubs.acs.org.

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