Enhanced Hydrolytic Degradation of Heterografted ... - ACS Publications

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Enhanced Hydrolytic Degradation of Heterografted Polyglycidols: Phosphonoethylated Monoester and Polycaprolactone Grafts Jens Köhler,† Fabian Marquardt,† Michael Teske,‡ Helmut Keul,*,† Katrin Sternberg,*,‡ and Martin Möller*,† †

Institute of Technical and Macromolecular Chemistry, RWTH Aachen University and Interactive Materials Research - DWI at RWTH Aachen e.V., Forckenbeckstr. 50, D-52056 Aachen, Germany ‡ Institute for Biomedical Engineering, University of Rostock, Friedrich-Barnewitz-Straße 4, D-18119 Rostock, Germany S Supporting Information *

ABSTRACT: Novel biodegradable materials with tunable hydrolytic degradation rate are prepared by grafting of phosphonoethylated polyglycidols with polyesters. First, the hydrolytically degradable polyester grafts are attached to polyglycidols partially grafted with phosphonoethylated diethyl esters through chemical-catalyzed grafting using tin(II) octanoate, then the diethyl ester groups are chemoselectively converted to the corresponding monoester (mixed phosphonate/ phosphonic acid) using alkali metal halides. The products are characterized by means of 1H, 13C, and 31P NMR spectroscopy, as well as size-exclusion chromatography and differential scanning calorimetry. The in vitro degradation of the copolymers is studied in phosphate buffered solution at 55 °C. The copolymers are of the same architecture, molecular weight, and crystallinity, only differing in the pendant phosphonate and mixed phosphonate/phosphonic acid groups, respectively. On the basis of mass loss, decrease of the molecular weight, and morphological analysis of the copolymers, the strong impact of mixed phosphonate/ phosphonic acid groups on the hydrolytic degradation rate is demonstrated.

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INTRODUCTION Biodegradable synthetic polymers, such as aliphatic polyesters are a compelling class of materials for biomedical applications.1−4 Compared to naturally occurring polymers, which typically degrade through enzyme-catalyzed pathways, aliphatic polyesters are degradable through enzymatically and chemically catalyzed hydrolytic routes. Prominent examples of polyesters used are polyhydroxyalkanoates, polylactide, polyglycolide, poly(ε-caprolactone), and copolymers.5−8 Continuous progress in the synthesis of polyesters has led to a diversity of hydrolytically degradable systems, which meet current and future demands for sustainability and ecology. Synthetic polyesters are valuable materials as they allow for tailored performance characteristics such as thermal properties, mechanical strength and degradation behavior. Poly(ε-caprolactone) (PCL) is one of the most widely applied artificial polyesters since PCL degrades in aqueous environment through hydrolysis mediated by acids, bases and enzymes.9 Commonly, PCL is prepared through ring-opening polymerization of ε-caprolactone. Catalysts, such as tin(II) octanoate are applied together with mono- or multifunctional alcohols as initiators.10−12 However, when the hydrolytic degradation rate is considered, PCL degrades much slower compared to other polyesters, like polylactide or polyglycolide, due to its hydrophobic nature. This apparent limitation is beneficial when long-term applications are desired, and the biodegradation should take several years.13,14 © 2013 American Chemical Society

The design of novel biodegradable materials is still a challenge in synthetic polymer chemistry. Especially the prediction and adjustment of their degradation rate is of crucial importance to adopt a biodegradable polymer for a specific application. The hydrolytic degradation of polyesters proceeds under carboxylic acid formation, which leads to an autocatalyzed degradation mechanism. The rate depends on various factors, such as molecular weight, hydrophilicity of the material, and the crystallinity of the polymer and its architecture. Recently, different routes have been developed to tune the degradation rate of PCL-based systems. Copolymerization with hydrophilic comonomers, such as 1,5dioxepan-2-one and trimethylene carbonate or lactide,15−18 as well as blending with additives, has been reported to increase the hydrophilicity of the system and thus to enhance the wateruptake in the hydrophilic domains.19,20 For polylactide it has been reported that the introduction of acidic end-groups leads to enhanced water uptake and accelerated degradation rates.21−23 More sophisticated macromolecular architectures have been designed to tune the degradation rate by decreasing the crystallinity of PCL, for example, block, multiblock, star-shaped copolymers, or cross-linked networks.24−27 In previous publications, our group has focused on the preparation of linear and star-shaped poly(glycidol-graft-εReceived: July 24, 2013 Published: October 2, 2013 3985

dx.doi.org/10.1021/bm401428b | Biomacromolecules 2013, 14, 3985−3996

Biomacromolecules

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Size-Exclusion Chromatography. Molecular weights (Mn,SEC and Mw,SEC) and molecular weight distributions (Mw/Mn) were determined by size-exclusion chromatography (SEC). SEC analyses were carried out with tetrahydrofuran (THF), DMF, or water as eluent. For THF SEC a high pressure liquid chromatography pump (Knauer 6420) with a RI detector (Jasco RI-2031 plus) at 30 °C was used. The eluent was THF (HPLC grade, Carl Roth) and a flow rate of 1.0 mL/min was used. Four columns with MZ SDplus gel were applied. Length of each column was 300 mm, diameter 8 mm and nominal pore widths of the gel particles were 50, 100, 1000, and 10000 Å. The calibration was achieved with commercially available poly(methyl methacrylate) (PMMA) standards. For DMF SEC, a high pressure liquid chromatography pump (Knauer K-1001 Wellchrom) with a dual RI-/Visco detector (WGE ETA-2020) at 30 °C was used. The eluent was DMF (optigrade, Promochem) with 1 mg/mL LiBr and a flow rate of 1.0 mL/min was used. Four columns with PSS GRAM gel were applied. Length of each column was 300 mm, diameter 8 mm, and nominal pore widths of the gel particles were 30, 100, 1000, and 3000 Å. Narrow distributed PMMA standards were used for calibration. For water SEC an Agilent 1100 system equipped with a MWD-UV detector and an Optilab DSP RI detector (Wyatt) was used. The eluent was H2O (HPLC grade, Carl Roth) with 0.1 mol/L sodium nitrate (NaNO3) + 0.01 wt % sodium azide (NaN3) and a flow rate of 1.0 mL/min was used. Three columns with Suprema gel were applied. Length of each column was 300 mm, diameter 8 mm, and nominal pore widths of the gel particles were 30, 1000, and 3000 Å. Narrow distributed poly(ethylene glycol) standards were used for calibration. Results were evaluated using the PSS WinGPC Unity software (version 7.1). Differential Scanning Calorimetry. The polymer samples (n = 5 samples per time point) of selected time points were analyzed by differential scanning calorimetry (DSC). DSC was performed on a Netzsch DSC 204. The measurements were performed under nitrogen with a heating rate of 10 K/min. Calibration was achieved using indium and cyclohexane. Cytototoxicity Measurements. Before cytotoxicity tests the probes were washed for 24 h in 0.1% Tween 20 (w), for further 24 h in 0.05% Tween 20 (w), then for 24 h in pure water and finally for 72 h in methanol. After washing, the probes were dried in a vacuum chamber for 24 h at 40 °C. For disinfection, samples were put into ethanol for 5 min, followed by three rinses in sterile water, and transferred to the bottom of the wells of a 96-well microtiter plate. The biocompatibility of materials was tested using CellQuanti-Blue assay (BioAssay Systems, Hayward, CA), as described elsewhere.36 L929 mouse fibroblasts (ATCC number CCL1) were purchased from DSMZ (Braunschweig, Germany) and incubated for 24 h at 37 °C and 5% CO2 in DMEM. The cells were seeded (4000 per well) in a volume of 200 μL of DMEM. After a 48 h incubation period, the extracts were removed by aspiration and replaced by 200 μL DMEM with 10% CellQuanti-Blue reagent. After 2 h incubation at room temperature fluorescence (590 nm) of samples was measured in a microtiter plate reader (reader FLUOstar Optima, BMG Labtech, Offenburg, Germany). The cell viability was reported relative to the absorbance of the untreated control (NC), which was set to 100%. Cells, incubated with 10−2 M TETD (tetraethylthiuramdisulfid) served as positive control (PC). For statistical analysis, one-way Anova was performed using GraphPad Prism version 5.00 (GraphPad Software, San Diego California, U.S.A.). Contact Angle Measurements. Contact angles were measured by sessile drop method (Contact Angle System, OCA 20, Dataphysics Instruments GmbH, Filderstadt, Germany) with water at 23 °C. Water drops were dispersed with 5 μL/s and had a volume of 11 μL. Mean values and standard deviations were calculated from five samples with n = 2 measurements per sample (one topside and one backside). Syntheses. Poly(ethoxy ethyl glycidyl ether) P(EEGE) (1) and polyglycidol (PG) (2) were synthesized according to literature procedure.28 The results of the chemical analysis for P(EEGE)26 and PG26 are summarized in Table S1, Supporting Information.

caprolactone)s (P(G-g-εCL)) by means of enzymatic and chemical catalysis.28−30 The copolymers prepared through lipase-catalyzed grafting show enhanced degradation, which has been related to the hydrophilic polyglycidol (PG) backbone, because the majority of the hydroxymethyl groups of PG remain unreacted in the enzymatic grafting. This assures fast water uptake in the highly hydrophilic domains. By contrast, linear P(G-g-εCL) copolymers prepared through chemical catalysis lack hydrophilicity, because almost all hydroxyl groups participate in the grafting. Thus, they are not hydrolytically degradable within a reasonable time frame which limits their use for non-degradable biomaterials. Recently, we extended the P(G-graft-εCL) library through lipase-catalyzed grafting from polyglycidols with pendant diethylphosphonatoethyl groups (DEPE), P(GDEPE-co-G).31,32 These well-defined polyether graft polyesters are a powerful class of materials for biomedical applications, because polyglycidols as well as polyphosphonates are biocompatible.33,34 Additionally, the phosphonate residues are versatile precursors for phosphonic acids and they promote adhesion to hard tissue, such as dentin, enamel and bones. However, to introduce P(GDEPE-co-(G-graf t-εCL)) as biomaterials the hydrolytic degradation profile of the phosphonoethylated graft copolymers must be established. Here we focus on chemically catalyzed grafting of εCL from phosphonoethylated polyglycidols, where all hydroxyl groups of PG participate in the grafting process.28 This allows us to precisely evaluate the influence of the DEPE residues on the hydrolytic degradation rate. Moreover, the pendant DEPE groups are addressed as intramolecular acid generators. Ethylphosphonatoethyl groups (EPE) are obtained through a postpolymerization modification using alkali metal halides with the purpose to facilitate hydrolytic degradation of the PCL grafts by mono phosphonic acid residues in the P(GEPE-co-(Ggraf t-εCL)) copolymers.

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EXPERIMENTAL SECTION

Materials. Potassium tert-butoxide (1 M solution in THF, Aldrich), N,N-dimethylformamide (DMF, dry, over molecular sieves, Aldrich), dichloromethane (DCM, p.a., VWR), acetone (p.a., VWR), 2hexanone (p.a., Aldrich), sodium iodide (NaI, p.a., Merck), lithium bromide (LiBr, ≥ 97%, Aldrich), tin(II) octanoate (Sn(oct)2, 95%, Aldrich), and PCL pellets (Capa 6800, Mw = 80000 g/mol, Perstorp) were used as received. Diglyme was distilled over sodium before use. 3Phenyl-1-propanol (≥98%, Fluka) was reacted with small amounts of sodium and distilled. Ethoxy ethyl glycidyl ether (EEGE) was synthesized according to Fitton et al.,35 purified by distillation and stored under nitrogen atmosphere over molecular sieves (3 Å). Diethyl vinylphosphonate (95+%, Aldrich) was stirred with calcium hydride (CaH2) for 24 h, distilled under reduced pressure and stored under nitrogen atmosphere over molecular sieves (3 Å). ε-Caprolactone (εCL, 99%, ABCR) was stirred with CaH2 for 24 h, distilled under reduced pressure, and stored under nitrogen atmosphere over molecular sieves (3 Å). Measurements. NMR Spectroscopy. 1H, 13C, and proton decoupled 31P NMR spectra (31P{1H}) were recorded on a Bruker DPX-400 FT-NMR spectrometer at 400, 101, and 162 MHz, respectively. Deuterated chloroform (CDCl3), dimethyl sulfoxide (DMSO-d6), or water (D2O) were used as solvents. Tetramethylsilane (TMS; CDCl3) or residual solvent signal (DMSO-d6, D2O) served as internal reference. End group signals are marked with an E. 31P{1H} NMR spectra were referenced against 85% phosphoric acid (H3PO4) as external standard. The amphiphilic graft copolymers were analyzed using solvent mixtures (80% DMSO-d6 and 20% DCM (v/v)). The coupling constants Jxy are given in Hz. 3986


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Table 1. Synthesis of P(GDEPE-co-(G-g-εCL)-co-G) (4a,b), (5a,b), and P(GDEPE-co-(G-g-εCL)) (6) Using 3a−c as Macroinitiators (t = 22 h, T = 120 °C, Conversion of εCL = 100%)a macroinitiator

a

polymer

No.

g, mmol OH

εCL g, (mmol)

4a 4b 5a 5b 6

3a 3a 3b 3b 3c

0.191, 0.256, 0.263, 0.273, 0.828,

0.743, (6.508) 1.991, (17.466) 0.539, (4.718) 1.118, (9.795) 18.890, (165.50)

(1.627) (2.180) (1.179) (1.224) (4.138)

yieldb (%)

Sn(oct)2 g, (mmol) 0.042, 0.042, 0.042, 0.042, 0.056,

(0.10) (0.10) (0.10) (0.10) (0.13)

91 93 87 89 94

Reagent ratios and yields obtained after purification. bAfter purification by precipitation in pentane.

Phosphonoethylated polyglycidols, P(GDEPEx-co-Gy)s (3) were synthesized according to an established literature protocol.31,32 The results of the chemical analysis for P(GDEPE4-co-G22) (3a), P(GDEPE10co-G16) (3b), and P(GDEPE9-co-G17) (3c) are presented in Table S2, Supporting Information. Synthesis of Poly(glycidol diethylphosphonatoethyl-co(glycidol-graf t-ε-caprolactone)-co-glycidol), P(GDEPE-co-(G-gεCL)-co-G); 4a,b, 5a,b, and 6. P(GDEPE10-co-G16) (3b; 0.273 g, 1.224 mmol OH) and εCL (1.118 g, 9.795 mmol) were heated to 50 °C in order to obtain a homogeneous solution. Sn(oct)2 (0.042 g, 0.10 mmol) was added, and the mixture was heated to 120 °C. After stirring for 22 h, the polymerization was quenched by addition of methylene chloride. The product was isolated by precipitation in pentane and dried under reduced pressure. 5b: Yield 89%. 1H NMR (DMSO-d6/ DCM): δ 1.25 (tr, 6H, 3JHH = 6.95 Hz, POCH2CH3), 1.27−1.38 (m, 2H, OCOCH2CH2CH2), 1.38−1.48E (m, 2H, CH2CH2CH2OH), 1.48−1.66 (m, 4H, OCOCH2CH2CH2CH2), 1.76−1.84 (m, 2H, ArCH2CH2), 1.96−2.10 (m, 2H, 2JHP = 18.1 Hz, CH2OCH2CH2P), 2.27 (tr, 2H, 3JHH = 7.33 Hz, OCOCH2CH2), 2.57−2.65 (m, 2H, ArCH2), 3.34−3.70 (m, 19H, ArCH2CH2CH2, OCH2CH(CH2OH)O, OCH 2 CH(CH 2 OCH 2 CH 2 P)O, OCH 2 CH(CH 2 OCOCH 2 )O, CH2CH2OH), 3.90−4.08 (m, 6H, POCH2CH3, CH2CH2OCO), 4.12−4.32 (m, 2H, CHCH2OCO), 4.35−4.50 (br s, CHCH2OH groups), 7.10−7.29 (m, 5H, Ar). 31P NMR (DMSO-d6/DCM): δ 28.3. The polymers 4a,b, 5a, and 6 were synthesized in analogy to 5b. The reagent ratios and yields are listed in Table 1. Postpolymerization Modification of Phosphonoethylated Polyether-graf t-polyesters. P(GDEPE4-co-G22) (3a; 1.123 g, 1.757 mmol DEPE) was reacted in 2-hexanone (30.0 mL) with LiBr (0.183 g, 2.107 mmol, 1.2 equiv). The mixture was stirred under reflux for 24 h. The solvent was removed and the product 7a was purified by precipitation in acetone. 7a: Yield 84°%. 7b was prepared in analogy to 7a with 1.2 equiv of NaI as dealkylation reagent. Moreover, graft copolymer 5b was used as substrate for the dealkylation of the DEPE groups with LiBr or NaI in presence of PCL grafts. The products 8a,b were analyzed without further purification. Finally, using 6 as starting material P(GEPE9-co-(G-g-εCL33)17) (9) was prepared with NaI following the same protocol. The reagent ratios are summarized in Table 2. 7a: 1H NMR (D2O): δ 1.23 (tr, 3H, 3JHH = 6.85 Hz, POCH2CH3), 1.82−2.06 (m, 4H, ArCH2CH2, CH2OCH2CH2P), 2.68 (tr, 2H, 3JHH = 7.27 Hz, ArCH 2 ), 3.44−3.82 (m, 14H, ArCH 2 CH 2 CH 2 , OCH2CH(CH2OH)O, OCH2CH(CH2OCH2CH2P)O), 3.88 (quin, 2H, 3JHH = 6.98 Hz, POCH2CH3), 7.0−7.41 (m, 5H, Ar). 13C NMR (D2O): δ 16.0 (d, 3JCP = 4.9 Hz, POCH2CH3), 27.1 (d, 1JCP = 131.8 Hz, CH2OCH2CH2P), 30.6 (ArCH2CH2), 31.5 (ArCH2), 60.6, 60.7 (POCH2CH3, OCH2CH(CH2OH)O), 66.5 (CH(CH2OCH2CH2P)O), 68.6−69.8 (ArCH2CH2CH2, OCH2CH(CH2OCH2CH2P)O, OCH 2 CH(CH 2 OH)O), 78.1 (OCH 2 CH(CH 2 OCH 2 CH 2 P)O), 79.5−79.7 (OCH2CH(CH2OH)O), 126.1 (Ar), 128.6 (Ar), 142.2 (Ar). 31P NMR (D2O): δ 23.0. 8b: 1H NMR (CDCl3): δ 1.20 (m, 3H, POCH2CH3), 1.26−1.46 (m, 2H, OCOCH2CH2CH2), 1.48−1.74 (m, 4H, OCOCH 2 CH 2 CH 2 CH 2 ), 1.70−2.00 (br s, 4H, ArCH 2 CH 2 , CH2OCH2CH2P), 2.28 (tr, 2H, 3JHH = 7.49 Hz, OCOCH2CH2), 3.20−3.80 (m, 21H, ArCH 2 CH 2 CH 2 , OCH 2 CH(CH 2 OH)O,

Table 2. Synthesis of P(GEPE4-co-G22) (7a,b), P(GEPE10-co(G-g-εCL11)12-co-G4) (8a,b), and P(GEPE9-co-(G-g-εCL33)17) (9) (t = 24 h; T = 130 °C; Solvent: 2-Hexanone): Reagent Ratios and Yields salta

macroinitiator polymer

No.

7a 7b 8a 8b 9

3a 3a 5b 5b 6

g, (mmol DEPE) 1.123, 1.225, 0.287, 0.304, 4.589,

(1.757) (1.917) (0.143) (0.150) (0.603)

g, (mmol) LiBr NaI LiBr NaI NaI

0.183, 0.344, 0.015, 0.027, 0.109,

(2.106) (2.300) (0.172) (0.180) (0.727)

yield (%) 84b 79b 89c 91c 90d

a

1.2 equiv with respect to DEPE groups. bAfter purification by precipitation in acetone. cNo purification was conducted. dAfter precipitation in basic water (pH = 8−9, adjusted by adding 44 g/L NaHCO3). OCH 2 CH(CH 2 OCH 2 CH 2 P)O, OCH 2 CH(CH 2 OCOCH 2 )O, CH 2 CH 2 OH, POCH 2 CH3 ), 3.88 (tr, 2H, 3 J HH = 6.64 Hz, CH2CH2OCO), 3.90−4.30 (br s, 2H, CHCH2OCO), 7.10−7.17 (m, 3H, Ar), 7.20−7.27 (m, 2H, Ar). 31P NMR (CDCl3): δ 22.7. 9: The NMR spectrum is the same as reported for 8b, only differing in the relative intensity of the signals. Accelerated In Vitro Degradation Study. For the degradation parallelepiped specimens of P(GDEPE9-co-(G-g-εCL33)17 (6), P(GEPE9co-(G-g-εCL33)17 (9), and the PCL reference were prepared by compression molding with a sample geometry in the range of 10 mm length, 5 mm width, and 0.1 mm thickness. The polymers were heated for 2 min at 80 °C by heating the plates of the hydraulic press. Then, the polymers were compressed to foils by applying a pressure of 2 tons for 1 min. Finally, the obtained foils were cut to specimens of appropriate size. For the investigation of the in vitro degradation behavior each polymer sample (n = 11 samples per time point) was placed in a test tube containing 2 mL of Sørensen buffer (0.1 M, pH 7.4) and kept at 55 °C under gentle shaking. Samples were periodically removed, washed with distilled water and dried in vacuum before analysis. The buffer solution was changed twice per week. Gravimetry. To evaluate the polymer fragmentation by determination of mass loss the washed and dried samples (n = 5 samples per time point) were weighted using a special accuracy balance (UMX 5 Mettler Toledo, Switzerland). The mass loss was determined as the mass at time t divided by the initial mass multiplied by 100. Molecular Weight Analysis. The degradation-induced reduction of the molecular weight of the polymer samples (n = 5 samples per time point, three measurements in parallel) was determined at 30 °C using a PSS SECcurity SEC system (Polymer Standard Services GmbH, Mainz, Germany), including a RI detector combined with a WGE Dr. Bures η 2010 viscosity detector (WGE Dr. Bures GmbH, Dallgow, Germany). The size separation process was performed with three PSS SDV columns (103, 105, and 106 A°, respectively). Chloroform stabilized with ethanol was used as eluent at a flow rate of 1 mL/min. The different polymer samples were dissolved in chloroform (1.5 mg/ mL) containing hexyl benzene as internal standard. The injection volume was 0.1 mL. The molecular weights were calculated by the 3987


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Table 3. P(GDEPEx-co-Gy) Macroinitiators 3a−c: Degree of Functionalization with Diethylphosphonatoethyl Groups (FDEPE), Percentage of Glycidol Repeating Units, Number Average Molecular Weights Determined by 1H NMR and SEC Analysis, and Molecular Weight Distributions P(GDEPEx-co-Gy)

FDEPEa x, (%)

glycidola y, (%)

Mn,NMRb (g/mol)

Mn,SECc (g/mol)

Mw/Mn,SECc

P(GDEPE4-co-G22) (3a) P(GDEPE10-co-G16) (3b) P(GDEPE9-co-G17) (3c)

4, (15) 10, (39) 9, (35)

22, (85) 16, (61) 17, (65)

2583 3567 3403

2900 3600 3900

1.3 1.4 1.4

a

According to 1H NMR analysis. bAbsolute molecular weight according to 1H NMR analysis. The accuracy of integration in 1H NMR spectra is ±5%. cMolecular weight and molecular weight distribution determined by SEC using narrow distributed poly(methyl methacrylate) standards and DMF as eluent.

Scheme 1. Chemical Structures of P(EEGE)26 (1) and PG26 (2) (Top) and Preparation of P(GDEPE4-co-(G-g-εCLn)m-co-Gp) (4a,b), P(GDEPE10-co-(G-g-εCLn)m-co-Gp) (5a,b), and P(GDEPE9-co-(G-g-εCLn)m) (6) (Bottom)

universal calibration method using 12 polystyrene calibration standards in a range between 376 and 2570000 g/mol. pH Value Measurement. Polymer samples (n = 3) were stored in 2 mL of pure water at 55 °C under gentle shaking and the pH values were measured at different time points by means of pH meter (Seven Easy pH Meter S20, Schwerzenbach, Switzerland). Oven-Based Karl Fischer Titration for the Determination of Water Uptake. Oven-based Karl Fischer (KF) titration was conducted according to Petersen et al.37 Before the KF measurements, each sample was weighted using a microbalance (UMX5, Mettler Toledo, Giessen, Germany) in order to enable the indication of water uptake per mg polymer. The polymer probes (10 × 5 × 0.1 mm) were contacted for 24 h with 2 mL of Sørensen buffer at 50 °C and thereafter carefully dried to remove water droplets from the surface. Reference samples were dried in a vacuum chamber at 40 °C and 40 mbar for two days, were dipped for two seconds in Sørensen buffer and finally water droplets were removed. For each polymer n = 4 samples were used. Samples were subsequently placed in the sample holder of the Drying Oven D0308 (Mettler Toledo, Gießen, Germany) and heated up to 120 °C for PCL and 160 °C for 6 and 9 in an argon atmosphere. Finally, the water content was determined using a Karl Fischer Coulometer C20 (Mettler Toledo, Gießen, Germany) with the Hydranal-Coulomat AG KF reagent (SigmaAldrich, St. Louis, U.S.A.). For statistical analysis, Mann−Whitney test was performed using SPSS version 15.01 with α = 0.05. Documentation of Degradation-Induced Fragmentation. The proceeding fragmentation process (n = 1 sample per time point) was documented by macro photography.

with pendant phosphonate groups through lipase-catalyzed grafting of εCL from phosphonoethylated polyglycidols. Grafting densities up to 80% have been achieved, depending on the reaction parameters.32 In the present work, we are focused on establishing the hydrolytic degradation profile of this new class of graft copolymers. First, we investigate the grafting from reaction catalyzed by Sn(oct)2, in order to achieve side chain growth from all initiation sites of the macroinitiator. This assures that the hydrolysis of the copolymers is not enhanced through residual hydrophilic hydroxyl groups at the PG backbone, which has been reported in the past.30 The microstructural analysis of the graft copolymers is accomplished by means of NMR spectroscopy and SEC. This section is completed with the preparation and characterization of two prototype polymers, carrying phosphonate and phosphonate/phosphonic acid residues, which are investigated in the degradation study. In the second part the results of the accelerated degradation study of poly((glycidol diethylphosphonatoethyl)-co-(glycidolgraf t-ε-caprolactone), P(GDEPE9-co-(G-g-εCL33)17) (6), and poly((glycidol ethylphosphonatoethyl)-co-(glycidol-graf t-ε-caprolactone), P(GEPE9-co-(G-g-εCL33)17) (9) in phosphatebuffered solution are presented. Because both polymers are of the same architecture and have similar molecular weights and crystallinities, the impact of the phosphonate and mixed phosphonate/phosphonic acid residues on the degradation profile can be precisely determined. Synthesis of Phosphonoethylated Macroinitiators 3a−c. The synthetic protocol as well as the characterization of the macroinitiators has been reported in detail.31,32 The

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RESULTS AND DISCUSSION Polymers decorated with phosphonate residues are valuable candidates for a multitude of different applications. Recently, we reported on the preparation of polyether graft polyesters 3988


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Figure 1. 1H NMR spectrum of P(GDEPE9-co-G17) (3c) in DMSO-d6 and P(GDEPE9-co-(G-g-εCL33)17) (6) in CDCl3; #DCM.

Table 4. Synthesis of P(GDEPE4-co-(G-g-εCLn)m-co-Gp) (4a,b), P(GDEPE10-co-(G-g-εCLn)m-co-Gp) (5a,b), and P(GDEPE9-co-(G-gεCL33)17) (6) (T = 120 °C, Conv. = 100%)a polymer (initiator)

[CL]/ [OH]b

CL unitsc (v)

Mn, NMRd

Mn, SECe

Mw/Mn, SECe

4a (3a) 4b (3a) 5a (3b) 5b (3b) 6 (3c)

4 8 4 8 40

97 191 63 144 567

10872 24384 10758 20003 68120

15100 24700 9100 20000 95100

1.4 1.5 1.6 1.4 1.3

IEf m; (%) 14; 16; 10; 12; 17;

(64) (73) (63) (75) (100)

Pn, SCg, n

wt % of free PCLh

7 12 6 12 33

3.0 4.0 2.9 3.2 1.5

a Macroinitiator used, ratio of εCL repeating units per OH group of the macroinitiator, εCL repeating units per macroinitiator, molecular weights obtained from 1H NMR and SEC, molecular weight distributions determined by SEC analysis, initiation efficiencies of the macroinitiators, degree of polymerization of the side chains and weight fraction of free PCL contained in the samples. bEquivalents of εCL per hydroxyl group of the macroinitiator in the feed. cNumber of εCL units (v) grafted from the macroinitiator according to 1H NMR analysis. dAbsolute molecular weight (Mn,NMR) determined by 1H NMR analysis. eNumber average molecular weight (Mn,SEC) and molecular weight distribution (Mw/Mn) determined by SEC analysis using narrow distributed PMMA standards and THF as eluent. fIE: Initiation efficiency of the macroinitiator. gPn,SC: Degree of polymerization of the side chains. Calculated from the number of εCL units (v) grafted from the macroinitiator divided by the number of sites which initiated the grafting. hWeight fraction of free PCL has been determined with WinGPC Unity software.32

S2). The 1H NMR spectra of the graft copolymers are all similar, only differing in the relative intensity of the signals. The spectrum of macroinitiator 3c and copolymer 6 is shown in Figure 1 as representative example. The microstructural analysis of the graft copolymers was conducted according to our recent report.32 The results of the microstructural analysis of copolymers 4−6 by means of NMR and SEC analysis are summarized in Table 4. SEC analysis reveals the preparation of copolymers with narrow molecular weight distributions ranging from 1.3 ≤ Mw/ Mn ≤ 1.6. The graft copolymers with a low PCL concentration show a tailing to lower molecular weights (Supporting Information, Figure S3). This has been explained with a decreased THF solubility of the polymers.32 Traces of water, which are bound to the highly hydrophilic macroinitiators lead to the formation of free PCL as side product. They can be removed from the products through precipitation in pentane. Thus, the concentration of remaining PCL in the product depends on the quality of the purification step.32 PCL concentrations ranging from 1.5 to 4.0 wt % are found.

microstructures of phosphonoethylated poylglycidols which are used in this study are presented in Table 3. The SEC traces of the macroinitiators in comparison to the parent polyglycidol, PG26 (2) are shown in Supporting Information, Figure S1. Grafting of ε-Caprolactone from 3a−c. The chemical ring-opening polymerization of εCL is conducted in bulk using Sn(oct)2 as catalyst at 120 °C (Scheme 1). P(GDEPE4-co-G22) (3a) and P(GDEPE10-co-G16) (3b) have been used to study the feasibility of our synthetic approach. The corresponding graft copolymers 4a,b and 5a,b are prepared by applying ratios of εCL to initiating hydroxyl groups [CL]/[OH] = 4 and 8, respectively. Finally, P(GDEPE9-co-G17) (3c) has been applied to prepare P(GDEPE9-co-(G-g-εCL33)17) (6) with [CL]/[OH] = 40, which finds application in the hydrolytic degradation study. The chemical grafting experiments showed quantitative monomer conversion for all reactions conducted as proven by 1H NMR analysis prior to the workup. The chemical analysis of the graft copolymers reveals that Sn(oct)2 tolerates the DEPE residues at the PG backbone, because no transesterification is observed. This is proven by comparing the 31P NMR spectra of the graft copolymers with those of the macroinitiators (Supporting Information, Figure 3989


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reached within 24 h, according to quantitative 31P NMR analysis. (Supporting Information, Figure S4). The 1H NMR spectra of P(GDEPE4-co-G22) (3a) and P(GEPE4-co-G16) (7a) are shown in Figure 2. The signals 12−14 show a significant high-field shift in the product 7a. Especially, signal 12 of the methylene group adjacent to the phosphorus atom shifts from δ = 2.2 ppm to δ = 2.0 ppm in 7a. The intensity of the signals 7−11 and 15−17 of the polyether backbone is the same as in 3a. This demonstrates the feasibility of the procedure, since the PG backbone is not affected by the dealkylation reagent. In the following, this concept is extended to the DEPEcontaining graft copolymers. P(GDEPE10-co-(G-g-εCL12)12-coG4) (5b) is chosen to study the reaction. Again, NaI and LiBr are applied in 2-hexanone as solvent to achieve the monodealkylation of the DEPE groups. The products 8a,b are analyzed by means of NMR and SEC without purification. Contrary to the results obtained for 3a as substrate, the efficiency of the salts is different here. With LiBr P(GDEPE2-coGEPE8-co-(G-g-εCL12)12-co-G4) (8a) with 82% of EPE groups is obtained, whereas NaI leads to quantitative conversion of the DEPE groups resulting in P(GEPE10-co-(G-g-εCL12)12-co-G4) (8b). The conversions of the DEPE groups are confirmed by 31 P NMR spectroscopy (Supporting Information, Figure S5). In Figure 3, the 1H NMR spectra of 5b and 8b are shown as an example. The ethylphosphonate/phosphonic acid-containing product 8b shows the similar high field shifts for the signals of the EPE groups as reported for 7a. The intensity of signal 19 remains constant within the error of integration, proving that the PCLgrafts are not cleaved during the reaction. This is further confirmed by the SEC-traces of P(GDEPE10-co-(G-g-εCL12)12-coG4) (5b), P(GDEPE2-co-GEPE8-co-(G-g-εCL12)12-co-G4) (8a), and P(GEPE10-co-(G-g-εCL12)12-co-G4) (8b; Supporting Information, Figure S6). The absence of signals in the low molecular weight region (40 mL ≤ Vel ≤ 45 mL) shows that no scission of the PCL grafts occurs. As already discussed for the SEC traces of the graft copolymers 4−5, the amphiphilicity of 8a,b affects the SEC analysis. In comparison to 5b, a tailing to higher molecular weights (lower elution volumes) is observed for 8a,b. However, the molecular weight distributions remain in the same range (Mw/Mn = 1.5), indicating that no side reactions occur. The phosphonoethylated PGs can be dealkylated quantitatively either with lithium bromide or sodium iodide, whereas for the graft copolymers, sodium iodide is superior. For that reason, the dealkylation of the prototype copolymer 6 is only conducted with sodium iodide. The results of the chemical analysis of the dealkylation experiments are summarized in Table 5. P(GDEPE9-co-(G-g-εCL33)17) (6) is quantitatively dealkylated to yield P(GEPE9-co-(G-g-εCL33)17) (9) (Supporting Information, Figure S7). These polymers are subjected to the hydrolytic degradation study, to investigate the impact of the DEPE groups and the ionic EPE residues on the course of the degradation. Before the results of the degradation experiments are presented, the chemical analysis of the copolymers 6 and 9 and the investigation of their thermal properties is summarized. Characterization of the Prototype Polymers 6 and 9. Before the degradation study is performed, the prototype polymers P(GDEPE9-co-(G-g-εCL33)17) (6) and P(GEPE9-co-(Gg-εCL33)17) (9) are characterized by means of SEC analysis and differential scanning calorimetry (DSC). Additionally, the

The graft copolymers 4a,b, which have been prepared starting from 3a decorated with 4 DEPE groups possess 64% (4a) and 73% (4b) of converted hydroxyl groups. When the phosphonate concentration is raised to 10 DEPE groups as in P(GDEPE10-co-G16) (3b) no increase in the IE is observed for 5a and 5b. However, the results show that the IE increases from 64% to 75% with increasing εCL concentration in the feed, independent from the phosphonate concentration of the macroinitiator (Table 4: 4a → b; 5a → b). Further increase of the εCL concentration in the feed should allow the formation of densely grafted copolymers with quantitative conversion of the pendant hydroxyl groups at the PG backbone. Indeed, graft copolymer 6, which has been prepared by grafting from P(GDEPE9-co-G17) (3c) with a ratio of [CL]/[OH] = 40, carries no remaining hydroxyl groups as deduced from 1H NMR analysis. P(GDEPE9-co-(G-g-εCL33)17) (6) finds application as model polymer in the hydrolytic degradation study, to elucidate the influence of the DEPE groups on the degradation rate of the PCL grafts. Monodealkylation of Phosphonate Functionalized PGs and Graft Copolymers. The introduction of DEPE groups to PG is effective to increase the hydrophilicity of the corresponding graft structures. However, the hydrophilicity could be further improved through postpolymerization modification of the DEPE residues, since phosphonic acids or mixed ethyl phosphonate/phosphonic acid groups (EPE) can be synthesized from the diethyl phosphonate precursor. When the graft copolymers are considered, the DEPE modification can not be achieved through a simple hydrolysis, because harsh reaction conditions are usually required to achieve a (mono)dealkylation, like refluxing in concentrated alkali or 6 M hydrochloric acid. Under these conditions, the PCL grafts of the copolymers would hydrolyze as well. Different mild and chemoselective procedures have been developed in the past to convert phosphonates to the corresponding (mono)acids. Treatment of phosphonates with iodo- or bromotrimethylsilane leads to phosphonic acids,38,39 however, the conversion of the DEPE residues of the polyether-graf t-polyesters with iodo- or bromotrimethylsilane in the presence of excess PCL ester groups can not be achieved chemoselectively. By contrast, monophosphonic acids can be prepared by a straightforward approach using sodium iodide or lithium bromide as dealkylating reagent.40−42 This method is highly selective and tolerates many functional groups such as ethers and esters.40 Therefore, we decide to apply alkali metal halides to prepare polyether-graft-polyester copolymers with ionic ethyl phosphonatoethyl side groups (EPE). Macroinitiator 3a is used as a model compound in order to elucidate the applicability of the synthetic procedure to our polymeric substrates (Scheme 2). P(GDEPE4-co-G22) (3a) is reacted with LiBr or NaI. In both experiments, conversions of the DEPE groups ≥98% are Scheme 2. Preparation of P(GEPE4-co-G22) (7) by Monodealkylation of P(GDEPE4-co-G22) (3a) Using either LiBr or NaI

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Figure 2. 1H NMR spectrum of P(GDEPE4-co-G22) (3a; top) and P(GEPE4-co-G22) (7a; bottom) recorded in D2O; *acetone.

Figure 3. 1H NMR spectrum of P(GDEPE10-co-(G-g-εCL12)12-co-G4) (5b; top) and P(GEPE10-co-(G-g-εCL12)12-co-G4) (8b; bottom) recorded in CDCl3; *acetone.

cytoxicity of the copolymers is investigated. Water contact angle measurements are performed to evaluate the hydrophilicity of the materials prior to the degradation study. SEC analysis using THF as eluent shows monomodal elution curves for both polymers with narrow molecular weight distributions (Mw/Mn = 1.3; Figure 4). However, switching the microstructure from DEPE to EPE residues leads to a remarkable decrease of the number average molecular weight: Mn,SEC = 65900 g/mol for 9 compared to Mn,SEC = 95100 g/mol for 6. This is because the PG backbone is highly collapsed in THF when the ionic EPE groups are formed. No hydrolysis of the PCL-grafts is observed. The copolymers are characterized by DSC measurements to elucidate possible effects of the postpolymerization modifica-

tion of the DEPE residues on the thermal properties of the polymers. The second heating and cooling curves of 6 and 9 are shown in Figure 5. The melting temperatures, the melting enthalpies, the crystallization temperature and the crystallization enthalpy are shown in Table 6. Both 6 and 9 are semicrystalline copolymers with rather sharp melting transitions at 54.8 and 54.7 °C, respectively. The crystalline domains of 6 seem to be a bit larger, as suggested from the melting enthalpies of 89 J/g for 6 and 84 J/g for 9. The degree of crystallinity is calculated from the heat of fusion for a 100% crystalline PCL of 139.5 J/g (Table 6).43 Based on the DSC results, the DEPE-containing copolymer 6 has 64% of crystalline domains, compared to 60% crystallinity for copolymer 9 with EPE residues. For both polymers 3991


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Table 5. Dealkylation of P(GDEPE4-co-G22) (3a), P(GDEPE10co-(G-g-εCL12)12-co-G4) (5b), and P(GDEPE9-co-(G-gεCL33)17) (6): Conversion of DEPE Groups, Number Average Molecular Weights Determined by 1H NMR and SEC and Molecular Weight Distributions polymer

substratea

salt

Conv.b DEPE (%)

7a 7b 8a 8b 9

3a 3a 5b 5b 6

LiBr NaI LiBr NaI NaI

98 98 82 100 100

Mn, NMRb (g/mol)

Mn, SECc (g/mol)

Mw/Mnc

2494 2557 19804 19948 68065

2500 2700 25800 25900 65900

1.5 1.8 1.5 1.5 1.3

Table 6 reveals that both polymers have the same molecular weight, they contain similar weight fractions of PCL (around 95 wt %) and have the same thermal properties. These are excellent findings, since influences on the hydrolytic degradation rate due to changes in chemical composition or degree of crystallinity can be excluded. Moreover, no nonfunctionalized PG repeating units with pendant hydroxyl groups are present in 6 and 9, which could facilitate the water-uptake and, thus, enhance hydrolysis.30 Thus, we are able to evaluate the influence of DEPE and EPE residues on the hydrolytic degradation rate precisely because the polymers are only differing in their microstructures. All examined polymers were considered as noncytotoxic, according to the DIN EN ISO 10993-5:2009-10. This was expected due to the fact of the high mass percentage of PCL in copolymer 6 and 9, which is often used as biomaterial.4 The cells on PCL exhibited 69% viability relative to negative control (NC). When compared to PCL, the viability of cells was increased, reaching 82% and 77% for copolymer 6 and 9, repectively. By comparison of all groups, statistically significant difference was only observed between PCL and copolymer 6 (p = 0.0226; Figure S8, Supporting Information). No significant differences between the contact angle of the copolymers and PCL are observed. For the PCL reference a contact angle of 76.4 ± 2.7 is found and copolymers 6 and 9 show contact angles of 79.9 ± 4.8 and 75.1 ± 4.9, respectively. Thus, the hydrophilicity of the materials is similar, which is referred to the high mass percentage of PCL and the low concentration of functional groups of the polyglycidol backbone. In Vitro Degradation. The in vitro degradation of copolymers 6 and 9 is performed in Sørensen phosphate buffer (pH 7.4) at 55 °C. The advantage of this accelerated in vitro degradation study is that it allows a screening of slower degradable polymers in a short time frame. Therefore, this screening facilitates a selection of suitable copolymers, which can then be further investigated under more physiological conditions at 37 °C. For copolymer 9 mass loss is already detected after 7 days, while for copolymer 6 mass loss is first observed after 63 days of in vitro degradation (Figure 6). A total mass loss of 8% (6) and 9% (9), respectively, has been detected at the end of the study. The mass loss progress does not follow a [mathematical] particularistic function, which suggests bulk degradation.44,45 Meanwhile, the PCL reference shows only minor mass loss during the whole degradation study. The results for the mass loss are reflected by the analysis of the degradation-induced changes in the molecular weights (Figure 7), since the course of the molecular weight loss is rather similar to the mass loss curve. Polymer 9 which contains ionic EPE groups shows enhanced water-uptake and degradation occurs already after 7 days, as proven by a Mn decrease of 26%. By contrast, the DEPE-containing polymer 6 takes up water more slowly, since no significant decrease in the average Mn is observed until day 35. The average Mn of the PCL reference does not change throughout the investigation, Mn = ∼91% after 147 days (Figure 7). For copolymer 6 Mn = ∼15% and for copolymer 9 Mn = ∼33% of the initial molecular weight has been determined at the end of the in vitro study. The fact that both polymers show no further molecular weight loss from day 119 to day 147, indicates that highly crystalline domains have already been formed and thus lowering the degradation rate.

a

3a: P(GDEPE4-co-G22); 5b: P(GDEPE10-co-(G-g-εCL12)12-co-G4); SEC (DMF): Mn = 23100 g/mol, Mw/Mn = 1.4. 6: P(GDEPE10-co-(G-gεCL33)17); SEC (THF): Mn = 95100 g/mol, Mw/Mn = 1.3. bAccording to 1H and 31P NMR analysis. cNumber average molecular weight (Mn,SEC) and molecular weight distribution (Mw/Mn) determined by SEC analysis using poly(ethylene glycol) standards and water as eluent for 7a,b and narrow distributed PMMA standards and DMF as eluent for 8a,b. THF was used as eluent for 9.

Figure 4. THF-SEC traces of P(GDEPE9-co-(G-g-εCL33)17 (6) and P(GEPE9-co-(G-g-εCL33)17 (9) measured with THF as eluent.

Figure 5. DSC second heating and cooling curves of P(GDEPE9-co-(Gg-εCL33)17 (6; gray line), P(GEPE9-co-(G-g-εCL33)17 (9; blue line), and the PCL reference (black line) measured at a heating rate of 10 K/min.

recrystallization occurs upon cooling at Tc (6) = 28.8 °C and Tc (9) = 28.1 °C, respectively. Under the experimental conditions applied, no glass transitions have been observed. 3992


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Table 6. DSC Analysis of Copolymers 6 and 9 and the PCL Reference: Melting Temperature (Tm), Melting Enthalpy (ΔHm), Crystallization Temperature (Tc), and Crystallization Enthalpy (ΔHc; Results from the Second Heating/Cooling Cycle) polymer PCL 6 9

Mn,NMR (g/mol) 80000 68120 68065

a

PCLb (wt%)

Tm (°C)

ΔHm (J·g−1)

crystallinityc (%)

Tc (°C)

ΔHc (J·g−1)

100 95.0 95.1

56.5 54.8 54.7

77.4 89.0 84.1

55 64 60

25.3 28.8 28.1

−63.4 −77.6 −72.3

a

According to product data sheet. bWeight fraction of PCL in the polymers. cCalculated from the heat of fusion using the fusion of 100% crystalline PCL of 139.5 J/g.43

Figure 6. Degradation-induced mass loss of PCL (black square), P(GDEPE9-co-(G-g-εCL33)17 (6; gray square), and P(GEPE9-co-(G-gεCL33)17 (9; blue square) in Sørensen buffer (0.1 M, pH 7.4) at 55 °C.

Figure 8. Molecular weight distribution of P(GDEPE9-co-(G-g-εCL33)17 (6; gray line) and P(GEPE9-co-(G-g-εCL33)17 (9; blue line) in Sørensen buffer (0.1 M, pH 7.4) at 55 °C after 0 (−), 63 (···), and 147 (---) days.

distribution could be observed over the degradation time (Figure S9, Supporting Information). Furthermore, the evolution of the pH values during the degradation study has been investigated. For that purpose, the polymer specimens have been immersed in pure water at 55 °C. The aqueous medium was not changed throughout the study. For copolymer 9 a relatively continuous pH value decrease has been observed. This observation suggests the release of acidic products throughout the duration of the study. Contrary for PCL and copolymer 6, a rather sporadic decrease is detected (Figure 9). However, choosing pure water with a conductance lower than 1 μS/cm makes the media very sensitive for pH Figure 7. Degradation-induced changes in molecular weight (Mn) of PCL (black square), P(GDEPE9-co-(G-g-εCL33)17 (6; gray square), and P(GEPE9-co-(G-g-εCL33)17 (9; blue square) in Sørensen buffer (0.1 M, pH 7.4) at 55 °C.

The molecular weight distribution of the copolymers 6 and 9 are shown after 63 and 147 days as representative examples (Figure 8). Copolymer 6 shows only minor changes in its molecular weight distribution maximum. However, a shoulder to lower molecular weight at around 2.4 × 104 Da is observed at day 63 and 147, pointing out for degradation products of lower molecular weight (Figure 8). The investigation of copolymer 9 over the degradation time reveals a distinct shift of the molecular weight maximum. At day 0 a maximum at 14.5 × 104 Da has been measured whereas after 63 days the maximum was 8.5 × 104 Da and after 147 days 7.5 × 104 Da. The formation of degradation products is proven by the broadening of the curves after day 63 and day 147. For the PCL reference no changes in the molecular weight

Figure 9. pH values of the water surrounding PCL (black square), P(GDEPE9-co-(G-g-εCL33)17 (6; gray square), and P(GEPE9-co-(G-gεCL33)17 (9; blue square) during accelerated in vitro degradation at 55 °C. 3993


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changes. So any byproducts or degradation products that leached into the medium will result in pH changes, and low variances in pH value should not be overestimated. So the pH increase for PCL on day 21 does not seem to be relevant, because several studies report a slow degradation.46 Additionally, data of molecular weight and weight loss approve this assumption. Oven-based Karl Fischer titration (KF) is performed with polymer samples, which have not been applied in the degradation study to approach the role of EPE moieties for the observed differences in polymer hydrolysis. The KF measurements of PCL show no water uptake into the bulk within the first 24 h. There is no difference in the Sørensen buffer stored and Sørensen buffer dipped samples. Copolymer 6 shows little more but no significant differences in water uptake of dipped and stored samples. One can hence assume a faster and higher water uptake into the bulk of copolymer 6 occurs than for PCL, even no significant differences were detected in comparison to PCL. The DEPE moieties seem to enhance the water uptake and thus the degradation by hydrolysis, which could be shown by mass loss and molecular weight analysis. Copolymer 9 shows a significant water uptake into the bulk after immersion compared to the dipped samples, which enhances the suggestion of an influence of the EPE moieties and the corresponding cation. The higher water uptake (Table 7) confirms the results of molecular weight analysis and mass loss, which indicate a faster decrease of copolymer 9 compared to copolymer 6 and PCL.

Table 8. DSC Analysis of 6 and 9 after 0, 63, and 147 Days of Degradation: Melting Temperature (Tm), Melting Enthalpy (ΔHm), and Degree of Crystallinity of the Copolymers polymer

day

Tma (°C)

ΔHma (J·g−1)

crystallinityb (%)

6

0 63 147 0 63 147

54.8 55.0/69.0 56.7/69.1 54.7 67.5 67.5

89.0 98.1 97.1 84.1 91.7 97.9

64 70 70 60 66 70

9

a

Day 0: Results obtained from second heating curve. Days 63 and 147: Results of first heating curve. bCalculated from the heat of fusion using the fusion of 100% crystalline PCL of 139.5 J/g.43

out for inhomogeneous degradation, which is further strengthened by the molecular weight distributions in Figure 8. For copolymer 9, only one endothermal peak is observed at 67.5 °C after 63 and 147 days. This assumes a more homogeneous degradation as already indicated by the steady mass loss and decrease of molecular weight. The melting enthalpies as well as the degrees of crystallinity are comparable for 6 and 9 at the end of the degradation study. However, the absolute changes in the enthalpy are higher for 9 (day 0, 84 J/g; day 147, 98 J/g) than for 6 (day 0, 89 J/g; day 147, 97 J/g). Within the first 91 days, cracks begin to appear on copolymer 9. Moreover, already 50% of the samples of 9 are fragmented. During the remaining degradation time, all samples fragment further and gradually degrade (Figure 10). For PCL and

Table 7. Results of Water Uptake Determination by KF Titration (n = 4) for PCL, P(GDEPE9-co-(G-g-εCL33)17 (6), and P(GEPE9-co-(G-g-εCL33)17 (9) Prior to Degradation in Mass Percent polymer

water uptake (%) stored for 24 h in Sørensen buffer

water uptake (%) dipped in Sørensen buffer

PCL 6 9

3.79 ± 1.61 5.54 ± 1.57 8.66 ± 1.09

3.54 ± 1.31 3.68 ± 0.57 3.20 ± 0.97

Figure 10. Macrophotographs of P(GEPE9-co-(G-g-εCL33)17 (9) after (A) 0, (B) 91, and (C) 147 days of degradation.

copolymer 6 no changes have been observed (photographs not shown). The results of the macroscopic analysis of the specimens reveal an accelerated degradation for copolymer 9, which is in agreement with the findings of the analysis at the molecular level. The ratio of the PCL grafts to the PGDEPE and PGEPE backbones of copolymers 6 and 9 have been determined at the end of the study by 1H NMR spectroscopy and compared to the initial ratio at day 0. The analysis reveals that the ratio for copolymer 6 remains constant during the study (Figure S11, Supporting Information). This finding is in accordance with the molecular weight distributions shown in Figure 8. The degradation products formed are of high molecular weight (Figure 8), and thus, they are not water-soluble and do not diffuse from the bulk material. Consequently, no change in the PCL/PG DEPE backbone ratio could be observed. The phosphonate residues of 6 are not affected during the hydrolysis, as proven by 31P NMR analysis after 147 days. The spectrum shows a single peak at δ = 28.3 ppm, which is the same chemical shift as meaured for 6 prior to the study. Hence, the DEPE groups of 6 do not catalyze the hydrolysis of the polyester grafts, however, they promote the water-uptake resulting in faster degradation in comparison to the PCL reference.

The copolymers 6 and 9 have been analyzed by means of DSC at specific degradation times. Samples from days 63 and 147 have been chosen because, as discussed previously, at these time points, significant mass loss and molecular weight decrease have been observed. The first heating curves of the samples removed at days 63 and 147 are compared to the second heating curves of the polymers at day 0. The results are summarized in Table 8. The heating scans are shown in Figure S10, Supporting Information. During the course of the degradation study, the melting transition of both copolymers 6 and 9 is shifted from 55 to 67− 69 °C after 63 days. Additionally, the melting enthalpies increase, which reflects higher crystallinity of the copolymers. This is in accordance with our expectations, because hydrolysis occurs initially in the amorphous domains of the polymers and the degree of crystallinity increases. For the DEPE-containing polymer 6, two endothermal peaks are observed after 63 and 147 days. The endothermal peak at lower temperature (55 and 56.7 °C) corresponds well to the melting temperature of 6 prior to the degradation study (day 0). If oligo(εcaprolactone)s would have formed, the melting temperature should be lower. The results obtained for copolymer 6 point 3994


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The 1H NMR analysis of copolymer 9 shows a slight decrease of the PCL/PGEPE backbone ratio after 119 days of degradation (Figure S12, Supporting Information). This is in line with an accelerated degradation rate and degradation products of lower molceular weight. Starting from day 7, a continuous mass loss is observed, reflecting the strong impact of the EPE groups on the degradation rate. Thus, the degradation products become water-soluble and diffuse from the bulk material. The diffusion could be facilitated by the fragmentation of the specimen, which is shown in Figure 10. The 31P NMR spectrum recorded after 119 days of degradation shows two broad signals located at δ = 24.9 and 20.7 ppm, respectively (Figure S13, Supporting Information). The latter signal corresponds to the signal of the EPE groups at day 0 (δ = 20.6 ppm). The new signal at δ = 24.9 ppm corresponds quite well to the signal of phosphonic acid groups attached to PG as reported recently by our group.31 This is a further hint on the participatiton of the EPE groups, however, further experiments are required to verify this hypothesis.

6, SEC traces of 4a and 5a, 31P NMR spectra of 3a and 7a, 31P NMR spectra of 5b and 8b, SEC traces of 5b and 8a,b, 31P NMR spectra of 6 and 9, relative viability of L929 fibroblasts grown on PCL, copolymers 6 and 9, SEC traces of PCL reference after 0, 63, and 147 days, DSC analysis of 6 and 9 after 0, 63, and 147 days, 1H NMR analysis of 6 after 0 and 147 days, 1H and 31P NMR analysis of 9 after 0 and 119 days. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Corresponding Author

*E-mail: [email protected]; [email protected]; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the DFG for financial support (scholarship for J.K., International Research Training Group “Selectivity in Chemoand Biocatalysis” - SeleCa). The authors are also grateful to Dr. Marina Hovakimyan for assistance in cytotoxicity studies, Dr. Svea Petersen for help in the water uptake studies, and Dr. Thomas Reske in performing GPC measurements of samples after degradation.

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CONCLUSIONS Polyglycidol-graf t-ε-caprolactone copolymers with pendant phosphonate groups are prepared by chemical catalyzed grafting of εCL from phosphonoethylated polyglycidols. NMR and SEC analysis reveals the formation of well-defined, narrow distributed copolymers. The initiation efficiency of the macroinitiators increases with increasing monomer concentration in the feed. A straightforward postpolymerization functionalization is presented using alkali metal halides to convert the diethylphosphonatoethyl residues chemoselectively to ionic ethylphosphonatoethyl groups. This approach has not yet been applied for polymer modifications. Polymer characterization reveals no degradation of the polyester grafts and no changes in the crystallinity of the polymers. Poly((glycidol diethylphosphonatoethyl)-co-(glycidol-graf tε-caprolactone), P(GDEPE9-co-(G-g-εCL33)17) (6) and poly((glycidol ethylphosphonatoethyl)-co-(glycidol-graf t-ε-caprolactone), P(GEPE9-co-(G-g-εCL33)17) (9) have been prepared as prototypes, which contain 95 wt % of degradable polyester grafts. They are of the same architecture, molecular weight, and crystallinity, only differing in the DEPE and EPE groups, respectively. The influence of DEPE and EPE residues on the hydrolytic degradation rate has been investigated by an in vitro study in phosphate-buffered solution at 55 °C. The copolymer 9, which carries EPE groups, starts to degrade already after 7 days. A continuous mass loss and molecular weight loss is observed. An accelerated degradation of copolymer 9 is also proven by macroscopic analysis of the specimens. For copolymer 6 decorated with DEPE groups lasts up to 63 days, before changes in polymer mass and molecular weight are detected. Thus, the functionalization of polyglycidol-graf t-ε-caprolactone copolymers with EPE groups shows a great potential for the design of novel hydrolytically degradable materials. Certainly, when the weight fraction of PCL in the copolymers is decreased the impact of the EPE groups will be more pronounced.

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AUTHOR INFORMATION

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REFERENCES

(1) Vert, M. Biomacromolecules 2005, 6, 538. (2) Gomes, M. E.; Reis, R. L. Int. Mater. Rev. 2004, 49, 261. (3) Gomes, M. E.; Reis, R. L. Int. Mater. Rev. 2004, 49, 274. (4) Woodruff, M. A.; Hutmacher, D. W. Prog. Polym. Sci. 2010, 35, 1217. (5) Chandra, R.; Rustgi, R. Prog. Polym. Sci. 1998, 23, 1273. (6) Nair, L. S.; Laurencin, C. T. Prog. Polym. Sci. 2007, 32, 762. (7) Li, S. J. Biomed. Mater. Res. 1999, 3, 342. (8) Ikada, Y.; Tsuji, H. Macromol. Rapid Commun. 2000, 21, 117. (9) Castilla-Cortázar, I.; Más-Estellés, J.; Meseguer-Dueñas, J. M.; Escobar Ivirico, J. L.; Marí, B.; Vidaurre, A. Polym. Degrad. Stab. 2012, 97, 1241. (10) Labet, M.; Thielemanns, W. Chem. Soc. Rev. 2009, 38, 3484. (11) Albertsson, A.-C.; Varma, I. K. Biomacromolecules 2003, 4, 1466. (12) Storey, R. F.; Sherman, J. W. Macromolecules 2002, 35, 1504. (13) Middleton, J. C.; Tipton, A. J. Biomaterials 2000, 21, 2335. (14) Gunatillake, P. A.; Adhikari, R. Eur. Cells Mater. 2003, 5, 1. (15) Hakkarainen, M.; Höglund, A.; Odelius, K.; Albertsson, A.-C. J. Am. Chem. Soc. 2007, 129, 6308. (16) Hakkarainen, M.; Adamus, G.; Höglund, A.; Kowalczuk, M.; Albertsson, A.-C. Macromolecules 2008, 41, 3547. (17) Bat, E.; Plantinga, J. A.; Harmsen, M. C.; Van Luyn, M. J. A.; Zhang, Z.; Grijpma, D. W.; Feijen, J. Biomacromolecules 2008, 9, 3208. (18) Fernández, J.; Larrañaga, A.; Etxeberría, A.; Sarasua, J. R. Polym. Degrad. Stab. 2013, 98, 481. (19) Renouf-Glauser, A. C.; Rose, J.; Farrar, D. F.; Cameron, R. E. Biomacromolecules 2006, 7, 612. (20) Odelius, K.; Ohlson, M.; Höglund, A.; Albertsson, A.-C. J. Appl. Polym. Sci. 2013, 127 (1), 27. (21) Lee, S.-H.; Kim, S. H.; Han, Y.-K.; Kim, Y. H. J. Polym. Sci., Part A: Polym. Chem. 2001, 39, 973. (22) Wiggins, J. S.; Hassan, M. K.; Mauritz, K. A.; Storey, R. F. Polymer 2006, 47, 1960. (23) Huffman, K. R.; Casey, D. J. J. Polym. Sci., Part A: Polym. Chem. 1985, 23, 1939. (24) Pospiech, D.; Komber, H.; Jehnichen, D.; Häussler, L.; Eckstein, K.; Scheibner, H.; Janke, A.; Kricheldorf, H. R.; Petermann, O. Biomacromolecules 2005, 6, 439.

ASSOCIATED CONTENT

S Supporting Information *

Synthesis of polyglycidol, PG26 (2), synthesis of P(GDEPEx-coGy) (3a−c), SEC traces of 2, 3a−c, 31P NMR spectra of 3c and 3995


Biomacromolecules

Article

(25) Stridsberg, K.; Albertsson, A.-C. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 1774. (26) Xian, J. L. J. Appl. Polym. Sci. 2013, 127, 2046. (27) Meseguer-Dueñas, J. M.; Más-Estellés, J.; Castilla-Cortázar, I.; Escobar Ivirico, J. L.; Vidaurre, A. J. Mater. Sci.: Mater. Med. 2011, 22, 11. (28) Hans, M.; Gasteier, P.; Keul, H.; Möller, M. Macromolecules 2006, 39, 3184. (29) Hans, M.; Keul, H.; Heise, A.; Möller, M. Macromolecules 2007, 40, 8872. (30) Hans, M.; Keul, H.; Möller, M. Biomacromolecules 2008, 9, 2954. (31) Köhler, J.; Keul, H.; Möller, M. Chem. Commun. 2011, 47, 8148. (32) Köhler, J.; Marquardt, F.; Keul, H.; Möller, M. Macromolecules 2013, 46, 3708. (33) Frey, H.; Haag, R. Rev. Mol. Biotechnol. 2002, 90, 257. (34) Monge, S.; Canniccioni, B.; Graillot, A.; Robin, J.-J. Biomacromolecules 2011, 12, 1973. (35) Fitton, A. O.; Hill, J.; Jane, D. E.; Millar, R. Synthesis 1987, 12, 1140. (36) Löbler, M.; Behrend, D.; Maletz, R.; Schmitz, K. P. Adv. Eng. Mater. 2008, 10, 12. (37) Petersen, S.; Kaule, S.; Teske, M.; Minrath, I.; Schmitz, K.-P.; Sternberg, K. J. Chem. 2013, DOI: 10.1155/2013/587875. (38) McKenna, C.; Schmidhauser, C. J. Chem. Soc., Chem. Commun. 1979, 739. (39) Blackburn, G. M.; Ingleson, D. J. Chem. Soc., Perkin Trans. I 1980, 5, 1150. (40) Krawczyk, H. Synth. Commun. 1997, 27 (18), 3151. (41) Boutevin, B.; Hervaud, Y.; Jeanmaire, T.; Boulahna, A.; Elasri, M. Phosphorus, Sulfur Silicon Relat. Elem. 2001, 174, 1. (42) Yokomatsu, T.; Shioya, Y.; Iwasawa, H.; Shibuya, S. Heterocycles 1997, 46, 463. (43) Ye, W. P.; Du, F. S.; Jin, W. H.; Yang, Y.; Xu, Y. React. Funct. Polym. 1997, 32, 161. (44) Burkersroda, V. F.; Schedl, L.; Göpferich, A. Biomaterials 2002, 23, 4221. (45) Göpferich, A. Biomaterials 1996, 17, 103. (46) Sun, H.; Mei, L.; Song, C.; Cui, X.; Wang, P. Biomaterials 2006, 27, 1735.

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