Poly(ether-ester) Conjugates with Enhanced Degradation

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Biomacromolecules 2008, 9, 2954–2962

Poly(ether-ester) Conjugates with Enhanced Degradation Marc Hans, Helmut Keul,* and Martin Moeller* DWI an der RWTH Aachen e.V. and Institute of Technical and Macromolecular Chemistry, RWTH Aachen, Pauwelsstr. 8, D-52056 Aachen, Germany Received July 10, 2008; Revised Manuscript Received July 21, 2008

When a linear or a four arm star-shaped polyglycidol is used as macroinitiator, densely grafted poly(glycidolgraft--caprolactone) and poly(glycidol-graft-L-lactide) and loosely grafted poly[(glycidol-graft--caprolactone)co-glycidol] copolymers have been synthesized by chemical or, in the latter case, by enzymatic catalyzed ringopening polymerization of -caprolactone and L-lactide. The well-defined copolymers possess similar molecular weights, but differ in their architecture, microstructure and chemical composition. The hydrolytic degradation behavior was studied in a phosphate buffer solution at pH 7.4 and 37 °C for up to 90 days. After different time periods, the mass loss was determined and the degraded copolymers were analyzed by means of NMR, size exclusion chromatography, and scanning electron microscopy. Compared to linear poly(-caprolactone), poly[(glycidol-graft--caprolactone)-co-glycidol] shows a change of the degradation mechanism and a tremendous enhancement of polymer degradation. As this effect is attributed to the high concentration of hydroxy groups at the polyglycidol backbone, this work points out a new possibility to tailor the degradation profiles of polyesters by the introduction of functionality into the polymeric material.

Introduction Aliphatic polyesters derived from cyclic monomers such as lactide (LA), glycolide (GA), and -caprolactone (CL) have been extensively investigated for their use in biomedical applications because of their ease of manufacturing and their biocompatibility.1-3 For the successful implementation in applications such as surgical sutures, drug delivery systems, and tissue engineering scaffolds, the hydrolytic degradation and erosion profiles of these polymers are of crucial importance.4,5 The most important steps involved in the erosion process are (i) the diffusion of water into the bulk polymer, which might be accompanied by swelling of the material, (ii) the polymer degradation leading to the creation of oligomers and monomers, and (iii) the weight loss via diffusion of degradation products from the bulk material. In general, two types of erosion are distinguished depending on the diffusivity of water inside the matrix: heterogeneous (surface) erosion and homogeneous (bulk) erosion.6 For the latter, water penetrates throughout the entire material before hydrolysis occurs. As a consequence, chain scission happens homogeneously throughout the entire speciman, inducing the decrease of molecular weight, the reduction in mechanical properties, and the loss of weight. This type of erosion behavior is generally shown by poly(lactic acid), poly(glycolic acid), and poly(-caprolactone). If the diffusion of water into the polymer is slower than the degradation of the polymer, the material is subjected to heterogeneous erosion. Only the region close to the surface is affected by hydrolysis. As the surface is eroded and removed, the hydrolysis front advances toward the material core. The bulk material maintains its properties. Polymers such as poly(ortho ester)s, poly(anhydride)s, and some poly(carbonate)s undergo surface erosion.7,8 Regarding the importance of the hydrolytic degradation, intensive research efforts have been made to understand and to control the degradation rate and the release of degradation products. The majority of the studies have been conducted in * To whom correspondence should be addressed. E-mail: moeller@ dwi.rwth-aachen.de.

simulating conditions prevailing inside the human body, that is, in vitro studies in buffer solution at 37 °C. Factors that influence the degradation include molecular weight, material hydrophilicity, morphology, cross-link density, and surface chemistry.9-12 To tailor the degradation properties, different routes have been employed: (i) The copolymerization of different monomers has been largely used to modify the hydrolysis rate. In this respect hydrophilic monomers like 1,5-dioxepan-2-one, ethylene glycol or hydroxyethyl acrylate have been incorporated into the polymers to enhance the degradation rate.13-16 (ii) New chemical macromolecular architectures and microstructures, for example, triblock, multiblock, star-shaped polymers, porous scaffolds, and cross-linked networks have been developed.17-19 It has been shown that the release of acidic degradation products, a possible cause of inflammatory responses, is controllable through the macromolecular design of the material.11 (iii) The effect of additives such as monomers, acidic, or basic compounds has been explored. For example, the blending with lauric acid leads to an acceleration of the polymer degradation.20,21 (iv) Recent studies have proven, that the modifcation of end groups also affects degradation behavior. The presence of free carboxylic acid moieties at the chain end has been shown to enhance moisture uptake and, hence, to accelerate the degradation.22-24 The latter possibility, the introduction of functionality into the polymeric structure, has been scarcely investigated, but has in our opinion a great potential to regulate degradation rates. Regarding functionality, polyglycidol has been shown to be an interesting and versatile polymer.25-30 The chemical and enzymatic grafting of polyglycidol with -caprolactone leads to densely and loosely grafted copolymers, poly(glycidol-graft-caprolactone), and poly[(glycidol-graft--caprolactone)-coglycidol].31 The latter show interesting properties due to the vicinity of hydroxy groups to the grafts. It is expected that building blocks in the polymer chain, which are able to adsorb water will strongly influence the degradation mechanism and

10.1021/bm8007499 CCC: $40.75  2008 American Chemical Society Published on Web 09/20/2008

Poly(ether-ester) Conjugates with Enhanced Degradation

consequently the degradation rate. Thus, by choosing the proper concentration of these groups, the polymer degradation can be tuned, a property needed in release systems. As polyglycidol is considered to be biocompatible,32,33 these polymers present also potential candidates for biomedical applications. The first part of this work is going to present the synthesis and characterization of copolymers with the same molecular weight but different chemical composition, architecture, and microstructure. It will be followed by the analysis of the hydrolytic degradation behavior of these polymers to see the influence of the polymer architecture and microstructure.

Experimental Section Materials. -Caprolactone (CL, g99%, Fluka) was stirred with CaH2 for 24 h, distilled under reduced pressure, and kept in a Schlenk flask under nitrogen until use. L-Lactide (LLA, >98%, Aldrich) was purified by recrystallization in ethyl acetate and sublimation in vacuum at 80 °C. Novozyme 435 (Lipase B from Candida antarctica immobilized on a macroporous resin, 10000 U g-1, Sigma) was dried under vacuum at room temperature for 24 h and stored under nitrogen. Potassium tert-butoxide (1 M solution in THF, Aldrich), and tin(II) 2-ethyl hexanoate (Sn(oct)2, 97%, ABCR) were used as received. 3-Phenylpropanol (3-PP, g98%, Fluka) was distilled over sodium and di(trimethylolpropane) (diTMP, 97%, Aldrich) was purified by condensation. Diglyme was distilled over sodium. Potassium methanolate was synthesized by reacting potassium with methanol in toluene. The solvent was removed and a white powder was obtained after drying in vacuum at 50 °C. Ethoxy ethyl glycidyl ether was synthesized from 2,3-epoxypropan-1-ol (glycidol) and ethyl vinyl ether according to Fitton et al.34 and purified by distillation. A fraction with a purity exceeding 99.8 GC% was used. All reactions were carried out in nitrogen atmosphere. Nitrogen (Linde, 5.0) was passed over molecular sieves (4 Å) and finely distributed potassium on aluminum oxide. Synthesis. Linear Polyglycidol (1a). 3-Phenylpropanol (2.33 mL, 17.1 mmol) was dissolved in diglyme (60 mL) and potassium tertbutoxide (1.71 mL of a 1 M solution in tetrahydrofuran (THF), 1.71 mmol) was added. The tert-butanol formed was removed by distillation. Ethoxy ethyl glycidyl ether (60.0 g, 0.41 mol) was added and the mixture was stirred for 16 h at 100 °C. The solvent was removed in vacuum at 80 °C and a viscous liquid was obtained. The product was dissolved in THF (110 mL/1 g polymer) and aqueous 32% HCl (3.3 g/1 g polymer) was added. The polyglycidol precipitated as an oil. The solvent was removed and the polyglycidol was dried in vacuum at 80 °C. Yield: 90%. Mn,SEC ) 6.200, Mw/Mn ) 1.10, monomodal. 1H NMR (DMSO-d6): δ 1.88 (qui, J ) 7.0 Hz, ArCH2CH2), 2.67 (t, J ) 7.6 Hz, ArCH2CH2), 3.45-3.95 (m, CH2OCH2CH(CH2OH)O), 4.53 (s, OH), 7.12-7.33 (m, Ar). 13C NMR (DMSO-d6; end group signals are marked with an E): δ 30.9, 31.6, 60.9, 63.0E, 69.3, 69.5, 70.7E, 71.7E, 80.0, 125.7, 128.2, 128.3, 141.7. From the 1H NMR spectrum a degree of polymerization of DPn ) 24 was determined (Mn,NMR ) 1912). Star-Shaped Polyglycidol (1b). DiTMP (2.02 g, 8.06 mmol) was dissolved in diglyme (34 mL) and potassium methanolate (230 mg, 3.28 mmol), dissolved in methanol (2.30 mL), was added. The methanol was removed by distillation. Ethoxy ethyl glycidyl ether (28.2 g, 0.20 mol) was added and the mixture was stirred for 20 h at 120 °C. The same workup procedure as for 1a led to the star-shaped polyglycidol 1b. Yield: 89%. Mn,SEC ) 6.100, Mw/Mn ) 1.05, monomodal. 1H NMR (DMSO-d6): δ 0.81 (t, J ) 7.4 Hz, CH3CH2C), 1.30 (m, CH3CH2C), 3.07-3.27 (m, CCH2O), 3.27-3.70 (m, OCH2CH(CH2O)O), 4.55 (s, OH). 13C NMR (DMSO-d6): δ 7.6, 22.8, 42.9, 60.8, 63.0E, 69.3, 69.5, 70.7E, 71.6E, 79.8. From the 1H NMR spectrum a degree of polymerization of DPn ) 23 was determined (Mn,NMR ) 1952). Poly(glycidol-graft--caprolactone) (2a,b). Synthesis of polymer 2a: Polyglycidol 1a (1.17 g, 0.61 mmol) and -caprolactone (6.38 g, 55.9 mmol) were heated to 130 °C. Sn(oct)2 (8 mg, 20 µmol) was added

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and the mixture was stirred for 20 h at 130 °C. The polymerization was stopped by adding methylene chloride, and the polymer was isolated by precipitation in hexane. After drying under vacuum at 50 °C, a viscous polymer was obtained. Yield: 100%. Mn,SEC ) 14400, Mw/Mn ) 1.14, monomodal. 1H NMR (DMSO-d6): δ 1.20-1.42 (m, CH2CH2CH2), 1.46-1.64 (m, CH2CH2CH2), 1.65-1.84 (m, ArCH2CH2), 2.27 (t, J ) 7.2 Hz, OCOCH2CH2), 2.60 (t, J ) 7.6 Hz, ArCH2CH2), 3.27-3.80 (m, ArCH2CH2CH2, OCH2CH(CH2OH)O, OCH2CH(CH2OCO)O, CH2CH2OH), 3.98 (t, J ) 6.4 Hz, CH2CH2OCO), 4.08-4.38 (m, CHCH2OCO), 4.40-4.60 (s, not converted CHCH2OH groups), 7.12-7.32 (m, Ar). 13C NMR (DMSOd6; end group signals are marked with an E): δ 24.0, 24.4E, 24.8, 25.0E, 27.8, 32.1E, 33.3, 33.5E, 60.5E, 63.4, 172.7, 172.8E (the peaks of the polyglycidol backbone are not distinguishable from the noise of the baseline). The synthesis of 2b was performed in analogy to 2a. Yield: 90%. Mn,SEC ) 15100, Mw/Mn ) 1.09, monomodal. Poly[(glycidol-graft--caprolactone)-co-glycidol] (3a,b). Synthesis of polymer 3a: Polyglycidol 1a (0.91 g, 0.48 mmol) and -caprolactone (4.98 g, 43.6 mmol) were heated to 80 °C to obtain a homogeneous solution. Novozyme 435 (400 mg, 8 wt %) was added and the mixture was stirred for 20 h at 80 °C. The polymerization was stopped by adding methylene chloride, the enzyme was removed by filtration, and the polymer was isolated by precipitation in hexane. After drying in vacuum at 50 °C, a waxy polymer was obtained. Yield: 75%. Mn,SEC ) 17700, Mw/Mn ) 1.28, monomodal. The NMR spectra were identical with those of the polymer 2a, except for the intensities of the signals related to the esterification of the polyglycidol hydroxy groups. The synthesis of 3b was performed in analogy to 3a. Yield: 80%. Mn,SEC ) 16200, Mw/Mn ) 1.27, monomodal. Poly(glycidol-graft-L-lactide) (4a,b). Synthesis of polymer 4b: Polyglycidol 1b (1.01 g, 0.52 mmol) and L-lactide (5.03 g, 34.9 mmol) were heated to 130 °C, the catalyst Sn(oct)2 (8.0 mg, 20 µmol) was added, and the mixture was stirred for 20 h. The polymerization was stopped by adding methylene chloride, the polymer 4b was isolated by precipitation in hexane and dried in vacuum. Yield: 99%. Mn,SEC ) 8100, Mw/Mn ) 1.15, monomodal. 1H NMR (DMSO-d6): δ (ppm) ) 0.77 (m, CH3CH2), 1.26-1.32 (m, CH3CH2), 1.28 (d, 3J ) 6.8 Hz, COCH(CH3)OH), 1.35-1.55 (m, COCH(CH3)O), 3.05-3.25 (m, OCH2C), 3.25-3.80 (m, OCH2CH(CH2OCO)O), 3.95-4.40 (m, CHCH2OCO, COCH(CH3)OH), 4.90-5.25 (m, COCH(CH3)O). 13C NMR (DMSO-d6; end group signals are marked with an E): δ 16.4, 20.3E, 65.5E, 67.7E, 68.6, 169.1, 169.6E, 174.0E (the peaks of the polyglycidol backbone are not distinguishable from the noise of the baseline). The synthesis of 4a was performed in analogy to 4b. Yield: 97%. Mn,SEC ) 8000, Mw/Mn ) 1.17, monomodal. Measurements. 1H NMR and 13C NMR spectra were recorded on a Bruker DPX-300 spectrometer at 300 and 75 MHz, respectively. Deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide (DMSO-d6) were used as a solvent, and tetramethylsilane served as internal standard. For the polyglycidols (1a,b), size exclusion chromatography (SEC) analyses were carried out at 80 °C using a high-pressure liquid chromatography pump (Bischoff HPLC 2200) and a refractive index detector (Waters 410). The eluting solvent was N,N-dimethylacetamide (DMAc), with 2.44 g L-1 LiCl and a flow rate of 0.8 mL min-1. Four columns with MZ-DVB gel were applied. The length of each column was 300 mm, the diameter 8 mm, the diameter of the gel particles 5 µm, and the nominal pore widths were 100, 1000, and 10000 Å. Calibration was achieved using narrow distributed poly(methyl methacrylate) standards. For all other polymers, SEC analyses were carried out at 35 °C using a high-performance liquid chromatography pump (ERC HPLC 64200) and a refractive index detector (ERC-7215a). The eluting solvent was THF (HPLC grade) with 250 mg L-1 2,6-di-tert-butyl-4-methylphenol and a flow rate of 1 mL min-1. Five columns with MZ gel were applied. The length of the first column was 50 mm and 300 mm for the other four columns. The diameter of each column was 8 mm, the diameter

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Figure 1. Poly(ether-ester) conjugates with different composition, microstructure, and architecture by means of enzymatic and chemically catalyzed ring-opening polymerization of -caprolactone (CL) or L-lactide (LLA) using linear (1a) and star-shaped (1b) polyglycidol as macroinitiator.

of the gel particles 5 mm, and the nominal pore widths were 50, 50, 100, 1000, and 10000 Å, respectively. Calibration was achieved using narrow distributed poly(methyl methacrylate) standards. Differential scanning calorimetry (DSC) analyses were performed on a Netzsch DSC 204 under nitrogen using a 10 K min-1 scan rate (first heating) and a 5 K min-1 scan rate (second heating). The samples (3-12 mg) were subjected to two cooling-heating cycles from -100 to 80 °C for the PCL copolymers and -100 to 200 °C for the PLLA copolymers. Lipase Activity Test. The samples were weighed into safe-lock tubes and layered with a 0.1 M Tris-HCl buffer solution (1.9 mL, pH ) 8). After incubation for a certain time at 37 °C the aqueous solution was isolated and the substrate solution (0.1% para-nitrophenyl laurate in acetonitrile, 20 µL) was added. After 15 min, the amount of paranitrophenol produced was determined by measuring the extinction of the solution at 400 nm with a Varian Cary 100 Bio spectrophotometer. Amounts, incubation times, and extinction values are listed in Table 1. Table 1. Lipase Activity Test: Amounts, Incubation Times, and Extinction Values sample

mass [mg]

incubation time [h]

extinction

novozyme 3b 3b 2b

0.5 2 50 50

0.25 0.25 65 65

0.45 0.02 0.02 0.02

Degradation Studies. The copolymers were subjected to compression molding. The polymer was filled in the compression tool between two Teflon discs and kept for 2 min at 80 °C by heating the plates of the hydraulic press. Finally, the copolymers were compressed to foils by applying a pressure of 2 tons for 30 s. The copolymers 3a,b gave foils with a thickness of 0.11 ( 0.01 mm. Due to the low melting and glass transition temperature, the copolymers 2a,b and 4a,b, respectively, gave sticky materials without defined shapes. The samples were subjected to hydrolytic degradation in a phosphate buffered saline (pH ) 7.4, Sigma-Aldrich). The polymers (60-80 mg) were weighed into safe-lock tubes and topped with 1.5 mL of the buffered saline solution. The tubes were placed in a Heidolph (Unimax 1010) incubator at 37 °C. At each degradation time, three specimens were removed per

polymer and thoroughly washed with distilled water. The specimens were freeze-dried (2 days) and vacuum-dried (2 days), weighed, and analyzed. The mass loss (∆m) for each sample was calculated as follows

∆m )

mi - m t mi

where mi and mt are the initial sample weight and the dry sample weight after degradation time t, respectively. The reported mass loss values are an average determined from three different samples. The data points in Figures 4 and 5 result from the measurements of two different samples.

Results and Discussion In the past we presented the chemically and the enzymatically catalyzed grafting of -caprolactone from a multifunctional hydroxy macroinitiator.31 The microstructures of the resulting polymers were significantly different. By chemical catalysis, all hydroxy groups of the polyglycidol initiated the polymerization resulting in a densely grafted copolymer. By enzymatic catalysis, however, the initiation efficiency of the hydroxy groups was much lower and gave a comb polymer with free hydroxy groups remaining at the polyglycidol backbone. Based on these results well-defined copolymers with different architecture, microstructure and chemical composition are synthesized (Figure 1). Linear and star-shaped polyglycidols are used as macroinitiators for the ring-opening polymerization of -caprolactone or L-lactide via chemical catalysis and -caprolactone via enzymatic catalysis. Moreover, the effect of architecture and microstructure on the degradation and erosion behavior of the poly(ether-ester) conjugates is studied. Synthesis. Polyglycidols with different architectures were prepared in two steps.31 In the first step, anionic ring-opening polymerization of ethoxy ethyl glycidyl ether using 3-phenylpropanol or di(trimethylolpropane) as initiator led to linear and four arm star-shaped poly(ethoxy ethyl glycidyl ether). The alcohol groups of the initiators were activated with potassium tert-butoxide or potassium methoxide, respectively. It is important to remove all emerging tert-butanol or methanol from



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Scheme 1. Ring-Opening Polymerization of -Caprolactone or L-Lactide by Means of Enzymatic and Chemical Catalysis Using Linear (1a) and Star-Shaped (1b) Polyglycidol as Macroinitiator

Table 2. Number Average Molecular Weights, Polydispersity Indices, Initiation Efficiencies, and Side Chain Lengths of the Copolymers Synthesized by Chemical or Enzymatic Ring-Opening Polymerization of -Caprolactone (CL) or L-Lactide (LLA) Using Linear (1a) or Star-Shaped (1b) Polyglycidol (PG) as Macroinitiator Nr

PG

monomer

2a 2b 3a 3b 4a 4b

1a 1b 1a 1b 1a 1b

CL CL CL CL LLA LLA

catalyst

Mn,theorya

Mn,NMRb

Mn,SECc

Mw/Mnc

IEd [%]

DPn,sce

Sn(oct)2 Sn(oct)2 novozyme novozyme Sn(oct)2 Sn(oct)2

12400 12000 12400 12000 11900 11700

12200 11900 10900 10800 11400 11400

14400 15100 17700 16200 8000 8100

1.14 1.09 1.28 1.27 1.17 1.15

90 78 35 37 100 100

4.2 4.9 9.4 9.1 2.7 2.9

a Calculated molecular weight at full conversion using Mn,NMR of 1a,b. b Number average molecular weight (Mn) determined by NMR. c Mn and molecular weight distribution (Mw/Mn), determined by size exclusion chromatography (SEC) against narrow distributed poly(methyl methacrylate) standards using THF as eluent. d Initiation efficiency of the multifunctional macroinitiator. e Degree of polymerization of the side chains.

Table 3. Glass Transition Temperature (Tg), Melting Temperature (Tm), and Melting Enthalpy (∆Hm) of the Copolymers 2a,b, 3a,b, and 4a,b, Determined by DSC (Results from the Second Heating Curve)

Tg [°C] Tm [°C] ∆Hm [J/g]

2a

2b

3a

3b

-62 21/29 48

-56 22/29 48

48/51 57

47/51 57

4a

4b

25

25

the system before the monomer is added and the polymerization initiated by heating the solution to 100 °C. A monomer to initiator molar ratio of [M]/[I] ) 24 was selected for this investigation. In the second step, acid-catalyzed removal of the protecting groups resulted in the linear and four arm star-shaped polyglycidols. 1 H NMR spectra showed only the expected signals (Figure S1, Supporting Information). The linear and star-shaped architecture has already been confirmed before by quantitative 13C NMR spectroscopy.31 The high number average molecular weights determined by SEC analysis are only relative values because poly(methyl methacrylate) standards were used for calibration. The high values are probably due to interactions of the hydroxy groups with the columns or due to intermolecular hydrogen bonds, which leads to association of the multifunctional polymers. For both polymers narrow monomodal elution curves are obtained (Figure S2, Supporting Information). The linear (1a) and the four arm star-shaped (1b) polyglycidols are used as macroinitiators for the ring-opening polymerization of -caprolactone or L-lactide via chemical (Sn(oct)2) or enzymatic (Novozyme 435) catalysis (Scheme 1). In a previous work,35 the conditions for the enzymatic grafting reaction of -caprolactone from polyglycidol have been optimized. Comb-shaped polymers with grafts of different lengths and free remaining hydroxy groups at the backbone have been synthesized successfully. To obtain polymers with a similar molecular weight (Mn ≈ 12000 g/mol), the following monomer to initiating hydroxy group ratios were used: CL/OH ) 3.8 and

LLA/OH ) 2.9. Upon complete conversion of the polyglycidol hydroxy groups side chains with the corresponding degrees of polymerization (DPn,sc) are obtained. The polymerization was carried out in bulk. The macroinitiator and monomer were heated to form a homogeneous solution, before the catalyst was added. After precipitation in hexane the polymers were obtained as waxy solids. In Table 2 the molecular weights, the polydispersity indices, the initiation efficiencies and the side chain lengths (DPn,sc) of the copolymers 2a,b, 3a,b, and 4a,b are listed. For all chemical catalyzed polymerizations nearly quantitative monomer conversion is observed. Average molecular weights determined by end group analysis are in good agreement with the theory. Monomodal elution curves are obtained for all polymers (Figure S3, Supporting Information) and low values of the polydispersity indices are observed. It can be assumed that no adventitious initiator was active. The lower number average molecular weight of the poly(glycidol-graft-L-lactide) 4a,b results from the fact that the PLLA side chains are shorter and more compact than the PCL chains of poly(glycidol-graft-caprolactone) 2a,b. Moreover, it might be due to a lower solubility of these copolymers in THF. Thus, the hydrodynamic radius of the polymer coil of the CL grafted polymers is larger. The initiation efficiency of the polyglycidol hydroxy groups was determined by 1H NMR spectroscopy, as already described before.31 Despite the low ratio of monomer to initiating hydroxy groups CL/OH ) 3.8, high initiation efficiencies were achieved for the copolymers 2a,b, 90 and 78%, respectively. Although the hydroxy groups at the growing poly(-caprolactone) chain ends are supposed to be more easily accessible, the reactivity of the primary hydroxy group at the polyglycidol backbone has to be higher. From the molecular weights (Mn,NMR) and the initiation efficiencies (IE) determined by NMR, the degree of polymerization of the side chains (DPn,sc) was calculated by using eq 1, exemplarily shown for copolymer 2a (molar mass of -caprolactone (MCL) ) 114.15 g/mol)

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DPn,Sc(2a) )

Mn,NMR(2a) - Mn,NMR(1a) IE(2a) MCL · DPn(1a) · 100

Hans et al.

(1)

For the copolymers 2a,b, side chain lengths of DPn,sc ) 4.2 and 4.9 are obtained. -Caprolactone conversion was close to 100%, and the initiation efficiency was in the range of 80-90%, thus the DPn,sc values are slightly higher than the theoretical values. To determine the monomer to hydroxy group ratios in the copolymers the term IE/100 in eq 1 is omitted: for the copolymers 2a,b CL/OH ) 3.8 is obtained. In the case of the copolymers 4a,b, the complete conversion of the polyglycidol hydroxy groups was observed from the integrals in the 1H NMR spectrum (Figure S4, Supporting Information). This is attributed to the higher reactivity of the primary hydroxy groups at the polyglycidol backbone compared to the secondary hydroxy groups at the growing PLLA chain ends. The side chain lengths of DPn,sc ) 2.7 and 2.9 are in good agreement with the theoretical values. As all hydroxy groups initiated the polymerization, the LLA/OH ratios are identical with the DPn,sc values. The initiation efficiency of the polyglycidol hydroxy groups using Novozyme 435 as catalyst and -caprolactone as monomer is only about 35%, which is due to steric hindrance. The growing poly(-caprolactone) side chains shield the remaining polyglycidol hydroxy groups and prevent them from initiation. Thus copolymers with PCL side chains of DPn,sc ) 9.4 and 9.1 are obtained. Due to the low initiation efficiency of the polyglycidol hydroxy groups, the degrees of polymerization of the side chains are higher than the theoretical ones. The discrepancy between the theoretical molecular weight and the molecular weight determined by NMR is due to incomplete conversion of the monomer for the given reaction time. Consequently, the monomer to hydroxy group ratio is slightly lower in the copolymers than in the feed: for 3a, CL/OH ) 3.3; and for 3b, CL/OH ) 3.4. Although having a slightly lower molecular weight than the copolymers synthesized by chemical catalysis, the number average molecular weights determined by SEC analysis are higher for 3a,b. Due to the lower grafting efficiency these polymers are less compact and have longer side chains. Consequently they have a larger hydrodynamic volume. The SEC elution curves are monomodal and the polydispersity indices are slightly higher than for the polymers synthesized by chemical catalysis. These results confirm the successful synthesis of six polymers with similar molecular weight, but with different chemical composition, architecture and microstructure. The copolymers were analyzed by means of differential scanning calorimetry. In Table 3, the glass transition temperature, the melting temperature, and the melting enthalpy of the copolymers 2a,b, 3a,b, and 4a,b are listed. In Figure 2, the corresponding second heating curves are shown. The chemically synthesized PCL copolymers 2a,b show a double melting peak around 25 °C, with a melting enthalpy of 48 J/g and a glass transition temperature at -62 or -57 °C, respectively. The appearance of two melting peaks is attributed to different crystallite sizes. Due to the lower grafting efficiency, the enzymatically synthesized copolymers 3a,b possess longer PCL chains. These are able to form larger crystallites, resulting in higher melting temperatures around 50 °C, with a melting enthalpy of 57 J/g. A glass transition temperature was not observed under the used experimental conditions. The short PLLA side chains of the copolymers 4a,b are not able to crystallize and show only a glass transition temperature at 25 °C.

Figure 2. DSC second heating curves of the poly(glycidol-graft-caprolactone) (2a,b), poly([glycidol-graft--caprolactone)]-co-glycidol] (3a,b), and poly(glycidol-graft-L-lactide) (4a,b) copolymers.

Degradation. Poly(-caprolactone) takes an outstanding position among aliphatic polyesters due to its well documented biodegradability and biocompatibility, its high thermal stability and good permeability for many therapeutic drugs. Its low degradation rate makes it suitable for drug delivery systems that remain active for more than 1 year.1 PCL is subjected to degradation by a bulk erosion mechanism. The water penetrates the material faster than the polymer is converted into water soluble fractions. So the molar mass of PCL decreases continously during biodegradation and is accompanied by a broadening of the molar mass distribution. In biotic environments, such as river and lake water, sewage sludge, or compost, molar mass and crystallinity are the dominant factors influencing the biodegradability of poly(-caprolactone).36,37 It has been shown, that degradation preferentially occurs in the amorphous domains because water cannot penetrate the crystalline zones.38 Upon degradation in compost or anaerobic sludge temperature strongly affects the degradation rate, what might be attributed to changes in the microflora.39,40 Under abiotic conditions high temperature and basic pH favor the degradation of poly(caprolactone).41,42 The degradation rate of PCL is not affected by its shape (i.e., film or mircoparticle).43,44 In the following, the hydrolytic degradation and erosion behavior of copolymers based on glycidol and -caprolactone are analyzed in relation to different architectures and microstructures and compared to copolymers based on glycidol and L-lactide with similar architectures and molecular weight. The copolymers are composed of about 15% nondedgradable polyglycidol and 85% degradable side chains. First the mass loss caused by the release of soluble fragments formed upon hydrolysis is monitored (Figure 3). The chemically synthesized PCL graft copolymers 2a,b show 3% weight loss in a three month period. Linear poly(caprolactone) chains show hardly any mass loss in such a short time scale.41,42 The branched architecture (graft copolymers) with short PCL side chains (4-5 repeating units) resulting in a semicrystalline material with a high concentration of hydroxy end groups and the hydrophilic polyether backbone are prone to enhance the water uptake.45 However, degradation experiments over a larger time scale have to be performed in order to confirm this presumption. The chemically synthesized PLLA copolymers 4a,b lose 53% (linear) and 59% (star-shaped) of their weight. Compared to the poly(glycidol-graft--caprolac-



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Figure 3. Mass loss as a function of time: poly(glycidol-graft--caprolactone) 2a,b, poly([glycidol-graft--caprolactone)]-co-glycidol] 3a,b, and poly(glycidol-graft-L-lactide) 4a,b.

tone) copolymers 2a,b, this result is in accordance with literature as PLLA is known to degrade faster than PCL. A slightly higher mass loss is observed for the star-shaped poly(glycidol-graftL-lactide). In comparison to the chemically synthesized PCL copolymers 2a,b, the enzymatically synthesized PCL copolymers 3a,b show a dramatically higher weight loss. While there is scarcely any mass loss for the copolymers 2a,b, the linear poly[(glycidol-graft--caprolactone)-co-glycidol] 3a loses 19% and the comb-shaped poly[(glycidol-graft--caprolactone)-coglycidol] 3b loses 65% of its weight after 3 months. From literature, it is known that amorphous PCL is degrading faster than crystalline PCL. Regarding the DSC results, the more amorphous, chemically synthesized, PCL copolymers 2a,b were expected to hydrolyze and erode at a higher rate than the enzymatically prepared copolymers 3a,b. The particularity of this result is emphasized by the fact that the weight loss of the semicrystalline PCL copolymer 3b is even higher than for the amorphous PLLA copolymers 4a,b. As copolymers 2a,b and 3a,b have similar molecular weight the differences in erosion behavior have to be connected to their different microstructure and architecture. For a better understanding of these results, the chemical composition of the residual copolymer was analyzed by means of NMR. As the polyether backbone is assumed nondegradable under these conditions,46 the PCL/PG and the PLLA/PG ratios were determined by end group analysis. For the polymers with a linear backbone the ratio of the degradable polymer to polyglycidol was calculated by comparison of the aryl signal of the polyglycidol end group at δ ) 7.2 ppm with the resonance signal of the methylene group of poly(-caprolactone) at δ ) 2.3 ppm or the CH signal of poly(L-lactide) at δ ) 5.0-5.5 ppm. For the polymers with a star-shaped polyglycidol backbone, the CH3 signal of the initiating group diTMP at δ ) 0.8 ppm was used as a reference. For all calculations the Mn values of the polyglycidols determined by NMR are used (Mn,NMR (1a) ) 1912 and Mn,NMR (1b) ) 1952). In Figure 4, the mass ratios of the degradable polymer to polyglycidol are represented. The chemically synthesized PCL copolymers 2a,b show barely any decrease of the mass ratio, which is in accordance with the fact that there is nearly no mass loss. The PLLA copolymers 4a,b show a significant decrease of the L-lactide content. From the ratios shown in Figure 4, it can be calculated that the PLLA content in the linear copolymer 4a drops from about 84 to 71%, and in the star-shaped copolymer

4b, from about 82 to 68%. Assuming that erosion only occurs by the loss of hydrolyzed side chain fragments, this decrease in L-lactide content would result in a mass loss of 43% for 4a and 45% for 4b, respectively. As the observed weight loss in Figure 3 is higher, the solubilization of the polyglycidol backbone is also contributing to the erosion of the material. Upon hydrolysis the hydroxy groups of the polyglycidol become available and at a certain point the partially hydrolyzed copolymers become water-soluble. The degradable polymer to polyglycidol ratio of the enzymatically synthesized copolymers 3a,b shows a slight increase at the beginning and remains constant in the further course of the experiment. The initial increase of the PCL to PG mass ratio is attributed to the solubilization of copolymer with a relatively high percentage of hydroxy groups and a low content of PCL. This assumption premises a fast water uptake, which can be explained by the numerous hydroxy groups at the polyglycidol backbone. In the linear statistically grafted copolymer areas with 2-3 consecutive repeating units with not converted hydroxy groups close to the polyether backbone create highly hydrophilic domains, where large amounts of water are absorbed and hydrolysis is likely to occur. The hydroxy groups may even act as neighboring groups enhancing the hydrolysis reaction. In line with these explanations the high erosion rate of the star-shaped copolymer 3b results from an even higher concentration of hydroxy groups at the core of the polyglycidol star, where enzymatic grafting is less probable due to steric hindrance. The partially hydrolyzed copolymers become water soluble and diffuse out of the bulk material. Thus, in contrast to the copolymers 2a,b the copolymers 3a,b show a completely different erosion profile although their composition is nearly the same. The effect of the hydroxy groups consists of a fast water uptake, thus an acceleration of the hydrolysis reaction and an enhanced solubilization of the partially degraded copolymers. More detailed investigations regarding the influence of architecture on the degradation profiles have to be performed in order to confirm the corresponding assumptions. Furthermore, the remaining polymer samples after hydrolysis have been subjected to SEC analysis. The Mn values are shown in Figure 5 and the polydispersity indices are represented in Figure S5 (Supporting Information). The chemically synthesized PCL copolymers 2a,b show hardly any decrease in the number average molecular weights and hardly any change in the polydispersity indices. Considering the mass loss curves, this

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Figure 4. Poly(-caprolactone) (PCL) or poly(L-lactide) (PLLA) to polyglycidol (PG) mass ratio as a function of time: poly(glycidol-graft-caprolactone) 2a,b, poly([glycidol-graft--caprolactone)]-co-glycidol] 3a,b, and poly(glyidol-graft-L-lactide) 4a,b.

Figure 5. Number average molecular weights of the copolymers poly(glycidol-graft--caprolactone) 2a,b, poly([glycidol-graft--caprolactone)]co-glycidol] 3a,b, and poly(glycidol-graft-L-lactide) 4a,b as a function of degradation time.

implies, that after 90 days the samples are still in the process of water uptake. Compared to the copolymers 3a,b the copolymers 2a,b have a different microstructure. The polyether backbone is shielded by numerous short PCL chains and the hydroxy groups are not locally arranged by the polymeric structure. Thus the creation of hydrophilic domains like in the case of the copolymers 3a,b is not possible. The PLLA copolymers 4a,b show a continuous decrease of the number average molecular weights from Mn ≈ 8000 to Mn ≈ 5000 and the polydispersity indices increase as a function of time. Thus, hydrolysis occurs throughout the material. All observations regarding the copolymers 4a,b fit the bulk degradation mechanism also described in literature. The average molecular weights and the polydispersity indices of the PCL copolymers 3a,b synthesized by enzymatic catalysis remain constant during the whole period of testing. The elution chromatograms also show a small fraction of oligomers confirming the occurrence of hydrolysis (Figure S6, Supporting Information). As this fraction remains small and the Mn values of the main peak remain constant despite significant mass loss these observations confirm our previous assumptions. Degradation does not occur homogeneously throughout the whole material as it is usually

described for PCL materials, but is localized. This probably results from an enhanced water penetration in certain domains, in which hydrolysis takes place and water soluble species erode from the material. Thus, the poly(ether-ester) conjugates 3a,b show a novel degradation profile, which is based on a fast water uptake and a combination of hydrolysis and solubilization. In Figure 6, scanning electron microscopy pictures of the copolymers 3a,b are represented. The other polymers were not subjected to visual surface analysis as their glass transition or melting temperature lie below 37 °C. The picture series for polymer 3a shows that the surface of the polymer remains intact, even at 19% mass loss. Nevertheless, the appearance of little pores at the surface is observed. With proceeding erosion, polymers change to more porous structures, which can be observed for copolymer 3b. After 42 days the mass loss was 47% and on the initially smooth surface large pores are visible. After 89 days and a mass loss of 65% a porous structure is observed. The pores result from the high water concentration in these areas, which lead to the erosion of the material through the ongoing solubilization of partially degraded copolymers. The copolymer 3b was tested for its lipase activity, in order to exclude the enhancement of degradation by residual lipase,



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Figure 6. Scanning electron microscopy: pictures of the polymers 3a,b after 0, 42, and 89 days of degradation.

eventually leached out from the Novozyme 435 beads upon synthesis. First the sample was incubated for a certain time at 37 °C with a Tris-HCl solution in order to extract the lipase. The aqueous solution was isolated and the substrate paranitrophenyl laurate was added. In the presence of lipase, the substrate is hydrolyzed and the concentration of the resulting para-nitrophenol is determined by measuring the absorbance Via UV-spectroscopy after 15 min. As a reference Novozyme 435 and polymer 2b was used. Novozyme 435 was incubated for 15 min and upon addition of the substrate lipase activity was clearly observed. For polymer 3b a negligeable extinction was measured. Incubation for 65 h before the addition of the substrate had no effect on the extinction value. As the results were analogue to those measured for polymer 2b, it can be presumed that there is no enzyme present in the copolymers 3a,b.

Conclusions Well-defined densely grafted poly(glycidol-graft--caprolactone) and poly(glycidol-graft-L-lactide) and loosely grafted poly[(glycidol-graft--caprolactone)-co-glycidol] copolymers have been prepared by chemical or, in the latter case, by enzymatic catalyzed ring-opening polymerization of cyclic esters from a linear and a star-shaped polyglycidol as macroinitiator. The polymers have similar molecular weight, but differ in their chemical composition, architecture of the backbone and their microstructure leading to different thermal properties. The degradation behavior of the branched copolymers synthesized by chemical catalysis is in agreement with literature. The PCL copolymers degrade barely in a three months period, while the PLLA copolymers are partially hydrolyzed. The poly[(glycidolgraft--caprolactone)-co-glycidol] synthesized by enzymatic catalysis show a different degradation mechanism and a significantly enhanced degradation rate compared to linear PCL materials, while their mechanical properties are expected to be similar. The free polyglycidol hydroxy groups are assumed

responsible for this effect: their presence leads to a fast water uptake and to highly hydrophilic domains, in which the hydrolytic reaction is favored. Additionally, they lead to a faster solubilization of the copolymers after partial degradation. This work points out a new possibility of tailoring polymer degradability and erosion. The combination of architecture, microstructure and functionality allows the design of novel biodegradable materials with new degradation properties. Considering the enormous potential of such copolymers, they will certainly gain a lot of attention in the future. Acknowledgment. This research has been supported by a Marie Curie Action RTN Biocatalytic Approach to Material Design BIOMADE (M.H. Contract No. MRTN-CT-2004505147). Additional financial support came from BMBF 13N8888 “Nano Inhale”. Supporting Information Available. 1H NMR spectra and SEC elution curves of the linear and star-shaped polyglycidols, SEC elution curves of the poly(ether-ester) conjugates, 1H NMR spectrum of poly(glycidol-graft-L-lactide), polydispersity indices of the copolymers as a function of degradation time, and SEC elution curves of the copolymer 3b monitored upon degradation. This information is available free of charge via the Internet at http://pubs.acs.org.

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BM8007499

Poly(ether-ester) Conjugates with Enhanced Degradation

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