Biomacromolecules 2002, 3, 194-200
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Amine-Reactive Biodegradable Diblock Copolymers Jo¨rg K. Tessmar,† Antonios G. Mikos,‡ and Achim Go¨pferich*,† Department of Pharmaceutical Technology, University of Regensburg, D-93040 Regensburg, Germany; and Department of Bioengineering, Rice University, Houston, Texas Received September 13, 2001; Revised Manuscript Received November 19, 2001
A new class of diblock copolymers was synthesized from biodegradable poly(lactic acid) and poly(ethylene glycol)-monoamine. These polymers were activated by covalently attaching linkers such as disuccinimidyl tartrate or disuccinimidyl succinate to the hydrophilic polymer chain. The polymers were characterized by 1H NMR spectroscopy, 13C NMR spectroscopy and gel permeation chromatography (GPC). These investigations indicated that the polymers were obtained with the correct composition, in high purities, and the expected molecular weight. By using dyes containing primary amine groups such as 5-aminoeosin as model substrates, it was possible to show that the polymers are able to bind such compounds covalently. The diblock copolymers were developed to suppress unspecific protein adsorption and allow the binding of bioactive molecules by instant surface modification. The polymers are intended to be used for tissue engineering applications where surface immobilized cell adhesion peptides or growth factors are needed to control cell behavior. Introduction There is great interest in developing biomimetic polymers that control interactions between the material and a biological system.1 This issue is particularly important in designing polymers for drug targeting2-4 or tissue engineering.1,5 To engineer a living tissue, for example, cells need in some cases to proliferate on biodegradable polymer scaffolds as the supply with cells is often limited.6,7 Among the many difficulties with this ambitious approach is the poor control of cell function with currently available biodegradable polymers. A major concern is the interaction of polymers with a biological environment that may lead to the nonselective adsorption of proteins, which may in turn trigger a number of nonspecific cellular responses. Although this appears to be an advantage, it may lead to uncontrollable tissue development and growth.8 It was the goal of this study to develop biodegradable polymers that can overcome these problems. We developed the following design strategy: primarily, the polymers need to be biodegradable. Furthermore, they should suppress the adsorption of proteins as this is a prerequisite to avoid an uncontrollable cell response. Finally, the polymers should allow for the covalent attachment of biologically active molecules, such as peptides that enhance cell adhesion9 or growth factors inducing a specific and desired cellular response.10 Immobilized peptides that control cell adhesion and maintain cell differentiation,8 as well as growth factors †
University of Regensburg. Rice University. * To whom correspondence should be addressed. Telephone: +49-941-943-4843. Fax: +49-941-943-4807. E-mail: achim.goepferich@ chemie.uni-regensburg.de. ‡
which once immobilized cannot be endocytosed or diffuse away, which then enable the better control of cell behavior.2,10,11 Currently available are different hydrophilic and nonionic polymers that are nonconducive to protein and peptide adsorption including poly(ethylene glycol), dextran, poly(vinyl alcohol), polyacrylamide, and others.12,13 These polymers are water-soluble and, therefore, not unambiguously suited to tissue engineering, because they are not able to form stable three-dimensional constructs for cell attachment and proliferation. However, when these materials are appropriately copolymerized, water-insoluble copolymers can be obtained that are still protein-repellent on the surface. An example of such a polymer is poly(lactic acid)-block-poly(ethylene glycol)-monomethyl ether (MeO-PEG-PLA), a well-established biodegradable diblock copolymer.14-20 These copolymers proved particularly suitable for the suppression of protein and peptide adsorption.21,22 However, these copolymers do not permit proteins and peptides to be tethered to the PEG chain end, which could serve as a spacer facilitating the formation of a receptor-ligand complex. Of the numerous ways to selectively modify the polymer surface5,24-26 most methods cannot be applied to MeOPEG-PLA matrixes without the loss of functionality. We therefore decided to develop a synthesis strategy by which the methyl ether group of the polymer is “exchanged” for a primary amine to which amine-reactive or thiol-reactive linkers can be attached. The chemical structure of these reactive polymers is illustrated in Figure 1. In this paper, we shall report on the synthesis of the biodegradable amine-reactive polymers starting from a modified poly(ethylene glycol) and poly(lactic acid). Reaction schemes were developed to obtain the polymers with
10.1021/bm015608u CCC: $22.00 © 2002 American Chemical Society Published on Web 12/21/2001
Biodegradable Diblock Copolymers
Figure 1. Structures of polymers derived from poly(D,L-lactic acid)block-poly(ethylene glycol): (a) succinimidyl tartrate PEG PLA (STNH-PEGxPLAy), (b) succinimidyl succinate PEG PLA (SS-NHPEGxPLAy), and (c) maleinimido propionate PEG PLA (MP-NHPEGxPLAy).
the desired molecular weights. Appropriate linkers were synthesized to obtain polymers that react with amine groups of bioactive molecules quickly. Concomitantly, we report on how attaching model compounds to the polymer can be seen as a first step toward the development of biomimetic polymers for tissue engineering applications. Materials Tetrahydrofuran (THF), toluene, diethyl ether, acetone, 1,4-dioxane, trifluoroacetic anhydride, tetramethylsilane (TMS), succinic acid and L-tartaric acid were purchased from Merck (Darmstadt, Germany) in analytical grade. Toluene was distilled prior to use and THF was dried over 4 Å molecular sieves (all from CARL Roth GmbH, Karlsruhe, Germany). 3,6-Dimethyl-1,4-dioxan-2,5-dion (D,L-lactide) was purchased from Aldrich (Steinheim, Germany) and used as received. Ethylene oxide, potassium bis(trimethylsilyl)amide, N-hydroxysuccinimide (NHS), and N,N′-dicyclohexylcarbodiimide (DCC) were purchased from Fluka Chemicals (Buchs, Switzerland). Methoxypoly(ethylene glycol), ethanolamine, and stannous 2-ethylhexanoate were purchased from Sigma Aldrich (Steinheim, Germany). Deuterated chloroform (chloroform-d1) and deuterated acetonitrile (acetonitrile-d3) were obtained from Deutero GmbH (Kastellaun, Germany). Chloroform, acetonitrile, methylene chloride, and N,N-dimethylformamide (DMF) were supplied by Carl Roth GmbH. 5-aminoeosin as well as 5- and 6-carboxytetramethylrhodamine, succinimidyl ester [TAMRA-SE], two fluorescent dyes, which were obtained from Molecular Probes/ Mobitec (Go¨ttingen, Germany). All solvents were of analytical grade and were used as received unless stated differently. Methods Polymer Synthesis. Synthesis of H2N-PEG-OH. Poly(ethylene glycol)-monoamine (H2N-PEG-OH) was synthesized as described by Yokoyama et al.27 For the synthesis of diblock copolymers the protocol was modified as follows: to obtain H2N-PEG2-OH of molecular weight 2000
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Da, 40 g ethylene oxide (EO, 910 mmol) was dissolved in 150 mL of THF (cooled to -79 °C). Then, 19.5 mL of a 0.5 M potassium bis(trimethylsilyl) amide solution in toluene (9.75 mmol) was slowly added. After 36 h of stirring, the solution was concentrated under vacuum until a viscous polymer solution was obtained. For purification, the product was dissolved in methylene chloride, filtered through a 0.2 µm solvent resistant regenerated nitrocellulose membrane filter (Spartan 30/A from Schleicher & Schuell, Dassel, Germany) and dropped into four portions of 300 mL diethyl ether of approximately 4 °C. The precipitated polymer was collected and vacuum-dried for 48 h. Synthesis of H2N-PEG-PLA. Poly(D,L-lactic acid) was attached to poly(ethylene glycol)-monoamine by a ringopening polymerization using stannous 2-ethylhexanoate as catalyst28-31 (Figure 2, reaction I). For the synthesis of H2NPEG2PLA20, a monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) with 2000 Da H2N-PEG block and a 20 000 Da PLA block, 2 g of H2N-PEG2-OH (1 mmol) was dissolved in 150 mL of toluene. After the solution was heated to reflux temperatures, 20 mL of toluene was distilled off to remove traces of water. The same procedure was used to obtain a dry solution of 20 g of D,L-dilactide (140 mmol) in 150 mL of toluene. Both solutions were mixed in a threeneck flask. To maintain a water-free environment, a constant stream of dry nitrogen was bubbled through the reaction mixture. After the solution was heated to reflux temperatures, the polymerization was initiated by adding 100 mg catalyst. To protect the amine group of H2N-PEG-OH, the trimethylsilyl groups that it carried from its own synthesis (see above, substance 1b, reaction Ib) were either left in place or 500 µL of glacial acetic acid was added to the reaction mixture to transform the amine end into nonreactive ammonium (substance 1a, reaction Ia). After 8 h, the solution was cooled to room temperature and toluene was removed by repeated distillation with 200 mL of methylene chloride under vacuum. Finally, the polymer was dissolved in 100 mL of acetone and precipitated at 4 °C in three beakers of 600 mL of water each to remove water soluble byproducts and eventually deprotect the amine group. This reaction scheme was used for all polymers independent of molecular weight or composition. Synthesis of Linkers. To obtain amine-reactive polymers, suitable linkers had to be synthesized starting from bifunctional carboxylic acids (reaction II). Amine-reactive esters were synthesized from either succinic or tartaric acid32,33 (Figure 2, reaction II). For a typical synthesis, 40 g of N,N′dicyclohexylcarbodiimide (194 mmol) was dissolved in 150 mL of a dry 1,4-dioxane/ethyl acetate (4:1) mixture at 35 °C. Then, 12.0 g of tartaric acid (80 mmol) and 20.14 g of N-hydroxysuccinimide (175 mmol) were separately dissolved in 500 mL of the same solvent mixture. The carbodiimide solution was then added to the solution of the acid under constant cooling in an ice bath and kept at 0 °C for 18 h under stirring. The precipitated raw product was isolated by filtration and washed 3 times with 100 mL of 1,4-dioxane. The final product (substance 3) was obtained by extraction three times with 500 mL of acetonitrile at 35 °C. From the united acetonitrile phases, the solvent was distilled off under
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Figure 2. Reaction scheme of the polymer synthesis.
vacuum until a white crystalline powder of the active ester was obtained. The amine-reactive dicarbonic acids, disuccinimidyl tartrate [DST] and disuccinimidyl succinate [DSS] were characterized by 1H NMR spectroscopy as described below. Synthesis of Amine-Reactive Polymer. As disuccinimidyl tartrate is not water soluble, it was attached to H2N-PEGPLA (substance 2) in organic solvents (Figure 2, reaction III). To optimize the reaction conditions, different solvents were investigated to increase the yield and reduce the amount of residual solvent. For a typical synthesis protocol, 10 g H2N-PEG2-PLA20 (0.45 mmol) was dissolved together with 1.0 g DST (2.9 mmol) or DSS (3.2. mmol) linker in 150 mL solvent. The solution was kept at reflux temperatures for 2 h and stirred for 15 h at room temperature. The solvent was removed under vacuum. The polymer was obtained from the precipitate by extraction with 2 × 100 mL acetone and subsequent precipitation in approximately 600 mL water of 4 °C. The activated polymer (ST-NH-PEG2PLA20 or SSNH-PEG2PLA20, substance 4) was removed by filtration, immediately vacuum-dried, and stored under vacuum over phosphorus pentoxide until further use. Polymer Characterization. NMR Spectroscopy of Polymers and Linkers. To record NMR spectra, 20 mg of polymer or 10 mg of linker was dissolved in 1 mL of CDCl3 or 1 mL of acetonitrile-d3, respectively. 1H NMR spectra were taken on a Bruker AC250 spectrometer with dual sample head and autosampler (Bruker, Rheinstetten, Germany) at 250.13 MHz using TMS as internal standard. For the detection of the polymer amine group, D2O was added to the polymer solution, and spectra were recorded after proton exchange. In addition, spectra were taken after adding
100 µL of trifluoroacetic anhydride to allow for the acylation of the amine and the hydroxy end group. 13C NMR spectra were recorded from the same polymer samples at 400.13 MHz, using a Bruker ARX400 spectrometer (Bruker, Rheinstetten, Germany). Gel Permeation Chromatography (GPC). The synthesized polymers were investigated by gel permeation chromatography (GPC). A 5 µm particle size Phenogel precolumn (50 × 7.8 mm, Phenomenex, Torrance, CA) in combination with various analytical columns were used as a stationary phase. For measurements below 3000 Da, such as for the investigation of poly(ethylene glycol)-monoamine (H2NPEG-OH), the use of a single 50 Å Phenogel column (5 µm, 300 × 7.8 mm, Phenomenex) proved to be sufficient. For molecular mass measurements up to 15 000 Da, such as when ethanolamine-derived H2N-Et-PLA and its fluorescence labeled derivates were analyzed, a 500 Å Phenogel column (5 µm, 300 × 7.8 mm, Phenomenex) was used. Finally, for the investigation of the higher molecular weight polymers up to 75 000 Da, two 1000 Å Phenogel columns (5 µm, 300 × 7.8 mm, Phenomenex) were switched in series. In all cases, chloroform with a flow rate of 1.0 mL/min served as mobile phase. Chromatograms were recorded on a 10AVP HPLC system (Shimadzu, Duisburg, Germany). The columns were kept at 35 °C using a CTO-10ACVP column oven (Shimadzu). For a typical run, 10 mg of polymer was dissolved in 2 mL of CHCl3 and filtered through a 0.2 µm chloroform resistant regenerated nitrocellulose membrane filter (Spartan 30/A from Schleicher & Schuell, Dassel, Germany). After injection of 50 µL of sample solution, signals were recorded by a RID 10A refractive index detector (Shimadzu).
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Polymer molecular weights of H2N-PEG-OH samples were calculated from the retention time of poly(ethylene glycol)-monomethyl ether (MeO-PEG) standards (Polymer Laboratories, Darmstadt, Germany). The calculations were performed using the Class VP GPC software package contained in the Class VP 5.03 Software from Shimadzu. Molecular weight calculations were performed without the use of Mark-Houwink constants; therefore, the obtained values of the molecular weights are always relative to the used polymer standards. The labeling of H2N-PEG-PLA with amine-reactive fluorescent dyes such as TAMRA-SE or the attachment of 5-aminoeosin to amine-reactive polymers (ST-NH-PEGPLA or SS-NH-PEG-PLA) was also assessed by GPC. For a typical experiment, 10 mg of polymer and 1 mg of dye were dissolved in 1 mL of DMF and incubated for 2 h at 37 °C. Excessive dye was precipitated by adding 9 mL of chloroform to the reaction mixture. After filtration (see above), GPC chromatograms were recorded by three detectors in series: a RID 10 refractive index detector, a SPD 10AVVP UV-detector and a RF-355 fluorescence detector (all from Shimadzu). The data were analyzed using the HPLC software Class VP 5.03 (Shimadzu) without further calculation of the molecular weights. Elemental Analysis. To determine the composition of linkers and polymers, about 20 mg of the substances were investigated by elemental analysis in a Elementar Vario EL III from Elementar Analyzensysteme GmbH (Hanau, Germany). Results Synthesis of H2N-PEG-OH. First poly(ethylene glycol)monoamine was synthesized with an intended molecular mass of 2000 Da. The GPC chromatograms of the synthesized H2N-PEG-OH revealed a peak retention time between the MeO-PEG4 and MeO-PEG1 molecular weight standards. The calculation of the molecular mass yielded 1950 Da (PI ) 1.19) using a GPC calibration with MeOPEG standards. The 1H NMR spectra confirmed the polymer structure. Figure 3 shows the characteristic proton signals of H2NPEG2-OH. Following the acylation of the amine group and the hydroxy group with trifluoroacetic anhydride, the numberaverage molecular weight was calculated from the 1H NMR spectra (Figure 3). The signals of the two ethylene protons neighboring the hydroxy group shifted from 3.6 to 4.5 ppm and were unequivocally identified. A number-average polymer molecular weight, MWn, was calculated by integration of the 1H NMR signal h with respect to the chemical shift s according to eq 1:
∫4.03.4h ds 44 + 22 MW[Da] ) 4.3 2 ∫4.5 h ds
(1)
44 and 22 are the molecular weight and half the molecular weight of one monomer respectively while 2 is the number of protons that are subject to a chemical shift by acylation.
Figure 3. Characterization of poly(ethylene glycol)-monoamine: (I) 1H NMR spectrum in CDCl prior to acylation with trifluoroacetic 3 anhydride; (II) 1H NMR spectrum in CDCl3 after acylation with trifluoroacetic anhydride.
The average molecular mass of H2N-PEG2-OH determined from the NMR spectra was 1400 Da. Synthesis of H2N-PEG-PLA. A crucial aspect of H2NPEG-PLA synthesis was to make sure that only diblock copolymers with a free amine end were obtained. To reach this goal, H2N-PEG-PLA was first synthesized from ethanolamine (H2N-Et-OH) which served as a model compound for H2N-PEG2-OH. When the resulting H2NEt-PLAy polymers were characterized with 1H NMR, their structure was confirmed completely (Figure 4a). The methylene and the methyl protons of lactic acid gave a signal at 5.1 and 1.6 ppm respectively. At 4.0 and 4.5 ppm, a peak appeared for the methylene protons of ethanolamine and at 2.75 ppm a signal for the amine group was observed. This amine group was also identified by adding deuterated water to the polymer solution (Figure 4b). Due to the proton exchange, the signal at 2.75 ppm disappeared and a signal for semideuterated water became visible at 4.8 ppm (Figure 4b). Additional peaks in the spectra result from residual toluene (7.1 ppm), acetone (2,1 ppm), and the humidity in the spectrometer (3.5 ppm). The signal at 6.5 ppm may be attributed to an amide group, for which, however, no proton exchange was observed upon D2O addition. The same results were obtained when the polymer was synthesized in the absence of acids. 13C NMR spectra concomitantly revealed only a weak amide signal at 170 ppm (Figure 4c). After the optimal conditions for the formation of diblock copolymers in the presence of a free primary amine group had been developed, ethanolamine was replaced by H2NPEG2-OH for the synthesis of H2N-PEG2-PLA20 diblock copolymers according to reaction I in Figure 2. As the absolute molecular weight of such polymers cannot be correctly determined using simple GPC analysis,34 the average molecular weight was again calculated from 1H NMR data. The calculations were based on the average molecular weight determined for H2N-PEG2-OH. The integration of 1H NMR spectra (Figure 7) allowed the calculation of a number-average molecular weight MW according to eq 2:
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Figure 5. GPC chromatograms obtained for H2N-PEG2PLA20 and MeO-PEG2PLA20 before and after reaction with TAMRA (UV detection at λ ) 530 nm): (a) H2N-PEG2PLA20 with TAMRA; (b) H2NPEG2PLA20 without TAMRA; (c) MeO-PEG2PLA20 with TAMRA; (d) MeO-PEG2PLA20 without TAMRA.
Figure 6. 1H NMR spectrum of disuccinimidyl tartrate (DST) in acetonitrile-d3.
Figure 4. NMR spectra of ethanolamine-poly(D,L-lactic acid) (NH2ET-PLA2) in CDCl3: (a) 1H NMR spectrum prior to proton exchange with D2O: (b) 1H NMR spectrum after proton exchange with D2O; (c) 13C NMR spectrum.
MW[Da] )
128‚
5.55 h ds ∫4.84
∫3.34.05 h ds
72 + 1400
(2)
Here, 128 is the number of PEG protons calculated according to eq 1, 72 is the molecular weight of one lactide subunit, and 1400 is the molecular weight of the PEG chain. The molecular mass that we obtained from these calculations was 17200 Da. Unfortunately, it was impossible to detect the terminal amine group via 1H NMR because of the high molecular weight of the polymer (Figure 7-inactive polymer). To confirm the presence of the primary amine group that is essential to the synthesis of amine-reactive polymers, H2N-PEG2-PLA20 was incubated with TAMRA-SE, an
Figure 7. 1H NMR spectra obtained for activated polymers (STNH-Et-PLA2): Comparison of two 1H NMR spectra of ST-NH-PEG2PLA20 (active) and H2N-PEG2PLA20 (inactive).
amine-reactive fluorescent dye, in DMF. To exclude a reaction of the dye with the hydroxy end group of PLA MeO-PEG2-PLA20 served as control. The resulting dye/ polymer conjugates were separated from nonattached fluorescent dye (MW ) 528.6) by GPC. Figure 5 shows the GPC chromatograms of H2N-PEG2-PLA20 prior to and after incubation with TAMRA-SE. From H2N-PEG2PLA20 incubated with TAMRA-SE, a marked signal at a retention time of 14.5 min was obtained under UV detection, which is indicative of polymer chains carrying an additional chromophore. Dye-free H2N-PEG2PLA20 solutions as well
Biodegradable Diblock Copolymers
as the MeO-PEG2PLA20 mixtures showed no UV signals at low retention times and, therefore, no molecular weight increase. From these results we can conclude that a free terminal amine group is present even in the high molecular weight polymers and that the dye is not able to bind at the hydroxy end of the PLA chain. Synthesis of Amine-Reactive Linkers. Amine-reactive esters of tartaric (DST) and succinic acid (DSS) were synthesized as shown in Figure 2 (reaction II). The 1H NMR spectra taken from DSS and DST after purification contained the methylene protons signals at 5.1 ppm and the hydroxy group proton signal at 4.5 ppm, as well as a peak for the four succinimidyl protons at 2.75 ppm (Figure 6). From the fact that no signals were obtained that might be attributed to a carboxylic acid, one can conclude that both carboxy groups were transformed into succinimidyl esters. The integrals showed a close agreement between theoretical and experimental values [DST, 1H NMR (CDCl3) δ 5.1 (d, 2H), 4.5 (d, 2H), 2.8 (s, 8H); DSS, 1H NMR (CDCl3) δ 3.1 (s, 4H), 2.8 (s, 8H)], confirming the molecular composition of the linkers. Synthesis of Amine-Reactive Polymer. After DSS and DST had been synthesized, they were attached to H2NPEG-PLA to obtain amine-reactive polymers (Figure 2, reaction III). To evaluate the optimum reaction conditions, ethanol amine derived H2N-Et-PLAy was used as a model compound that allowed us to characterize the reaction products by 1H NMR spectroscopy (Figure 7). Different solvents were examined for their potential to co-dissolve H2N-Et-PLAy or H2N-PEG-PLA and their linkers. 1H NMR investigations showed that the use of different solvents resulted in different active polymers with different amounts of residual solvent. With acetone, ethyl acetate, and acetonitrile, the signal of the amine group was still able to be detected at 2.75 ppm, which indicates that the reaction was incomplete. The best results were obtained with a combination of acetonitrile and traces of triethylamine, in which the latter served as a base to deprotonate the amine group of the polymer. With this solvent, a maximum degree of conversion was achieved as a strong signal at 2.8 ppm, which results from the succinimidyl group demonstrate. The polymer’s molecular composition investigated by elemental analysis revealed that the block copolymers were obtained in the desired composition [Anal. Calcd for H2N-PEG2PLA20: C, 50.35; H, 5.87; N, 0.06. Found: C, 49.96; H, 6.56; N, 0.07. Anal. Calcd for ST-HN-PEG2PLA20: C, 50.35; H, 5.87; N, 0.12. Found: C, 50.06; H, 6.52; N, 0.13]. However, the nitrogen content was below the detection limit of the method and should, therefore, be interpreted with the necessary circumspection. Investigation of the Polymer’s Amine Reactivity. To evaluate the successful attachment of linkers to higher molecular weight H2N-PEG-PLA, we used GPC to confirm the presence of succinimidyl groups in the polymer chains. An increase of UV absorption in the polymer fraction was indicative of amine-reactive polymer (data not shown). As 1H NMR failed to detect the linker at the end of the PEG chain due to the high molecular weight of the polymer, we tested the amine-reactivity of polymers by incubation with
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Figure 8. GPC chromatograms of the amine-reactive ST-NH-PEG2PLAy and its reaction product with 5-aminoeosin (fluorescence detection λex ) 523 nm, λem ) 542 nm): (a) ST-HN-PEG2PLA40 with 5-aminoeosin; (b) ST-HN-PEG2PLA20 with 5-aminoeosin; (c) ST-HN-PEG2PLA40; (d) ST-HN-PEG2PLA20.
appropriate fluorescent dyes such as 5-aminoeosin. Therefore, an active polymer was incubated with dye and the fluorescent product was separated from nonattached 5-aminoeosin using GPC. The chromatograms showed a fluorescence signal at a retention time of 13.1 or 14 min, which is indicative of polymers carrying a fluorescence label. Additional signals at 26 min stemmed from the excess of fluorescent dye not attached to the polymer (Figure 8). Discussion A new class of amine-reactive diblock copolymers was synthesized. The synthesis involved the reaction of asymmetric poly(ethylene glycol)-monoamine that was synthesized from ethylene oxide. The polymer molecular weight was determined by GPC analysis using MeO-PEG of narrow molecular weight distribution as standards. The results obtained by GPC were confirmed by 1H NMR analysis after modification of H2N-PEG-OH with trifluoroacetic anhydride. The exact calculation of the numberaverage molecular weight was possible based on the chemical shift of protons next to the ester bonds formed under these conditions as proven by the model MeO-PEG samples (data not shown). For the synthesis of H2N-PEG-PLA, ethanolamine was used as a simple model compound to find appropriate reaction conditions. Its reaction with dilactide and stannous 2-ethylhexanoate catalyst revealed the preferential polymerization at the hydroxy end when the amine group was protected in an appropriate way. The amine group was able to be detected with 1H NMR spectroscopy only in the case of low molecular weight polymers such as polymers derived from ethanolamine. For high molecular weight products such as H2N-PEG2-PLA20, the primary amine end was detected by the attachment of amine-reactive dyes such as TAMRASE which led to an increase of UV absorption of the polymer fraction in GPC chromatograms (Figure 5). Unfortunately, no fluorescence detection was possible due to fluorescence quenching of chloroform. Negative controls with diblock copolymer containing no amine group showed that the hydroxy group of the polymers were not modified with TAMRA-SE.
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To activate H2N-PEG-PLA for the reaction with proteins and peptides, amine-reactive linkers were synthesized from succinic and tartaric acid according to the literature.32 Identity and purity of DSS and DST were confirmed with 1H NMR measurements. The 1H NMR spectra revealed the correct composition (Figure 6) and the absence of products resulting from the catalyst dicyclohexylcarbodiimide. Amine-reactive diblock copolymers were obtained from H2N-PEG2-PLA20 by incubation with DST or DSS, respectively. 1H NMR spectra proved that the conversion rate was at its highest when acetonitrile was used with traces of triethylamine. A further advantage of acetonitrile over other solvents such as DMF or DMSO was its higher vapor pressure, which facilitated its removal. Investigation of the UV absorption of the polymer revealed a change due to the incorporation of the succinimidyl group. The successful covalent attachment of 5-aminoeosin in solution of polymer and dye indicates the ability of the polymer to bind substances without any further activation. This might be especially useful if three-dimensional polymer scaffolds are incubated with aqueous solutions of growth factors, resulting in covalently immobilized proteins on the scaffold surface. Preliminary experiments with EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid, sodium salt) indicate that approximately 50% of the end groups are amine reactive (data not shown). By changing the type of linker, it will be possible to bind proteins and peptides not only by their amine groups but also by their thiol groups. The reactive polymers can be processed to any desirable type of polymer matrix such as porous scaffolds for tissue engineering applications, or even particles for drug delivery purposes and can bind substrates by simple incubation in aqueous solution. These polymers look very promising for use as biomimetic materials incorporating bioactive proteins and peptides such as growth factors. They will allow us to gain insight into the modulating cellular function for tissue regeneration. Conclusions New polymers were developed that will allow for the covalent attachment of proteins and peptides to their surface. The materials are diblock copolymers with a water-insoluble chain that is biodegradable and ensures that the material erodes in a biological environment. The second chain is hydrophilic and is designed to suppress unspecific protein adsorption to control the biomaterial-cell interactions through specific receptor-ligand coupling. As a model poly(D,L-lactic acid)-block-poly(ethylene glycol)-monoamine was synthesized and activated for reaction with amines by attaching disuccinimidyl tartrate or disuccinimidyl succinate. Incubation with an amine group containing dye confirmed that the resulting polymers were amine reactive without any further chemical activation, allowing for the covalent immobilization
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