Synthesis and Characterization of Biodegradable Poly (ester amide) s

Biomacromolecules 2009, 10, 3037–3047

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Synthesis and Characterization of Biodegradable Poly(ester amide)s with Pendant Amine Functional Groups and In Vitro Cellular Response Mingxiao Deng,† Jun Wu,‡ Cynthia A. Reinhart-King,‡ and Chih-Chang Chu*,†,‡ Department of Fiber Science and Apparel Design, and Department of Biomedical Engineering, Cornell University, Ithaca, New York 14853-4401 Received June 9, 2009; Revised Manuscript Received September 4, 2009

The purpose of this study was to use a convenient synthetic strategy to prepare a new family of biodegradable amino acid-based poly(ester amide)s (PEAs) with pendant amine groups along the polymer backbone, and investigate the applications of the new polymers in the biomedical area. Two amino acids, L-phenylalanine (Phe) and L-lysine (Lys), were used as the model amino acid compounds to illustrate the synthesis, characterization, and biological property of this new family of functional PEAs. These new PEAs were obtained by two-step reactions, the ring-opening reaction of ε-(benzyloxycarbonyl)-L-lysine N-carboxyanhydride (Z-LysNCA) with L-phenylalanine hexane-1,6-diol diester p-toluenesulfonate (Phe-6), and subsequently solution polycondensation with di-p-nitrophenyl sebacoyl (NS). The benzyloxycarbonyl (Z) protective groups of the resulting polymer (PEAZ-Lys) were completely removed to produce the new functional PEAs having free pendant amine groups (PEALys-NH2). The level of the pendant amine groups on the PEA-Lys-NH2 could be tailored by adjusting the Phe-6 to Z-LysNCA feed ratio. Analyses of FTIR, 1H NMR, 13C NMR spectra, and DSC revealed the desired chemical structures and thermal property of PEA-Z-Lys as well as the final functional PEA-Lys-NH2. The free pendant amine groups were used to chemically conjugate a fluorescent dye to demonstrate the utility of this new family of functional PEA. An in vitro cell culture study of these functional PEAs showed that they supported the proliferation of bovine aortic endothelial cell slightly better than gelatin-coated glass coverslips. This new family of biodegradable functional PEA with free amine groups may have great potential applications for biomedical and pharmacological fields.

Introduction Amino acid-based biodegradable poly(ester amide)s (PEA) have been widely investigated for several years due to their good biocompatibility, biodegradability, and a wide range of mechanical and thermal properties. The amide and ester bonds in the polymer backbone have provided PEAs with combined favorable properties of both polyesters and polyamides.1-24 Biodegradable PEAs were usually synthesized by a solution polycondensation reaction of R-amino acids, aliphatic dicarboxylic acids (or dichloride of dicarboxylic acids), and diols. In the past 10 years, our group has established a general methodology for synthesizing PEAs by a solution polycondensation between di-p-toluenesulfonic acid salts of bis-L-R-amino acid R,ω-alkylene diesters (as bis-nucleophiles) and active dip-nitrophenyl esters of dicarboxylic acids (as bis-electrophiles).1,12-14 Due to the saturated nature of these diacids, diols, and the type of amino acids used (e.g., L-phenylalanine, L-leucine, L-valine, DL-methionine), the resulting PEA homopolymers reported before 20005 did not have any built-in functional groups located either along the PEAs backbone chain or as pendant groups. The availability of functional pendant groups could significantly expand the utility of PEAs. For example, built-in functional groups could allow further chemical conjugation with a wide variety of drugs or biological agents, thereby providing * To whom correspondence should be addressed. Tel.: +1 607 255 2938. Fax: +1 607 255 1093. E-mail: [email protected]. † Department of Fiber Science and Apparel Design. ‡ Department of Biomedical Engineering.

a novel route toward functionalized biomaterials.25 The builtin functional groups in PEAs could also provide us with an efficient and powerful method of tailoring the properties of PEAs, such as hydrophilicity, biodegradation rate, and mechanical and thermal properties. Although PEAs consisting of different sequential structures (e.g., types of diols and diacids) have been synthesized and characterized by numerous research groups, the efforts of synthesizing functional PEAs were not materialized and reported until in early 2000. The first reported functional PEA synthesis was based on the copolymer approach24,26 and the pendant functional group provided is a carboxylic acid, which is located in the L-lysine segments of the PEA copolymers. Another approach to provide functionality to PEAs was reported by Guo et al.,1,12,14,27 in which the functional group is the photoreactive carbon-to-carbon double bonds along the PEA backbones. These >CdC< reactive sites were introduced into PEAs by using unsaturated diacids and diols. The availability of these >CdC< bonds in the PEA backbone permits the fabrication of hydrogels by photogelation of PEA precursors that prior generations of PEA based upon saturated diacids and diols could not achieve. The resulting PEA-based hydrogels have been tested as drug carriers.28 Glilies et al. have recently reported another method of synthesizing functional PEA with free pendant amino groups.29 The authors incorporated bis(L-lysine) R,ω-alkylene diester monomer into the PEA, and the pendant amine group can be recovered after deprotection reaction. However, based on their report, the starting material is very expensive; the synthesis and purification steps also appear to be complicated.

10.1021/bm9006437 CCC: $40.75  2009 American Chemical Society Published on Web 10/07/2009

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In this paper, we report the synthesis and characterization of a new family of biodegradable PEA having free amine groups by using a modified copolymer method of our prior published approach. This new copolymer method involves a ring-opening reaction of a protected amino acid derivative like ε-(benzyloxycarbonyl)-L-lysine N-carboxyanhydride (Z-LysNCA) with di-p-toluenesulfonic acid salts of bis-L-phenylalanine, followed by the traditional solution polycondensation of di-p-toluenesulfonic acid salts of bis-L-amino acid with di-p-nitrophenyl sebacoylate. The pendant free amine groups on PEA copolymers can be easily regenerated by a subsequent deprotection under a simple mixed acid treatment. The content of amine groups of the resulting functional PEA copolymers could be controlled by adjusting the feed ratio of Phe-6 to Z-LysNCA monomers. This new copolymer approach may be a universal synthetic route with a broad applicability for the preparation of PEAs with different pendant functional groups, such as carboxylic acid groups or hydroxyl groups, depending on the type of amino acids used. An example of using this pendant free amine group in PEA copolymers was also given in this paper. The in vitro cellular response to these new functional PEA copolymers was studied to determine the cellular biocompatibility for their future potential biomedical applications. The presence of pendent functional groups as well as a possibility of coupling drugs and biologically active agents is of great potential interest for the biomedical and pharmacological applications.

Experimental Section Materials. The L-phenylalanine (L-Phe), p-toluenesulfonic acid monohydrate (TosOH), p-nitrophenol, sebacoyl chloride, 1,6-hexanediol (Alfa Aesar, Ward Hill, MA), and ε-(benzyloxycarbonyl)-L-lysine (HLys(Z)-OH, from Fluka) were used as received. 5-(and 6)-Carboxyfluorescein succinimidyl ester (NHS-fluorescein, excitation maximum 491 nm and emission maximum 518 nm) was purchased from Pierce (Rockford, IL). Triethylamine (NEt3) and ethyl acetate (Fisher, Fairlawn, NJ) were dried over calcium hydride and distilled prior to use. All other chemicals and solvents, such as N,N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), trifluoroacetic acid, acetone, tetrahydrofuran, methanol, and chloroform, were purchased from VWR Scientific (West Chester, PA) and used as received unless otherwise noted. The gelatin powder was purchased from Sigma. Measurements. 1H and 13C NMR spectra were recorded on a Varian (Palo Alto, CA) Unity Inova 400 MHz Spectrometer, with the residual proton resonance or the carbon signal of the deuterated solvent as the internal standard. The number-average and weigh-average molecular weight of the resultant polymers were determined with a Waters 410 size-exclusion chromatography equipped with two Waters Styragel columns (HT6E, HT3) and a differential refractometer detector. Chloroform was used as the eluent (1.0 mL/min) and the average molecular weight of the polymers was calculated based on calibrations using polystyrene standards. Infrared spectra were recorded with a Perkin Elmer (Madison, WI) Nicolet Magana 560 FTIR spectrometer, using KBr plates. The NHS-fluorescein attached PEA sample was examined with Olympus BX41 fluorescent microscope. The thermal properties of the monomers and polymers were analyzed with a TA Instruments DSC 2920 differential scanning calorimenter (TA Instruments, New Castle, DE). DSC samples were analyzed over the temperature range of -42 to 270 °C, with a scan rate of 10 °C/min. The reduced viscosity of the resultant polymers was determined with a Cannon-Ubbelohde viscometer in DMSO solution at a concentration of 0.25 g/dL at 25 °C. Synthesis of the Monomers and Polymers. The new family of PEA with pendant amine groups were synthesized from the following five major steps: (1) synthesis of protected ε-(benzyloxycarbonyl)-L-lysine N-carboxyanhydride (Z-LysNCA); (2) synthesis of di-p-toluenesulfonic acid salts of bis-L-phenylalanine ester (Phe-6) and its derivative

Deng et al. monomer with Z-LysNCA (Z-Lys-Phe-6); (3) synthesis of di-pnitrophenyl esters of dicaboxylic acids (NS); (4) solution polycondensation of monomers Z-Lys-Phe-6, Phe-6, and NS; and (5) deprotection of the resulting polymer (PEA-Z-Lys). The synthesis of Phe-6 and NS was performed according to the previously published method, as shown in Scheme 1.14 Synthesis of ε-(Benzyloxycarbonyl)-L-lysine N-Carboxyanhydride (Z-LysNCA). The synthesis of ε-(benzyloxycarbonyl)-L-lysine Ncarboxyanhydride was prepared by the Fuchs-Farthing method using triphosgene.30 In brief, a suspension of the protected H-Lys(Z)-OH (6.00 g, 21.40 mmol) in 150 mL of ethyl acetate was reflux in a nitrogen atmosphere. A solution of triphosgene (2.37 g, 8.00 mmol) dissolved in 30 mL of ethyl acetate was added to the stirred reaction mixture. When the reaction mixture started to become transparent, a stream of nitrogen was bubbled through the solution to removed HCl. After the reaction was complete, the solvent was evaporated under vacuum to give a colorless oily residue which crystallized upon cooling in a refrigerator. The product obtained, Z-LysNCA, was further purified by recrystallization three times in a mixture of ethyl acetate/petroleum ether and dried in vacuo. The yield was 87%. Synthesis of Amine-Protected Di-p-toluenesulfonic Acid Salt of N-Benzyloxycarbonyl-L-lynsyl-bis-L-phenylalanine Hexane-1,6-diester Monomer (Z-Lys-Phe-6). The synthesis of Z-Lys-Phe-6 monomer was performed according to a modified method of Knobler et al.31,32 The amounts of Z-LysNCA monomer to be incorporated into PEA would depend on the desired content of the pendant amine groups on the final PEA polymer, which can be controlled via the molar ratio of Phe-6 to Z-LysNCA. The different molar combinations of Phe-6 and Z-LysNCA in this work are summarized in Table 1 and illustrated in Scheme 1. A typical experimental procedure for the synthesis of Z-Lys-Phe-6-50 monomer is given here. Z-LysNCA (2.43 g, 7.93 mmol) was added to a solution of Phe-6 (6.00 g, 7.93 mmol) in 30 mL of N,N-dimethylacetamide (DMA). The reaction mixture was stirred at 40 °C for 3 h and the solution temperature was raised to 80 °C for 24 h in a nitrogen atmosphere. The reaction was subsequently cooled to a room temperature and used in the next stage polycondensation reaction without further purification. 1 H NMR (DMSO-d6, 400 MHz): δ (ppm) 1.06 (br, 4H, (CH2)2CH2CH2(CH2)2), 1.38 (br, 8H, CH2CH2(CH2)2CH2CH2, NHCH2CH2CH2CH2CH), 1.69 (br, 2H, NH(CH2)3CH2CH), 2.29 (s, 6H, ArCH3), 2.97-3.19 (m, 4H, ArCH2, NHCH2(CH2)3CH), 3.78 (t, 1H, ArCH2CHNH), 4.00 (t, 4H, CH2(CH2)4CH2), 4.29 (t, H, NH3+CHCOO), 4.54 (t, H, NHCOCHNH3+), 5.00 (s, 2H, ArCH2OCONH), 7.11-7.51 (m, 23H, ArH), 8.44 (s, 6H, NH3+CHCOO, NHCOCHNH3+). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 20.85 (ArCH3), 21.29 (NH(CH2)2CH2CH2CH), 24.74, 24.85 ((CH2)2CH2CH2(CH2)2), 27.67, 27.89 (CH2CH2(CH2)2CH2CH2), 29.11 (NHCH2CH2(CH2)2CH), 30.94 (NH(CH2)3CH2CH), 36.26 (ArCH2CHNH), 36.59 (ArCH2CHNH3+), 40.11 (NHCH2(CH2)3CH), 52.05 (NH(CH2)4CH), 53.47 (ArCH2CHNH3+), 54.15 (ArCH2CHNH), 64.67 (ArCH2OCONHCH2), 65.21, 65.48 (CH2(CH2)4CH2), 124.28, 125.61, 126.75, 127.27, 127.75, 128.31, 128.40, 128.58, 129.11, 129.36, 134.78, 136.90, 137.30, 138.32, 144.84 (ArC), 156.15 (ArCH2OCONH), 169.00 (NHCOCHNH3+), 169.09 (NH3+CHCOO), 171.06 (CH2OOCCHNH); mp)216 °C. Synthesis of Amine-Protected Poly(ester amide)s, PEA-Z-Lys. As shown in Scheme 2, a solution polycondensation was performed based on our previously published procedures.1,13,14 An example of the synthesis of PEA-Z-Lys-50 (sample 6 in Table 1) is illustrated by the following synthesis procedures. NS (3.52 g, 7.93 mmol) and dry NEt3 (2.41 mL, 17.45 mmol) were added to a solution of Z-Lys-Phe6-50 (8.08 g, 7.93 mmol) in 30 mL of DMA under a nitrogen atmosphere. The reaction solution was stirred at room temperature for several min and subsequently at 80 °C for 24 h. The resulting solution was cooled to room temperature, diluted with 30 mL of DMA and precipitated into an excess of cold ethyl acetate. Purification was performed by dissolving the polymer in dichloromethane and slowly adding into an excess of cold ethyl acetate. The tar-like polymer was

Synthesis of Biodegradable Poly(ester amide)s


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Scheme 1

filtered off and dried in vacuo at 50 °C. The composition of PEA-ZLys was determined by 1H and 13C NMR in DMSO-d6. This polymer was used for the preparation of deprotected PEA-Lys-NH2.

Synthesis of Amine-Pendant Poly(ester amide)s, PEA-Lys-NH2. The protective groups (Z groups) of the side-chain amine groups in the Lys units were removed by utilizing trifluoroacetic acid/methanesulfonic

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Table 1. Monomer Combinations for the PEA-Z-Lys and PEA-Z-Lys-NH2 Synthesis monomer feed ration Z-LysNCA

Phe-6

NS

expected Z-lysine content (%)

Z-Lys-Phe-6

protected polymer

deprotected Polymer

0 1 1 1 1 1

1 19 5.7 3 1.9 1

1 19 5.7 3 1.9 1

0 5 15 25 35 50

Z-Lys-Phe-6-0 Z-Lys-Phe-6-05 Z-Lys-Phe-6-15 Z-Lys-Phe-6-25 Z-Lys-Phe-6-35 Z-Lys-Phe-6-50

PEA-Z-Lys-0 PEA-Z-Lys-05 PEA-Z-Lys-15 PEA-Z-Lys-25 PEA-Z-Lys-35 PEA-Z-Lys-50

PEA-Lys-NH2-05 PEA-Lys-NH2-15 PEA-Lys-NH2-25 PEA-Lys-NH2-35 PEA-Lys-NH2-50

Scheme 2

acid/anisole mixture. The PEA-Z-Lys-50 (5.00 g) was dissolved in 20 mL of trifluoroacetic acid and stirred for 1 h at room temperature. Subsequently, methanesulfonic acid (1.30 mL) was dissolved in 2.60 mL of anisole and added to the solution of PEA-Z-Lys-50. After stirring for an additional 1 h, the solution was precipitated into an excess of cold diethyl ether. To remove the excess acids, the polymer was dissolved in DMA, neutralized with triethylamine, and then precipitated into an excess of ethyl acetate. The resultant polymer, PEA-Lys-NH2, was filtered, and dried in vacuo at 50 °C. Fluorescent Dye Attachment, PEA-Lys-Dye. As shown in Scheme 3, the NHS-fluorescein dye was attached onto the free amine site of PEA-Lys-NH2-05 (sample 3 in Table 2) to demonstrate the existence and usefulness of the pendant amine groups on the functional PEA polymer chain. A typical experimental procedure of preparing a

fluorescent dye-tagged PEA (PEA-Lys-05-Dye) is given here. A solution of PEA-Lys-NH2-0.5 (1.00 g) and NHS-fluorescein dye (10 mg) in 15 mL of DMSO were stirred at room temperature. After 6 h, the solution was precipitated into distilled water. The polymer was sequentially washed with distilled water to remove any physically absorbed fluorescent dye and dried in vacuo overnight. The dried polymer (0.10 g) was dissolved in 10 mL of chloroform, and the solution was cast onto glass coverslips. The coated slides were dried in vacuo for 12 h and used for fluorescent testing. Cell Adhesion and Proliferation Assay. Bovine aortic endothelial cells (BAEC, primary cells) were purchased from VEC Technologies.33 BAECs were maintained at 37 °C in 5% CO2 in Medium 199 (Invitrogen, Carlsbad, CA) supplemented with 10% Fetal Clone III (HyClone, Logan,



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Scheme 3

UT), and 1% each of penicillinstreptomycin, MEM amino acids (Invitrogen, Carlsbad, CA), and MEM vitamins (Mediatech, Manassas, VA). BAECs were used from passages 8-12. Media was changed every two days. Cells were grown to 70% confluence before splitting or harvesting. The evaluation of the BAEC cell attachment capability on the polymer surface and polymer cytotoxicity in the media was performed by cell proliferation assay with subsequent MTT assay. The round micro glass coverslips (diameter, 12 mm, no. 2, VWR, West Chester, PA) were coated with the PEA polymer by dipping the coverslips into the polymer/DMF solution (5 wt %) and vacuum drying. This coating and drying procedures

were repeated for three times. After the final vacuum drying, the PEALys-NH2-25 coated glass coverslips were placed into cell culture plates and treated with 2 wt % gelatin aqueous solution. For comparison, gelatincoated tissue culture wells were also prepared. In brief, gelation solution was prepared by dissolving gelatin powder into distilled water to make a 2 wt % solution. This gelatin solution was sterilized before use. For the gelatin coating, 24-well cell culture plates were coated with gelatin by adding 0.3 mL of gelatin solution to each well. After 5-10 min, the gelatin solution was removed and the coated plate was ready for use. Untreated wells were used as controls.

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Table 2. Fundamental Properties of PEA-Z-Lys and PEA-Lys-NH2 polymer

yield (%)

PEA-Z-Lys-0 PEA-Z-Lys-05 PEA-Lys-NH2-05 PEA-Z-Lys-15 PEA-Lys-NH2-15 PEA-Z-Lys-25 PEA-Lys-NH2-25 PEA-Z-Lys-35 PEA-Lys-NH2-35 PEA-Z-Lys-50 PEA-Lys-NH2-50

82 79 78 76 75 77 75 73 72 69 70

lysine molar ratioa (mol %) 2 10 25 33 46

Mnb (kg/mol)

Mwc (kg/mol)

Mw/Mn

ηredd (g/dL)

Tg (°C)

68.0 67.0 45.3 69.5 23.6 44.1 12.0 47.4 10.7 48.7

89.9 104.0 80.0 99.9 48.9 73.0 19.5 95.6 18.4 92.0

1.32 1.55 1.77 1.44 2.07 1.66 1.38 2.02 1.71 1.89

0.58 0.58 0.54 0.55 0.59 0.45 0.75 0.48 0.79 0.50

32.35 28.95 30.95 23.87 25.42 19.85 32.39 24.37 28.74 20.44 29.78

a Lysine molar ratio in polymer determined by 1H NMR. b Mn determined by GPC. c Mw determined by GPC. d Measured in DMSO at 25 °C (concentration ) 0.25 g/dL).

Cultured cells were seeded onto each test well at the same cell density concentration (20000 cells/well) in 24-well plates and then incubated in a 37 °C, 5% CO2 incubator. Cell media was changed every day. After the predetermined incubation periods (24, 48, and 72 h), the cell culture plates were removed from the incubator. The media from the wells was aspirated, and 0.5 mL fresh media was added to each well. A total of 40 µL of MTT solution (5 mg/mL) was subsequently added to each well, followed by 4 h incubation at 37 °C and 5% CO2. The cell culture medium was carefully removed and 400 µL of acidic isopropyl alcohol (with 0.1 M HCl) was added to dissolve the formed formazan crystals. The plate was gently shaken for 30 min, and 100 µL solution was transferred from each well to a 96-well plate. Optical density (OD) of each well was measured at 570 nm (subtract background reading at 690 nm) by using a microplate reader.

Results and Discussion Monomers Synthesis. In this study, three types of monomers have been synthesized for preparing PEA with pendant free amine groups. Phe-6 and NS have been synthesized based on our previously published procedures. Both of these two monomers can be prepared with high yields and easily purified by recrystallization. The chemical structure of Phe-6 and NS has been confirmed by NMR, FTIR, and DSC. All the analysis data are consistent with our prior studies.13,14 All the three monomer reactions and their subsequent solution polycondensation have been found to proceed smoothly without producing any byproducts. The amine-protected Lys/Phe-based toluenesulfonic acid salt monomer (Z-Lys-Phe-6) was first synthesized by modifying the synthesis method of Knobler et al.31,32 The Phe-6 (Phe-based p-toluenesulfonic acid salt) was used as the model monomer to couple with Z-Lys-NCA to synthesize a new corresponding monomer Z-Lys-Phe-6. In this reaction, Z-LysNCA can react stoichiometrically (1:1) with Phe6, and the molar ratio of Z-LysNCA to Phe-6 can be easily controlled by the feed molar ratio. The structure of the Z-Lys-Phe-6 was confirmed by 1H and 13 C NMR. The 1H NMR peaks marked with numbers from 1 to 18 are assigned to the corresponding protons of Z-Lys-Phe-6 and Phe-6 as shown in Figure 1. When comparing with the 1H NMR of Phe-6, the distinct peak at 5.00 ppm on the spectrum of Z-Lys-Phe-6 was assigned to the ArCH2 protons derived from the protective groups of L-lysine segments. An identical observation was made in the 13C NMR spectra (Figure 2), which showed the peak at 64.67 ppm, corresponding to the carbon atoms of ArCH2. In the carbonyl region, the peaks at 156.15, 169.00, 169.09, and 171.06 ppm were attributed to the different carbon of CdO from L-phenylalanine and L-lysine segments. It is important to note that the existence of L-lysine segments has broken the symmetrical structure of methylene carbons of

Figure 1. 1H NMR spectra of two monomers: (a) Phe-6, (b) Z-LysPhe-6; 191 × 279 mm (300 × 300 DPI).

diol part and every methylene carbons shown two split peaks in 13C NMR spectrum of Z-Lys-Phe-6. All these evidence have strongly support the anticipated molecular structure of Z-LysPhe-6. The degree of incorporating L-lysine segments per Phe-6 molecule was calculated from the ratio of integration value of 5.00 ppm assigned to methylene proton signal of Z group to that of 4.00 ppm assigned to methylene proton signal of Phe-6 in the 1H NMR spectrum. As a result, the level of L-lysine unit incorporated into Phe-6 unit in Z-Lys-Phe-6 monomer could be quantitatively controlled by changing the feed molar ratio of Phe-6 to Z-LysNCA reactants (Table 1).


Figure 2.


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C NMR spectra of two monomers: (a) Phe-6, (b) Z-Lys-Phe-6; 223 × 351 mm (300 × 300 DPI).

13

Synthesis of Protected PEA-Z-Lys. As shown in Scheme 2, the amine-protected PEAs (PEA-Z-Lys) were prepared by solution polycondensation of Z-Lys-Phe-6 and Phe-6 with NS monomers under similar optimized conditions as our prior studies. The polycondensation proceeded smoothly with a high yield of PEA-Z-Lys. In this polymerization reaction, triethylamine was used as an acid receptor for toluenesulfonic acid, which was produced during the regeneration of amino groups from the Z-Lys-Phe-6 and Phe-6 monomers. The main advantage of using NS, instead of diacyl chloride, as a diacid monomer is that NS is a stable and solid monomer, therefore, the stoichiometric balance of amine and carboxyl groups in the polycondensation can be precisely controlled by accurately weighted monomers. Table 2 shows the polymerization yields and the ηred of PEA-Z-Lys, which were found to be similar among the five types of PEA-Z-Lys, even though the Z-Lys contents ranged from 5 to 50%, as shown in Table 1. The reason

of this phenomenon was explained below. All the PEA-Z-Lys obtained were solid and insoluble in methanol but soluble in common organic solvents, such as chloroform, DMF, and THF. Both FTIR and NMR confirmed the structure of the new amine-protected PEA-Z-Lys polymer. The FTIR spectrum of PEA-Z-Lys-25, shown in Figure 3, had the ester carbonyl stretch (1737 cm-1), amide I bond (1644 cm-1), and amide II bond (1534 cm-1). In the 1H NMR spectra (Figure 4), the distinct peaks assigned to the methylene groups of protecting group of L-lysine segments can be still observed at 5.00 ppm. Figure 5 shows a comparison of carbonyl region of the 13C NMR spectra for both the PEA-Z-Lys-0 (without L-lysine segments) and the PEA-Z-Lys-25. The peaks at 171.67 and 172.27 ppm are associated with the (Phe)NHCO and (Phe)COO, respectively. The three additional peaks observed in the carbonyl region are attributed to (Z-Lys)NHCO, (Z-Lys)CONH and Z group at 171.33, 171.98, and 156 ppm, respectively. These FTIR and

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Figure 3. FTIR spectra of three representative PEAs: (a) PEA-ZLys-0, (b) PEA-Z-Lys-25, (c) PEA-Lys-NH2-25; 381 × 261 mm (72 × 72 DPI).

Figure 4. 1H NMR spectra of three representative PEAs: (a) PEAZ-Lys-0, (b) PEA-Z-Lys-25, (c) PEA-Lys-NH2-25; 223 × 274 mm (300 × 300 DPI).

NMR spectra confirm the presence of Z-Lys unit on the PEAZ-Lys backbone. The GPC data of the PEA polymers are summarized in Table 2. All of PEA-Z-Lys polymers have similar molecular weight and molecular weight distribution, which were consistent with our prior report.5 When comparing with interfacial polymerization technique, we obtained much higher molecular weights of PEAs from the polycondensation reaction.11 The GPC traces are unimodal with no signal of coexisting low or high molecular weight species that may be produced from an uncontrolled polycondensation. The composition of the PEA-Z-Lys, determined by 1H NMR, in some cases slightly deviate from intended composition (Table 1). The deviation became more pronounced at a lower L-lysine unit content. This

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deviation is caused by the removal of lower molecular weight polymers having a high L-lysine content during the purification process. Deprotection of PEA-Z-Lys. The benzyloxycarbonyl (Z) protecting group on amino group of L-lysine can be easily removed either by catalytic hydrogenolysis or catalytic transfer hydrogenation34 However, the Z group on ω-amino position is usually removed under strong acid hydrolysis or catalytic hydrogenolysis. In this study, the removal of the Z groups at the N-terminal of the L-lysine units of PEA-Z-Lys was performed by immersion of the PEA-Z-Lys in the mixed solvent of methanesulfonic acid, anisole, and trifluoroacetic acid for 1 h followed by neutralization with triethylamine. 1H NMR analysis of the PEA-Lys-NH2 demonstrated near a complete removal of the protecting Z groups as evident in the absence of proton peaks of Z groups at 5.00 ppm (Figure 4). In the 13C NMR spectrum, the disappearance of carbonyl carbon signal of Z groups at 165 ppm confirmed the complete deprotection (Figure 5). This acid-based deprotection method, however, showed some molecular weight loss of the PEAs as shown in Table 2. The data in Table 2 shows that the molecular weight of PEAs (PEAZ-Lys-05, PEA-Lys-NH2-05, Table 2) was reduced from 67000 to 45300 (32% molecular weight reduction) by the treatment with a mixed acid deprotection medium. A similar range of molecular weight reduction was also reported by Barrera who used catalytic hydrogenolysis deprotective method.25 The reduction in molecular weight of PEA-Lys-NH2 became more profound as the L-lysine content increased. This PEA molecular weight reduction upon the acidic-based deprotection procedure could be attributed to (1) the loss of the Z protecting groups and (2) the partial hydrolysis of the ester bonds in PEA backbone under the strong acid deprotection condition. However, due to the intermolecular association caused by hydrogen bonding among the amine groups, the PEA-Lys-NH2 obtained in this study has a relative higher reduced viscosity ηred than the corresponding PEA-Z-Lys before deprotection. To directly demonstrate the existence and use of free amine groups in the PEA-Lys-NH2, the NHS-fluorescein dye attached PEA has been synthesized as shown in Scheme 3. The left image in Figure 6 shows that the NHS-fluorescein attached PEA sample displayed the characteristic green color of the NHSfluorescein dye. In contrast, the right image of control sample (PEA-Z-Lys-05) shows a distinct black color. This new family of PEA-Lys-NH2 differs from our previously synthesized PEA copolymers in two main aspects. First, PEALys-NH2 family has two amino acids linked via an amide bond within the same block, while our prior PEA copolymers have one amino acid in each repeating block, and a copolymer approach was used to incorporate two different amino acids in two different repeating blocks.23,24,34 Second, PEA-Lys-NH2 family provides pendant functional amine groups and exhibits positive charge, while our prior PEA copolymers have pendant functional carboxylic acid groups and exhibits negative charge in a physiological pH. In the Gillies et al. reported study of PEA with amine functional groups,22 they introduced the L-lysine derivative onto the PEA backbone, and the amine group in the L-lysine unit can be recovered after deprotection reaction. Their synthesis method is basically the same as our previously published studies of PEA copolymer.24 The only difference is that Glilies et al. made a new bis(L-lysine) R,ω-alkylene diester monomer in diamine instead of in di-p-toluenesulfonic acid salt, which we have used. In this paper, we established a new synthesis method by introducing ε-(benzyloxycarbonyl)-L-lysine N-carboxyanhy-



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Figure 5. 13C NMR spectra of three representative PEAs: (a) PEA-Z-Lys-0, (b) PEA-Z-Lys-25, (c) PEA-Lys-NH2-25; 228 × 383 mm (300 × 300 DPI).

Figure 6. Fluorescent microscope images PEA film on glass coverslips: (a) PEA-Lys-05-Dye film on glass coverslip, (b) PEA-Lys-NH205; 531 × 220 mm (72 × 72 DPI).

dride into making functional PEA. The chemical structure of our functional PEA with free amine groups is also different from that of Glilies et al.’s. In our functional PEAs, the two R-amino acids, Phe and Lys, are directly connected by a peptide bond instead of separated by diacids spacer on the PEA backbone as in the Glilies et al. study. It is important to point out that our new methodology can also be easily extended to introduce other functional groups like carboxyl or hydroxyl groups onto PEAs as we are currently investigating. Thermal Property. The glass transition temperatures (Tg) of the PEA polymers are shown in Table 2. All PEA polymers having L-lysine content were amorphous and exhibited Tgs

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Deng et al.

Figure 7. DSC traces of three representative PEAs: (a) PEA-Z-Lys0, (b) PEA-Z-Lys-25, (c) PEA-Lys-NH2-25; 203 × 212 mm (300 × 300 DPI).

ranging from 18 to 32 °C; no melting temperature was observed. Typical DSC traces of these PEAs are shown in Figure 7. For the PEA-Z-Lys-0 (PEA without L-lysine units), the Tg is 32.35 °C, which is consistent with our previous report.5 The Tg of the protected PEA-Z-Lys-25 was 19.85 °C, about 12 °C lower than that of PEA-Z-Lys-0. The reason for this significant reduction in Tg is attributed to the presence of the bulky pendent protective group in the L-lysine unit, which could act as an internal plasticizer, that is, increasing free volume of polymer chains and hence lower Tg.35,36 After deprotection, the recovered pendant amine group in the L-lysine unit strengthened the

intermolecular interaction among the PEA chains via hydrogen bonds. Therefore, the deprotected PEA-Lys-NH2-25 exhibited a higher Tg (32.39 °C) than its protected PEA-Z-Lys-25 counterpart. As shown in our prior study,5,14,37 the chemical structure of PEA had a profound effect on Tg. For example, the introduction of CdC double bond into the diols and dicarboxylic acid parts of PEA increases the rigidity of PEA polymer chain, and raises the Tg up to 109 °C.14 However, this rigid unsaturated PEA chains resulted in their poor solubility in common organic solvents.1,13,14 For the saturated PEA, the methylene chain length of PEA backbone could also affect the flexibility of the polymer chain and Tg. Therefore, to obtain a PEA with a good balance between Tg and solubility, the length of methylene chain was kept at 6 and 8 on diols and dicaboxylic acid parts, respectively in this study. Solubility. Table 3 shows the solubility of PEA-Z-Lys and PEA-Lys-NH2 having different L-lysine contents at room temperature. All the protected PEA-Z-Lys were completely soluble in polar solvents, such as CHCl3, DMF, and DMSO but could not dissolve in ethyl acetate, acetone and water. All PEA-Lys-NH2 were soluble in DMF and DMSO but not in water. The PEA-Lys-NH2 having higher L-lysine contents, such as PEA-Lys-NH2-50, tends to form strong intermolecular hydrogen bonds; once it dried, it was difficult to be redissolved. Cell Adhesion and Proliferation. From the microscope images (Figure 8), both the gelatin-coated and PEA-Lys-NH225 coated groups, showed confluent BAEC cells after 3 days, while the untreated control groups was less than 50% confluence. The MTT assay was used to test the cytotoxicity of the new functional PEAs. The data in Figure 9 illustrate that the BAEC proliferation rate of the coated groups (functional PEA and gelatin) showed clear higher proliferation rates than the untreated control, and the PEA-Lys-NH2-25 coated group showed the same or slightly better cell proliferation than the gelatin-coated group. We hypothesized that the positive amine groups in the functional PEAs could facilitate endothelial cell attachment and

Table 3. Solubility of PEA-Z-Lys and PEA-Lys-NH2 at Room Temperature (25 °C)a PEA-Z-Lys-0 PEA-Z-Lys-05 PEA-Lys-NH2-05 PEA-Z-Lys-15 PEA-Lys-NH2-15 PEA-Z-Lys-25 PEA-Lys-NH2-25 PEA-Z-Lys-35 PEA-Lys-NH2-35 PEA-Z-Lys-50 PEA-Lys-NH2-50 a

H2O

DMF

DMSO

THF

methanol

ethyl acetate

chloroform

acetone

-

+ + + + + + + + + + (

+ + + + + + + + + + (

+ + ( + + + + -

( (

-

+ + + + + + + + ( + -

-

+, soluble; -, insoluble; (, partially soluble or swelling.

Figure 8. Proliferation Assay of BAEC. Group: (a) blank control (without any treatment); (b) gelatin-coated; and (c) PEA-Lys-NH2-25-coated; 254 × 163 mm (600 × 600 DPI).


Figure 9. Proliferation Assay of BAEC. Group: (a) blank control (without any treatment); (b) gelatin-coated; and (c) PEA-Lys-NH2-25coated; 254 × 163 mm (600 × 600 DPI).

proliferation. In recent years, many efforts have been directed toward the creation of varieties of biocompatible materials with the ability of controlling and promoting cell attachment, migration, proliferation, differentiation, and long-term viability. For endothelial cell culture, collagen or gelatin coatings have been widely used to facilitate cell attachment. However, the current gelatin coating can not meet the rapid development of tissue engineering. This newly developed positively charged functional PEA could provide a viable alternative to gelatin to promote cell adhesion and proliferation. In this study, we also found that the quality of PEA-Lys-NH2-25 coating could have a profound impact on cell culture outcomes. An uneven coating could lead to lower cell attachment. In a subsequent separate study we will report whether the BAEC adhesion on these functional PEAs is primarily nonspecific and integrin-mediated adhesion.

Conclusions Synthetic amino acid-based biodegradable poly(ester amide)s containing reactive free amine functional groups in the L-lysine unit (PEA-Lys-NH2) have successfully been synthesized via ring-opening reaction and solution polycondensation (two-step reactions). By adjusting the protected L-lysine derivative (ZLysNCA) contents, the free amine group contents of the resulting functional PEA-Lys-NH2 polymers can be well controlled. All the chemical structures of monomers and polymers have been confirmed by NMR and FTIR. The thermal and physical properties of this new family of functional PEALys-NH2 were studied. The preliminary data of cell proliferation and cytotoxicity of these new functional and positive charged PEAs (PEA-Lys-NH2) show that these new functional PEAs support bovine aortic endothelial cell proliferation without cytotoxicity. Acknowledgment. This study was partially supported by a Morgan tissue engineering seed grant to C.-C.C. and C.A.R.K. and a research assistantship to J.W. from the Dept. of Biomedical Engineering.

References and Notes (1) Guo, K.; Chu, C. C. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 1595–1606.


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BM9006437

Synthesis and Characterization of Biodegradable Poly (ester amide) s

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