Synthesis of Polyethylene Glycol (PEG) Derivatives and PEGylated

Biomacromolecules 2003, 4, 1055-1067

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Synthesis of Polyethylene Glycol (PEG) Derivatives and PEGylated-Peptide Biopolymer Conjugates Jing Li and W. John Kao* Division of Pharmaceutical Sciences of the School of Pharmacy and Department of Biomedical Engineering of the College of Engineering, University of WisconsinsMadison, Madison, Wisconsin 53705 Received March 11, 2003

We synthesized a library of 50 poly(ethylene glycol) (PEG) derivatives to expand the extent of conjugation with biologically active molecules (biopolymers, peptides, drugs, etc.) and biomaterial substrates. The formation of PEG derivatives was confirmed with HPLC, 1H and 13C NMR. PEG derivatives were polymerized into networks in order to study the role of PEG and terminal functional groups in modulating the hydrophilicity of biomaterials and cell-biomaterial interaction. The resulting surface hydrophilicity and the number of adhered fibroblasts were primarily dependent on the PEG concentration with the molecular weight and the terminal functional group of PEG derivatives being less important. One of PEG derivatives, PEG-bisglutarate, was utilized to link peptide sequences to gelatin backbone in the formation of novel biomedical hydrogels. PEG-peptide conjugates were characterized by mass spectroscopy. PEG-peptide modified gelatins were characterized by gel permeation chromatography. Introduction Poly(ethylene glycol) (PEG) is employed extensively in pharmaceutical and biomedical areas. PEG is subject to ready chemical modification and attachment to other molecules and surfaces. When attached to other molecules, PEG modulates the solubility and increases the size of the attached molecules. A wide range of applications of PEG can be categorized as follows: (i) purification of proteins and nucleic acids,1-3 (ii) purification of biological materials due to the formation of aqueous polymer two-phase systems,4 (iii) conjugation with proteins resulting in a reduction of immunogenicity, antigenicity and an increased serum half-life,5-9 (iv) surface modification for retarding protein adsorption, platelet adhesion and thrombogenicity,10-16 and (v) drug conjugation and drug release.17-21 Because the repeating ethylene oxide units of PEG possess no reactive side moieties, PEG is bound to other compounds through terminal functional groups. Two approaches are commonly used for the functionalization of PEG:22-26 (i) alteration of the terminal hydroxyl group through a series of reactions to a more active functional group and (ii) reaction of PEG under controlled conditions with difunctional compounds so that one of the functional groups reacts with PEG and the other remains active.21 In most cases, several steps are conducted to achieve the expected derivatization. Currently, many biomolecules have been modified with PEG, however, only a few classes of compounds are routinely used as substrates for PEG conjugation. To expand the extent of conjugation with biologically active molecules * To whom correspondence should be addressed. School of Pharmacy, University of WisconsinsMadison, 777 highland Ave., Madison, WI 53705. Phone: (608) 263-2998. Fax: (608) 262-5345. E-mail: wjkao@ pharmacy.wisc.edu.

(biopolymers, peptides, drugs, etc.) and biomaterial substrates, we synthesized a library of 50 PEG derivatives (Figure 1). A large part of the chemistry was adopted from previous research work to ensure the synthesis of derivatives. We also combined synthesis procedures in new sequences to create a few novel heterodifunctional PEGs. They were CN-PEG-Ac, Tos-PEG-Ac, PT-PEG-Ac, COOHPEG-Ac, Glu-PEG-Ac, and PEG-peptide modified gelatin. The procedures were optimized and simplified to obtain the highest degree of conversion. However, the purification of a specific family of PEG derivatives was beyond the scope of the current study and was not performed. To evaluate the biological function of PEG derivatives, we copolymerized a group of acrylated PEG derivatives with TMPTA and acrylic acid to study the role of PEG and the terminal functional group in determining the hydrophilicity of biomaterials and cell-biomaterial interaction, the result of which would help us to design biopolymers with favorable chemical and biological properties. To illustrate one application of PEG derivatives, we conjugated PEG-bis-glutarate, one of PEG derivatives, to a peptide sequence forming a PEG-peptide conjugate which was used to modify gelatin backbone in the formation of novel biomedical hydrogels. Method and Materials All starting compounds were used as received without additional purification. All chemicals were purchased from Aldrich except for those specified. THF and MC were dried prior to use. Intermediate and final products were characterized by a reverse phase HPLC system (Gilson, 10% to 100% acetonitrile at a flow rate of 1 mL/min in 30 or 60 min coupled with UV/vis and ELSD detectors). Gelatin (Sigma) and modified gelatins were characterized by an Ultrahydrogel

10.1021/bm034069l CCC: $25.00 © 2003 American Chemical Society Published on Web 05/17/2003

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Biomacromolecules, Vol. 4, No. 4, 2003

Li and Kao

PEG Derivatives and PEGylated Peptide Biopolymer

Biomacromolecules, Vol. 4, No. 4, 2003 1057

Figure 1. Structures and names of PEG derivatives.

column in a GPC system (Waters, 80% 0.1 M NaNO3 and 20% acetonitrile at a flow rate of 0.7 mL/min in 60 min coupled with an RI detector). An established method based on TNBS was used to characterize the percent modification of lysyl groups on gelatin.27-29 Synthesis of PEG Derivatives. Synthesis of acrylated PEG derivatives is summarized in Figure 2. Synthesis of

COOH-PEG-Ac is presented in detail in Figure 3. Products were characterized and verified by HPLC (Table 1) and NMR (Table 2, 13C NMR). All HPLC runs lasted 30 min except for those specified. The synthesis of MPEG derivatives is not demonstrated to minimize redundancy, because the chemistry of MPEG derivatization is the same with the acrylated PEG derivatives.

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Figure 2. Chemical reactions and structures of critical intermediate and final products resulting from the synthesis of acrylated PEG derivatives in the formation of TMPTA networks. Side products are numbered and shown according to Figure 1.

Figure 3. Formation of COOH-PEG-Ac, intermediate and final products.

MPEG-Ac and PEG-bis-Ac. A total of 1 eq. mol MPEG (2K Da) was dissolved in dry THF followed by the addition of 2 eq. mol AC and 2.5 eq. mol TEA, stirred under Ar at room temperature for 3 h, dried using the drying procedure in which the solution was precipitated in cold hexane, filtered, and the filtrate was precipitated in cold hexane, filtered, and dried in a vacuum oven to obtain MPEG-Ac.30 Following the same procedure, a mixture of PEG (2K Da), HO-PEG-Ac, and PEG-bis-Ac was obtained when the molar ratio of PEG:AC:TEA was 1:1:1.2 (Figure 2, Table 1). PEG-bis-Ac (575 Da and 1, 2, 3.4, 4.6, and 8K Da)

were synthesized resulting in approximately 98% yield in a similar fashion by varying the molar ratios and reaction time as follows: PEG 400:AC:TEA ) 1:3:4, 30 min; PEG 1K: AC:TEA ) 1:2.5:3, 30 min; PEG 2K:AC:TEA ) 1:4:5 3 h; PEG 3.4K:AC:TEA ) 1:5:6, 3 h; PEG 4.6K:AC:TEA ) 1:6: 7, 4 h; PEG 8K:AC:TEA ) 1:6:7, 4 h. PT-PEG-Ac. To synthesize HO-PEG-Tos, 1 eq. mol PEG (2K Da) and 2 eq. mol pToSC were dissolved in MC followed by the addition of 2 eq. mol TEA, stirred under Ar at room temperature for 8 h, followed by the drying procedure as described above and a mixture of PEG, HO-


PEG Derivatives and PEGylated Peptide Biopolymer Table 1. Comparison of HPLC Retention Time, Normalized Peak Area, Percent Conversion, and Yield for PEG Derivatives and Intermediates Synthesized from PEG 2Ka

compounds

retention normalized contime peak area version yield (min) (Ni) factor (%)

PEG

10.6

1

PEG PEG HO-PEG-Ac PEG-bis-Ac

10.6 11.8 13.1

1 10.8 22.8

0.03 0.31 0.66

3 no 30 medium 65 medium

PEG HO-PEG-Tos PEG-bis-Tos

10.6 13.5 16.5

1 1.3 0.3

0.38 0.50 0.12

34 no 49 strong 12 strong

PEG-bis-Ac Tos-PEG-Ac PEG-bis-Tos

13.1 14.8 16.4

1 4.4 2.8

0.12 0.54 0.34

11 medium 51 strong 32 strong

PEG-bis-Ac PT-PEG-Ac PEG-bis-PT

13.1 14.0 14.9

1 4.1 9.9

0.07 0.27 0.66

6 strong 24 strong 59 strong

PEGb HO-PEG-CNb PEG-bis-CNb

17.7 19.1 20.9

1 2.2 0.9

0.25 0.53 0.22

25 no 52 weak 22 weak

PEG-bis-Acb CN-PEG-Acb PEG-bis-Acb

20.9 22.8 23.9

1 2.5 1.2

0.21 0.54 0.25

20 medium 50 medium 23 weak

PEG HO-PEG-EtAt PEG-bis-EtAt

10.6 12.9 13.2

N/A

0 0.41 0.59


PEG HO-PEG-COOH PEG-bis-COOH

10.6 8.2 7.1

1 7.1 12.3

0.05 0.35 0.60


PEG-bis-COOHb HO-PEG-COOHb COOH-PEG-Acb PEGb HO-PEG-Acb PEG-bis-Acb

11.5 13.7 15.2 17.7 20.6 23.2

5.1 0 14.4 1 1.7 1.6

0.22 0 0.60 0.04 0.07 0.06

11 0 30 2 4 3

PEG-bis-Glu

8.0

1

UV signal

100 no

weak weak medium no medium medium

1

1

98 weak

HO-PEG-Glu Glu-PEG-Ac HO-PEG-AcGlu Ac-PEG-AcGlu

10.5 11.8 12.7 14.1

1 0.7 0.4 0.1

0.45 0.33 0.17 0.05

40 30 15 5

PEG-bis-TES

13.1

1

1

93 no

PEG-bis-NSuGlu

13.9

1

1

92 medium

PEG-bis-NSuGlu NSuGlu-PEG-TrpGlu PEG-bis-TrpGlu

13.9 10.2 8.5

1 8.4 5.7

0.07 0.55 0.38

6 medium 50 strong 35 strong

PEG-bis-NSuGlu NSuGlu-PEG-GGGGlu PEG-bis-GGGGlu

13.6 9.2 7.0

1 1.4 0.3

0.37 0.53 0.10

34 medium 49 medium 9 weak

PEG-bis-NSuGlu NSuGlu-PEG-RGDGlu PEG-bis-RGDGlu

13.6 9.4 7.6

1 0.8 0.1

0.53 0.41 0.05

49 weak 38 weak 5 weak

weak medium medium medium

b 60-min run. a All values expressed as mean of chromatograms of 3 independent synthesis (n ) 3). All peaks were normalized with the signal from the ELSD detector of the internal PEG 2K, if applicable, to calculate normalized peak area and conversion factor (%, Ni/ΣN), where Ni is normalized peak area for each peak and N is the sum of all of the Ni. Signals from various HPLC detectors were utilized to identify the chemical structure of each individual peak of a given chromatogram.

PEG-Tos, and PEG-bis-Tos was obtained31 (Figure 2, Table 1). PEG-bis-Tos was obtained with approximately 99% yield when the molar ratio of PEG:pToSC:TEA was 1:5:5. To synthesize Tos-PEG-Ac, 1 eq. mol mixture of PEG, HO-PEG-Tos, and PEG-bis-Tos was dissolved in dry THF followed by the addition of 4 eq. mol AC and 5 eq. mol TEA, stirred at room temperature for 4 h, followed by the drying procedure and a mixture of PEG-bis-Tos,

Tos-PEG-Ac, and PEG-bis-Ac was obtained (Figure 2, Table 1). To synthesize PT-PEG-Ac, 1 eq. mol mixture of PEG-bis-Tos, Tos-PEG-Ac, and PEG-bis-Ac was dissolved in toluene with 2 eq. mol potassium phthalimide, stirred under reflux at 50 °C for 20 h, cooled, followed by the drying procedure and a mixture of PEG-bis-PT, PTPEG-Ac, and PEG-bis-Ac was obtained24 (Figure 2, Table 1). CN-PEG-Ac. To synthesize HO-PEG-CN, 1 eq. mol PEG (2K Da) was dissolved in dry MC, added to 1.5 eq. mol sodium powder under Ar followed by the addition of 1 eq. mol acrylonitrile, stirred under Ar at room temperature for 1 day, filtered, concentrated under vacuum with Rotavapor (BUCCI R-114, Switcherland), precipitated in cold hexane, filtered, and dried in a vacuum oven, and a mixture of PEG-bis-CN, HO-PEG-CN, and PEG was obtained32,33 (Figure 2, Table 1). To synthesize CN-PEG-Ac, 1 eq. mol mixture of PEG-bis-CN, HO-PEG-CN, and PEG was dissolved in dry THF, followed by the addition of 3 eq. mol AC and 4 eq. mol TEA, stirred at room temperature for 1.5 h, followed by the drying procedure and a mixture of PEGbis-CN, CN-PEG-Ac, and PEG-bis-Ac was obtained (Figure 2, Table 1). COOH-PEG-Ac. To synthesize HO-PEG-EtAt, 4 eq. mol sodium and 4 eq. mol naphthalene was dissolved in dry THF, stirred under Ar at room temperature for 1 h. A total of 1 eq. mol PEG (2K Da) was dissolved in dry THF followed by the addition of sodium/naphthalene solution dropwise, stirred under Ar at room temperature for 1 h followed by the addition of 4 eq. mol ethyl bromoacetate, stirred under Ar at room temperature for 4 h, followed by the drying procedure, and a mixture of PEG, HO-PEGEtAt and PEG-bis-EtAt was obtained34 (Figure 2, Figure 3, Table 1). To synthesize HO-PEG-COOH, 1 eq. mol mixture of PEG, HO-PEG-EtAt, and PEG-bis-EtAt was dissolved in DI water followed by the addition of 4 eq. mol sodium hydroxide, stirred for 1 h, and extracted by MC twice. The pH of the aqueous phase solution was adjusted to 3 by 0.1 M HCl. The solution was extracted again by MC three times. The organic phase solution was evaporated using Rotavapor, and a mixture of PEG, HO-PEG-COOH, and PEG-bis-COOH was obtained23 (Figure 2, Figure 3, Table 1). To synthesize COOH-PEG-Ac, 1 eq. mol mixture of PEG, HO-PEG-COOH and PEG-bis-COOH was dissolved in dry THF followed by the addition of 4 eq. mol AC and 5 eq. mol TEA. The solution was stirred at room temperature for 1.5 h, followed by the drying procedure, and a mixture of PEG, PEG-bis-COOH, HO-PEG-COOH, COOH-PEG-Ac, HO-PEG-Ac, and PEG-bis-Ac was obtained (Figure 2, Figure 3, Table 1). Glu-PEG-Ac. To synthesize HO-PEG-Glu, 1 eq. mol PEG (2K Da) was dissolved in dry THF followed by the addition of 3 eq. mol glutaric anhydride and 0.03 eq. mol glutaric acid, stirred under reflux at 40 °C for 1 day, followed by the drying procedure, and HO-PEG-Glu was obtained35,36 (Figure 2, Table 1). PEG-bis-Glu was obtained with 98% yield when the molar ratio of PEG:glutaric anhydride was 1:4 under reflux at 70 °C. To synthesize GluPEG-Ac, 1 eq. mol HO-PEG-Glu was dissolved in dry

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Figure 4. Formation of PEG-peptide conjugate modified gelatin. PEG was first modified to PEG-bis-Glu having carboxyl groups at both terminals. Carboxyl groups were then activated by N-hydroxysuccinimide forming PEG-bis-NSuGlu. One activated terminal was conjugated to peptide forming PEG-peptide conjugate, and the other activated terminal was conjugated to lysyl groups on gelatin forming the PEG-peptide gelatin conjugate. Table 2. 13C NMR Chemical Shifts (ppm) for MPEG-Ac, PT-PEG-Ac, CN-PEG-Ac, COOH-PEG-Ac, and Glu-PEG-Ac Synthesized from PEG

THF followed by the addition of 2 eq. mol AC and 2 eq. mol TEA. The solution was stirred at room temperature for 3 h, followed by the drying procedure, and a mixture of HOPEG-Glu, Glu-PEG-Ac, HO-PEG-AcGlu, and AcPEG-AcGlu was obtained (Figure 2, Table 1). PEG-bis-TES. A total of 1 eq. mol PEG-bis-acrylate (2K Da) was dissolved in dry THF followed by the addition of 40 eq. mol triethoxysilane and a grain of chloroplatinic acid, stirred under reflux at 50 °C for 3 days, cooled, followed by the drying procedure and PEG-bis-TES was obtained37-39 (Table 1). Gelatin Conjugated with PEGylated Peptide. Synthesis of PEG-peptide conjugates and PEG-peptide modified gelatin is illustrated in Figure 4. All intermediates and final PEG-peptide conjugates were characterized and verified by HPLC (Table 1) and 1H NMR (Table 3). Glu-PEG-

GGGGlu and Glu-PEG-RGDGlu were verified by MS spectroscopy (IONSPEC HIRESMALDI FT-Mass Spectrometer, Agilant). PEG-bis-NSuGlu. A total of 1 eq. mol PEG-bis-Glu synthesized as described above was dissolved in dry THF followed by the addition of 4 eq. mol N-hydroxysuccinimide. A total of 4 eq. mol N,N′-dicyclohexylcarbodiimide was dissolved in a small amount of THF, added to the PEG solution dropwise, stirred at room temperature for 4 h, and filtered. The filtrate was precipitated in cold hexane, dried in a vacuum oven for 4 days, and PEG-bis-NSuGlu resulted as a white and sticky solid and was kept at 4 °C under Ar40-43 (Figure 4, Table 1). NSuGlu-PEG-TrpGlu and Glu-PEG-TrpGlu, NSuGlu-PEG-GGGGlu and Glu-PEG-GGGGlu, NSuGluPEG- RGDGlu and Glu-PEG-RGDGlu. A total of 1 eq.



Table 3. 1H NMR Chemical Shifts (ppm) for PEG, PEG-bis-Glu, PEG-bis-NSuGlu, and PEG-bis-TrpGlu Synthesized from PEG 2K

Table 4. Percent Modification of Gelatin Lysyl Groups by PEG-Peptide Conjugates types of gelatin gelatin only gelatin-Glu-PEG-Glu gelatin-Glu-PEG-GGGGlu gelatin-Glu-PEG-RGDGlu gelatin-Glu-PEG-TrpGlu

% modification (n ) 3) 0 94∼98 68∼74 94∼98 71∼77

mol PEG-bis-NSuGlu was dissolved in DMF followed by the addition of 2 eq. mol Trp, 2 eq. mol GGG (Bachem) dissolved in 0.1 M MES (Sigma), or 3 eq. mol RGD (Bachem) dissolved in 0.1 M MES, respectively, stirred at room temperature under Ar for 1, 4, or 7 day, respectivley. A mixture of (a) PEG-bis-NSuGlu, NSuGlu-PEG-TrpGlu, and PEG-bis-TrpGlu; (b) PEG-bis-NSuGlu, NSuGluPEG-GGGGlu, and PEG-bis-GGGGlu; or (c) PEG-bisNSuGlu, NSuGlu-PEG-RGDGlu, and PEG-bis-RGDGlu resulted, respectively. The mixture was either (i) diluted by DI water, dialyzed against DI water (MW 1000 Da cutoff), and lyophilized to obtain a mixture of (a) PEG-bis-Glu, Glu-PEG-TrpGlu, and PEG-bis-TrpGlu; (b) PEG-bisGlu, Glu-PEG-GGGGlu, and PEG-bis-GGGGlu; or (c) PEG-bis-Glu, Glu-PEG-RGDGlu, and PEG-bis-RGDGlu in powder form, respectively, or (ii) added with 1% gelatin water solution, adjusted to pH 8, stirred for 1 h, dialyzed against DI water (cutoff MW 60 000∼80 000 Da) for overnight at 55 °C, and lyophilized to obtain the Glu-PEGTrpGlu, Glu-PEG-GGGGlu, or Glu-PEG-RGDGlu modified gelatin, respectively (Figure 4). The percentage of gelatin lysyl groups modified by PEG-peptide conjugates was characterized by GPC and determined using an established TNBS method in triplicates27-29, 44(Table 4). Synthesis and Characterization of Networks of Acrylic Acid-co-TMPTA-co-PEG Derivatives. The acrylated PEG derivatives were conjugated into a previously developed network.45-48 Briefly, PEG derivatives dissolved in TMPTA with five concentrations (0.2, 0.4, 0.8, 1.25, and 2.5 g/ml) were photopolymerized with TMPTA and acrylic acid dissolved in TMPTA (60 µL/ml) in the presence of a photo initiator, 2,2-dimethoxy-2-phenyl-actone dissolved in TMP-

TA (9 mg/mL). The networks were placed in DMF to leach out unreacted materials. After 5 h, the networks were transferred to water for equilibration under sterile condition for at least 12 h followed by contact angle measurement and cell culture study. The hydrophilicity of the resulting networks was quantified with an underwater air bubble captive system.44 Based on our system, the higher the angle, the lower the hydrophilicity of the substrate. A total of 75 000 human neonatal dermal fibroblasts (Clonetics) in 1 mL fibroblast basal medium supplemented with basic human fibroblast growth factor, insulin, and 5% fetal bovine serum (Clonetics) were cultured with the networks as well as TCPS films. After 2, 24, or 48 h, networks were fixed with Wright stain (Sigma), and the adherent cell morphology and density were qualified using a computer-assisted video analysis system (MetaMorph V.4.1) coupled to an inverted light microscope (PHOTOMETRICS, SenSys). All experimental results were expressed in mean ( standard deviation (S.D.). Each sample was independently repeated 3 times (n ) 3). Comparative analyses were performed with Statview 4.5 using analysis of variance and Fisher’s protected least significant difference test at 95% confidence level (p < 0.05). Results and Discussion PEG-bis-Ac. PEG acrylate was a starting material for synthesizing other PEG derivatives. The acrylate group was converted to triethoxysilane through a hydrosilation procedure30-32 and to aldehyde by a procedure adapted from Wirth.49 PEG acrylate also functioned as a photopolymerizable component. MPEG-Ac was used to form a copolymer with acrylic acid to create an adhesive film for the buccal delivery of butorphanol tartrate, an opinoid analgesics.50 PEG-bis-Ac formed an interpenetrating polymer network with alginate for encapsulation of islets of Langerhans.51 PEG acrylate was used to form hydrogels in numerous applications to deliver bovine serum albumin and horseradish peroxidase to increase the efficacy of implanted biosensors and other devices,52 5-fluorouracil in the treatment of superficial basal cell epithelium and multiple actinic keratoses,53 and nitric

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oxide for the prevention of thrombosis and restenosis following procedures such as balloon angioplasty.54 Using excess amount of TEA over AC was important in the optimal synthesis of PEG acrylate, because excess AC would hydrolyze in the presence of moisture during workup. The acid thus produced could form a complex with the unsaturated bond of PEG acrylate. Using an excess amount of TEA could not only move the equilibrium to product side but also prevent the formation of complex between double bond and acid. PEG (2K Da) eluted off around 10.6 min in the HPLC chromatogram. From the structures of the three components of the mixture, PEG-bis-Ac was more hydrophobic than HO-PEG-Ac, which in turn was more hydrophobic than PEG. Hence, peaks eluted off at a time around 11.8 and 13.1 min represented HO-PEG-Ac and PEGbis-Ac, respectively. UV signals were observed on the two peaks that were caused by acryloyl group(s) possessing double bond(s) (Table 1). The presence of double bonds in the products was verified by 1H and 13C NMR (Table 2). PT-PEG-Ac. Substituting hydrogen in the hydroxyl group with the p-toluenesulfate group, the resulting tosyl group became a better leaving group that could undergo substitution reaction and produce PEG derivatives more easily. Tos-PEG-Ac was an intermediate in the synthesis of other acrylated PEG derivatives, such as PT-PEG-Ac and PEG amines.55-56 The PEG-Tos conjugate was used in the formation with oxazoline in a process demonstrated to be a living polymerization.57 The phthalimide terminal group on the PEG-PT conjugate can be converted to amine group by hydrozinolysis forming PEG-NH258,59 which in turn can be converted to other reagents such as a PEG-bound carbodiimide as a catalyst for converting carboxylic acids to anhydrides.58 PEG-NH2 was also converted to a PEG polymer with a terminal 5-methyl-5-(4′-methylphenyl)hydantoin group that could be used as a surface-anchored biocidal film.60 From the structures of the three components of the mixture, PEG-bis-Tos was more hydrophobic than HO-PEG-Tos, which in turn was more hydrophobic than PEG. Peaks eluted off around 13.5 and 16.4 min represented HO-PEG-Tos and PEG-bis-Tos, respectively. UV signals were observed on peaks that were caused by tosyl groups (Table 1). Comparing the elution times of HO-PEG-Tos (13.5 min) and HO-PEG-Ac (11.8 min) on the HPLC chromatogram, tosyl groups were more hydrophobic than acrylate groups. So, the hydrophobicity of the compounds were PEG-bisTos > Tos-PEG-Ac > PEG-bis-Ac. Hence, the peaks eluted off at 14.8 min and 13.1 denoted Tos-PEG-Ac and PEG-bis-Ac respectively (Table 1). After phthalimide group substituted tosyl group the two peaks denoting PEG-bis-Tos (16.4 min) and Tos-PEG-Ac (14.9 min) shifted to new positions, 14.0 and 14.8 min, denoting PEG-bis-PT and PT-PEG-Ac respectively (Table 1). Both double bond and phthalimide groups in the products were verified by 1H and 13C NMR (Table 2). CN-PEG-Ac. PEG-CN can also be converted to the PEG-NH2 conjugate by reacting with LiALH4 followed by hydrolysis.61 From the structure of the three components in the mixture, PEG-bis-CN was more hydrophobic than HO-

Li and Kao

Figure 5. HPLC chromatograms of (a) ELSD signals and (b) UV signals at 254 nm for PEG and various acrylated PEG derivatives.

PEG-CN which in turn was more hydrophobic than PEG. Because hydrophilicities of CN and OH were very close, 60-min HPLC runs were used for a better separation. The elution time for PEG (2K Da) on a 60-min run was 17.7 min. HO-PEG-CN and PEG-bis-CN eluted off around 19.2 and 21.0 min, respectively (Table 1). The presence of CN in the products was verified by 1H and 13C NMR (Table 2). Similarly, the peaks eluting off around 21.9 min and 22.7 represented CN-PEG-Ac and PEG-bis-Ac, respectively, after acrylation (Figure 5, Table 1). COOH-PEG-Ac. There was no oxidant agent strong enough to directly oxidize the OH group on PEG to COOH, because strong oxidants would cleave the backbone of PEG as demonstrated by Johansson using permangamate (KMnO4).24,62 The approach we chose was to conjugate to the terminal OH group a small molecule that was converted to carboxylic acid after the conjugation. Carboxylated PEG has a wide range of applications, such as conjugation directly to 5-fluorouracil for the treatment of lymphocytic leukemia63 and to doxorubicin for the increase of the antitumor activity.64 Paclitaxel (Taxol), another anticancer drug, was also modified in the same way with increased water solubility and decreased cytotoxicity.65 In the mixture of PEG-bis-COOH and HO-PEGCOOH, both hydroxyl and carboxyl groups could be acrylated. Considering all of the possibilities of acrylation reactions, a mixture of products would be obtained. Comparing the chromatograms of acrylated carboxyl PEG with PEG, acrylated PEG and HO-PEG-COOH mixture, the elution times for each compounds were as follows (based on 60min runs of HPLC): 11.5 min (product 36), 13.7 min (product 18), 15.2 min (product 28), 18.2 min (PEG), 20.6 min (product 13), 23.2 min (product 30) (Figure 3, Figure 5 and Table 1). The presence of COOH in the products was verified by 1H and 13C NMR (Table 2). Glu-PEG-Ac. PEG glutarate is another PEG derivative with terminal COOH group. This was an easier and less


Figure 6. HPLC chromatograms of ELSD signals of PEG-peptide conjugates and intermediates.

complicated method to introduce terminal COOH group(s) to PEG with higher yield compared to PEG carboxylate. The linkage between PEG and glutarate was an ester bond spaced by three methylene groups with the terminal COOH. The inherent structure difference determined the differences in the physical, chemical, and biological properties of COOHPEG-Ac and Glu-PEG-Ac. Both PEG derivatives can be further conjugated to amino acids, peptides or other biological effective molecules. The ester bond in the PEG-glutarate conjugate is susceptible to hydrolytic cleavage,66 which could be advantageous in an application where the attached protein should detach slowly from PEG, thus releasing free protein or lightly PEGylated protein into solution. If the release rate is controlled, the protein activity level in the serum could be maintained even as the total amount of protein in the serum decreases by clearance.67 Vestling and co-workers have taken advantage of this hydrolytic instability to tag lysines with succinate groups and thus determine the position of PEGylation by mass spectrometry.68 The formation of Glu-PEG-Ac was confirmed by HPLC (Figure 5, Table 1) as well as 1H and 13C NMR (Table 2). The formation of PEG-bis-Glu was confirmed by HPLC (Figure 6, Table 1) as well as 1H and 13C NMR (Table 3). 13C NMR chemical shifts for MPEG-Ac, PT-PEG-Ac, CN-PEG-Ac, COOH-PEG-Ac, and Glu-PEG-Ac synthesized from PEG 2K were listed in Table 2. For all samples, the methyl stretch carbon and the β carbon of PEG chains were observed around 68 to 72 ppm, whereas the R carbon shift was highly dependent on the terminal group (X). The assigned carbon showed signals at the corresponding chemical shift. The final acrylated products had a general chemical structure of X-CH2CH2O(CH2CH2O)nCH2CH2OCOCHCH2, where X was -OCH3, -PT, -CN, -OCH2COOH, or -OCO(CH2)3COOH. We observed three unique chemical shifts that corresponded to the three carbons of the acrylate group. Specifically, the chemical shifts for -COOstretch and -CHCH2 stretch were observed at 165.3 to 170.6 and 128.0 to 130.9 ppm, respectively. In addition, appropriate chemical shifts were observed for the assigned carbon for each terminal group (X). PEG-bis-TES. Siloxane polymers have many useful characteristics such as high chain flexibility, low-temperature elasticity, transparency, high oxidative and thermal stability, and excellent resistance to radiation. We explored PEGbis-TES synthesis in which PEG linked to triethoxysilane through hydrosilation reaction. PEG-bis-TES could further cross-link with tetraethoxysilane in the formation of novel


biomaterials. Siloxane polymer with PEG side chain was found to have higher conductivity than siloxane itself.44 We aim to explore the application of PEG modified siloxane in the biomedical areas. The formation of PEG-bis-TES was monitored by HPLC. The hydrophilicity of triethoxysilane and acrylate were very close. As the reaction continued, the UV signal around 13.1 min diminished and disappeared after 3 days (Table 1). Because the retention time of PEG-bis-TES was close to that of PEG-bis-Ac and because PEG-bis-TES was not associated with the UV signal, the diminishing UV signal indicated the formation of PEG-bis-TES. PEG-bis-NSuGlu. N-Hydroxysuccinimide was used to activate the carboxyl group to further conjugate with the N terminus of peptides or amine groups on proteins. This approach is used extensively in modifying enzymes to increase enzyme stability, decrease thermal sensitivity, and enhance process versatility.69 Lipase from Candida cylindracea, after modified with PEG-NSu, was soluble in organic solvents such as benzene and catalyzed an ester exchange to synthesize eicosapentaenoyl phosphatidylcholines from dipalmitoylphosphatidylcholine and eicosapentaenoic acid.70 Doxorubicin was also modified by PEG-NSu.71 Superoxide dismutase modified with PEG-NSu was effective in lowering the elimination rate of superoxide dismutase from the blood circulation without any change in the distribution pattern of organs other than the kidney.72 Recombinant methioninase, an effective antitumor agent, was modified by PEG-NSu resulting in lowered immune reactions and prolonged serum half-life.73 PEG-NSu was also used to modify monoclonal antibodies N12 and L26 specific to the ErbB2 oncoprotein to enhance penetration into growing solid tumors and extend antitumor effects.74 Arginine deiminase modified with PEG-NSu showed 20-folds increase in circulating halflife and enhanced tumor inhibition of human hepatomas and human melanomas.75 In our study, PEG-bis-NSuGlu was very sensitive to water and hydrolyzed readily. Storage at 4 °C under Ar prevented the hydrolysis. The formation of PEG-bis-NSuGlu was confirmed by HPLC (Figure 6, Table 1) as well as 1H and 13C NMR (Table 3). 1H NMR chemical shifts for PEG, PEG-bis-Glu, PEGbis-NSuGlu, and PEG-bis-TrpGlu synthesized from PEG 2K were listed in Table 3. For all samples, the methyl stretch proton and β proton of PEG chains were observed around 3.6 to 3.8 ppm, whereas the R proton shift was highly dependent on the terminal group (X). Final products had a general chemical structure of X-CH2CH2O(CH2CH2O)nCH2CH2-X, where X was - OH, -Glu, -NSuGlu, or -TrpGlu. The appropriate chemical shifts were observed for the assigned proton for each terminal group (X). Glu-PEG-TrpGlu, Glu-PEG-GGGGlu, and GluPEG-RGDGlu. Our previous investigation revealed that the extracellular protein fibronectin derived oligopeptide RGD and PHSRN sequences played an important role in modulating human leukocyte behavior (i.e., adhesion, protein release, fusion to form foreign body giant cells) in vitro and in vivo.45,76-77 Leukocytes are critical in host inflammation and immune response, which directly influence drug delivery device efficacy and biomaterial biocompatibility. Hence, by

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Li and Kao Table 5. Surface Hydrophilicity of TMPTA Networks Containing XPEG-Aca XPEG-Ac type

Figure 7. GPC chromatograms of PEG-peptide conjugate modified gelatin.

conjugating these bioactive peptides to biopolymers such as gelatin using PEG as a linker, we endeavor to create novel biomedical hydrogels with favorable host response. Experiment conditions for conjugating activated PEG with peptide varied with the peptide identity. For example, PEG could conjugate with tryptophan in DMF whereas conjugation with GGG and RGD could not occur in DMF. A combination of solvent DMF and 0.1 M MES had to be used. Furthermore, the volume ratio of DMF and MES had to be 2:1 or higher so that the hydrolysis of activated PEG was not significant during the conjugation. Formation of PEG peptide conjugates was characterized by HPLC (Figure 6, Table 1). For Glu-PEG-RGDGlu and Glu-PEG-GGGGlu conjugates, the products were a mixture of conjugates and hydrolysis products. Peaks of the conjugates were composed of several peaks close together. The formation of Glu-PEGTrpGlu was verified by 1H and 13C NMR (Table 3), whereas the formation of Glu-PEG-GGGGlu and Glu-PEGRGDGlu was verified by MS spectrometry. The predicted Mp of PEG-bis-GGGGlu 2K was 2570 Da and the experiment data was 2593 Da. The predicted Mp of Glu-PEGRGDGlu 2K was 2742 Da and the experiment data was 2743 Da. Characterization of PEGylated-Peptide Modified Gelatin. Gelatin is a water-soluble functional protein obtained from acidic or alkaline hydrolysis of collagen,78-79 the safety and biocompatibility of which have been established and the material is approved in the United States for use in food and pharmaceutical products.80-81 After modified by PEGbis-Glu, Glu-PEG-TrpGlu, Glu-PEG-GGGGlu, and GluPEG-RGDGlu conjugates, the GPC elution time for the modified gelatin decreased indicating increased molecular weight of gelatin (Figure 7). The percentage modification of lysyl groups on gelatin was determined with TNBS method (Table 4). The modification by the PEG-peptide conjugates was up to 96%. The percentage of modification was controlled by varying the molar ratio of gelatin versus PEG-peptide conjugates. Characterization of Acrylic Acid-co-TMPTA-co-PEG Derivative Networks. The hydrophilicity of networks varied with three factors: concentration, molecular weight, and terminal functional groups of PEG derivatives (Table 5). For networks containing CN-PEG-Ac 2K and 5K, PT-PEGAc 5K, or COOH-PEG-Ac 5K, the hydrophilicity increased to a constant value with increasing PEG concentration. For networks containing MPEG-Ac 5K, PT-PEG-

2K (Da) MPEG-Ac PT-PEG-Ac CN-PEG-Ac COOH-PEG-Ac Glu-PEG-Ac 5K (Da) MPEG-Ac PT-PEG-Ac CN-PEG-Ac COOH-PEG-Ac Glu-PEG-Ac

XPEG-Ac concentration in the network formulation (g/mL) 0.2

0.4

0.8

1.25

2.5

37 ( 8 23 ( 4b 46 ( 4 44 ( 3 34 ( 4

34 ( 6 45 ( 4b,c 32 ( 2c 42 ( 6 47 ( 3

37 ( 4 40 ( 6c 36 ( 5c 38 ( 6c 44 ( 4

34 ( 2 38 ( 2c 37 ( 2c 46 ( 7b 48 ( 2

29 ( 5 41 ( 2b,c 39 ( 2b,c 43 ( 4b 45 ( 3

41 ( 6 46 ( 4d 46 ( 5 51 ( 3b,d 43 ( 3

45 ( 6d 46 ( 1 32 ( 2b,c 42 ( 2c 49 ( 4

51 ( 7d 51 ( 7 36 ( 7b,c 39 ( 1b,c 44 ( 5

42 ( 5d 40 ( 3c 37 ( 1b,c 43 ( 4c 38 ( 5

47 ( 1d 39 ( 2b,c 39 ( 3b,c 44 ( 4c 40 ( 3

a All values are expressed in degree (°, mean ( S.D., n ) 3). b The value that is different (p < 0.05) vs respective value of MPEG-Ac given the same XPEG-Ac concentration and molecular weight. c The value that is different (p < 0.05) vs respective value at 0.2 g/mL. d The value that is different (p < 0.05) vs respective value of 2K Da molecular weight given the same XPEG-Ac concentration and type.

Ac 2K, or Glu-PEG-Ac 2K, the hydrophilicity decreased to a constant value with increasing PEG concentration. However, for networks containing MPEG-Ac 2K, the hydrophilicity did not change with increasing PEG concentration. For networks containing COOH-PEG-Ac 2K or GluPEG-Ac 5K, the hydrophilicity increased to a maximum at a concentration between 0.4 and 0.8 g/ml and dropped down with increasing PEG concentration. The density of adherent fibroblasts dropped sharply on the networks containing PEG derivatives at a concentration between 0.8 and 1.25 g/mL (Table 6). For networks containing PEG derivatives with the same functional terminal group and concentration, adherent cell density was not affected by molecular weight. For networks containing different types of PEG derivatives with the same molecular weight and concentration, the adherent cell density varied little with different functional groups. The adherent cell density was lower on all PEG containing TMPTA networks than that on TCPS controls. Higher PEG concentration resulted in lower adherent cell density. This phenomenon can be attributed to PEG’s unique properties. Because of the ether linkage, PEG is hydrophilic and highly solvated in aqueous solutions.82 Surface bound PEG has high mobility that is entropically favored. Any approaching protein or macromolecule to adsorb onto the surface would have to constrain this motion, thereby decreasing the entropy of the system.82 Because protein adsorption is a major determinant for cell adhesion, reduced protein adsorption due to increased PEG concentration would definitely result in reduced adherent cell density. All adherent fibroblasts showed polar cell body morphology after 24 h culture (Figure 8). There was no correlation between network surface hydrophilicity and adherent cell density. Some research groups also have the same finding.83-89 However, the other research groups found that higher hydrophilicity correlated to higher cell adhesion.90-96 We argue that there is actually no direct relationship between surface hydrophilicity and adherent cell density. Cellular



Table 6. Adherent Human Dermal Fibroblast Density on TMPTA Networks Containing XPEG-Aca culture time 2h XPEG-Ac type

0.2

0.4

0.8

2K (Da) MPEG-Ac PT-PEG-Ac CN-PEG-Ac COOH-PEG-Ac Glu-PEG-Ac TCPS

3(2 2(1 5(3 2(2 2(1 4(1

3(2 2(1 2(1 1(0 1(1

0 1(1 1(1 1(1 1(1

a

24 h

48 h

XPEG-Ac concentration in the network formulation (g/mL) 1.25 2.5 0.2 0.4 0.8 1.25 2.5 0.2 0 1(0 0 0 0

0 0 0 0 0

5(2 1(0 3(2 3(1 3(2 6(0

2(1 3(2 4(2 2(1 2( 0

0 3(2 1(1 6(4 1(1

0 0 1(1 0(0 0

0 0 0 0 0

3(1 3(1 5(3 3(2 2(2 5(1

0.4

0.8

1.25

2.5

4(2 2(1 4(3 1(1 2(1

1(1 3(3 3(1 1(1 1(1

0 0(0 5(2 1(1 0

0 0 0 0 0

All values are expressed in ×100 cells/mm2 (rounded-off for clarity, mean ( S.D., n ) 3).

Figure 8. Representative adherent human dermal fibroblast density and morphology on networks. (a) Networks containing COOH-PEG 2K with different concentration at 2 h after cell culture (40×). (b) Networks containing CN-PEG 2K at concentration of 0.2 g/mL at different time periods after cell culture (40×).

function mediated by adsorbed proteins and substrate chemicophysical properties is a complex and dynamic interrelationship. From our experimental design, the contribution of the PEG terminal moiety, COOH groups of the acrylic acid component, surface morphology, and topography in mediating the extent of cell adhesion should all be considered in conjunction with surface hydrophilicity. Conclusions We established, optimized, and simplified the synthesis of a library of PEG derivatives that were further conjugated with biologically active molecules and biomaterial substrates. PEG-peptide modified gelatin was demonstrated in the further formation of biomedical gelatin-based materials. The network hydrophilicity and the number of adhered fibroblasts

were primarily dependent on the PEG concentration with the molecular weight and terminal functional groups of PEG derivatives being less important. List of Abbreviations AC, acryloyl chloride AC-PEG-AcGlu, R-acryloly-ω-acryloylglutarate-PEG CN-PEG-Ac, R-cyanoethyl-ω-acryloyl-PEG COOH-PEG-Ac, R-carboxy-ω-acryloyl-PEG ELSD, evaporative light scattering detector Glu-PEG-Ac, R-glutarate-ω-acryloyl-PEG Glu-PEG-GGGGlu, R-glutarate-ω-GGGglutarate-PEG Glu-PEG-RGDGlu, R-glutarate-ω-RGDglutarate-PEG Glu-PEG-TrpGlu, R-glutarate-ω-tryptophanglutaratePEG GPC, gel permeation chromatogram HO-PEG-Ac, R-hydroxy-ω-acryloyl-PEG

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HO-PEG-AcGlu, R-hydroxy-ω-acryloylglutarate-PEG HO-PEG-CN, R-hydroxy-ω-cyanoethyl-PEG HO-PEG-COOH, R-hydroxy-ω-carboxy-PEG HO-PEG-EtAt, R-hydroxy-ω-ethyl acetate-PEG HO-PEG-Glu, R-hydroxy-ω-glutarate-PEG HO-PEG-Tos, R-hydroxy-ω-tosyl-PEG HPLC, high performance liquid chromatogram MC, methylene chloride MES, 2-(N-Morpholino) ethanesulfonic acid Mp, peak molecular weight MPEG, poly(ethylene glycol) monomethyl ether MPEG-Ac, MPEG-acrylate NSuGlu-PEG-GGGGlu, R-N-succinimidylglutarate-ωGGGglutarate-PEG NSuGlu-PEG-RGDGlu, R-N-succinimidylglutarate-ωRGDglutarate-PEG NSuGlu-PEG-TrpGlu, R-N-succinimidylglutarate-ω-tryptophanglutarate-PEG OD, optic density PEG, poly(ethylene glycol) PEG-bis-EtAt, PEG-bis-ethyl acetate PEG-bis-Ac, PEG-bis-acrylate PEG-bis-AcGlu, PEG-bis-acryloylglutarate PEG-bis-CN, PEG-bis-cyanoethylate PEG-bis-COOH, PEG-bis-carboxylate PEG-bis-Glu, PEG-bis-glutarate PEG-bis-NSuGlu, PEG-bis-N-succinimidyl glutarate PEG-bis-PT, PEG-bis-phthalimide PEG-bis-TES, PEG-bis-triethoxysilane PEG-bis-Tos, PEG-bis-tosylate PEG-bis-TrpGlu, PEG-bis-tryptophan glutarate PEG-bis-GGGGlu, PEG-bis-GGG glutarate PEG-bis-RGDGlu, PEG-bis-RGD glutarate pToSC, p-toluenesulfonyl chloride PT-PEG-Ac, R-phthalimide-ω-acryloyl-PEG RI, refractive index TCPS, tissue culture polystyrene TEA, triethylamine TMPTA, trimethylolpropane triacrylate TNBS, trinitrobenzenesulfonic acid Tos-PEG-Ac, R-tosyl-ω-acryloyl-PEG

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Synthesis of Polyethylene Glycol (PEG) Derivatives and PEGylated

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