Conjugate Addition Reactions Combined with Free-Radical Cross

Biomacromolecules 2001, 2, 430-441

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Conjugate Addition Reactions Combined with Free-Radical Cross-Linking for the Design of Materials for Tissue Engineering Donald L. Elbert and Jeffrey A. Hubbell* Department of Materials and Institute for Biomedical Engineering, ETH-Zurich and University of Zurich, Moussonstrasse 18, CH-8044 Zurich, Switzerland Received October 20, 2000; Revised Manuscript Received January 16, 2001

PEG-diacrylamide was synthesized and utilized to make materials for tissue engineering. Acrylamide groups readily react with thiol groups, and peptides containing a single thiol group were coupled to the PEGdiacrylamide in aqueous solution at room temperature in about 2 h. If only a portion of the acrylamide groups were targeted for reaction with peptide, sufficient amounts of PEG-diacrylamide remained to be polymerized by free-radical mechanisms via photoinitiation. The photopolymerization step can be performed in contact with cells, providing a means to produce bioactive scaffolds for tissue engineering. The photopolymerization conditions and precursor composition greatly affect the stiffness of the materials, which subsequently affected cell spreading. The kinetics and extent of cell spreading on the bioactive materials were measured and compared to cell spreading on tissue culture polystyrene. Although the PEG materials resist protein adsorption, the experiments suggest that the cells can secrete extracellular matrix that can adhere to the gels. Introduction A new method for attaching biological molecules to hydrogels is described, using a chemical scheme that is rapid, requires no purification steps, and can be performed in contact with proteins, cells, and tissues. This is accomplished using a conjugate addition reaction, also called a Michaeltype reaction, which is very well suited for the modification of polymers that will subsequently be used for the formation of a hydrogel material via a free-radical cross-linking reaction. Such free-radical cross-linkable polymers are desirable in tissue engineering because they can be polymerized in direct contact with cells and tissues, either in solution with homogeneous initiation,1,2 or in very thin layers on the surface of a tissue by interfacial photoinitiation.3,4 These materials may be useful to promote the regrowth of endothelial cells onto the surface of an artery after a balloon angioplasty procedure or the regrowth of mesothelial cells on the surface of an internal organ after abdominopelvic surgery.5, 6 Researchers in the field of tissue engineering seek to provide signals to biological systems that prompt certain cells to regenerate tissues. Although soluble signals such as growth factors are of vital importance for the regeneration of tissue, it is likely that some form of solid scaffold will also be required to control tissue regrowth. The solid scaffold can profoundly influence the phenotype of cells that regrow through or on top of the material, yet it is very difficult to control the surface properties of most artificial materials used for tissue engineering. This is due to nonspecific protein * To whom correspondence should be addressed. E-mail: hubbell@ biomed.mat.ethz.ch.

adsorption onto the surfaces of the materials, which usually leads directly to the emergence of a wound healing phenotype for cells in contact with the material. Additionally, the presence of nonspecifically adsorbed proteins on a material will make the design of cell-specific materials extremely difficult. The materials described herein are hydrogels of poly(ethylene glycol) (PEG) and are largely resistant to protein adsorption. Thus, if biological molecules are conjugated to the material, the cells will respond only to the incorporated ligand, substantially unaffected by spuriously adsorbed serum proteins. The adhesive behavior of endothelial cells seeded upon artificial surfaces containing adsorbed proteins has been described.7,8 The important parameters for the growth of an endothelial cell monolayer on top of a material are the strength of cell adhesion, the rate of cell migration, and the rate of cell proliferation. These parameters are strongly dependent upon changes in protein surface concentration and protein identity. It will be very important to optimize these parameters when using cell adhesion peptides immobilized on surfaces to promote tissue regrowth. However, the study of cell adhesion peptides on surfaces is often complicated by the presence of nonspecifically adsorbed serum proteins on the surfaces containing the cell adhesion-promoting peptides, leading to a mixture of signals for the cell. Celladhesion promoting peptides have been attached to many artificial materials, but most of these surfaces allow nonspecific protein adsorption.9-13 These materials in contact with blood would promote both platelet and leukocyte adhesion within a few hours of implantation. Other materials are resistant to protein adsorption,14-18 but cannot be polymerized in contact with living tissue due to harsh cross-

10.1021/bm0056299 CCC: $20.00 © 2001 American Chemical Society Published on Web 03/03/2001

Conjugate Addition Reactions

linking conditions. Still other materials may be resistant to protein adsorption only at relatively short time points.19-22 In contrast, the materials described herein are particularly well suited for the study of endothelial cell biology both in vitro and in vivo. In this article, we describe a new method to couple biological molecules to polymers. We also describe the synthesis and characterization of a photopolymerizable macromonomer, PEG-diacrylamide, that exhibits unique properties. Endothelial cell behavior on the materials was assessed by the extent of cell spreading on the materials, and the important parameters that affect cell spreading on these materials are described. Materials and Methods Poly(ethylene glycol)-Diacrylate. PEG mol wt 8000 (50 g, 12.5 mmol -OH, Aldrich, Milwaukee, WI) was azeotropically distilled in 500 mL toluene under argon, removing about 200 mL of toluene. The solution was cooled in a roomtemperature bath under Ar and then cooled in an ice bath. Anhydrous dichloromethane (Aldrich) was added until the solution became clear, about 100 mL. Triethylamine (2.61 mL, 18.75 mmol, Aldrich) was added dropwise with stirring, followed by the dropwise addition of 2.03 mL of acryloyl chloride (18.75 mmol, Aldrich). The reaction proceeded overnight in the dark under argon. The solution was filtered through paper under vacuum until clear, followed by precipitation in diethyl ether. The product was collected by filtration and dried under vacuum. Twenty grams of the product was then dissolved in 200 mL deionized water, with 10 g of sodium chloride. The pH was adjusted to pH 6 with NaOH, and extracted 3 times with 100 mL dichloromethane (some product remains in the water phase as an emulsion). The dichloromethane washes were combined, and the product was precipitated in diethyl ether and dried under vacuum. The product was stored at -20 °C under argon: PEGdiacrylate mol wt 8000, 17 g. 1H NMR (DCCl3): 3.6 ppm (789.35 H, PEG), 4.3 ppm (t, 11.74 H, -CH2-CH2-OCO-CHdCH2), 5.8 ppm (dd, 2.42 H, CH2dCH-COO-), 6.1 ppm, 6.4 ppm (dd, 4.12 H, CH2dCH-COO-). PEGdiacrylate mol wt 3400 was made similarly from PEG mol wt 3400 (Aldrich). NMR: 1H NMR (DCCl3): 3.6 ppm (311.32 H, PEG), 4.3 ppm (t, 4.54 H, -CH2-CH2-O-COCHdCH2), 5.8 ppm (dd, 2.10 H, CH2dCH-COO-), 6.1 ppm, 6.4 ppm (dd, 4.08 H, CH2dCH-COO-). The number of end groups in the PEG was determined by a previously published method.23 Poly(ethylene glycol)-Diamine. PEG mol wt 3400 (100 g, 58.8 mmol -OH) was azeotropically distilled in 700 mL toluene under argon, removing about 300 mL of toluene. The solution was cooled in a room temperature bath under Ar and then cooled in an ice bath. Anhydrous dichloromethane (Aldrich) was added until the solution become clear, about 100 mL. Triethylamine (24.6 mL, 176.5 mmol, Aldrich) was added dropwise with stirring, followed by the dropwise addition of 13.65 mmol mesyl chloride (176.5 mmol, Aldrich). The reaction proceeded overnight under argon. The solution was filtered through paper under vacuum

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until clear, followed by precipitation in diethyl ether. The product was collected by filtration and dried under vacuum. The PEG-dimesylate product was added to 400 mL 25% aqueous ammonia solution in a 1 L Nalgene bottle. The lid was tightly closed and sealed with Parafilm, and the reaction was vigorously stirred for 4 days at room temperature. The lid was then removed and the ammonia allowed to evaporate for 3 days. The pH of the solution was raised to 13 with 1 N NaOH, and the solution was extracted with 100 mL dichloromethane 3×. The dichloromethane washes were combined and concentrated in vacuo. The product was precipitated in diethyl ether, and dried under vacuum: PEGdiamine 82 g. 1H NMR (DCCl3): 3.6 ppm (310.4 H, PEG), 2.9 ppm (t, 4 H, -CH2-CH2-NH2). The presence of an amine group on the PEG terminus was detected by NMR, based on the presence of a triplet at 2.9 ppm, corresponding to the protons on the carbon in the alpha position relative to the amine. By comparison with the PEG backbone peak (3.6 ppm), the product was calculated to contain 99.6% PEG diamine and 0.4% PEG R-monoamine, ω-monohydroxyl. Poly(ethylene glycol)-Diacrylamide. PEG-diamine mol wt 3400 (20 g, 11.76 mmol amine) was azeotropically distilled in 400 mL of toluene under argon, removing about 100 mL of toluene. The solution was cooled in a roomtemperature bath under Ar and then cooled in an ice bath. Anhydrous dichloromethane (Aldrich) was added until the solution become clear, about 50 mL. Triethylamine (2.46 mL, 17.64 mmol, Aldrich) was added dropwise with stirring, followed by the dropwise addition of 1.43 mL of acryloyl chloride (17.64 mmol, Aldrich). The reaction proceeded overnight in the dark under argon. The solution was filtered through paper under vacuum until clear, followed by precipitation in diethyl ether. The product was collected by filtration and dried under vacuum. The product was then dissolved in 200 mL of deionized water, with 10 g of sodium chloride. The pH was adjusted to pH 6 with NaOH and extracted 3 times with 100 mL of dichloromethane (some product remains in the water phase as an emulsion). The dichloromethane washes were combined and the product was precipitated in diethyl ether, and dried under vacuum. The product was stored at -20 °C under argon: PEG-diacrylamide mol wt 3400, 17 g. 1H NMR: (DCCl3) 3.6 ppm (168 H, PEG), 5.6 ppm (dd, 1 H, CH2dCH-CON-), 6.1 ppm, 6.2 ppm (dd, 2.27 H, CH2dCH-CON-). The infrared spectrum contained amide I and II peaks at 1540 and 1674 cm-1 and a very small ester peak at 1720 cm-1. Peptide Synthesis and Purification. Peptides were synthesized on a Perseptive Biosystems (Farmington, MA) Pioneer peptide synthesizer, using Fmoc/HBTU/HOBT chemistry, with Novasyn TGR resin (Novabiochem, Laeufelgfingen, Switzerland). All amino acids and activators were from Novabiochem, solvents were from Perkin-Elmer Europe B. V. (Rotkreuz, Switzerland), and the N-terminal amino acid of the peptides was always N-acetylglycine. Peptides were cleaved from the resin using 8.8 mL of trifluoroacetic acid (Perseptive Biosystems), 0.5 mL of deionized water, 0.5 mL of phenol (Aldrich), and 0.2 mL of triisopropylsilane (Aldrich) per gram of resin, for 2 h at room temperature. The resin was removed by filtration, and the solution was

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precipitated in ether, recovered by filtration, and dried under vacuum. Peptides were purified by C18 chromatography (Prep Nova-Pak HR C18 6 µm 60 Å, 19 × 300 mm column, Waters, Milford MA) using a Perseptive Biosystems Biocad 700E. A gradient elution was used with the following solvents: (A) 0.1% trifluoroacetic acid in deionized water and (B) 0.1% trifluoroacetic acid in acetonitrile. The column was equilibrated to 95% A /5% B, and the peptide was eluted from the column by a gradient to 75% A /25% B over 10 min. Peaks were collected automatically, and the peak containing the desired product was identified by MALDITOF mass spectrometry. Peptides were stored under argon at -20 °C. Analysis of Conjugate Addition Reaction. Reaction kinetics were followed using Ellman’s reagent. Ellman’s reagent (40 mg 5,5′-dithiobis(2-nitrobenzoic acid), Sigma, St. Louis, MO) was dissolved in 10 mL 0.1 M phosphate buffer, pH 8.0. This was added (100 µL) to 3 mL of 0.1 M phosphate buffer, pH 8.0, containing 0.05-0.15 mmol of thiol-containing compound. The thiol concentration was estimated using an extinction coefficient of 14 150 at 412 nm.24 Peptides conjugated to PEG were detected on a Voyager Elite (Perseptive Biosystems) MALDI-TOF mass spectrometer, using a matrix of R-cyano-4-hydroxycinnamic acid, 10 mg/mL in 50% acetonitrile, and 50% 1% trifluoroacetic acid in water. The polymer-peptide conjugates in 1% trifluoroacetic acid in water were added to the matrix at a ratio of 1 mol polymer per 200 mol R-cyano-4-hydroxycinnamic acid. Polymer-peptide conjugates were also analyzed using a Nova-Pak 3.9 × 150 mm C18 column (Waters), using a Waters 2690 pump, a Waters 996 photodiode array detector, and a PL-EMD 960 evaporative mass detector (Polymer Laboratories, Amherst, MA). A gradient from 95% 0.1% trifluoroacetic acid in water/5% acetonitrile to 40% 0.1% trifluoroacetic acid in water/60% acetonitrile in 9.5 min was used. Peptide Coupling and Photopolymerization of Materials. To 39.4 mL of 10 mM HEPES buffered saline (HBS) (8 g/L NaCl, 2.603 g/L Sodium HEPES, pH 7.4) was added 612 mL of triethanolamine (TEOA) and 260 µL of 6.0 N HCl, yielding a buffer of pH 8.0 (10 mM HBS/115 mM TEOA buffer, pH 8.0). PEG-diacrylate or PEG-diacrylamide (230 mg) was dissolved in 770 µL of 10 mM HBS/ 115 mM TEOA buffer, pH 8.0, and air bubbles were eliminated by centrifugation at 500 g for 5 min. For PEGdiacrylate, peptides containing a single cysteine residue were dissolved in 115 µL of 1 mM MES buffered saline (195.2 mg/L 2-(N-morpholino)ethanesulfonic acid, 8.75 g/L NaCl, pH 5.8) which was then added to 870 µL of the PEGdiacrylate solution. This was allowed to react for 10 min in the dark at room temperature. To this mixture was added N-vinylpyrrolidone (NVP, Aldrich) and 10 µL of a 10 mM solution of Eosin Y (Sigma) in 10 mM HBS, pH 7.4, to yield a mixture ready for photopolymerization. For PEG-diacrylamide, any acrylate groups present on the PEG were quenched by the addition of 15 µL of 235.3 mM cysteine in 1 mM MES buffered saline, pH 5.8 (equivalent to 3% of the end groups of PEG mol wt 3400 dissolved at 20% w/v). This was allowed to react for 30 min at room temperature

Elbert and Hubbell

in the dark. This was followed by the addition of the peptides containing a single cysteine residue in 100 µL of 1 mM MES buffered saline, pH 5.8, which was allowed to react for 2 h at 37 °C in the dark. To this mixture was added 11.8 µL of NVP and 10 µL of 10 mM solution of Eosin Y (Sigma) in HBS, to yield a mixture ready for photopolymerization. Photopolymerization was accomplished with a filtered lamp producing 100-150 mW of light between 480 and 520 nm (ILC Technology, Sunnyvale, CA). The PEG solution (300 mL) was pipetted into a well of a 24-well plate, and was exposed to 75 mW/cm2 for 1 min (PEG-diacrylate mol wt 8000) or 50 mW/cm2 for 2 min (PEG-diacrylamide mol wt 3400 and PEG-diacrylate mol wt 3400). Gels were removed from the wells and allowed to swell in 10 mM HBS, pH 7.4, for 36 h at room temperature. The gels were then washed twice with 10 mM HBS, pH 7.4, and were then cut into a circular shape with a custom-made stainless steel hole punch with a diameter identical to the diameter of a well of a 24well plate. The gels were placed into wells of a 24-well plate and were incubated with cell culture medium containing serum and placed in a 37 °C cell culture incubator for 30 min. The meniscus shape was retained in the polymerized gel, and thus the side of the gel that had been polymerized against air could be distinguished from the side of the gel that had been polymerized in contact with polystyrene. The air-polymerized side of the gel was used for cell culture studies. Mechanical properties of the photopolymerized materials were investigated by photopolymerizing the PEG-diacrylate within the fluid space of a rheometer (Bohlin Instruments CVO 120 High-Resolution rheometer, Gloucestershire, England, U.K.) with a lower plate that allowed the fiber optic access to the solution in the rheometer. Photopolymerization was otherwise the same as described above. A gap of 0.2 mm was used with a parallel plate of diameter 2 cm, a constant strain of 0.05, and a frequency of 0.1 Hz, and photopolymerization was accomplished after setting of the gap thickness on the fluid precursor mixture. The free-radical polymerization of the material was further investigated by size exclusion chromatography of degradation products of the hydrogels. PEG-diacrylate mol wt 8000 was photopolymerized as described above, and the gels were allowed to swell overnight in 10 mM HBS, pH 7.4. Each gel was then placed in 1.5 mL of 0.1 M NaOH and placed at 80 °C for 65 h. The pH was adjusted by addition of 150 µL of 1 M HCl and 1.5 mL of 10 mM PBS, pH 7.4 (PBS). The resulting degradation products were then analyzed by aqueous size exclusion chromatography using a Waters 2690 pump, a Waters 996 photodiode array detector, a Waters 410 Differential refractometer, and a Shodex OHpak SB-804 HQ column with an exclusion limit of 106 Da (Alltech Associates, Deerfield, IL). The solvent was 0.2 µm filtered 10 mM PBS, pH 7.4 pumped at 0.4 mL/min. Poly(acrylic acid) standards were used to calibrate the column (American Polymer Standard, Mentor, OH). Cell Culture. Neonatal normal human dermal fibroblasts (Clonetics, San Diego, CA) were grown in fibroblast cell culture medium (Dulbecco’s Modified Eagle’s Medium, with 10% fetal bovine serum and 1% antibiotic-antimycotic,


Figure 1. Coupling of a biological molecule to PEG-diacrylamide by a conjugate addition reaction. The reaction can be performed under physiological conditions, and in contact with proteins, cells and tissues. The product can be subsequently free-radical polymerized to form a material.

GIBCO BRL, Life Technologies, Grand Island, NY) at 37 °C and 5% CO2. Fibroblast cells were removed from culture substrates using trypsin/EDTA (GIBCO BRL), centrifuged at 500g for 5 min, and resuspended in culture medium. Human umbilical vein endothelial cells (HUVEC, PromoCell, Heidelberg, Germany) were grown in Endothelial Cell Growth Medium (PromoCell) on tissue culture substrates that had been pretreated using a 3% gelatin solution. HUVECs were removed from culture flasks by removing the culture medium, washing the cells with 10 mM HBS, pH 7.4, and addition of 3 mL of 0.025% trypsin/0.01% EDTA solution (Clonetics, Walkersville, MD). After about 3 min, trypsinization was stopped by addition of 3 mL trypsin neutralizing solution (Clonetics). Cells were centrifuged at 250g for 5 min, resuspended in culture medium, and seeded onto the materials at 10 000 cells/cm2 in 1 mL of medium containing serum proteins. The culture medium was replaced daily. Cells on surfaces were imaged using a Zeiss Axiovert 135 inverted phase contrast microscope with a 10× objective (Zeiss, Oberkochen, Germany). Images of the cells were recorded at different time points, and the projected areas of cells were manually traced using image processing software (QwinPro v.2.1, Leica Imaging Systems, Ltd., Cambridge, England, U.K.). At least 50 cells per gel were traced, with three gels per treatment. All of the visible cells in a particular area were counted, and a number of different areas per gel were analyzed. Cell spreading was described in terms of the percentile of cells with projected area greater than a given value. The projected areas of cells from different treatments were analyzed by the nonparametric Kruskal-Wallis or Mann-Whitney U tests. The projected cell areas of cells in solution were measured using the same method but with endothelial cells that had settled onto but not attached to a poly(ethylene glycol) gel. Results Peptides were attached to acrylate or acrylamide groups on PEG molecules via a conjugate addition reaction (see Figure 1). If only a fraction of the PEG-diacrylate or PEGdiacrylamide molecules were targeted for reaction with the thiol-containing peptides, then a sufficient amount of PEGdiacrylate or PEG-diacrylamide molecules remain in the


mixture to form a cross-linked material via a free-radical polymerization mechanism, carried out after the peptide is grafted by conjugate addition. The peptide will be linked to one terminus of a PEG molecule, and the other terminus of the polymer will still contain an acrylate or acrylamide group. After polymerization, the peptide will thus be pendantly attached into the network, meaning that the peptide will be attached to the cross-linked network via a PEG spacer. Synthesis of PEG Derivatives. The use of PEGdiacrylamide in tissue engineering has not been described previously. An acrylamide group on PEG would be expected to have a similar rate of free-radical polymerization as an acrylate group, and a similar rate of free-radical termination. With PEG-diacrylamide, the ligand is attached through a stable amide bond, as opposed to PEG-diacrylate where the ligand is attached through a hydrolytically unstable ester bond. As such, for the present application where stability of attachment of the adhesion ligand is desired, reaction of the free thiol on the peptide with an acrylamide of PEGdiacrylamide is more advantageous than reaction with an acrylate of a PEG-diacrylate. However, the reaction of a thiol group with an acrylamide group is about 24 times slower than the reaction of a thiol group with an acrylate group.25,26 One could also employ a more reactive linker that yielded a stable linkage, such as a vinyl sulfone linkage, which reacts about 50 times faster than an acrylamide,25 but this group is not free radical polymerizable and would thus require the synthesis of a difunctional PEG, e.g., a PEG R-acrylate, ω-vinyl sulfone or a PEG R-acrylamide, ω-vinyl sulfone, neither of which are particularly straightforward to produce. As such, the compromise of carrying out the coupling reaction somewhat longer, using the PEG-diacrylamide, seems optimal, especially since the peptide coupling reaction would not be carried out in contact with the tissue. To produce PEG-diacrylamide, PEG-diamine was first synthesized. This was performed in a manner similar to a previously described procedure.27 Although PEG-diamines are commercially available, such as Jeffamine (Huntsman Corporation, Salt Lake City, UT), conversion of the alcohol groups on the PEG to amine groups has been commonly found to proceed to about 70-90%.28 This is unacceptable for the present purpose, because only a few percent of the end groups on the PEG will be targeted for reaction with thiol groups, and the presence of the more reactive acrylate groups in the product would lead to attachment of the peptide primarily to acrylate groups, even if they were present only in a minority. The conversion of the alcohol to a mesylated group is straightforward, and 100% conversion can be routinely achieved. Additionally, it was found that addition of PEG-mesylate to 25% ammonia water in a tightly closed container for about 1 week led to conversions of greater than 99% by NMR. Acrylation of the amine with acryloyl chloride is not substantially different than acrylation of an alcohol group,29 and NMR following acrylation of the PEG-diamine indicated the absence of amine groups or acrylate groups. Infrared spectroscopy indicated some acrylates present in the product, however. The acrylate groups were also detected by targeting a few percent of the end groups of the PEGdiacrylamide product for reaction with a thiol-containing

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Figure 2. Kinetics of the coupling between the thiol of a cysteine group in a peptide and an acrylate group on poly(ethylene glycol). The disappearance of thiol groups was measured using Ellman’s reagent. Using the cysteine-containing peptide Ac-GCGYGRGDSPG-NH2, the starting amount of thiols was 0.78 µmol, which was added to 2% w/v PEG-diacrylate mol wt 8000 (5 µmol acrylate groups) in buffered saline. The reaction was studied at pH 8 ()), pH 7.4 (O), and pH 6.4 (2). Loss of thiols due to disulfide bonding in the absence of acrylate groups was also studied (9).

compound. The reaction kinetics revealed that about 1% of the end groups in the product were acrylate groups. It was demonstrated that the product was mainly PEG-diacrylamide, however, by photopolymerizing a gel from a 20% solution of the PEG-diacrylamide product and placing the gel in 0.1 M NaOH, at 80 °C. Gels made from PEGdiacrylate were degraded in 3 h under these conditions, yet gels made from the more hydrolytically stable PEGdiacrylamide were intact for longer than 1 month under these conditions. Conjugate Addition Reaction. The reaction between an acrylate group and a thiol group is rapid under physiologic conditions. Although this is a second-order reaction, in these studies the end product of the coupling reaction will always be used in a subsequent photopolymerization reaction, and thus a large excess of acrylate groups will always be required. The coupling reaction will thus always be performed within the pseudo-first-order regime, and the reaction can be usefully modeled as a first-order reaction. A first-order rate constant can then be determined at a particular initial concentration of PEG-diacrylate and a particular pH. The pH is important because it will determine the percentage of the thiol groups that are in the very reactive ionized thiolate form. With strong buffering, the pH will not change, and the ratio of thiolate groups to protonated thiol groups will remain constant. The effects of PEG-diacrylate concentration and pH can thus be incorporated into a first-order rate constant. For PEGdiacrylate, it was found that even at dilute PEG-diacrylate concentrations (2% w/v PEG-diacrylate mol wt 8000) and at 6.4 equiv of acrylates per thiol, the reaction in buffered saline at pH 8 was too rapid to be measured reliably using Ellman’s reagent, with a pseudo-first-order half-life of about 30 s at room temperature. At pH 7.4, the pseudo-first-order half-life could be measured more accurately, and was estimated to be about 4 min. At pH 6.4, a pseudo-first-order half-life of 33.0 min was determined, and a plot of the logarithm of the normalized concentration of thiol vs time yielded a curve that was fitted well by linear regression, with a coefficient of determination of 0.998 (see Figure 2). Under

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these reaction conditions, disulfide bond formation between two peptides was found to be extremely slow. Reverse-phase HPLC was useful in demonstrating the reaction of the peptide with the PEG-diacrylate. Using the gradient as stated in the Methods section, the peptide AcGCGYGRDGSPG-NH2 eluted between 4.105 and 4.150 min, and the disulfide bonded dimer eluted between 4.5 and 4.530 min (the identities of the compounds in these peaks were verified by collection of the peaks and analysis by MALDI-TOF mass spectrometry). PEG and PEG derivatives (including peptide-PEG conjugates) eluted between 7.5 and 10 min. PEG-diacrylate does not absorb light at 273 nm but was detected at shorter wavelengths. Additionally, an evaporative mass detector was useful for demonstrating the elution of PEG derivatives. The free peptide eluted as a very sharp peak, but upon mixing of the thiol-containing peptide with the PEG-diacrylate, the free peptide peak was no longer present, and the absorbance of light at 273 nm (due to the tyrosine residue in the peptide) coeluted with the PEG. If the peptide Ac-GCGYGRGDSPG-NH2 was attached to PEG-diacrylate mol wt 3400 at a ratio of 1 peptide to 2 PEG-diacrylate, MALDI-TOF mass spectrometry could detect the peptide-PEG adduct, at the expected molecular weight. PEG derivatized with two peptides, i.e., peptide-PEG-peptide, was not a prominent peak in the MALDI-TOF mass spectrum. The reaction between a thiol-containing peptide and PEGdiacrylamide was substantially slower than the reaction with the PEG-diacrylate, and the kinetics of the coupling reaction could be followed by reverse-phase HPLC. At a 20% concentration of PEG-diacrylamide mol wt 3400 (117.6 µmol acrylamide/mL), pH 8 and 37 °C, a thiol-containing peptide (Ac-GCGYGRDGSPG-NH2, 3.66 µmol/mL) reacted with the acrylamide group with a pseudo-first-order half-life of 19.5 min, with a coefficient of determination of 0.989. For a 5% concentration of PEG-diacrylamide, with the other conditions the same, a pseudo-first-order half-life of 98.7 min was found, with a coefficient of determination of 0.999. Given that the concentrations of PEG-diacrylamide differed by a factor of 4, the pseudo-first-order rate constants for the different concentrations of PEG-diacrylamide should also differ by a factor of 4 and were found to differ by a factor of about 5. The rate constants for coupling of a thiol-containing peptide to PEG-diacrylamide were determined after quenching any acrylate groups in the PEG-diacrylamide product with the amino acid cysteine. In the absence of this quenching step, the amount of acrylates in the PEG-diacrylamide product could be measured by titrating with the cysteinecontaining peptide. The reaction of a thiol with an acrylate is practically instantaneous, so the presence of any acrylate groups can be determined by the rapid disappearance of a portion of the cysteine-containing peptide. It was found that if a small amount of the end groups on the PEGdiacrylamide were first reacted with the amino acid cysteine, then only acrylamide groups were available for reaction with the cysteine-containing peptide. Adding enough cysteine to react with 3% of the end groups of the PEG-diacrylamide at pH 8, room temperature for 30 min was sufficient to


quench all contaminating acrylate groups as evidenced by the reaction kinetics observed with reverse-phase chromatography. For photopolymerization, 20% PEG-diacrylamide solutions were typically used to produce gels, and thus the coupling of the thiol-containing peptide was performed with this concentration of PEG-diacrylamide. Since the half-life of the coupling reaction at this PEG-diacrylamide concentration was measured to be about 20 min at pH 8, 37 °C, coupling the peptide to the PEG-diacrylamide for 2 h allowed for 6 half-lives, or reaction of 98% of the peptide with the PEG. The remaining thiol-containing peptides will likely be incorporated into the gel during the photopolymerization step, with the thiol acting as a radical transfer agent during polymerization. These radical-reacted peptides will not be attached on a pendant PEG chain, but rather on the end of the terminated polyacrylamide nodes in the crosslinked polymer network, and thus may be hidden from interaction with cell-surface receptors.5 Additionally, it was found that the reaction of the thiol with the acrylamide occurs sufficiently slowly that disulfide bond formation between two peptides becomes apparent. Storage and dissolution of a peptide containing a single cysteine residue such as AcGCGYGRGDSPG-NH2 yielded about 2% of the peptide as dimers via disulfide bond formation. For reaction of AcGCGYGRDGSPG-NH2 with PEG-diacrylamide, about 5% of the peptide had formed dimers via disulfide bond formation after the 2 h coupling period. Immediately after dissolution of the peptide, about 2% of the peptide was found as dimer, as was expected, and the amount of dimerized peptide increased during the 2 h coupling period. The formation of the dimer should be a second-order reaction, with the rate proportional to the square of the concentration of the peptide. It was in fact observed that the rate of dimer formation decreased as the amount of thiol-containing peptide decreased. These peptide dimers do not become attached to the PEG and presumably can diffuse out of the gels. The gels were allowed to swell for 36 h before cells were seeded onto the gels, and the peptide dimers may diffuse out of the gel during this time. Hydrolysis of Ester Bonds. The peptides were attached to PEG-diacrylate via ester bonds. These bonds are prone to base-catalyzed hydrolysis. The rate of hydrolysis of the ester bond between the peptide and the PEG-acrylate was measured by reverse-phase HPLC. Immediately after reaction between the PEG-diacrylate and the thiol-containing peptide, no peaks associated with free peptide were observed. However, over time, a peak corresponding to free peptide emerged with first-order kinetics. A fraction containing the new peptide peak was collected and analyzed by MALDITOF mass spectroscopy, which revealed that this new product had a molecular weight equal to the original thiolcontaining peptide plus 72 mass units. This corresponded to the modification of the thiol group of the original peptide with proprionic acid. This was the expected product of the hydrolysis of the ester bond between the peptide and PEG. The rate of hydrolysis is a function of the pH, the temperature, and the chemical environment around the ester bond. A highly hydrated environment around the bond will


allow the hydrolysis reaction to occur at a maximal rate, and with PEG, it would be expected that the hydrolysis of this ester would be relatively rapid. At pH 8 and 37 °C, peptide was hydrolyzed from the PEG-acrylate with first-order kinetics, and a half-life of 3.5 days (0.1 M phosphate buffer, pH 8). At pH 7.4 and 37 °C, peptide was hydrolyzed from the PEG-acrylate with a half-life of 10.9 days (0.05 M PBS, pH 7.4). The hydrolysis reaction is actually second-order, but the second compound in the reaction is hydroxide ion, which should be at constant concentration in a well-buffered solution, leading to the observation of first-order kinetics. Thus, the rate constant should vary linearly with hydroxide ion concentration. The hydroxide concentrations for these two pH conditions differ by a factor of 3.98; the measured rate constants differ by a factor of 3.09. The hydrolysis of the amide bond between the peptide and PEG-acrylamide was also measured. After 5 days in 0.1 M phosphate buffer, pH 8, 37 °C, about 1% of the peptide had hydrolyzed from the PEG-diacrylamide, which was at the limit of detection of this method. The same result was found with another amide-based linking chemistry, based on reaction of amines with PEG activated as an NHS-ester, indicating that an amide bond between a peptide and PEG is very stable under physiological conditions (data not shown). Cell Interactions with Incorporated Peptides. Hydrogels were formed by photopolymerization of a 20% w/v solution of PEG-diacrylate mol wt 8000. The gels were placed in 10 mM HBS, pH 7.4, and the gels swelled about 36% in diameter after 24 h if the gel was formed from a precursor solution containing 0.28 NVP/acrylate, and swelled 26% and 22% in diameter with 0.65 and 0.94 NVP/acrylate, respectively. The modification of one out of eight of the PEGdiacrylates with a peptide via the conjugate addition reaction did not lead to a significant change in swelling of the gels. A series of materials incorporating varying amounts of adhesion peptide was produced as follows. Peptides containing a single thiol were reacted with a 20% w/v solution of PEG-diacrylate, mol wt 8000 in 10 mM HBS/115 mM TEOA buffer, pH 8 for 10 min. The amount of peptide in the gel was varied, and 1/8, 1/16, or 1/32 of the PEG-diacrylate molecules was derivatized with a peptide. The NVP and Eosin Y were added to the solution, with mixing by vortexing, and the solution was added to wells of a 24-well plate and exposed to light at about 500 nm for 1 min. The gels were allowed to swell for 1 h in the wells, and then moved to sterile tubes containing 25 mL HBS, pH 7.4. The gels were stored at room temperature for 1.5 days. PEG-based materials are often described as nonadhesive to proteins and cells, and this was tested by seeding fibroblast cells onto the gels. The gels were placed into wells of a 24well plate, and incubated with fibroblast medium containing 10% serum for 30 min. The fibroblast cells were harvested with trypsin/EDTA and seeded onto the gels in medium containing 10% serum. Under these conditions, fibroblast cells will attach and spread on almost all artificial surfaces, however, the fibroblast cells were unable to spread on the PEG-diacrylate and PEG-diacrylamide based materials. This was true both for the base PEG material (no peptide)

436


Elbert and Hubbell Table 1. Endothelial Cells Seeded on Hydrogels Made by the Photopolymerization of PEG-Diacrylate mol wt 8000a projected cell area (µm2) NVP 1.5 µL/mL 25% RGD 1/8 RGD 1/16 RGD 1/32 RDG 1/8 no peptide

760 585 450

887 929 750

Figure 3. Photomicrograph showing spreading of endothelial cells at 24 h postseeding on PEG-diacrylate, mol wt 3400 gels containing (A) Ac-GCGYGRGDSPG-NH2 and (B) Ac-GCGYGRDGSPGNH2, at a nominal peptide concentration of 3.68 pmol/cm2.

and for the material containing the peptide Ac-GCGYGRDGSPG-NH2. This peptide is not recognized by any cellular receptors and thus serves as a control peptide. Cell spreading was normal and substantial on tissue culture polystyrene, the normal surface for the culture of human cells. Fibroblasts and HUVECs have receptors for peptides containing the Arg-Gly-Asp (RGD) sequence. If gels were formed containing the peptide Ac-GCGYGRGDSPG-NH2, normal endothelial cell spreading could be observed on the PEG materials (see Figure 3). The extent of cell spreading was investigated as a function of NVP concentration, peptide concentration, and time. The projected cell areas on the surfaces were manually traced from images of cells on the surfaces, and the distribution was displayed using percentile graphs, showing the percent of cells with projected areas greater than a given value. For endothelial cells, most rounded cells in solution have projected areas of about 250400 µm2. Cells with projected areas greater than 1000 µm2 were defined as spread on the surface, and cells with projected areas greater than 2000 µm2 were defined as wellspread. Peptide concentrations were varied by changing the amount of peptide added to the PEG-diacrylate precursor solution, as described above. Peptide concentrations are reported in two ways: as the fraction of PEG molecules containing an attached peptide, and as the nominal surface concentration of peptide. By assuming that the upper 10 nm of the surface is available for interaction with the cell surface receptors, then 1 nmol of peptide/µL of precursor solution leads to a nominal surface concentration of 1 pmol/cm2.5 The presence of RGD peptide led to cell spreading on the materials, and a reduction of nominal peptide surface concentration from 3.125 to 0.781 pmol/cm2 led to only a small change in cell area (see Table 1). The effect of peptide concentration on cell spreading was investigated in an

1118 1056 687 357 397

tcps

50%

25%

50%

583 729 517 319 312

1680 1791 1133 387

1026 1086 671 308

498 597 487

2h 1210 1206 924

586 636 500 273 315

4h 1609 1462 1032 392 385

25% - 6559

tcps RGD 1/8 RGD 1/16 RGD 1/32 RDG 1/8 no peptide

25% 1h 1321 1114 867 368 357

25% - 5036


469 422 329

NVP 5 µL/mL

25% - 3698


50%

NVP 3.5 µL/mL

1061 1116 575 363 300

508 638 374 279 258

24 h 1604 1888 1159 414 330

25% - 7443

50% - 1766 671 728 548

1822 2101 1032 350 366

1005 1415 524 313 311

50% - 3372 921 843 661 321 282

2127 3058 1912 355 420

1338 1769 1099 278 340

50% - 4599 869 1038 585 300 247

2296 3702 1843 331 297

1256 2253 1182 251 240

50% - 5492

a The hydrogels contained the cell adhesion peptide Ac-GCGYGRGDSPG-NH2 or the control peptide Ac-GCGYGRDGSPG-NH2, which were coupled to the PEG-diacrylate via a conjugate addition reaction. Images of cells on the materials were obtained at 1, 2, 4, and 24 h, and the projected cell areas were manually traced, with at least 150 cells traced per condition. The projected cell areas of cells in the 25th or 50th percentile are given. The NVP concentrations (1.5, 3.5, and 5 µL/mL of gel precursor solution) correspond to 0.28, 0.65, and 0.94 NVP/acrylate, respectively. The fraction of PEG-diacrylates containing an attached peptide is indicated, with 1/8, 1/16, and 1/32 corresponding to nominal peptide surface concentrations of 3.12, 1.56, and 0.78 pmol/cm2, respectively.

additional experiment, and the treatments with different peptide concentrations were not significantly different by the nonparametric Kruskal-Wallis test. However, the concentration of NVP was an important parameter, and relatively modest changes in NVP concentration led to significant differences in cell spreading (see Table 1). NVP concentrations were 1.5, 3.5, and 5 µL/mL of precursor solution (0.28, 0.65, and 0.94 NVP/acrylate, respectively). For gels with 3.125 pmol/cm2 of the RGD peptide, projected cell areas at a given percentile were more than doubled by increasing the NVP concentration from 1.5 to 5 µL/mL (see Figure 4). The projected cell areas for gels with 1.5, 3.5, or 5 µL/mL were all significantly different from each other with p < 0.05 by the Kruskal-Wallis and Mann-Whitney nonparametric statistical tests, however, an increase of the NVP concentration to 7 µL/mL did not lead to a significant increase in projected cell area vs that observed with 5 µL/mL. Time of culture was also an important factor. On tissue culture



Table 2. NVP Concentration in the Gel Precursor Solution Having a Profound Effect on the Mechanical Properties of the Gelsa

NVP/ NVP in gel acrylate precursor in gel (µL/mL) precursor 1.5 3.5 5

Figure 4. Endothelial cell spreading on PEG-diacrylate mol wt 8000 materials containing cell adhesion promoting peptide, with one out of eight of the PEG-diacrylate molecules derivatized with a peptide: (A) kinetics of cell spreading on RGD-containing materials vs tissue culture polystyrene (TCPS), with NVP concentrations of 5 µL/mL of gel precursor solution (0.94 NVP/acrylate), where thick lines represent TCPS and thin lines represent RGD-containing gels; (B) effect of NVP concentration on cell spreading. The concentrations of NVP were 1.5, 3.5, or 5 µL/mL, corresponding to 0.28, 0.65, and 0.94 NVP/acrylate, respectively, and the cells were observed at 4 h post-seeding (thin lines) and 24 h post-seeding (thick lines).

polystyrene, projected cell areas of endothelial cells substantially increased at 1, 2, 4, 10, and 24 h as compared with the previous time point. However, on peptide containing gels, most of the increase in cell area occurred within the first 4 h post-seeding, and little increase in cell area was noted between 4 and 24 h (see Figure 4). However, spreading on the PEG-diacrylate gels was not as extensive as that found on tissue culture polystyrene at any time point. At a given percentile and time in culture, it was consistently found that projected cell areas on RGD-containing gels were less than 50% the value found for tissue culture polystyrene. The striking effect of NVP concentration on cell spreading was statistically significant and was further investigated. Even small changes in NVP content had substantial effects on gel mechanical properties, and this was investigated by photopolymerizing the PEG-diacrylate solution within a rheometer. The measured storage moduli (G′) are reported in Table 2. The loss modulus is very small, so the storage modulus can also be taken to be the shear modulus, and the Young’s modulus should be three times the value of the storage modulus (assuming Poisson’s ratio ) 0.5). An increase in the concentration of NVP in the gel precursor solution led to a substantial increase in the stiffness of the materials. The increase in stiffness of the materials was correlated with an increase in the spreading of cells on the materials, while maintaining a constant concentration of peptide within the gels. The effect of NVP incorporation on the gel was further assessed by degrading the gel with strong base and measuring the molecular weight distribution of the resulting degradation products. Since the gels result from an acrylate polymeri-

0.28 0.65 0.94

storage modulus, G′ (Pa) 4250 ( 1848 12667 ( 1159 32867 ( 12506

no. of PEG chains attached mol wt to each polypoly(AA-co-VP) (AA-co-VP) 12 100 28 800 37 300

131 242 267

a The storage modulus is essentially the shear modulus for these materials, and thus a Young’s modulus can be determined by the relation: E ) 2(1 + µ)G′, where µ is Poisson’s ratio (assumed to be the ideal value, 0.5, a reasonable estimate for cross-linked gels), or E ) 3G′. The reason for the change in the mechanical properties is a result of a change in the nanostructure of the gels, as reflected by the change in the mol wt of the poly(acrylic acid-co-NVP) chains contained within the hydrogel. The mol wt of the poly(acrylic acid-co-NVP) chains was measured by degrading the gel with strong base followed by size exclusion chromatography. The degree of polymerization of PEG-diacrylate in the gel could be determined by assuming that NVP and acrylate groups have the same reactivity, and thus the ratio of acrylic acid to NVP in poly(acrylic acid-co-NVP) is known. The number of PEG molecules originally attached to each poly(acrylic acid-co-NVP) could thus be determined.

zation, the gels consist of PEG molecules connected by polyacrylate chains. Hydrolysis of ester bonds in the gels then releases PEG-diol and poly(acrylic acid), or poly(acrylic acid-co-NVP) if the NVP is incorporated into the precursor solution. The molecular weight of the poly(acrylic acid-co-NVP) polymer chains was measured by size exclusion chromatography, using poly(acrylic acid) standards. The parameter reported is Mp, the molecular weight at the peak maximum. A trend was observed that the molecular weight of the poly(acrylic acid-co-NVP) chains increased with increasing NVP concentration (see Table 2), which was further confirmed by studying gels with a much higher concentration of NVP, 3.6 NVP/acrylate. In this case, an Mp of 100 200 was found. It was also determined that the important parameter is the NVP-to-acrylate ratio, and not the concentration of NVP in the precursor solution, which was determined by comparing the Mp of poly(acrylic acid)co-NVP derived from gels made from PEG-diacrylate mol wt 8000 or PEG-diacrylate mol wt 3400 (data not shown). It was hypothesized that the hydrolysis of the ester bond between the peptide and the PEG for the PEG-diacrylate system may adversely affect cell interactions with the peptide-containing gels over long periods of culture. Thus, a new type of linkage was produced, by using PEGdiacrylamide as the base polymer instead of PEG-diacrylate. Although the conjugate addition reaction between the thiolcontaining peptide and the PEG-diacrylamide was slower, the amide bond formed was much more stable, and the photopolymerization kinetics were unaffected by the change from acrylate to acrylamide termini. The PEG-diacrylamide that was synthesized was mol wt 3400, and this was compared with PEG-diacrylate of mol wt 3400. Gels were photopolymerized from PEG-diacrylamide mol wt 3400 and PEG-diacrylate mol wt 3400 at an NVP concentration of 11.8 µL/mL, or 0.94 NVP/acrylate, and thus should be comparable to gels made from PEG-diacrylate mol wt 8000 with 5 µL/mL of NVP.

438


Elbert and Hubbell Table 3. Endothelial Cells Seeded on Hydrogels Made by Photopolymerization of PEG-Diacrylate mol wt 3400 or PEG-Diacrylamide mol wt 3400a projected cell area (µm2)

Figure 5. Endothelial cell spreading on PEG-diacrylamide mol wt 3400 photopolymerized gels containing different concentrations of the cell adhesion promoting peptide, Ac-GCGYGRGDSPG-NH2, with 1/ , 1/ , or 1/ 16 50 160 of the PEG-diacrylamide molecules derivatized with peptide at 4 h post-seeding. This corresponds to nominal peptide surface concentrations of 3.68, 1.18, and 0.368 pmol/cm2, respectively. The concentration of NVP was 11.8 µL/mL or 0.94 NVP/ acrylate. TCPS ) tissue culture polystyrene.

Nominal peptide concentrations of 3.68, 1.18, and 0.368 pmol/cm2 were used for the gels made with PEG-diacrylamide or PEG-diacrylate mol wt 3400. Cell spreading was significantly affected by peptide concentration at 4 h postseeding on gels made from PEG-diacrylamide (see Figure 5). Cell spreading on materials made from PEG-diacrylamide mol wt 3400 was very similar to that found on materials made from PEG-diacrylate mol wt 3400 (see Table 3), and comparable to cell spreading observed on materials made from PEG-diacrylate mol wt 8000. HUVECs on both substrates were observed for 2 weeks. During this time, cells that were in contact with other cells formed monolayers with a cobblestone morphology, as is found in vivo and on tissue culture polystyrene. No change in the morphology of this HUVEC monolayer was observed on the PEG-diacrylate materials over this two week period, despite a greater than 50% reduction in peptide concentration in the material due to hydrolysis of the peptide. Discussion A great need exists for new materials for tissue engineering. At this stage in the development of materials to promote healing following balloon angioplasty, a variety of systems are under investigation.30,31,5,11 Each system has limitations, but the knowledge obtained from these materials will be invaluable in the design of biomaterials in the future. A biomaterial that will successfully promote the regeneration of a monolayer of cells in an animal must have properties that meet a number of constraints. In general, the material must be applied as a liquid and must be polymerizable in contact with living tissue. The precursor thus may not contain toxic chemicals. The precursor must be polymerizable in a very thin layer (about 20 µm) at the surface of the tissue. There must be some mechanism for the incorporation of biological molecules such as proteins or peptides within the material. The ability to deliver growth factors and other soluble factors from the materials is also of interest. The material perhaps should be degradable, either hydrolytically or enzymatically, although the optimal time course of this degradation is unknown. Alternatively, the material might not be degradable, but would then have to be stable in the

PEG-diacrylate

PEG-diacrylamide

25%

50%

25%

50%

RGD 1/16 RGD 1/50 RGD 1/160 RDG 1/16 no peptide

825 946 522

1h 456 595 389

1370 506

827 346

385

320


1054 1069 764

2h 596 551 467

1740 806

1074 535


1631 1543 1031 421 380

4h 1096 990 631 317 306

2146 1375 993 387 389

1291 816 647 331 330


1832 1577 1087

24 h 1215 1033 771

1915 1583 964

1262 968 564

a The hydrogels contained the cell adhesion peptide Ac-GCGYGRGDSPG-NH2 or the control peptide Ac-GCGYGRDGSPG-NH2, which were coupled to the PEG-diacrylate or PEG-diacrylamide via a conjugate addition reaction. Images of cells on the materials were obtained at 1, 2, 4, and 24 h, and the projected cell areas were manually traced, with at least 150 cells traced per condition. The projected cell areas of cells in the 25th or 50th percentile are given. The NVP concentration of 11.8 µL/ mL corresponds to 0.94 NVP/acrylate. The fraction of PEG-diacrylates containing an attached peptide is indicated, with 1/16, 1/50, 1/160 corresponding to nominal peptide surface concentrations of 3.68, 1.18, and 0.368 pmol/cm2, respectively. Before the 24 h measurement, the culture medium above the gels was changed, and too few cells were left on the “RDG 1/ ” and “no peptide” surfaces to measure projected areas. 16

body for many years. Ideally, the material should be nonadhesive to proteins, lest adsorbed proteins interfere with the incorporated biological molecules (however, materials that promote selective protein adsorption may be useful32). The mechanical properties must not be neglected, since the materials will be under shear flow, and it has also been demonstrated that the mechanical properties of a material affect cell behavior and spreading.33,34 The biological molecules will inevitably be released from the material as the material degrades, and this may be of importance, because soluble cell adhesion molecules can cause the detachment of cells from a surface. The cells will produce an extracellular matrix, which may interfere with cell adhesion to peptides, potentially causing the release of a sheet of cells from the material, if the extracellular matrix does not adhere to material. Finally, the identities of the biological molecules that should be incorporated into the material have not been definitively identified, complicated due to the inability to discern between the effects of intentionally attached biological molecules and unintentionally adsorbed proteins.


The materials presented here meet many of these constraints. It has already been demonstrated that such materials can be photopolymerized in thin layers in contact with tissue,6 without toxic effects. It has been demonstrated that biological molecules can be incorporated within these materials5 and that the materials can be used to deliver proteins.35 This study demonstrates that the materials are nonadhesive to proteins and that a wide range of mechanical properties can be obtained. Issues that remain to be resolved involve the degradability of the material, the influence of cell extracellular matrix production on long-term cell survival, and the identity of ligands to promote selective cell adhesion. The new coupling method described here to attach biological molecules to PEG-diacrylate is simple, allowing the rapid screening of various peptides in contact with cells. The synthesis of a peptide containing a single cysteine residue is straightforward and standard, and the cysteine residue can be maintained in the reduced state indefinitely at -20 °C under argon. The coupling procedure can be performed in less than 10 min, with only a few percent of the peptide lost to disulfide bonding. The similarity of cellular response to PEG-diacrylate materials and PEG-diacrylamide materials indicates that hydrolysis of peptides from the PEG-diacrylate materials does not affect cell behavior at less than 24 h post-seeding, and monolayers of cells can survive on both materials for greater than 2 weeks. Consequently, due to the dynamic nature of biological systems, the release of the peptide from the material may not hinder the use of these peptide-modified PEG-diacrylate materials in the animal. PEG-diacrylamide is an interesting new derivative for photopolymerizing in vivo. It should not be toxic, due to its high molecular weight and hydrophilicity, which will restrict access of the polymer to the cell’s interior. Acrylamide is very toxic, but this is because it is uncharged and small and can thus pass through cell membranes. If PEG-diacrylamide were to degrade, hydrolytically or enzymatically, it would not yield acrylamide but acrylic acid, which is much less toxic than acrylamide because of the charge on acrylic acid. PEG-diacrylamide undergoes rapid free-radical polymerization. Peptides can be attached to the polymer via a conjugate addition reaction in about 2 h, and the bonds formed between the peptide and the polymer are stable amide links. It is possible to produce PEG-diacrylamide with only a few percent acrylate bonds, which can be quenched by reaction with the amino acid cysteine. The PEG-diacrylamide materials are not degradable, either enzymatically or hydrolytically, but should be well tolerated in vivo, and degradability can be engineered into such gels.23 Cell spreading on PEG-diacrylate and PEG-diacrylamide peptide-containing materials was similar at both short and long time points. This indicates that initial cell spreading data is unaffected by the hydrolysis of peptide from the PEG-diacrylate material. Additionally, the long-term survival of cells (>2 weeks) on PEG-diacrylate materials may imply that the cells are attached to their own extracellular matrix and that this extracellular matrix can adhere well to PEG-based materials, even though these materials resist the adsorption of proteins from solution.


Endothelial cells do not spread on these materials to the same extent as on tissue culture polystyrene. This is an important comparison to make, since tissue culture polystyrene is a well-characterized substrate. Additionally, the measurement of projected cell areas is perhaps the only measurement of cell spreading that is sensitive enough to demonstrate such differences between surfaces. Thus, it should be utilized as a standard measure of cell spreading in order to allow comparisons between different surfaces produced by different research groups. However, it should not be implied that it is our primary goal to achieve the same degree of cell spreading as observed on tissue culture polystyrene, since this degree of cell spreading promotes a phenotype that leads to dedifferentiation of the cells. It has been demonstrated that the projected area of a cell is correlated with the density of cell attachment sites on a surface and is an important determinant of cell phenotype.36,37 However, the optimal projected cell area for endothelialization of the materials has not been determined, and data from our system implies a lower potency of small RGD peptides as compared with intact proteins such as fibronectin and vitronectin. Lo et al. show that projected cell areas are greater on stiffer materials, with projected cell areas increasing from 1740 to 2180 µm2 upon an increase in the Young’s modulus of the material from 14 000 to 30 000 Pa.34 This would correspond to a storage modulus of about 4666 and 10 000, respectively. This corresponds well to PEG-diacrylate gels made with 0.28 and 0.65 NVP/acrylate, respectively (see Table 2). The cell areas reported by Lo et al. are averages of spread cells. From our studies, using gels with the highest concentration of RGD, and averaging the projected cell areas only of spread cells at 24 h (i.e., only cells with projected cell areas greater than 1000 µm2), we found average projected cell areas of 1533 µm2 and 1989 µm2 for gels with storage moduli of 4250 and 12667 Pa, respectively. The agreement is remarkable, given the difference in cell types (NIH 3T3 vs endothelial cell), and the difference in adhesion signal (adsorbed Type 1 collagen vs RGD peptide). Another study by the same group showed normal cell spreading at a Young’s modulus of 70 000, but limited cell spreading at a Young’s modulus of 15 000.38 Some of our materials surpass the maximum Young’s modulus studied by Pelham and Wang, but cell spreading similar to tissue culture polystyrene was not achieved with our system. The limited cell spreading is likely due to the RGD peptide itself or due to the conjugate addition/photopolymerization system. With the current system, spreading comparable to that observed on tissue culture polystyrene may require more peptide on the surface, or peptides with higher binding affinities toward integrins. Data from Hern and Hubbell, Brandley et al.,39 and this work indicate that the assumption that the outer 10 nm of a gel is available for cell interactions is indeed not an accurate assumption (see Table 4). Compared with cell spreading data on glycophase glass containing covalently attached RGD peptides from Massia and Hubbell,40 this assumption appears to overstate the accessible peptide concentration at the surface by a factor of 40-100. Perhaps less than the upper 10 nm of the hydrogel surface is available to the cell, or it may

440


Elbert and Hubbell

Table 4. Survey of the Literature Revealing That Peptides Attached to Polymers Are Less Effective in Promoting Cell Spreading Than Peptides Attached to a Solid Surface, by a Factor of about 50a peptide required (fmol/cm2) well-spread cells Massia and Hubbell40 Brandley and Schnaar39 Hern and Hubbell5 this study Neff and Caldwell22

10 1000 368 470

spread cells 1 33 100 60 60

a Massia and Hubbell attached RGD peptides to glass, Brandley et al. attached RGD peptides to polyacrylamide gels, Hern and Hubbell attached RGD peptides to monoacrylate-PEG-NHS-ester, followed by photopolymerization with PEG-diacrylate, and Neff et al. attached RGD peptides to PEG-based surfactants, which were then adsorbed onto polystyrene.

also be that an entropic penalty results from attachment of the peptide to a flexible polymer chain opposed to a rigid surface, or that the transmission of force through the flexible polymer is inefficient. This is corroborated by the results of Neff et al., using a system in which peptides are attached to a monolayer of water-soluble polymer adsorbed on a rigid surface. In this system, about 470 fmol/cm2 are required for well spread cells, as opposed to Massia and Hubbell’s 10 fmol/cm2. Interestingly, with the system of Neff et al., spreading similar to that observed on tissue culture polystyrene is observed at 2.3 pmol/cm2, whereas in this study the highest concentration studied (3.6 pmol/cm2) did not lead to spreading as observed on tissue culture polystyrene. In future studies, it will be important to determine the mechanism for the difference in spreading observed between PEG gels and tissue culture polystyrene. The repertoire of integrins and cell surface receptors utilized, the binding affinity between ligands and integrins, or the dynamic nature of the PEG surface may be responsible. Such mechanisms may be determined using anti-integrin antibodies and cyclic RGD peptides with higher affinities for integrin receptors. Massia and Hubbell41 measured endothelial cell areas on glycophase glass containing 12.1 pmol/cm2 of RGD, and found that 25% of the endothelial cells had cell areas greater than 3000 µm2, and 50% of the cells had cell areas greater than 2679 µm2, suggesting that increasing the concentration of RGD may not be sufficient to mimic spreading on tissue culture polystyrene with endothelial cells. For the production of materials that promote reendothelialization following balloon angioplasty, additional parameters must be measured. Cell migration and proliferation will be necessary for reendothelialization, and both are affected by the concentration of peptide on the surface. Additionally, the materials should promote endothelial cell attachment, but not elicit a response from platelets or leukocytes. Thus, the identity of the peptide will be important, and peptides related to RGD, such as REDV, may be more specific toward endothelial cells.42 Ultimately, the phenotype of endothelial cells on artificial surfaces must be similar to that found on their natural substrate, i.e., the subendothelium in blood flow, and this must be verified by the measurement of the concentrations of a number of secreted proteins and cell surface proteins. Finally, the materials tested in this study were not polymerized via

interfacial polymerization, and the interfacial polymerization process in the presence of peptide may have to be optimized. References and Notes (1) Hill-West, J. L.; Chowdhury, S. M.; Sawhney, A. S.; Pathak, C. P.; Dunn, R. C.; Hubbell, J. A. Prevention of postoperative adhesions in the rat by in situ photopolymerization of bioresorbable hydrogel barriers. Obstet. Gynecol. 1994, 83 (1), 59-64. (2) Pathak, C.; Sawhney, A.; Hubbell, J. Rapid Photopolymerization Of Immunoprotective Gels In Contact With Cells And Tissue, J. Am. Chem. Soc. 1993, 115, 2548-2548. (3) Lyman, M. D.; Melanson, D.; Sawhney, A. S. Characterization of the formation of interfacially photopolymerized thin hydrogels in contact with arterial tissue. Biomaterials 1996, 17, 359-364. (4) Sawhney, A. S.; Pathak, C. P.; Hubbell, J. A., Interfacial photopolymerization of poly(ethylene glycol)-based hydrogels upon alginatepoly(l-lysine) microcapsules for enhanced biocompatibility. Biomaterials 1993, 14,: 1008-1016. (5) Hern, D. L.; Hubbell, J. A. Incorporation of adhesion peptides into nonadhesive hydrogels useful for tissue resurfacing. J. Biomed. Mater. Res. 1998, 39, 266-276. (6) West, J. L.; Hubbell, J. A. Separation of the arterial wall from blood contact using hydrogel barriers reduces intimal thickening after balloon injury in the rat: the roles of medial and luminal factors in arterial healing. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13188-13193. (7) DiMilla, P. A.; Stone, J. A.; Quinn, J. A.; Albelda, S. M.; Lauffenburger, D. A. Maximal migration of human smooth muscle cells on fibronectin and type IV collagen occurs at an intermediate attachment strength. J. Cell Biol. 1993, 122, 729-737.. (8) Bloom, L.; Ingham, K. C.; Hynes, R. O. Fibronectin regulates assembly of actin filaments and focal contacts in cultured cells via the heparin-binding site in repeat III13. Mol. Biol. Cell 1999, 10, 1521-1536. (9) Massia, S. P.; Hubbell, J. A. Covalent surface immobilization of ArgGly-Asp- and Tyr-Ile-Gly-Ser-Arg- containing peptides to obtain well-defined cell-adhesive substrates. Anal. Biochem. 1990, 187, 292-301. (10) Dee, K.; Andersen, T.; Bizios, R. Osteoblast population migration characteristics on substrates modified with immobilized adhesive peptides. Biomaterials 1999, 20, 221-227. (11) Mann, B.; Tsai, A.; Scott-Burden, T.; West, J. Modification of surfaces with cell adhesion peptides alters extracellular matrix deposition. Biomaterials 1999, 20, 2281-2286. (12) Pakalns T, H. K.; Fields, G. B.; McCarthy, J. B.; Mooradian, D. L.; Tirrell, M. Cellular recognition of synthetic peptide amphiphiles in self-assembled monolayer films. Biomaterials 1999, 20, 2265-2279. (13) Lin HB, G. C.; Asakura S, Sun W, Mosher DF, Cooper SL, Endothelial-Cell Adhesion On Polyurethanes Containing Covalently Attached RGD-Peptides. Biomaterials 1992, 13, 905-914. (14) Bearinger, J. P.; Castner, D. G.; Healy, K. E. Biomolecular modification of p(AAm-co-EG/AA) IPNs supports osteoblast adhesion and phenotypic expression. J. Biomater. Sci. Polym. Ed. 1998, 9, 629-652. (15) Drumheller, P. D.; Hubbell, J. A. Polymer networks with grafted cell adhesion peptides for highly biospecific cell adhesive substrates. Anal. Biochem. 1994, 222, 380-388. (16) Ozeki, E.; Matsuda, T. Development of an artificial extracellular matrix. Solution castable polymers with cell recognizable peptidyl side chain. ASAIO Trans. 1990, 36, M294-M296. (17) Griffith, L. G.; Lopina, S. Microdistribution of substratum-bound ligands affects cell function: hepatocyte spreading on PEO-tethered galactose. Biomaterials 1998, 19, 979-986. (18) Maheshwari, G.; Brown, G.; Lauffenburger, D. A.; Wells, A.; Griffith, L. G. Cell adhesion and motility depend on nanoscale RGD clustering. J. Cell Sci. 2000, 113, 1677-1686. (19) Moghaddam, M.; Matsuda, T. Molecular Design Of 3-Dimensional Artificial Extracellular-Matrix-Photosensitive Polymers Containing Cell Adhesive Peptide. J. Polym. Sci., Part A: Polym. Chem. 1993, 31, 1589-1597. (20) Kao, W. J.; Hubbell, J. A. Murine macrophage behavior on peptidegrafted polyethyleneglycol-containing networks. Biotechnol. Bioeng. 1998, 59, 2-9. (21) Rowley, J. A.; Madlambayan, G.; Mooney, D. J. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 1999, 20, 45-53. (22) Neff, J. A.; Tresco, P. A.; Caldwell, K. D. Surface modification for controlled studies of cell-ligand interactions. Biomaterials 1999, 20, 2377-2393.

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Biomacromolecules, Vol. 2, No. 2, 2001 441 (34) Lo, C. M.; Wang, H. B.; Dembo, M.; Wang, Y. Cell movement is guided by the rigidity of the substrate. Biophys. J. 2000, 79, 144152. (35) West, J. L.; Hubbell, J. A. Photopolymerized hydrogel materials for drug delivery applications. React. Polym. 1995, 25, 139-147. (36) Ingber, D. E. Fibronectin controls capillary endothelial cell growth by modulating cell shape. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3579-3583. (37) Ingber, D. E.; Folkman, J. Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix. J. Cell Biol. 1989, 109, 317-330. (38) Pelham, R. J.; Wang, Y. Cell locomotion and focal adhesions are regulated by substrate flexivility. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 13661-13665. (39) Brandley, B.; Schnaar, R..L. Covalent Attachment of an Arg-GlyAsp Sequence Peptide to Derivatizable Polyacrylamide Surfaces: Support of Fibroblast Adhesion and Long-Term Growth. Anal. Biochem. 1988, 172, 270-278. (40) Massia, S. P.; Hubbell, J. A. An RGD spacing of 440 nm is sufficient for integrin alpha V beta 3- mediated fibroblast spreading and 140 nm for focal contact and stress fiber formation. J. Cell Biol. 1991, 114, 1089-1100. (41) Massia, S. P.; Hubbell, J. A. Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials. J. Biomed. Mater. Res. 1991, 25, 223-242. (42) Hubbell, J. A.; Massia, S. P.; Desai, N. P.; Drumheller, P. D. Endothelial cell-selective materials for tissue engineering in the vascular graft via a new receptor. Biotechnology (N.Y.) 1991, 9, 568-572.

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Conjugate Addition Reactions Combined with Free-Radical Cross

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