Synthesis and Characterization of Proteoglycan-Mimetic Graft

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Synthesis and Characterization of Proteoglycan-Mimetic Graft Copolymers with Tunable Glycosaminoglycan Density Laura W. Place, Sean M. Kelly, and Matt J Kipper Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm501045k • Publication Date (Web): 29 Aug 2014 Downloaded from http://pubs.acs.org on September 1, 2014

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Synthesis and Characterization of ProteoglycanMimetic Graft Copolymers with Tunable Glycosaminoglycan Density Laura W. Place,† Sean M. Kelly,‡ and Matt J. Kipper*,†,‡ †

School of Biomedical Engineering and ‡Department of Chemical and Biological Engineering,

Colorado State University, 1370 Campus Delivery, Fort Collins, Colorado, United States ABSTRACT Proteoglycans (PGs) are important glycosylated proteins found on the cell surface and in the extracellular matrix. They are made up of a core protein with glycosaminoglycan (GAG) side chains. Variations in composition and number of GAG side chains lead to a vast array of PG sizes and functions. Here we present a method for the synthesis of a proteoglycan-mimetic graft copolymers (or neoproteoglycans) with tunable GAG side-chain composition. This is done using three different GAGs, hyaluronan, chondroitin sulfate, and heparin. Hyaluronan is functionalized with a hydrazide-presenting linker. Either chondroitin sulfate or heparin is grafted by the reducing end on to the hyaluronan backbone through reductive amination. PG mimics with heparin or chondroitin sulfate side chains and four different ratios of GAG side chain result in graft copolymers with a wide range of sizes. The chemistry is confirmed through ATR-FTIR and 1

H NMR. Effective hydrodynamic diameter and zeta potential are determined using dynamic

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light scattering and electrophoretic mobility measurements. Graft copolymers were tested for their ability to bind and deliver basic fibroblast growth factor (FGF-2) to mesenchymal stem cells (MSCs). The chondroitin sulfate-containing graft copolymers successfully deliver FGF-2 to cells from surfaces. The lowest graft density of heparin-containing PG mimic also performs well with respect to growth factor delivery from a surface. This new method for preparation of GAGbased graft copolymers enables a wide range of graft density, and can be used to explore applications of PG mimics as new biomaterials with tunable biochemical and biomechanical functions.

INTRODUCTION Proteoglycans (PGs) are proteins with polyanionic glycosaminoglycan (GAG) side chains.1-2 PGs are found on the cell surface and in the extracellular matrix (ECM) in many tissues.3-4 PGs come in a wide range of sizes, with protein cores ranging in size from 20 to 400 kDa, and having from just one or two GAG side chains (e.g. decorin, biglycan), to over 100 side chains (aggrecan).5-6 The GAG side chains of PGs are linear polysaccharides bearing carboxylate and sulfate ionic groups, and are bound do the core protein via their reducing ends. Chondroitin sulfate (CS) and heparan sulfate (HS) are the dominant GAGs found in large PGs, such as perlecan, aggrecan, and versican (Figure 1).7-9 Perlecan may have up to four HS, CS or keratan sulfate (KS) side chains.10-11 Human versican has various isoforms that might have from zero to 23 CS chains, and potentially many more N-linked and O-linked oligosaccharides.7,

9, 12-13

Aggrecan can have over 100 GAG side chains, which are also mostly CS.12, 14 Smaller PGs also present HS (glypican) or both CS and HS (e.g. syndecans). These smaller PGs are organized on the surfaces of endothelial cells where they present a high density of HS and CS GAGs in the


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glycocalyx. Another important GAG is hyaluronan. Hyaluronan is a high-molecular-weight, nonsulfated GAG and binds N-terminal affinity sights on versican and aggrecan in the ECM, creating hierarchical GAG assemblies.15 The GAG heparin is generally found associated with serglycin in intracellular granules of mast cells.16 Heparin is structurally similar to HS. Because of its similarity to HS and commercial availability, heparin is often used in place of HS in experimental work.17 The structures of GAGs are shown in Figure 1.

Figure 1. Structures of common PGs found in the ECM. Protein backbones are drawn in black, with each different family of GAG chains shown in different colors. Structures of the disaccharide repeating unit of each GAG chain are shown in the Structure Key. Gal, galactose; GlcA, glucuronic acid; IdoA, iduronic acid; GlcNAc, glucosamine; GalNAc, galactosamine. Bold text indicates the locations of possible sulfate groups; X = H or SO3−, Y = COCH3 or


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SO3−.18 Reprinted with permission from reference 18. Copyright 2013 The Authors Journal compilation Copyright 2013 FEBS. PGs have important biochemical and biomechanical functions that are governed in part by their structure. The high negative charge density of the GAG side chains results in a high degree of counterion complexation and high osmotic pressure.19-20 The high charge density and consequent high hydration of PGs imparts compressive strength to cartilage and intervertebral disc, lubricates articulating surfaces, and organize the nanostructure of other ECM components through electrostatic binding.5,

21

Their pendent GAGs also play a variety of roles in cell

signaling. GAGs bind and stabilize cytokines forming a reservoir and protecting them from proteolytic degradation.6, 17, 22-23 They can also form GAG-cytokine and GAG-cytokine-receptor complexes that regulate cytokine signaling.15, 24-32 In the endothelial glycocalyx, the GAG side chains of PGs regulate small molecule transport, protein adsorption, and cellular interactions of blood components with the blood vessel wall. 5, 33-36 Because of these many biological functions, there is an increase in interest in the synthesis of PG mimics or ‘neoproteoglycans’ for tissue engineering applications and for biochemical studies.18, 37 In their recent review Weyers and Linhardt categorize PG mimics into three broad types: protein- or peptide-GAG complexes, nanoparticle-GAG complexes, and polymer-GAG complexes.18 Examples of peptide-GAG complexes are the ‘peptidoglycans’ synthesized by the Panitch group and their collaborators.38-41 They demonstrated that collagen-binding peptides modified with an average of one dermatan sulfate GAG side chain bind to collagen, delay ﬁbrillogenesis, and increase mechanical properties of a collagen gel and of aligned collagen threads.38, 40 This group has also shown that CS modified with HA-binding peptides can modify the mechanical properties of collagen-based scaffolds and alter their biodegradation.41-42 The HA


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binding mimics the behavior of the PG aggrecan. The second type of PG mimics, nanoparticleGAG complexes, includes GAGs bound to inorganic nanoparticles and nanoparticles formed by the electrostatic complexation of GAGs with polycations. Our group has created polyelectrolyte complex nanoparticles (PCNs) through electrostatic interactions of the GAGs heparin, HA, and CS with the polycationic polysaccharide chitosan and modified chitosans.43-45 CS- and heparinbased PCNs have similar size and composition to the PG aggrecan.45 These PCNs can stabilize and deliver the heparin-binding growth factor FGF-2 for up to 30 days, and even perform better than aggrecan at maintaining growth factor activity.45-46 As examples of the third type of PG mimics, GAG-polymer complexes, Sarkar et al. functionalized a terminal group on CS with vinyl monomers that can be polymerized to form brush-like copolymers, and demonstrated surface modification.47 Polymers with oligosaccharide pendent groups, might be considered a fourth type of PG mimic. Lee et al. prepared glycopolymers with pendent disaccharides and a terminal biotin whereby they can be bound to surfaces.37 These were readily adaptable to a microarray platform for protein binding studies.37 The strategies reviewed above enable access to a broad range of PG mimics with different relative amounts of peptide and GAG functional groups. The ‘peptidoglycans’ have a single GAG side chain bound to one or more functional peptides.38-41 The PCNs from our previous work behave as colloidal nanoparticles with a very high density of GAG chains and no peptide functional groups, but do not afford control over the GAG side chain density.43-45 The GAGpolymers and glycopolymers are well-suited to surface modification.37, 47 In the present work, we hypothesize that PG mimics can be synthesized with control over the degree of GAG side chain functionalization over a broad range using graft copolymers. In this approach, end-functionalized GAGs are covalently bound to a complementary pendant


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functional group on a core polymer to form PG mimics. The density of GAG side chains can be tuned to mimic the broad range of GAG substitution found in natural PGs, by controlling the stoichiometry. As the core polymer, we use a functionalized hyaluronan; we demonstrate this graft copolymer strategy using both 80 kDa chondroitin sulfate and a much smaller 14 kDa heparin as GAG side chains, attached to the core HA molecule via their reducing ends. The chemistry and solution behavior (hydrodynamic radius and zeta potential) of these PG mimics is examined. Finally we adsorb these PG mimics to chitosan-heparin and chitosan-chondroitin sulfate polyelectrolyte multilayers to bind and deliver the heparin-binding growth factor FGF-2.

MATERIALS AND METHODS Materials. Chondroitin sulfate sodium salt (CS; from shark cartilage, 6 % sulfur, 6 sulfate / 4 sulfate = 1.24, Mw = 84.3 kDa; PDI = 1.94), N,N-dimethylformamide (DMF; 99.8 %), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide

hydrochloride

(EDC;

98%),

N-hydroxysuccinimide (NHS; 98%), MES sodium salt (99 %), sodium chloride (99 %), sodium hydroxide (97 %), cysteamine hydrochloride (98 %), and sodium triacetoxyborohydride (STAB; 97 %) were purchased from Sigma-Aldrich (St. Louis, MO). Heparin sodium (Hep; from porcine intestinal mucosa, 12.5 % sulfur, Mw = 14.4 kDa; PDI = 1.14) was purchased from Celsus Laboratories (Cincinnati, OH). Sodium hyaluronate (HA; 95%, Mw = 740 kDa), 5,5’-dithio-bis[2-nitrobenzoic

acid]

(Ellman’s

reagent),

N-[ß-maleimidopropionic

acid]

hydrazide

trifluoroacetic acid salt (BMPH), seamless cellulose dialysis tubing (12 kDa MWCO), sodium acetate,

sodium

phosphate

dibasic,

sodium

phosphate

monobasic,

agarose,

tris(2-

carboxyethyl)phosphine hydrochloride (TCEP-HCl), and Zeba Spin Desalting Columns (10 mL, 7K MWCO) were purchased from Thermo Fisher Scientific (Waltham, MA). Biotech cellulose


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ester membrane dialysis tubing (300 kDa MWCO) was purchased from Spectrum Laboratories (Rancho Dominguez, CA). Glacial acetic acid was purchased from Acros Organics (Geel, Belgium). Recombinant human FGF basic (FGF-2) 146 aa (carrier free) was purchased from R&D Systems (Minneapolis, MN). Chitosan (Chi; Protosan UP B 90/20, 5 % acetylated determined by 1H NMR, Mw = 80 kDa; PDI = 1.52) was purchased from Novamatrix (Sandvika, Norway). Human fibronectin was purchased from BD Biosciences (Bedford, MA). 4′6 Diamidino-2-phenylindole•2HCl

(DAPI)

was

purchased

from

Thermo-Scientific

(Rockford, IL). CellTiter-Blue® Cell Viability Assay was purchased from Promega (Madison, WI). Fetal bovine serum (FBS), 0.25 % trypsin with EDTA, low-glucose Dulbecco’s modified Eagle’s medium (D-MEM), minimum essential medium alpha (α-MEM; supplemented with

L-glutamine,

ribonucleosides, and deoxyribonucleosides), and Dulbecco’s phosphate

buffered saline (DPBS) without Ca2+ and Mg2+ were purchased from HyClone (Logan, UT). Antibiotic-antimycotic (anti/anti), 1 M HEPES buffer solution, and Dulbecco’s phosphate buffered saline with Ca2+ and Mg2+ were purchased from Gibco (Grand Island, NY). A Millipore Synthesis water purification unit (Millipore, Billerica, MA) was used to obtain ultrapure; 18.2 MΩ•cm water (diH2O) used for making all aqueous solutions. Synthesis of PG mimics. There are three reactions in the synthesis of these graft copolymers, thiolation of HA to form the HA-SH intermediate, BMPH-modification of HA-SH to form the functionalized HA-BMPH intermediate, and GAG coupling via reductive amination. HA-SH intermediate. The procedure used by Damodaran et al. to thiolate dextran was adapted for thiolating HA.48 250 mg of HA was added to 50 mL of MES activation buffer (0.1 M MES, 0.5 M NaCl, pH 6.0) in a 45 °C oil bath and allowed to dissolve overnight. After the solution cooled to room temperature, EDC-HCl (0.645 g 10× molar excess relative to carboxylate


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functional groups on HA) and NHS (0.967 g) were added and allowed to react for two hours to activate the carboxylate functional group on HA (Scheme 1). Following the two hour activation step, the pH was raised to 7.2 using sodium hydroxide. Cysteamine hydrochloride (0.88 g, 23× molar excess compared to HA starting material) was added to the mixture and the reaction was allowed to continue for five hours. The reaction mixture was then dialyzed for five days (using 12 kDa MWCO dialysis tubing) against decreasing concentrations of NaCl (0.5 M, 0.25 M, 0.1 M, 0.05 M NaCl and finally to diH2O) to remove excess cysteamine hydrochloride and EDC. Following dialysis, the product was lyophilized to recover a solid, thiolated HA (HA-SH). This intermediate was analyzed by Ellman’s reagent test to confirm the degree of thiolation, and characterized by 1H NMR and FTIR, described below. Unmodified HA has a 1H NMR spectrum similar to that reported by Pomin49: 1H NMR (D2O, 400 MHz): δ 4.70-4.30 (m, HC1), 3.95-3.25 (m, HC2-6), 2.02 (s, -C(O)-CH3). HA-SH intermediate48: 1H NMR (D2O, 400 MHz): δ 4.65-4.35 (m, HC1), 3.95-3.25 (m, HC2-6), 3.25-3.15 (m, -CH2-CH2-SH), 2.94-2.88 (m, -CH2-SH), 2.03 (s, -C(O)-CH3). Scheme 1. Synthesis of HA-SH intermediate.

HA-BMPH intermediate. HA-SH was reacted with TCEP to reduce disulfide bonds, by dissolving 100 mg of HA-SH in 20 mL of phosphate buffered saline (PBS) (0.1 M sodium phosphate, 0.15 M NaCl, pH 8). TCEP (114 mg) was dissolved in the solution and the reduction was carried out for one hour. After one hour, the reaction solution was passed through desalting


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columns to exchange the reaction solution with PBS (0.1M sodium phosphate, 0.15M NaCl, pH 7.2). Next, 73 mg of BMPH was added to the solution and the coupling reaction proceeded for two hours at room temperature. The solution was then dialyzed as in the thiolation reaction to remove any unreacted BMPH and lyophilized to obtain solid HA-BMPH intermediate. The HABMPH intermediate was characterized by 1H NMR (D2O, 400 MHz) and FTIR. HA-BMPH intermediate: 1H NMR (D2O, 400 MHz): δ 3.95-3.15 (m, HC2-6 and -CH2-CH2-S-), 3.25-3.15, 2.9-2.85 (m, -CH2-S-), (m, 2.48-2.40 (m, N-CH2-), 2.18-2.09 (m, -CH2-C(O)-hydrazide) 2.03 (s, -C(O)-CH3). (See Figure SI.3 in Supporting Information.)

Scheme 2. Synthesis of HA-BMPH intermediate

Graft copolymers. Eight different compositions of graft copolymers were synthesized using four different ratios of either CS or heparin to HA-BMPH. We nominally refer to these as 1:1, 1:3, 1:10, and 1:30 graft copolymers, where the 1:1 ratio indicates the highest graft density synthesized by adding a stoichiometric amount of CS or heparin to the HA-BMPH (one polymer chain per thiol group in the HA-SH intermediate). The 1:30 graft copolymers have only one thirtieth of the stoichiometric amount of CS or heparin added. The reductive amination chemistry used for the coupling was adapted from Sisu et al. and Dalpathado et al (Scheme 3).50-51 A


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similar chemistry has also been used to functionalize collagen surfaces with oligosaccharides.52 For each reaction, HA-BMPH (15 mg) and either CS or heparin were placed into a round-bottom flask, the flask was sealed, and 10 mL of anhydrous DMF were added. The flask was then purged with nitrogen. After purging, 350 µL of acetic acid was added and the reaction vessel was step-wise heated from 25 °C to 85 °C in 10 °C increments over a period of two hours. During this time, 1 g of sodium triacetoxyborohydride (STAB, 100× molar excess) was dissolved in 10 mL of anhydrous DMF in a separate round-bottom flask and purged with nitrogen. After the two hour heating, 356 µL of STAB was added to the reaction vessel, and every two hours for a total of ten hours (1.78 mL of STAB). The reaction was allowed to run overnight. After reaction, the solution was heated to 60 °C under vacuum to remove DMF, dissolved in diH2O and dialyzed as described above, then lyophilized and the recovered product was a white powder. Scheme 3. Coupling CS or heparin to HA-BMPH to form graft copolymers.

Chemical Characterization of PG Mimics. Neat polymers, synthesis intermediates, and PG mimics with different GAG ratios were analyzed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and proton nuclear magnetic resonance (1H NMR). ATR-FTIR was performed on a Nicolet 6700 spectrometer (Thermo Electron Corporation, Madison, WI) using a Smart iTR ATR sampling accessory with a ZnSe crystal to collect spectra


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from 4000 - 650 cm−1. For NMR, samples were dissolved in D2O at concentrations of 1-5 mg mL−1 depending on sample solubility. Spectra were collected (64 scans, 5 seconds relaxation time, and 25 °C) on a 400 MHz spectrometer (Agilent Varian 400MR) equipped with automated tuning and a 7600 sample changer. DLS and Zeta Potential. Hydrodynamic diameter of the graft copolymers and the neat polymers was measured by dynamic light scattering (DLS) using a 90Plus/BI-MAS (Brookhaven Instruments, Holtsville, NY). All samples were dissolved in PBS at 5 mg mL−1. Measurements were taken at 25 °C at a fixed angle of 90°, 1 min per measurement. For zeta potential, samples were dialyzed against diH2O (300 kDa MWCO) for 24 hours to remove any unreacted polymer, lyophilized and dissolved in PBS at 5 mg mL−1. The zeta potential was then measured using the same instrument as in DLS, via electrophoretic light scattering (ELS). Each sample was measured five times, 30 scans each, at 25 °C and the values reported are the mean zeta potential for each sample ± the standard error of the mean. Cell Harvest and Culture. The cell harvesting and expansion procedure is described in detail in our previous work.53 Bone marrow aspirates from the iliac crest of female sheep were centrifuged. The supernatant containing the nucleated cells was mixed with growth media (lowglucose D-MEM with 10 % FBS, 1 % anti/anti, and 2.5 % HEPES) and seeded into culture flasks. After 24 h, the media was changed to remove all non-adherent cells. The marrow stromal cell (MSC) colonies were allowed to grow for at least seven days, then the cells were trypsinized, counted, and reseeded in culture flasks using maintenance media for culture expansion (α-MEM with 10 % FBS, 1 % anti/anti, and 2.5 % HEPES). MSCs were cryo-preserved prior to seeding into experimental conditions. Cells were not used beyond the fifth passage.


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Preparing Surfaces for FGF Delivery. To test FGF-2 delivery from graft copolymers, a tissue culture polystyrene (TCPS) 96-well plate was coated with chitosan-heparin or chitosan-CS polyelectrolyte multilayers (PEMs) with chitosan, heparin, CS or different compositions of graft copolymers as the terminal layer as illustrated in Scheme 4. The PEM preparation was adapted from previous methods established in our laboratory for preparation of GAG-modified surfaces.43, 46, 54-56 The layer-by-layer deposition of PEMs is a facile method to coat a surface whereby the surface is exposed to alternating solutions containing oppositely charged polyelectrolytes until a desired thickness or number of layers is deposited. Chitosan, heparin, CS and graft copolymer solutions were prepared in acetate buffer (0.2 M, pH 5). Chitosan and heparin were dissolved at a concentration of 0.01 M (on a saccharide unit basis); CS was prepared at a concentration of 0.02 M. A rinse solution was prepared by adjusting the pH of diH2O to 4 with acetic acid. Chitosan, heparin, CS, and rinse solutions were filtered with a 0.22 µm polyvinylidene fluoride (PVDF) syringe filters (Fisher Scientific). Graft copolymers were dialyzed (300 kDa MWCO) against diH2O for 24 hours and lyophilized, then were dissolved at 5 mg mL−1. PEM construction was done by alternating adsorption and rinse steps; 100 µL of solution was added to each well and adsorbed under gentle agitation for five minutes. Five layers of either chitosan-heparin or chitosan-CS were adsorbed, the sixth layer contained either heparin, CS, or graft copolymer; chitosan-terminated samples were constructed with seven layers. Each condition shown in Scheme 4 was prepared in triplicate. Surfaces were then sterilized with 70 % ethanol for 15 minutes, and rinsed with sterile PBS prior to protein adsorption and cell seeding.


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Scheme 4. Conditions used to evaluate FGF-2 delivery using PG-mimics.

PEMs were prepared with and without adsorbed FGF-2. The experimental conditions included surfaces with no FGF-2 as negative controls and FGF-2 delivered in solution as positive controls FGF-2 adsorption was performed according to Almodóvar et al.53 Briefly, 100 µL (100 ng mL−1 in diH2O) was placed in each well of the 96-well plate and allowed to adsorb for two hours under gentle agitation. The FGF-2 solution was then aspirated, and the wells were rinsed with sterile PBS. We have previously shown that this results in a mitogenic dose of FGF-2 adsorbed to heparin-modified surfaces.53 Prior to cell seeding, all surfaces were coated with fibronectin (10 µg mL−1 in diH2O); 100 µL was placed in each well of the 96-well plate for one hour under static conditions.53 The fibronectin solution was then aspirated and the surfaces were rinsed with sterile PBS. MSC Response to FGF-2, Graft Copolymers, and FGF-2 bound to Graft Copolymers. MSCs were seeded at a density of 7000 cells cm−2 in α-MEM (10 % FBS) and allowed to attach for three hours. After three hours the media was aspirated and replaced with low-serum media


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(2.5 % FBS). We have previously determined that under these conditions ovine MSCs survive, but do not proliferate over four days unless stimulated by FGF-2.45-46,

53

In this low-serum

condition, the optimal mitogenic dose of FGF-2 for recovering the cell proliferation is 1 ng mL−1. Hence, these conditions have been established by our lab as a means of evaluating the function of FGF-2. Cultures were maintained for four days, with a medium change on day two with FGF-2 (1 ng mL−1) included in the media change for positive control samples, no additional FGF-2 was provided for samples with FGF-2 adsorbed on the surfaces. To evaluate FGF-2 delivery CellTiter-Blue Cell Viability Assay was performed on day two and day four. Additionally, on day four, cultures were evaluated via microscopy. Cells were fixed with glutaraldehyde (2 % in DPBS with Ca2+ and Mg2+) for 45 minutes at 4 °C, and then stained with DAPI (1 µg mL−1 in DPBS with Ca2+ and Mg2+) for 15 minutes at room temperature. The wells were rinsed with DPBS with Ca2+ and Mg2+ between each step, and protected from light. After fixing and staining, surfaces were stored dry, protected from light, at 4 °C until microscopy was performed. Images were obtained using a 4× objective on an Olympus IX70 epi-fluorescence microscope (Center Valley, PA) equipped with a QImaging Micropublisher camera with an appropriate filter for DAPI. Statistics. Data analysis was performed using Minitab (Minitab, Inc., State College, PA), version 16. For the CellTiter-Blue assay testing comparisons between groups were performed via analysis of variance (ANOVA) models with Tukey’s multiple comparison tests. Differences with p < 0.05 were considered statistically significant. CellTiter-Blue Data are expressed as the mean ± standard error of the mean (n = 3).

RESULTS AND DISCUSSION


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Chemical Characterization. The HA-SH intermediate had limited solubility in D2O, likely caused by increased hydrophobicity and potentially by some disulfide bond formation. This prohibited precise determination of the degree of thiolation by 1H NMR. Instead, Ellman’s reagent was used to calculate thiol content on the HA-SH intermediate. Approximately 50 % of the carboxylate groups, present on each disaccharide of HA, were modified with thiol. This result was the basis used to determine stoichiometric ratios of GAG side chain to available grafting sites on HA. The 1:1 ratio is one CS or heparin chain for every one thiol group on the HA-SH intermediate and 1:30 is one CS or heparin chain for every 30 thiols on the HA-SH intermediate. This notation will be used throughout the results. ATR-FTIR was used to characterize the chemistry of the HA-SH and HA-BMPH intermediates (Figure SI.1 in Supporting Information) and all eight of the graft copolymers (Figure 2). Figure 2 (A) shows an example of the FTIR peak assignments for the heparin 1:30 graft copolymer. The peak assignments are summarized in Table S.1 in the Supporting Information. Figure 2 (B and C) shows FTIR spectra of all of the copolymers stacked so that the changes with copolymer composition can be observed.


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Figure 2. (A) FTIR spectrum of the heparin 1:30 graft copolymer. The peak assignments are listed in Table S.1 of the Supporting Information. FTIR spectra of (B) heparin-containing and (C) CS-containing graft copolymers, shown with the spectra of the constituent neat polysaccharides. In B arrows indicate decreasing (down arrows) and increasing (up arrows) intensities of absorptions associated with the linker and the sulfate groups, respectively with


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increasing graft density. In both A and B the relative amount of the sulfated GAG graft (heparin or CS) increases from bottom to top. Note that reciprocal scaling is used on the wavenumber abscissa. All of the spectra contain the expected absorptions from the GAGs: broad OH stretching near 3280 cm−1, carbonyl stretching between 1600 and 1700 cm−1, amide N-H bending and OH bending near 1560 cm−1 and 1373 cm−1, respectively, ether stretching near 1150 cm−1, and the saccharide ring between 1000 and 1100 cm−1. Notable differences among the neat GAGs are that the hyaluronan lacks the very strong sulfate stretches between 1200 and 1250 cm−1 and near 980 cm−1 that are characteristic of the sulfated GAGs. The heparin spectrum is also much narrower in the carbonyl stretching region, exhibiting primarily the antisymmetric COO− stretch at 1611 cm−1, but no discernable amide at higher wavenumber. The spectra of the graft copolymers provide evidence of increasing graft density. These are marked with the arrows in Figure 2 B. The graft copolymer spectra all contain sharp CH2 stretches associated with the methylene groups in the cysteamine and BMPH; these absorptions are attenuated as the graft density increases (down arrows in Figure 2 B). The heparin-containing graft copolymers also exhibit a sharp absorption at 1537 that is not present in the neat GAGs. This absorption is likely associated with the hydrazide. However, in the CS-containing copolymers, the stronger and broader amide N-H bending near 1560 cm−1 makes this peak at 1537 cm−1 unresolvable. For both the heparin- and CS-containing graft copolymers the strong sulfate stretching absorptions at 1227 cm−1 and 987 cm−1 increase with increasing graft density (up arrows in Figure 2 B). To confirm stability of the graft copolymer coupling, FTIR spectra of copolymers before and after dialysis (300 kDa cut off) were obtained for the lowest (1:30) and highest (1:1) graft densities. Spectra in the region showing the sulfate and saccharide ring


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vibrations for the highest graft density (1:1) copolymers are shown in Figure 3. Dialysis for 24 hours against diH2O does not reduce the sulfate absorptions at 1227 cm−1 or 987 cm−1 indicating that the heparin and CS conjugation to the hyaluronan is successful. In fact the OH bending and sulfate stretching absorptions at (1373 and 1227 cm−1) are stronger after dialysis and lyophilization. Servaty et al. clearly demonstrates that these absorptions are sensitive to the amount of bound water.57 Therefore, their increase is likely caused by counterion exchange reducing the amount of bound water changing their hydrogen bonding environment.57 Similar spectra of the 1:30 copolymers show slight reductions in the sulfate absorptions for the heparin copolymer and no changes for the CS copolymer, after dialysis (not shown).

Figure 3. ATR FTIR spectra of the CS and heparin 1:1 copolymers before and after dialysis using 300 kDa cut off dialysis membranes to remove uncomplexed heparin or chondroitin sulfate, confirming stable addition of the graft copolymers.


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To confirm the change in composition with increasing graft density, the ATR-FTIR spectra for the graft copolymers were fit to a sum of hyaluronan and the corresponding GAG (heparin or CS) FTIR spectra, using a linear least squares algorithm, over the range from 950 to 1500 cm−1. The ratios of the GAG to hyaluronan spectra obtained from these fits are plotted in Figure 4 (A). These should not be interpreted as a precise determination of the copolymer composition, but they do illustrate the relative increase of GAG in the copolymer as the graft density increases. Figure SI.2 in the Supporting Information shows the fits for the heparin copolymers and their residuals. Residuals show evidence of the hydrazide at 1537 cm−1 and consumption of the carboxylic acid in hyaluronan at 1611 cm−1. 1H NMR of the graft copolymers also shows evidence of increasing ratio of GAG to hyaluronan with increasing graft density. (See Figure SI.3 in the supporting information for example spectra.)

Figure 4. (A) Spectral ratios from the fit of graft copolymers’ ATR-FTIR spectra to the sum of GAG and hyaluronan spectra. See Figure SI.2 in the Supporting Information, for fits. (B.) Zeta


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potential of graft copolymers. (C.) Histograms of heparin, CS, hyaluronan (“HA”), and graft copolymer hydrodynamic diameters obtained from DLS measurements. Top plot is heparin graft copolymers; bottom plot is CS graft copolymers.

Zeta Potential and Light Scattering. The GAGs hyaluronan, CS and heparin all have negative zeta potentials (-23 ± 0.9, -22 ± 1.3, and -26 ± 2 mV, respectively). The change in zeta potential with grafting density is shown in Figure 4 (B.). For the graft copolymers, the zeta potential becomes more negative with increasing CS or heparin graft density. DLS was conducted to determine the effective hydrodynamic diameter of each copolymer type and the neat GAGs. Histograms of the hydrodynamic diameters for the neat GAGs and the graft copolymers are shown in Figure 4 (C.). Hydrodynamic diameters increase with increasing graft density for both the CS- and heparin-containing graft copolymers (from about 60-120 nm for the 1:30 graft copolymers to about 400-700 nm for the 1:1 graft copolymers), which is comparable to that of aggrecan.58 Changes in graft copolymer zeta potential and size are consistent with increasing graft density of the graft copolymers. MSC Response to FGF-2 delivered in solution or adsorbed to surfaces. Graft copolymers were tested for their ability to deliver FGF-2 to MSCs when adsorbed to surfaces. Surfaces were first coated with either chitosan-chondroitin sulfate or chitosan-heparin polyelectrolyte multilayers. We have previously characterized these multilayers extensively and used them to deliver FGF-2 to MSCs.53, 59 The coatings were prepared with the terminal layer being, chitosan (“CS Chi” and “Hep Chi”), CS, or heparin (“Hep”), or one of the copolymers (“CS 1:1”, “CS 1:30”, “Hep 1:1”, or “Hep 1:30”), as illustrated in Scheme 4. FGF-2 was then adsorbed to surfaces of each type. MSCs were seeded onto these surfaces, onto surfaces without FGF-2


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adsorbed, but with FGF-2 delivered in solution, and onto surfaces with no FGF-2, and cultured for four days. Experiments for all conditions were performed in triplicate. Samples were evaluated for cell activity via CellTiter-Blue Cell Viability Assay and stained with DAPI for imaging. The CellTiter-Blue results are displayed in Figure 5.

Figure 5. CellTiter-Blue data for FGF-delivery to MSCs on (top row) CS-containing and (bottom row) heparin-containing surfaces. “*” indicates that the response is higher than the same condition with no FGF-2. “†” indicates that the response is higher than the Hep Chi, Hep, and Hep 1:1 conditions with the same mode of FGF-2 delivery. When no FGF-2 is delivered the cells survive, but do not proliferate over four days in this lowserum condition, consistent with our previous reports.45-46, 53 Cells also tend to respond relatively


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poorly to the surfaces that are terminated with chitosan (“Hep Chi” and “CS Chi” in Figure 5). Surfaces terminated with CS, the CS graft copolymers and the Hep 1:30 graft copolymers all support cell proliferation in response to FGF-2 delivered either in solution or by adsorption to the surfaces. In the CS case, the CS-terminated, CS 1:1-terminated, and CS 1:30-terminated surfaces do not perform any differently from each other, and on day 4 all perform better than the Chi-terminated samples. Additionally, all of these surfaces exhibit higher cell activity than their counterparts with no FGF-2. This indicates that both graft copolymer densities are able to present FGF-2 with similar effectiveness as CS coated on the surface and can successfully deliver FGF-2 to MSCs as well as delivery in solution. It is important to note that when FGF-2 is presented in solution, the FGF-2 is also added when the media is changed on day 2. However, in the conditions where FGF-2 is adsorbed, no additional FGF-2 is added during the media change on day 2. The surfaces containing heparin generally exhibit lower cell activity than the CS surfaces. However, in the Hep 1:30-terminated case activity levels are similar to those seen on CS surfaces. Additionally, FGF-2 adsorbed to the Hep 1:30 demonstrates similar levels as FGF-2 delivered in solution. Heparin has been reported to interfere with cell attachment.45, 53 Low cell attachment could result in the lower levels of cell activity observed here. The Hep 1:30terminated surface may contain less heparin than either the Hep or Hep 1:1 conditions resulting in higher initial cell adhesion, while still being capable of binding FGF-2 and presenting it to the cells. These results suggest that both grafting densities on the CS copolymer and the Hep 1:30 copolymer are able to bind FGF-2 and enhance cell activity. Alternatively, it is possible that the surfaces bind similar amounts of FGF-2, but the Hep 1:30 better presents the FGF-2 to the cell surface receptor.


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After culture, cells were fixed and stained with DAPI for imaging to further support the CellTiter-Blue data. The results from microscopy are in agreement with the cell activity assay. Images of the CS 1:30-terminated and the Hep 1:30-terminated samples with no FGF-2, FGF-2 delivered in solution, and FGF-2 adsorbed to the surface are displayed in Figure 6. Very few cells are seen in the samples with no FGF-2, and samples with FGF-2 delivered in solution look similar to those with FGF-2 adsorbed. This increase in cell number in the presence of FGF-2 indicates that the FGF-2 is active both when adsorbed to graft copolymers and when delivered in solution.


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Figure 6. Microscopy of cells cultured for four days on 1:30 ratio graft copolymer surfaces (A.) CS 1:30 no FGF-2, (B.) CS 1:30 FGF-2 delivered in solution, (C.) CS 1:30 FGF-2 adsorbed, (D) Hep 1:30 no FGF-2, (E.) Hep 1:30 FGF-2 delivered in solution, (F.) Hep 1:30 FGF-2 adsorbed. These results are similar to those shown previously by our group with the use of GAG-rich PCNs. We have bound FGF-2 to GAG-rich PCNs and delivered them in solution and from electrospun nanofibers to MSCs. The MSCs exhibited upregulated cellular activity when exposed to FGF-2 delivered from our PCNs and in some cases when delivered in solution.45-46 Delivery of growth factors in solution in vivo is impractical. Growth factors have a short half-life in the body on the order of minutes.45, 60-61 In our previous work we demonstrated that while FGF-2 delivered in solution lost activity over time, our GAG-rich PCNs were able to sustain activity over 30 days.45-46 The graft copolymers described in this work exhibit the ability to bind FGF-2 and have the potential for incorporation into an implant for delivery of growth factor to sites of healing.

CONCLUSIONS PGs are an important class of biomolecules with a wide range of structures and functions, related to their substitution with different numbers and types of GAG side chains.6, 18 Recently, interest in the development of PG mimics or ‘neoproteoglycans’ has led to a number of proposed applications of GAG-presenting materials in tissue engineering, drug delivery, biosensors, and biochemical

studies.

Neoproteoglycans

include

‘peptidoglycans’,

glycopolymers,

and

nanoparticle assemblies. These approaches do not afford control over the GAG functionality over a broad range of degree of GAG substitution. The peptidoglycans have peptide functionality attached to approximately one GAG chain.38,

40

Glycopolymers do not have high-molecular


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weight GAGs, like PGs, and GAG-nanoparticle complexes have a very high but relatively uncontrolled degree of GAG functionality. The aim of this work was to develop a new platform for development of PG mimics that enables formation of graft copolymers with a wide range of GAG side chain densities. The ability to tune graft density and to use different GAG side chains enables us to mimic a wide range of large ECM proteoglycans, which contain CS and HS. The heparin-based PG mimics may also be similar to the heparin-rich intracellular PG serglycin. The method presented here results in graft copolymers that mimic PGs by binding the reducing end of GAGs to a functionalized core polymer in different ratios. We have demonstrated that we can create stable graft copolymers with a broad range of graft densities. This strategy is demonstrated here using both a short, highly charged GAG (14 kDa heparin) and a larger GAG (80 kDa CS), both coupled to the same core polymer – a hydrazide-functionalized hyaluronan. We also demonstrate that these PG mimics can present the heparin-binding growth factor, FGF2, to cells from surfaces. In our FGF-2 activity assay the surfaces terminated with CS or the two CS-containing graft copolymers behaved similarly; cells on all three types of surfaces responded to FGF-2 either adsorbed to the surfaces or delivered in solution. The advantage of the surfaceadsorption is that the FGF-2 presentation can be sustained, and does not need to be included as a supplement during media changes. This could provide a model for the delivery of heparinbinding growth factors from the surfaces of implanted devices, which might need to present sustained cytokine signals to nearby cells. In the FGF-2 delivery study, the heparin-containing PGs demonstrate a potential advantage of the graft copolymer structure. While the CS-containing PGs performed similarly with respect to FGF-2 delivery as surfaces terminated with CS, the surface terminated with 1:30 heparincontaining graft copolymers performed better than either the surfaces terminated with 1:1


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heparin graft copolymer or heparin. This suggests that tuning the graft density can influence the biochemical properties of these mimics. Further work is needed to more fully characterize the structure of these surfaces, so that the structure-function relationships related to cell adhesion and growth factor delivery can be elucidated. In this work, we have focused on tuning the graft density of PG mimics. The protein cores of PGs are also important for governing some of their biochemical and biomechanical functions. For example, binding to other components of the ECM, susceptibility to enzymatic cleavage, and presentation on cell surfaces are functions that can all be potentiated by the protein core. The reductive amination chemistry that we use to bind the GAGs to modified hyaluronan in this work is similar to chemistry that has been used to end-functionalize oligosaccharides, and to glycosylate proteins.52 The strategy presented here could be readily expanded to aminecontaining peptide and protein core molecules. Supporting Information. ATR-FTIR spectra of the HA-SH and HA-BMPH intermediates; Table of FTIR peak assignments; ATR-FTIR spectra of the heparin-containing graft copolymers, showing linear least squares fits to obtain the spectral ratios; 1H NMR of hyaluronan, CS, and the CS-containing copolymers and confirmation of the graft copolymer coupling. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Fax: 970-491-7369; Tel.:970-491-0870; email: [email protected]. ACKNOWLEDGMENT


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We thank Prof. Patrick A. Johnson at the University of Wyoming for use of the dynamic light scattering and zeta potential instrument. We thank Prof. Melissa Reynolds for assistance with the FTIR spectroscopy, and Prof. Travis Bailey for helpful discussions. Funding for this work was provided by the National Science Foundation (DMR 0847641).

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Table of Contents Image:


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Figure 1. Structures of common PGs found in the ECM. Protein backbones are drawn in black, with each different family of GAG chains shown in different colors. Structures of the disaccharide repeating unit of each GAG chain are shown in the Structure Key. Gal, galactose; GlcA, glucuronic acid; IdoA, iduronic acid; GlcNAc, glucosamine; GalNAc, galactosamine. Bold text indicates the locations of possible sulfate groups; X = H or SO3−, Y = COCH3 or SO3−.18 Reprinted with permission from reference 18. Copyright 2013 The Authors Journal compilation Copyright 2013 FEBS. 81x100mm (200 x 200 DPI)


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Scheme 1. Synthesis of HA-SH intermediate. 161x27mm (300 x 300 DPI)


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Scheme 2. Synthesis of HA-BMPH intermediate 159x50mm (300 x 300 DPI)


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Scheme 3. Coupling CS or heparin to HA-BMPH to form graft copolymers. 173x53mm (300 x 300 DPI)


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Scheme 4. Conditions used to evaluate FGF-2 delivery using PG-mimics. 77x39mm (300 x 300 DPI)


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Biomacromolecules

Figure 2. (A) FTIR spectrum of the heparin 1:30 graft copolymer. The peak assignments are listed in Table S.1 of the Supporting Information. FTIR spectra of (B) heparin-containing and (C) CS-containing graft copolymers, shown with the spectra of the constituent neat polysaccharides. In B arrows indicate decreasing (down arrows) and increasing (up arrows) intensities of absorptions associated with the linker and the sulfate groups, respectively with increasing graft density. In both A and B the relative amount of the sulfated GAG graft (heparin or CS) increases from bottom to top. Note that reciprocal scaling is used on the wavenumber abscissa. 169x174mm (300 x 300 DPI)


Biomacromolecules

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Figure 3. ATR FTIR spectra of the CS and heparin 1:1 copolymers before and after dialysis using 300 kDa cut off dialysis membranes to remove uncomplexed heparin or chondroitin sulfate, confirming stable addition of the graft copolymers. 106x140mm (600 x 600 DPI)


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Biomacromolecules

Figure 4. (A) Spectral ratios from the fit of graft copolymers’ ATR-FTIR spectra to the sum of GAG and hyaluronan spectra. See Figure SI.2 in the Supporting Information, for fits. (B.) Zeta potential of graft copolymers. (C.) Histograms of heparin, CS, hyaluronan (“HA”), and graft copolymer hydrodynamic diameters obtained from DLS measurements. Top plot is heparin graft copolymers; bottom plot is CS graft copolymers. 92x52mm (600 x 600 DPI)


Biomacromolecules

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Figure 5. CellTiter-Blue data for FGF-delivery to MSCs on (top row) CS-containing and (bottom row) heparincontaining surfaces. “*” indicates that the response is higher than the same condition with no FGF-2. “†” indicates that the response is higher than the Hep Chi, Hep, and Hep 1:1 conditions with the same mode of FGF-2 delivery. 118x83mm (300 x 300 DPI)


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Biomacromolecules

Figure 6. Microscopy of cells cultured for four days on 1:30 ratio graft copolymer surfaces (A.) CS 1:30 no FGF-2, (B.) CS 1:30 FGF-2 delivered in solution, (C.) CS 1:30 FGF-2 adsorbed, (D) Hep 1:30 no FGF-2, (E.) Hep 1:30 FGF-2 delivered in solution, (F.) Hep 1:30 FGF-2 adsorbed. 126x144mm (600 x 600 DPI)


Biomacromolecules

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34x13mm (300 x 300 DPI)


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Synthesis and Characterization of Proteoglycan-Mimetic Graft

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