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Biomimetic Proteoglycans Mimic Macromolecular Architecture and Water Uptake of Natural Proteoglycans Katsiaryna Prudnikova, Robert W Yucha, Pavan Patel, Alicia Kriete, Lin Han, Lynn S. Penn, and Michele S. Marcolongo Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00032 • Publication Date (Web): 11 Apr 2017 Downloaded from http://pubs.acs.org on April 12, 2017

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Biomimetic Proteoglycans Mimic Macromolecular Architecture and Water Uptake of Natural Proteoglycans Katsiaryna Prudnikova†, Robert W. Yucha§⊥, Pavan Patel§, Alicia Kriete†, Lin Han§, Lynn S. Penn⁄⁄, Michele S. Marcolongo†*. †

Department of Material Science and Engineering, Drexel University, 3141 Chestnut Street, Philadelphia, Pennsylvania 19104, United States §

School of Biomedical Engineering, Science & Health Systems, Drexel University, 3141 Chestnut Street, Philadelphia, Pennsylvania 19104, United States

⁄⁄

Department of Chemistry, Drexel University, 3141 Chestnut Street, Philadelphia, Pennsylvania 19104, United States

Keywords: Proteoglycans, chondroitin sulfate, biomimetic, bottlebrush.

ABSTRACT

Aging and degeneration of human tissue come with the loss of tissue water retention and associated changes in physical properties partially due to degradation and subsequent loss of proteoglycans. We demonstrated a novel method of fabrication of biomimetic proteoglycans,

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which mimic the three-dimensional bottlebrush architecture and physical behavior of natural proteoglycans responsible for tissue hydration and structural integrity. Biomimetic proteoglycans are synthesized by an end-on attachment of natural chondroitin sulfate bristles to a synthetic poly(acryloyl chloride) backbone. Atomic force microscopy imaging suggested threedimensional core-bristle architecture, and hydrodynamic size of biomimetic proteoglycans was estimated at 61.3±12.3 nm using dynamic light scattering. Water uptake results indicated that biomimetic proteoglycans had a ~50% increased water uptake compared to native aggrecan and chondroitin sulfate alone. The biomimetic proteoglycans are cytocompatible in the physiological ranges of concentrations and could be potentially used to repair damaged or diseased tissue with depleted proteoglycan content.

Introduction Proteoglycans (PGs) make up a family of nano-scale biomacromolecules present in almost every tissue and are composed of negatively charged glycosaminoglycan (GAG) bristles attached to a protein core in a bottle-brush configuration.1-3 PGs differ in their architecture, such as protein core length and number of GAG bristles attached, and perform various biological functions including modulation of cell growth4,

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and collagen fibrillogenesis6,

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as well as

regulation of signal transduction.8, 9 Composing, in part, the extracellular matrix (ECM) in soft and connective tissues and integrating with other ECM proteins, PGs are also critical for maintaining tissue structural integrity as well as providing hydration and swelling pressure through charged GAGs, thus enabling tissue to withstand compressive loads.1, 10-14 The effect of PG macromolecular architecture on tissue hydration and mechanics can be understood in the context of cartilage and intervertebral disc, where electrostatic repulsion between densely spaced


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chondroitin sulfate chains on opposing proteoglycan aggrecan molecules contributes ~50% of tissue compressive stiffness.15-17 In natural tissue remodeling, proteoglycans are enzymatically digested along the protein core.13, 18-21 However during degeneration, the enzymatic digestion of proteoglycans outpaces the cellular synthesis, leading to loss of these important matrix macromolecules, which can result in a host of mechanical, hydration and nutritional deficits to tissue function that, in turn, are manifested clinically as degenerative conditions including arthritis, intervertebral disc degeneration, skin aging and urinary incontinence, among others.10, 22-24 In an attempt to restore proteoglycan content to the matrix, researchers have used tissue engineering25-27, genetic engineering28-31 and growth factors32-37 to stimulate intracellular proteoglycan synthesis, in addition to enzymatic inhibitors38-40 to suppress further degradation of the macromolecules in the matrix. While intriguing, these technologies are still not widely used clinically. In a different approach, researchers have invented novel materials that could introduce proteoglycan functionality into the tissue. For example, synthetic polymers and nanoparticles have been modified with sugar residues to achieve glycan functionality41-43 as the latter plays a significant biological role in cell communication and growth factor activity. To mimic elegant natural processes or molecular hierarchical applications, substantial work has been performed on GAG-based macromolecules and scaffolds. Elisseeff’s group has developed a novel tissue bioadhesive based on chondroitin sulfate functionalized with methacrylate and aldehyde groups in order to chemically attach it to tissue proteins.44, 45 Aggrecan mimics have been developed by Panitch and co-workers where hyaluronic acid (HA) binding peptide was conjugated to functional groups along chondroitin sulfate (CS) chains in order to facilitate attachment of CS molecules to HA.46 HA is a high-molecular weight nonsulfated polysaccharide which is


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abundant in connective tissue and forms aggregates with proteoglycans.47,

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48

By bringing

proteoglycan activity to a matrix, those aggrecan mimics have been further shown to decrease degradation of ECM46, 49, 50, improve bulk mechanical properties of ECM scaffolds46, 49, affect expression of type II collagen49 as well as lower catabolic gene expression in an ex vivo osteoarthritic model.50,

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Recently, Place et al. have developed a proteoglycan-mimetic

copolymers where they varied grafting density of chemically modified CS and heparin chains attached to a hyaluronic acid backbone via a reductive amination chemistry where amine groups were introduced on HA by first functionalizing it with a thiol followed by an attachment N-(βmaleimidopropionic acid) hydrazide linker.52 The resulting mimics were shown to successfully deliver fibroblast growth factor (FGF-2) to mesenchymal stem cells. With this strategy, however, non-specific modification of polysaccharide molecules during the synthesis may lead to ringopening and aldehyde formation along the CS and heparin chains resulting in creation of rather multiple randomly distributed attachment/anchor sites along polysaccharide molecules, thus making them a multifunctional cross-linker. To mimic multivalent presentation of CS on proteoglycans chains, Lee et al. designed a glycopolymer where oligosaccharides have been assembled into a long polymer using a “grafting-through” method. In this approach, oligosaccharides were first functionalized with norbornene end groups, which were then reacted via ring-opening polymerization.42 While in these approaches functionality of PGs associated with their negative ionic density and biological activity has been achieved, macromolecular bottlebrush architecture of natural molecules with GAG chains coupled end-on on a backbone has yet to be developed. In synthetic organic chemistry, synthesis of macromolecular bottle brushes has been extensively explored, as reviewed by Sheiko et al.53 With an advancement of living radical


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polymerization, significant work on bottlebrush polymers has been done by Matyjaszewski and co-workers who used a grafting-from approach in which side chains are polymerized from a backbone polyinitiator via atom transfer radical polymerization (ATRP).54-57 Rzayev and co-workers have synthesized dense bottlebrush block copolymers using this “grafting-from” method with poly(lactic acid) (PLA) and poly(styrene) (PS) side chains and explored their self-assembly in solution.58, 59 In a “grafting-from” approach, Bowden et al. have been able to achieve high molecular weights (1-60 MDa) for bottlebrushes by polymerizing macromonomers of PLA terminated with a reactive norbornene using Grubbs’ first and second generation catalysts.60 While these methods allow for high-molecular weight bottlebrushes with well-defined structures, they are not easily applicable to synthesis of biomacromolecules due to harsh synthesis conditions as well as poor solubility of GAGs in typical aprotic solvents commonly employed. Here, we demonstrate a novel method of fabrication of biomimetic proteoglycans by using a hybrid natural/synthetic polymer approach and a grafting-to reaction strategy (Figure 1). The resulting proteoglycan mimic is composed of natural glycosaminoglycan bristles (same as natural proteoglycans) covalently attached end-on to a synthetic polymer core (replacing the protein core of natural proteoglycans) thus replicating a three-dimensional organization of natural biomacromolecules that is critical to the molecular interactions necessary to support extracellular matrix. We utilize commercially available CS (~22 kDa), previously identified as having a single terminal primary amine group.61 We conjugate CS chains onto poly(acryloyl chloride) (PAC) backbone by reacting primary amines on CS with acyl chloride groups on PAC and characterize the structure of the resulting biomimetic proteoglycans, water uptake and cytotoxicity.


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Figure 1. Schematic of biomimetic proteoglycans fabrication: chondroitin sulfate (CS) molecules are grafted onto a polymer core by reacting terminal primary amine groups on CS with functional groups on a synthetic, enzymatically-resistant backbone. By varying core length and attachment density of chondroitin sulfate bristles, a family of biomimetic proteoglycans with a different macromolecular architecture can be fabricated. Our reaction strategy allows us to avoid additional functionalization of CS, thus maintaining its unaltered chemical structure. The design choice of a synthetic polymer core of the biomimetic proteoglycans is selected to resist the normal enzymatic degradation (MMPs and ADAMTs) that attacks the protein core associated with natural proteoglycans13,

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, potentially providing a

longer-lasting molecule in vivo protected from the aggressive degenerative environment. Experimental Section Materials Chondroitin sulfate sodium salt from bovine cartilage (CS), poly(acrylic acid) (PAA) (MW 250kDa, 35% wt in H2O), glycine, aggrecan from bovine articular cartilage, fluorescamine, 3aminopropyltriethoxysilane (APTES), 1X phosphate buffer saline (PBS), ethyl acetate (EtAc),


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sodium borate buffer (SBB), sodium hydroxide (NaOH), deuterium oxide (D2O), ethanol (EtOH),

Live/Dead

Staining

Kit,

MTT

Assay

Kit,

Chondroitinase

ABC,

tris(hydroxymethyl)aminomethane (Tris), bovine albinum serum (BSA) were purchased from Sigma-Aldrich, Saint Loius. Poly(acryloyl chloride) (PAC) (MW 10kDa, 25% solution in dioxane) was purchased from Polysciences, Warrington, PA. Dialysis membranes (RC6, MWCO 50 kDa and MWCO 1 kDa) were purchased from Spectrum Labs, Rancho Dominguez, CA. Hyaluronic acid sodium salt (HA) (MW 2,000 kDa) was obtained from Lifecore Biomedical, LLC, Chaska, MN. Blyscan Assay Kit was obtained from Accurate Chemical & Scientific Corporation, Westbury, NY. L929 fibroblast cell line was obtained from ATCC. RPMI Medium, minimum essential medium (MEM), fetal bovine serum (FBS) were obtained from Corning Mediatech, Inc., Manassas, VA. Aggrecan from fetal bovine knee articular cartilage (for AFM imaging) was isolated in our laboratory as described below. All chemicals were used as received. Biomimetic proteoglycan (BPG10) synthesis We have previously identified a functional primary amine (PA) on the terminal end of chondroitin sulfate (Sigma-Aldrich, MW ~22kDa).61 This functional group can undergo further reaction for incorporation into a larger molecule by grafting covalently to a linear, long chain polymer possessing reactive sites (“grafting-to”). Using this strategy, different macromolecular architecture of hybrid macromolecules can be achieved by varying the polymer core length and attachment density of CS bristles. Biomimetic proteoglycan (BPG10) was synthesized by coupling CS onto poly(acryloyl chloride) (PAC) liner polymer through the reaction between a primary amine on CS and acyl chloride groups on PAC at 3 different mol/mol ratios of CS molecules to acyl chlorides groups


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(AC) (1:1, 1:5, and 1:10) (Figure 2). In a control experiment, glycine (Gly) was conjugated to PAC at the same mol ratios and experimental conditions. To evaluate reaction kinetics, 0.0105 mM, 0.0517 mM, and 0.105mM solutions of PAC were prepared in EtAc. CS and Gly solutions were prepared in sodium borate buffer (SBB, 0.1M, pH 9.4) at 1.14 mM. Equal volumes of PAC and either CS or Gly solutions were combined for a total volume of 6 ml at ~ 108:1, 22:1 and 11:1 CS/Gly:PAC (mol/mol) ratio, or alternatively

Figure 2. (1) Natural chondroitin sulfate (CS) is grafted onto poly(acryloyl) chloride backbone (2) via a reaction between a terminal primary amine on CS and acyl chloride groups on PAC. Resulting polymer (3) consists of a synthetic linear backbone with natural CS bristles; unreacted acyl chloride groups are hydrolyzed into carboxyl groups at the water-EtAc interface during the synthesis.


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~1:1, 1:5 and 1:10 CS/Gly:AC (mol/mol), respectively. The two-phase system was vigorously stirred for 10 minutes, and then allowed to react for 3-96 h with continuous mixing at room temperature (Thermolyne Labquake). After the mixing was stopped and clear separation of immiscible layers had been observed (commonly within 1 h), EtAc (top layer) was carefully removed and the aqueous solution containing biomimetic proteoglycan was sampled for conjugation analysis. In a control experiment, CS and Gly samples were mixed with EtAc only (at the same volume ratios as described above) to ensure that there were no side reactions for a primary amine group and no adverse effect of EtAc on CS. BPG10 samples synthesized at 1:10 CS:AC (mol/mol) ratio for 24 h were chosen for further analysis by 1H-NMR, FTIR, DLS, AFM, water uptake and cytotoxicity studies. Fluorescamine Assay Conjugation of CS and Gly onto PAC core was confirmed with fluorescamine assay by monitoring concentration of primary amines in the solution.61, 62 A 50-µL aliquot of the 10 mM fluorescamine solution in DMSO was added to 150 µL of the tested BPG10 or Gly solution in a 96 well plate (Corning). The solution was allowed to react for 5 min while on a shaker plate. Fluorescence intensity was read with a Tecan 2000 spectrophotometer (excitation 365 nm, emission 490 nm). Each solution was tested at least in triplicate. Conjugation of CS or Gly to a polymer backbone was determined by comparing signal intensity of conjugated samples to control non-reacted CS or Gly solutions (mixed with EtAc) of the same concentration:

% =

,


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where Iinit and Iconj correspond to signal intensity of fluorescently tagged primary amines of the non-reacted control CS (or Gly) and conjugated samples, respectively. Dialysis and Lyophilization After reaction, samples were dialyzed using RC6 membrane (MWCO 50 kDa) against DI water for 96 h. The dialysis membrane was pre-equilibrated in DI for 2 hrs prior to dialysis, and water was changed daily. Following dialysis, samples were frozen and lyophilized (Labconco, FreeZone benchtop system) at -90°C, 0.189 mBar. For 1H-NMR, DLS and water uptake analysis, CS material was subject to the same synthetic, processing and purification conditions; dialysis was performed using RC6 membrane with MWCO 1 kDa. Purified samples were reconstituted at predetermined concentrations and used for further characterization. CS Stability Commercial Blyscan Assay was used to confirm no degradation of CS in basic conditions at pH 9.4. The Blyscan Assay is dimethylmethylene blue based dye-binding method for analysis of non-degraded sulfated glycosaminoglycans. CS solutions in 1X PBS (positive control) and SBB buffers were prepared at 25 mg/ml, kept at room temperature for up to 96 h, and non-degraded GAG content was quantified every 24 h. Validity of the assay was confirmed with CS sample degraded by Chondroitinase ABC (ChsABC) (0.5 U/ml) in a buffer containing 50mM Tris, pH 8.0, 60 mM sodium acetate and 0.02% BSA at T = 37°C (negative control). Non-degraded GAG content in CS/Tris samples (with and without ChsABC) was quantified for 24 h only due to full degradation of CS within 24 h. The Blyscan Assay was performed according to the manufacturer’s protocol, and solution absorbance was measured at 656 nm with Tecan 2000 spectrophotometer (n=4).


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1

H-NMR

Lyophilized CS and BPG10 samples were solubilized in deuterium oxide (D2O) at 25 mg/ml. 1

H -NMR spectra were taken on a 300 MHz NMR spectrometer (UNITYNOVA, McKinley

Scientific, Sparta, NJ) at 64 scans and at ambient temperature. ATR-FTIR CS, BPG10 and PAC (25% solution in dioxane) were analyzed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) on a Nicolet 6700 Mid-IR spectrometer. Dynamic Light Scattering (DLS) Hydrodynamic sizes of CS and BPG10 samples were measured with a Nanobrook Omni Particle Sizer, using BIC particle solutions software. CS samples processed under similar conditions as BPG10 were used for DLS measurements. Samples were dissolved in 1X PBS buffer (pH 7.4) at 1 mg/ml concentration, and hydrodynamic size was measured at 90° angle at room temperature. Two samples of each type were tested, and each experiment was run in triplicate. Atomic Force Microscopy (AFM) Imaging Natural aggrecan was extracted from fetal bovine knee articular cartilage and purified, following the established guanidine hydrochloride method.63 Imaging of the macromolecular architecture was performed following established procedures.64 Briefly, a freshly cleaved, 1 cm × 1 cm mica surface was functionalized with positive charges by 30 minute incubation of 50 µL 0.03% APTES solution in a closed container at room temperature. Following incubation, the surface was rinsed gently in a stream of MilliQ water and air-dried. A 50 µL droplet of 25 µg/ml solutions of natural aggrecan or BPG10 were incubated on the functionalized mica surface for 15


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minutes to allow the adsorption of individual molecules. Afterwards, samples were rinsed gently in a stream of MilliQ water to remove un-adsorbed molecules and dried in air. AFM images were obtained via tapping mode imaging using nanosized silicon tips (nominal end radius < 10 nm, NCHV-A, BrukerNano) and a Multimode VIII AFM (BrukerNano). Water uptake Water uptake measurements (n=3) were performed on CS, natural aggrecan (Sigma-Aldrich), hyaluronic acid, and BPG10 using a thermal gravimetric analyzer at 90% relative humidity, 370C over 24 h immediately after drying samples in a vacuum oven at 370C for three days. All samples reached the maximum hydration state within 4 hrs. Water uptake (WU) was estimated as: , % =

( )

× 100%,

where WRH90 is the fully hydrated sample weight at 90% relative humidity, and W0 is the dry sample weight. For BPG10 sample, weight of GAG bristles (estimated from conjugation results) was used as the W0. Cytotoxicity L-929 fibroblasts were seeded at a density 13,000 cell/cm2 on 12-well tissue culture plates (RPMI media, 5% fetal bovine serum, L-glutamine and 1% pen/strep) and allowed to attach for 24 h before dosing with 0.2 mg/ml, 2 mg/ml and 10 mg/ml solutions of CS and BPG10 (sterilized via exposure to UV light 254 nm for 1 hr). Cell viability was investigated after 48 hrs using LIVE/DEAD Viability Cytotoxicity Kit (Invitrogen). Images were collected on an inverted fluorescent microscope and processed with ImageJ software. For cell metabolic activity study, L-929 fibroblasts were seeded at 50 × 104 cell/ml density on 96-well tissue culture plates (MEM with 10% FBS) and allowed to attach for 24 h. The MTT


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assay kit was used to colorometrically monitor cell viability after 24 h following dosing with CS and BPG10 at 0.02, 0.2 and 2 mg/ml concentrations (sterilized via exposure to UV light 254nm for 1 h). Metabolically active cells convert the water soluble dye to insoluble purple colored formazan compound which is then solubilized in DMSO and its concentration determined by optical density at 540 nm on a spectrophotometer. Statistical Analysis One way ANOVA test was used to analyze the results of water uptake experiments and MTT cell viability results (CS and BPG10 were compared at each concentration, with sample type being a variable). The post hoc Tukey test was used with significance level of 0.05. Results and Discussions Biomimetic Proteoglycan Design and Synthesis We have designed, synthesized and characterized a novel hybrid macromolecule that mimics the three-dimensional architecture of natural proteoglycans. Natural PGs are composed of a protein core and a various number of GAG bristles. While performing a number of biological functions in tissue, PGs are also essential for tissue hydration and mechanical strength through GAG charge density and dense, comb-like macromolecular architecture. In natural aggrecan, chondroitin sulfate bristles are linked to a serine residue on the protein core via a glycosidic bond.65 When CS chains are isolated from the proteoglycan core via enzymatic digestion, they are released with an intact terminal amino acid residue.66 Previously, we identified a terminal primary amine on CS and conjugated CS to amine-reactive vinyl monomers as well as immobilized it on epoxy-functionalized surfaces61. We have synthesized biomimetic proteoglycans using a grafting-to strategy where (CS) bristles were assembled end-on to a linear poly(acryloyl chloride) (PAC) core through the use of a


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reaction between a terminal primary amine on the CS and acyl chloride groups on PAC. In prior literature on PG mimics and GAG scaffolds, chemical groups along CS chain were modified to attain desired functionality for further cross-linking reaction or attachment to HA. Here, by using a terminal primary amine on CS, we avoid CS functionalization, which simplifies synthesis of a PG mimic and preserves chemical structure of CS bristles in a resulting macromolecule. Reaction occurred at the interface between non-aqueous phase consisting of PAC dissolved in ethyl acetate and an aqueous SBB phase containing CS. The reaction of acyl chloride with primary amines in basic conditions (pH > 9) is widely used for fabrication of polyamide (e.g. nylon) and nylon composites via in situ interfacial polymerization67,

68

and is known to be

almost instantaneous with the reaction constant k ~ 104-105 L/(mol s).67 Similarly, acyl chlorideamine chemistry has been used to synthesize polyacrylamide compounds with glucose and galactose pendant groups.43 CS has been previously found to be stable in neutral conditions at 30°C for up to 1000 h, and no appreciable depolymerization was observed in highly acidic (0.1M HCl, pH 1.5) and very basic (0.1M NaOH, pH 12.1) conditions for up 900 h at 30°C.69 However, increase in temperature up to 60°C resulted in accelerated degradation after approximately 96 and 190 h in acidic and basic environments, respectively. We used the Blyscan Assay to confirm stability of CS in SBB buffer at pH 9.4 for 96 h (Figure SI. 1). As expected, CS did not show degradation at pH 9.4 similarly to the positive control at neutral pH 7.4 (1X PBS). Validity of the assay was confirmed with a negative control of CS, which was digested with Chondroitinase ABC (MES buffer, pH 8.0) and full degraded within 24 h (Figure SI. 1). Amine-acyl chloride reaction and hydrolysis of acyl chloride groups are two competing reactions in the system, with the latter producing carboxyl acids along the initial PAC core.


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When PAC is added to CS/Gly aqueous phase, an intermediate hazy layer is formed in between two phases suggesting presence of partially soluble PAA-PAC chain. This layer slowly disappears within ~3 hours indicating increased solubility of CS-PAA/PAC polymer. Thus, our final biomimetic molecule is composed of natural CS bristles attached end-on to a poly(acrylic acid) core (PAA). PAA has been used extensively in biomaterials research.70, 71, 72 To evaluate the conjugation efficacy of CS to the PAC backbone, we used a fluorescamine assay. The CS-PAC reaction was carried out for 96 h, and samples were taken from the aqueous phase at 3, 24, and 96 h. A control experiment in which glycine was used instead of CS was also conducted. Because glycine is so much smaller than CS, this control experiment allowed an evaluation of the effect of molecular size on conjugation efficacy. The Gly-PAC reaction was carried out for 72 h, and samples were taken from the aqueous phase periodically. For both GlyPAC and CS-PAC, the fluorescamine assay followed the disappearance of primary amine from the aqueous phase; reduction in fluorescence intensity is associated with loss of primary amine as it is converted to an amide linkage.

The fluorescence intensities of conjugation reactions at

various times were compared with fluorescence intensities of solutions of known concentrations of non-conjugated CS and non-conjugated Gly in a mixed solvent of SBB and EtAc.

These

control samples were subject to the same synthesis conditions to ensure that estimated change in primary amine concentration is a result of CS conjugation to PAC rather than an effect of media/solvents. Figure 3 shows the percent conjugation from the fluorescence assay for the Gly-PAC conjugation (A) and for the CS-PAC conjugation (B). Comparison of 3A with 3B shows that, for the two mol ratios in which the acyl chloride groups are in excess (1:5 and 1:10), the small glycine molecule is conjugated with greater efficacy than is the large CS molecule. The figure


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also shows that the conjugation results for the 1:1 mol ratio of reactive groups are erratic and lower, suggesting that consistent and reliable results require an excess of acyl chloride groups. This is not surprising, since an excess of acyl chloride would help offset whatever steric problems may be inherent in the interfacial reaction of free molecules with side groups on the carbon backbone of the PAC polymer. Figure 3 shows significant increase in conjugation for CS-PAC at 96 h as compared to 24 h. However, given high reaction rates between acyl chlorides and primary amines, most conjugation is supposed to occur at initial stages. One of the possible reasons could be increased aggregation of CS in presence of BPG10 over long periods of time making some unreacted CS chains inaccessible to the fluorescamine dye. This, in turn, may lead to low measured signal intensities resulting in overestimation of conjugation efficiency. This hypothesis, however, will require further investigation. The large size of the CS molecule apparently does not prevent achievement of a high level of conjugation to the PAC backbone – up to 85% for a 10-fold molar excess of acyl chloride groups over the terminal amine-bearing CS molecules. While it is easy to visualize the easy access of a small molecule to the interface, large molecules also have adequate access.

Access for

molecules of any size is enhanced by enlargement of the interfacial area itself due to by stirring, Also, like the Gly, the CS has half of the three-dimensional volume that encloses the interface in which to diffuse and rotate in order to bring the an acyl chloride and a terminal amine into reaction distance. Additionally, reaction is performed in 0.1M SBB buffer where salt concentration is comparable with effective charge density on CS (at 25 mg/ml) leading to effective charge screening.


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Figure 3. Conjugation of glycine and CS to PAC backbone at 1:1 1:5 and 1:10 Gly:AC or CS:AC mol:mol ratios, respectively, as estimated with the fluorescamine assay: A) conjugation of glycine to PAC as estimated over 72 hrs. B) Conjugation of CS to PAC over 96 hrs. Data points shown represent an average ± SD (n = 3). Similar to glycine, CS samples achieved high conjugation rates fairly early along the reaction time axis. This, too, confirms that the energy barrier for positioning of molecules, even large ones, at the interface for chemical reaction is not large. While it would be interesting to explore earlier time points, presence of an intermediate layer of partially soluble PAC at the liquid interface at earlier stages adversely affects accuracy of sampling for the fluorescamine assay.


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From the results in Figure 3 we determined the 24-hr time point to be the most efficient synthesis duration that would allow sufficient conjugation of CS into PAC and hydrolysis of the remaining acyl chloride groups in a competing reaction. Figure 4 shows comparison between Gly and CS conjugation efficacy to PAC at 24 h. For both Gly and CS, a non-linear increase in conjugation efficacy with the increase of PAC amount in the reaction mixture was observed, most likely due to a concomitant increase in hydrolysis of available pendant acyl chloride groups. It should be noted that CS conjugation is significantly lower than that of Gly at each mol ratio. This highlights the ultimate effect of the larger molecular weight of CS that affects diffusion, and spatial orientation of the molecules to a greater extent.

Figure 4. Comparison of glycine and CS conjugation to PAC for all tested mol ratios at 24 hrs: larger MW of CS leads to a decrease in reaction efficacy between primary amine groups on CS and PAC as compared to Gly-PAC. Data points shown represent an average ± SD (n = 3) (* - P ≤ 0.05, ** - P ≤ 0.01). Through conjugation analysis, we estimated that at 24-hr time point the resulting molecules had ~ 20, ~ 14, and ~ 7-8 CS bristles attached to PAC for 1:1, 1:5 and 1:10 CS:AC ratios, respectively. For further structural, physical and biological characterization, we chose to


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synthesize biomimetic proteoglycan BPG10 at 1:10 CS:AC ratio running the reaction for 24 hrs. These conditions allow high yield of the resulting hybrid macromolecule with a lower amount of unreacted CS. Bristle density and spacing calculations suggest that BPG10 contained ~7-8 CS bristles on the 10 kDa PAA core with bristles spaced at 3-4 nm and a total estimated MW of ~ 160-180 kDa.73 This compares to 2-3 nm bristle spacing on natural aggrecan molecule.13 The molecular weight of the BPG in our experiment, for the most part, was limited by the length of the commercially available PAC polymer, conceivably this could be modified with custom-prepared PAC polymers of different lengths. By having a synthetic polymer in place of a protein backbone, our molecule is designed to resist enzymatic degradation by MMPs and aggrecaneses, the major enzymes responsible for degrading natural PGs in vivo. Naturally, several cleavage points for these (and other) enzymes exist throughout the large PG, aggrecan, core protein resulting in differently sized fragments of aggrecan. These aggrecan fragments vary in functional capacity, such as electrostatic repulsion and osmotic potential, as well as the increased tendency to migrate out of the tissue related to the reduced size of the fragments.10 Designing a three-dimensional biomimetic molecule with an enzymatically resistant backbone may help to improve material residence time in tissue and prolong functional activity. 1

H-NMR

Chemical structures of CS and biomimetic proteoglycan were confirmed via 1H-NMR showing characteristic peaks for CS for all purified compounds (Figure 5). As mentioned before, in order to maintain the beneficial natural biological function of GAG bristles, we avoided any chemical modification/functionalization of CS in our reaction strategy. Extensive review of chemical shifts for CS can be found elsewhere.74,

75

As seen in Figure 5, no degradation of CS bristles was


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observed for both BPG10 and control CS sample subjected to the same experimental conditions, indicating that the processing methods were protective of CS. Some peak broadening occurs at ~ 2 and ~ 4.5 ppm which could be due to incorporation of a synthetic backbone. Due to insolubility of PAC in D2O, which is the only solvent for CS and BPG10, direct comparison of spectra is not possible between PAC and BPG10 samples.

Figure 5. 1H-NMR spectra shows characteristic CS peaks for all compounds, where GlcA and GalNAc correspond to D-glucuronic acid and N-acetyl-D-galactosamine monosaccharides, respectively: A) original CS (Sigma); B) processed CS subjected to the same experimental conditions as BPG10; C) BPG10. No degradation of CS bristles during the course of the synthesis is observed; peak broadening for BPG10 sample at ~2 ppm and 4.5 ppm could be due to a synthetic backbone incorporation.


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However, from PAA 1H-NMR spectrum, it could be expected that typical chemical shifts corresponding to protons on a PAA polymer backbone would be present in 1.5-2.5 ppm peak region76, and may not be well resolved in our case due to a small amount of PAA polymer in a final product as well as an overlap with a signal from CS in that region. FTIR. ATR-FTIR was used to characterize chemical composition of CS, BPG and PAC (25% solution in dioxane). FTIR spectrum of CS shows characteristic absorptions of GAG chains77: broad OH stretching at 3000-3600 cm-1, carbonyl stretching near 1600 cm-1, N-H bending near 1550 cm-1, characteristic sulfate group stretching near 1220 cm-1 and absorptions of the saccharide unit at 950-1100 cm-1 (Figure 6). Similar spectrum is observed for BPG10 sample suggesting that composition of GAG bristles in our mimic was not altered.

Figure 6. FTIR spectra of a) PAC (25% solution in dioxane), b) CS, c) BPG10. While primary amines are known to produce two N-H stretch absorptions and a secondary amide only one, their frequencies are found near 3300-3500 cm-1. This region is obscured by broad O-H stretching in GAGs, and N-H stretch absorptions cannot be successfully resolved to monitor conversion of primary amines. However, increase in N-H bending absorption relative to


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carbonyl stretching in a BPG sample may be indicative of an amide bond formation between GAG bristles and a synthetic core. IR spectrum obtained for PAC is similar to those reported in literature.78, 79 Absorptions observed near ~ 1100-1400 and 2800-3000 cm-1 are characteristic of dioxane solvent. As shown in Figure 6, a characteristic peak corresponding to C=O stretching absorption of an acyl chloride group at 1770 cm-1 is no longer present in a BPG sample. Instead, carbonyl C=O stretching shifts to a lower wavenumber (~1600 cm-1) similar to C=O stretching in CS, what is consistent with expected conversion of acyl chlorides to carboxylic acid groups. Additionally, C-Cl stretching absorption near ~700 cm-1 is not resolved in the BPG spectrum. DLS. Dynamic light scattering measurements were performed on CS (dialyzed and lyophilized) and BPG10 samples in 1X PBS solution at pH 7.4. As seen in Figure 7, CS hydrodynamic size was measured as 9.7±0.8 nm, which is comparable to previously reported values.52 On the other hand, size of BPG10 was estimated at 61.3±12.3 nm. Both CS and BPG samples were measured at 1 mg/ml concentration, at which fixed charge density of GAG chains is effectively screened in 1X PBS buffer. Therefore, increase in hydrodynamic size of BPG10 as compared to CS in these high salt conditions suggests covalent attachment of CS to PAC rather than aggregation via electrostatic interactions. Atomic Force Microscopy Atomic force microscopy imaging shows the bottlebrush architecture of the largest and most dense proteoglycan, aggrecan, where ~100 CS bristles are attached to a protein core of ~ 250 kDa (Figure 8A). In our study, we used a smaller core (MW 10 kDa), and AFM results suggest an expected ‘star’/’blob’ architecture of CS-PAC molecules consisting of a number of longer


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Figure 7. DLS measurements show increase in hydrodynamic size for BPG10 as compared to CS. The inset shows an example of an autocorrelation function for BPG10. bristles (22 kDa) attached to a shorter core (10 kDa) (Figure 8B). If CS molecules were grafted by reaction of side groups along the chain, large cross-linked GAG aggregates would likely form due to multi-functionality of CS. This is the first observation of an end-on attachment of CS bristles to a core polymer for a biomimetic PG.

Figure 8. AFM image of A) natural fetal bovine knee cartilage aggrecan (~ 2,000 kDa), B) BPG10 (~ 160-180 kDa): BPG10 samples are shown to have a ‘star’/’blob’ form consistent with ~7 CS bristles (22 kDa) attached to a polymer core (10 kDa).


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Water Uptake Water uptake measurements were performed on natural aggrecan (Sigma), CS (processed), HA sodium salt and BPG10 by means of thermogravimetric analysis at 90% RH at 37°C over 24 hours (Figure 9).

Figure 9. Water uptake results: A) representative water uptake curves for CS, natural aggrecan, HA and BPG10; B) HA and BPG10 had an increased water uptake compared to natural aggrecan alone with P ≤ 0.01 and P ≤ 0.001, respectively. Data points shown in 7B represent an average ± SD (n = 3). All samples reached the maximum hydration state within 4 hrs (Figure 9A). CS and HA showed water uptake of ~ 41 and 49% (Figure 9B), respectively, similar in magnitude to


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previously reported results, where number of adsorbed water molecules per disaccharide unit for both CS and HA was determined with IR spectroscopy.80 While sulfated CS is expected to have a higher water uptake compared to nonsulfated HA, presence of salt in our HA sample could lead to an additional increase in water absorption, as HA salt, for example, was previously shown to have an increased water content compared to a saltfree sample.81 Water uptake of natural aggrecan (~38%) was similar to that of CS, but it should be noted that aggrecan also contains a non-charged protein core and ionic density of the sample would depend on the number of GAG chains and their length. Biomimetic proteoglycan, BPG10, has a water uptake of ~ 62% per initial GAG weight, where GAG content of a biomimetic sample was estimated from CS-PAC conjugation results. This interesting finding demonstrated that biomimetic aggrecan showed a 50% increase in water uptake than natural aggrecan or CS alone, perhaps due in part to the charges along the polymer core, which are not present in aggrecan protein core (~16% of additional charged groups per BPG molecule). Cytocompatibility Cytocompatibility of BPG10 has been found to be concentration-dependent and comparable with CS at physiologically relevant concentrations. Cell viability study showed that BPG10 did not have an adverse effect on cell metabolic activity at concentrations of 0.02 and 0.2 mg/ml, as compared to natural CS (Figure 10). However further increase in BPG10 concentration to 2 mg/ml (~ 4 × 10-6 mg of PG/cell) led to ~20% decrease in the number of metabolically active cells what could be caused by higher effective charge density of BPG10 due to additional ionic groups present on a polymer backbone.


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Figure 10. Cell viability results as determined with MTT assay: ratio of metabolically active cells (normalized to live control) is compared for CS and BPG10 at 3 dose concentrations. Data points shown represent an average ± SD (n = 6) ( *- P ≤ 0.05). Similar trend was observed in cytotoxicity experiments, where, as indicated by Live/Dead Assay, both CS and BPG10 maintained cytocompatibility up to a concentration of 2 mg/ml (Figure 11). In prior work, GAG-based scaffolds were shown to be biocompatible when seeded with chondrocytes at comparable GAG/cell ratio.44 Both CS and BPG10 exhibit cytotoxicity at a concentration of 10 mg/ml. The range of 50-200 × 106 cell/ml has been reported for cartilage (for ≤ 0.5 mm thickness).82 With ~20-80 mg/ml concentration of PGs in cartilage83, the natural PG/cell ratio can be estimated as 0.1-1.6 × 10-6 mg of PG/cell, which confirms that for high dosing concentration of BPG (such as 10 mg/ml in Live/Dead Assay), cells were subject to increased osmotic pressure and ionic conditions not common for native tissues. These results indicate that for highly ionic materials (even those naturally occurring in human tissue, such as CS), cytocompatibility is compromised when the effective dose significantly exceeds native GAG/cell ratio found in vivo.


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Figure 11. Cytotoxicity results (Live/Dead Assay) for CS and BPG10 samples at 0.2-10 mg/ml dose concentrations. Similar trends observed for both neat CS and BPG10 indicate that synthesis conditions, processing and presence of a synthetic backbone did not adversely affect cytocompatibility of our mimic. While macromolecules fabricated from functionalized CS have been successfully used in various tissue engineering projects, our novel biomimetic material could be useful for applications, where unaltered CS structure coupled with a bottle-brush architecture may be preferred. These PG mimics could be potentially used to molecularly engineer soft tissues depleted of PGs by increasing tissue osmotic pressure and mechanical stability through intra- and intermolecular interactions between negatively charged GAG bristles. At the same time, they can


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also serve as drug delivery systems, where drugs can be coupled to negatively charged GAG bristles via either physical encapsulation or a covalent bond. Biomimetic proteoglycans can also be used as model systems to directly study the biological activity and functions of GAG bristles in the absence of a protein core while maintaining their macromolecular architecture.

Conclusions Biomimetic proteoglycans with full glycosaminoglycan bristles (chondroitin sulfate) and a synthetic polymer core in bottlebrush architecture have been synthesized. These molecules mimic the three-dimensional structural and water uptake of natural proteoglycans and can be modulated to mimic the broader family of proteoglycans. Our synthesis design enables fabrication of various biomimetic proteoglycans by varying the length of an enzymatically resistant, synthetic polymer core, GAG bristle density and type of GAG chains (depending on its primary amine content). A “grafting-to” approach, where GAG chains are coupled onto a polymer core through the reaction of a primary amine on a GAG would allow to avoid additional functionalization of disaccharide units commonly used in current synthesis strategies, thus maintaining GAG chemical structure.

Associated content Supporting Information Results of CS stability in 0.1 M SBB buffer at pH 9.4. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors


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* E-mail: [email protected]. Present Addresses ⊥ Takida Pharmaceuticals, 35 Landsdowne Street, Cambridge, Massachusetts 02139, United States. Author Contributions K.P., L.S.P. and M.S.M. developed the synthesis strategy and provided guidance for the project. K.P. synthesized biomimetic proteoglycans and analyzed structure (1H-NMR) and water uptake. K.P. and R.W.Y. conducted in vitro cytotoxicity experiments. L.H. and P.P. conducted AFM experiments. A.K. conducted DLS measurements. K.P. and M.S.M. prepared the manuscript with contribution provided by all authors. All authors have read and given approval to the final version of the manuscript. ACKNOWLEDGMENT We acknowledge Wallace H. Coulter Foundation for financial support. The authors also thank Yuesheng Ye for his assistance with TGA experiments. REFERENCES 1.

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