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Tailoring Stimuli Responsiveness using Dynamic Covalent Crosslinking of Poly(vinyl alcohol)-Heparin Hydrogels for Controlled Cell and Growth Factor Delivery Justine J Roberts, Pratibha Naudiyal, Lauriane Jugé, Lynne E Bilston, Anthony Michael Granville, and Penny Jo Martens ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00321 • Publication Date (Web): 10 Nov 2015 Downloaded from http://pubs.acs.org on November 18, 2015

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ACS Biomaterials Science & Engineering

Tailoring Stimuli Responsiveness using Dynamic Covalent Crosslinking of Poly(vinyl alcohol)-Heparin Hydrogels for Controlled Cell and Growth Factor Delivery

Justine J. Roberts, Pratibha Naudiyal, Lauriane Jugé, Lynne E. Bilston, Anthony M. Granville, and Penny J. Martens*

Dr. J. J. Roberts Graduate School of Biomedical Engineering, UNSW Australia, Sydney, 2052, New South Wales, Australia

P. Naudiyal Graduate School of Biomedical Engineering, UNSW Australia, Sydney, 2052, New South Wales, Australia

Dr. L. Jugé Neuroscience Research Australia and School of Medical Sciences, UNSW Australia, Sydney, 2052, New South Wales, Australia

Prof. L. E. Bilston Neuroscience Research Australia and Prince of Wales Clinical School, UNSW Australia, Sydney, 2052, New South Wales, Australia

ACS Paragon Plus Environment

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Dr. A. M. Granville School of Chemical Engineering, UNSW Australia, Sydney, 2052, New South Wales, Australia

Dr. P. J. Martens Graduate School of Biomedical Engineering, UNSW Australia, Sydney, 2052, New South Wales, Australia

E-mail: [email protected]


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ABSTRACT Heparin-based hydrogels are attractive for cell encapsulation and drug delivery because of the ability of heparin to bind native proteins. However, heparin-based hydrogels have received little attention for their potential as stimuli-sensitive materials. Biosynthetic, poly(vinyl alcohol) (PVA)-heparin hydrogels were formed using dynamic, covalent crosslinking. Hydrogel stimulisensitivity was tailored by tuning the concentration of heparin to PVA. Relatively thermally and pH stable hydrogels were produced when formed from only the synthetic, non-ionic PVA polymer crosslinked via hydrazone bonds. Crosslinking in the ionic biopolymer heparin, to form PVA-heparin gels, has a profound impact on thermal stability, with degradation ranging from over 6 months to only 4 days across 25-50 °C. PVA-heparin hydrogels degrade within 18 days at basic pH (10), while not fully degrading over 6 months at lower pH (4, 7.4). This finding is attributed to the anionic repulsion of carboxyls and sulfates in heparin. PVA-heparin macromers were cytocompatible and enabled mild cell encapsulation, in addition to providing pH-controlled growth factor release. Overall, it is demonstrated that the biopolymer heparin can be used to create pH and temperature responsive hydrogel biomaterials for cell and drug delivery.

KEYWORDS: hydrogel, heparin, poly(vinyl alcohol), stimuli responsive, hydrazone crosslinking


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1. INTRODUCTION Heparin plays an important role in many biological processes, as it is a native polysaccharide found in close proximity to cell surfaces and extracelluar matrix proteins.1 As such, it is popular to form hydrogels comprising heparin because they exhibit attractive properties, including binding and stabilizing proteins, such as growth factors.2 Many groups have used heparin-based hydrogels to provide sustained release of heparin-binding growth factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), to mediate cell behavior (e.g., angiogenesis, chemotaxis) for a broad array of wound healing and tissue engineering applications.3-7 A unique characteristic that is thought to contribute to the growth factor binding ability of heparin is that it is anionic at physiological pH, due to its high density of carboxyl and sulfate groups.1, 8 Ionizable pendant groups, such as carboxyls and sulfates, can accept or donate protons leading to conformational and/or solubility changes, in response to stimuli (pH, temperature).9 Heparin-based hydrogels have been explored in great depth for their proteinbinding capabilities; however, they have not been explored in depth for their potential as stimuli sensitive biomaterials.4 It is hypothesized that heparin-based hydrogels will introduce stimuliresponsiveness to pH and temperature due to heparins anionic nature, while also introducing biofunctionality for cell encapsulation and controlled growth factor release. Stimuli-responsive hydrogels are attractive because they can provide for the spatially and temporally controlled release of drugs or cells to the desired tissue based on stimuli such as, pH, temperature, ionic strength, electric fields, magnetic fields, light, chemical stimuli, and biological stimuli.10 Particularly interesting are the dual temperature- and pH-responsive hydrogels that are attracting increasing attention recently for their advantages in biomedical applications. Natural polymers have been extensively used to make temperature- and pH-sensitive hydrogels because


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they commonly contain acidic or basic functional groups.10, 11 The most prevalent temperatureand pH-responsive natural hydrogels are based on proteins, such as gelatin,11,

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or

polysaccharides such as chitosan13-16 and dextran11, 17 which contain a high density of amino or carboxyl groups. Similar to other stimuli-responsive polysaccharides, heparin has a high charge density making it ideal for use in stimuli-responsive gels. Heparin crosslinked with a combination of non-covalent and covalent crosslinking has demonstrated reversibly temperatureresponsive rheological behavior.18-20 However, a full exploration of the potential for heparin to be used as a dual, temperature- and pH-responsive material has not been undertaken. Therefore, in this study we aim to explore heparin crosslinked with reversible, covalent bonds to evaluate if these materials are responsive to multiple stimuli. Although heparin has many advantages, hydrogels designed from purely natural materials are often weak and can have uncontrolled enzymatic degradation in vivo. Therefore, heparin has been combined with synthetic polymers such as poly(ethylene glycol),6 Pluronic,7 and poly(vinyl alcohol) (PVA),21 to form biosynthetic hydrogels, which provide increased control over hydrogel mechanical and chemical properties.4 PVA is a cytocompatible synthetic polymer commonly used to form hydrogels for cell encapsulation and drug delivery. PVA is hydrophilic due to its pendant hydroxyl groups, which can be easily modified for crosslinking with biopolymers.22, 23 In addition, PVA has previously been combined with a variety of ionizable biopolymers to form stimuli responsive hydrogels thus making it a good candidate for a dual stimuli responsive gel by copolymerizing it with the heparin. For example, chitosan-PVA biomaterials have been designed which respond to stimuli such as temperature15 and pH change13, 14 due to chitosan’s high density of cationic amino groups. Gelatin-PVA blends have been shown to be responsive at varying pH because of the carboxyl and amino groups present in gelatin.12 Heparin-PVA materials have not


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been explored in depth for their response to the stimuli pH and temperature, despite the high density of ionizable groups in heparin. Instead, PVA and heparin polymers have previously been combined via methods such as chemical crosslinking,24 photocrosslinking,21 and pressurization,25 and explored for reducing thrombogenicity24, 25 and for their ability to activate growth factors.21 The aim of this study was to design hydrogels that provided tunability of temperature- and pH- responsiveness using the ionic biopolymer, heparin. Heparin was crosslinked with PVA via reversible, covalent hydrazone bonds to form biosynthetic hydrogels. Hydrazone bonds are advantageous for biomedical applications because they can be injected and can form in situ via the orthogonal addition of hydrazide and aldehyde groups in a mild reaction where the only byproduct is water.26 To evaluate hydrogel material properties, the ratio of heparin to PVA was varied, while the initial hydrogel crosslinking density was kept constant, and gelation and degradation behavior were monitored over time. Gelation behavior was monitored using in situ rheology to observe the impact of heparin content on rate of gelation. The impact of heparin concentration, temperature, and pH on the hydrogels in aqueous solution was characterized via mass loss, swelling and mechanical properties to evaluate time dependent behavior (e.g., degradation). To probe if heparin within the hydrogels ionized at varying pH, hydrogels were characterized with Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR). To evaluate if the synthesized macromers were compatible, fibroblasts were plated with the individual macromers and a cell growth inhibition assay was performed. To assess if the crosslinking reaction (gelation) was cytocompatible, fibroblasts were encapsulated within the hydrogels for several days and a Live/Dead assay was performed. To investigate if heparincontaining hydrogels had pH-controlled drug release a model protein therapeutic, basic fibroblast growth factor (bFGF), was encapsulated and released for several weeks. Overall, this study


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evaluates the incorporation of heparin to make biosynthetic PVA-heparin hydrogels, towards the design of both bioactive and stimuli-responsive hydrogels for a variety of biomedical applications (e.g., cell and drug delivery).

2. EXPERIMENTAL SECTION 2.1. Materials. PVA (13–23 kDa, 98% hydrolysed), glycine ethyl ester hydrochloride (99%), triethylamine (≥99%), 1,1’-carbonyldiimidazole (CDI, ≥95%), hydrazine monohydrate (98%), 3amino-1,2-propanediol (97%), sodium periodate (NaIO4, ≥99%), ammonium hydroxide solution (28-30% NH3), heparin (heparin sodium salt from porcine intestinal mucosa, grade I-A, 17–19 kDa), N-hydroxybenzotriazole (HOBt), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, ≥98%), sodium chloride (NaCl, ≥99.5%), hydrochloric acid (HCl, 36%), cellulose membrane dialysis tubing (MWCO 12 kDa), Dulbecco’s Phosphate Buffered Saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), bovine serum albumin (BSA), Penicillin-Streptomycin, Trypsin-EDTA Solution 1X, calcein-AM (≥96%), and propidium iodide (≥94%) were purchased from Sigma-Aldrich. Dimethyl sulfoxide (DMSO) dried over 4Å molecular sieves (100%), ethanol (100%), and diethyl ether (100%) were bought from Ajax Chemicals. Fetal bovine serum (FBS) was purchased from Moregate Biotech. Basic fibroblast growth factor and the bFGF enzyme-linked immunosorbent assay (ELISA) kit were purchased from ThermoFisher Scientific. 2.2. Macromer Synthesis. Hydrazide-modified PVA (PVA-HY),27, 28 aldehyde-modified PVA (PVA-AL),27,

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and aldehyde-modified heparin (heparin-AL)3 were synthesized as described

previously. Hydrazide-modified PVA was made with 5 HY groups per chain. In a typical experiment, PVA (1 g, 0.0625 mmol) was dissolved in dry DMSO (20 mL). CDI (254 mg, 1.563 mmol) was added


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to the PVA solution under a nitrogen atmosphere and then stirred for 3 hours. Glycine ethyl ester hydrochloride (44 mg, 0.313 mmol) followed by triethylamine (44 µL, 0.313 mmol) was added and the reaction was stirred at room temperature for 21 hours. Hydrazine monohydrate (1 mL) was added and the mixture was stirred at room temperature for 24 hours. Modified PVA was precipitated in a 10-fold excess of 80/20 diethyl ether and ethanol. The polymer was dissolved in water, dialyzed against deionized water and lyophilized. The incorporation of the hydrazide groups could not be determined directly from the 1H NMR spectra of PVA-HY, since the peaks of the methylene protons of the hydrazide modification overlapped with the protons of the PVA backbone. Therefore, to determine the hydrazide groups PVA-HY was reacted with excess formaldehyde to form a hydrazone bond which enables quantification, as described previously.27 The number of hydrazide groups per chain was determined by comparing the area of the protons of the resulting hydrazone bond (i.e., 6.5 and 7 ppm) to the area of the protons in the PVA backbone (i.e., 4.1-3.8 and 1.8-1.4 ppm). 1H NMR (D2O, 300 MHz): δ - 7 (d, 1H, CONHN = CH2), 6.5 (d, 1H, CONHN = CH2), 4.1–3.8 (m, 1H, CH of PVA backbone), 1.8–1.4 (m, 2H, CH2 of PVA backbone). To synthesize PVA-AL, PVA was modified with 5 AL groups per chain. Briefly, PVA (1 g, 0.0625 mmol) was dissolved in dry DMSO (20 mL). CDI (254 mg, 1.563 mmol) was added to the PVA solution and then stirred in a nitrogen atmosphere for 3 hours. A solution of 3-amino1,2-propanediol (118 mg, 1.25 mmol) in DMSO (1 mL) and triethylamine (1.25 mmol) was added and the reaction was stirred at 60 °C for 18 hours. Concentrated aqueous NH3 (1.4 mL) was then added and stirred at room temperature for 1 hour. Modified PVA was precipitated in 10-fold excess of a 80/20 mixture of diethyl ether/ethanol, redissolved in deionized water, dialyzed against deionized water, and lyophilized. To create the aldehyde-modified PVA by


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oxidation, the amino-glycerol-modified PVA (1 g) was dissolved in water (20 mL) and the solution was cooled to 0 °C. NaIO4 (0.15 M, 12.5 eq per amino-glycerol group) was added to the PVA solution and the mixture was stirred for 1 hour. The solution was then dialyzed against deionized water, lyophilized, and the product was characterized by 1H NMR spectroscopy. The aldehyde functionality (-CH2CH = O) is completely hydrated in D2O, since only a signal corresponding to the 1,1-diol form (CH2CH(OH)2) at 3.2 ppm was observed and the aldehyde signal (-CH = O) at 9.6 ppm was almost undetectable. The number of aldehyde groups per chain was determined by comparing the area under the integral for the methylene protons adjacent to the hydrated aldehyde group protons to the methylene peaks on the PVA backbone (i.e., 4.1-3.8 and 1.8-1.4 ppm). 1H NMR (D2O, 300 MHz): δ - 4.1–3.8 (m, 1H, CH of PVA backbone), 3.24 (m, 2H, CH2-CH(OH)2), 1.8–1.4 (m, 2H, CH2 of PVA backbone). Heparin was modified with 5 AL groups per chain. Heparin (1 g, 1.67 mmol of disaccharide repeating units) was dissolved in deionized water (167 mL). HOBt (255 mg; 1.67 mmol) and 3amino-1,2-propanediol (152 mg; 1.67 mmol) were added and stirred until dissolution. The pH of the reaction mixture was then adjusted to 6.0 and EDC (64 mg; 0.33 mmol) was added and the pH was maintained at 6.0 until it stabilized and the reaction was stirred overnight. The solution was dialyzed against 0.1 M NaCl at pH 3.5 (2 x 4 L, 48 hours), followed by dilute HCl (pH 3.5) (4 L, 24 hours), and then dionized water (4 L, 24 hours). The solution was lyophilized. The modified heparin was then oxidized to form heparin-AL. In a typical experiment, aminoglycerol-modified heparin (1 mmol of repeating units) was dissolved in 90 mL of deionized water. Predissolved NaIO4 (214 mg, 1 mmol dissolved in 1 mL water) was added to the reaction mixture and stirred for 30 minutes. The reaction was quenched with excess ethylene glycol (320 µL, 5 mmol) and stirred for 1 hour. The solution was dialyzed against deionized water and


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lyophilized to obtain heparin-AL. 1H NMR could not be used due to the random sulfation pattern and the absence of a reference peak. Instead, amino-glycerol modified heparin and native heparin were monitored on a UV spectrophotometer at an absorbance of 290 nm for the rate of periodate consumption, which is used as a measure of aldehyde formation.3 The control, native heparin, was used to demonstrate that there was not any appreciable oxidation of the native polymer backbone under the reaction conditions (

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