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Biopolymer Molecular Weight Can Modulate the Wound Healing Efficacy of Multivalent Sonic Hedgehog-Hyaluronic Acid Conjugates Taylor A Holstlaw, Mavish Mahomed, Livia W Brier, David M Young, Nancy J Boudreau, and Wesley M Jackson Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00553 • Publication Date (Web): 05 Jul 2017 Downloaded from http://pubs.acs.org on July 7, 2017
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Biopolymer Molecular Weight Can Modulate the Wound Healing Efficacy of Multivalent Sonic Hedgehog-Hyaluronic Acid Conjugates Taylor A. Holstlaw1, Mavish Mahomed1, Livia W. Brier1, David M. Young2, Nancy J. Boudreau2, and Wesley M. Jackson.1,*
1. Valitor, Inc. Berkeley, CA 94710, USA. 2. Department of Surgery, UC San Francisco, San Francisco, CA 94110, USA.
KEYWORDS: drug development, pharmacodynamics, hyaluronic acid, growth factor, protein conjugates, Sonic hedgehog, protease, proteolytic protection, wound healing, diabetic ulcer
ABSTRACT:
There is a clinical need for new therapeutics to improve healing of chronic impaired wounds. Thus, we investigated how biopolymer conjugation could be used to improve the wound healing performance of a key growth factor for tissue regeneration: Sonic hedgehog (Shh). We generated two multivalent Shh conjugates (mvShh) using hyaluronic acid with two different MWs, which exhibited equivalent potency and proteolytic protection in vitro. Using db/db
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diabetic mice, we showed that mvShh made with smaller HyA MW resulted in more rapid and robust neovascularization compared to mvShh made with larger MW HyA. Further, smaller mvShh conjugates resulted in faster wound resolution compared to the unconjugated Shh. This study is the first to show how the wound healing efficacy of a multivalent protein-polymer conjugates is sensitive to the polymer MW, and our findings suggest that this parameter could be used to enhance the efficacy of growth factor conjugates.
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INTRODUCTION: Protein-based drugs have enormous potential to promote wound healing,1 but the clinical translation of these therapies has proven challenging. The clinical performance of recombinant proteins in the wound space is limited in part by their rapid proteolysis and clearance from wound bed in vivo, often resulting in an inadequate window of therapeutic activity after administration.2-5 For example, due to its short in vivo half-life (~30 minutes6), the development of vascular endothelial growth factor (VEGF) was halted in phase II clinical trials despite its positive safety profile,7 as frequent administration was required to yield only a modest treatment effect.8 As a second example, becaplermin (platelet derived growth factor, Smith and Nephew) received FDA approval, but it has demonstrated poor cost-efficacy,9-10 and has struggled with clinical adoption. Thus, better control over the duration of their treatment effect is critical to advance the development of protein therapeutics as effective wound healing products. A variety of biomaterial and nanotechnology strategies have been implemented to improve the tissue-level distribution and stability of recombinant proteins after clinical administration.11 One commonly employed strategy uses polyethylene glycol (PEG) conjugated to a protein-based drug (i.e., PEGylation) to prevent deactivation by immune cells and proteolytic enzymes via steric inhibition.12 This method also modulates the size of the resulting macromolecular entity, thereby enabling control over its vascular permeability, tissue diffusion, and elimination routes. However, these methods to develop protein-based drugs primarily utilize a single-parameter design approach. As such, improvements made to one parameter of protein pharmacology are made at the expense of another. Using PEGylation as an example, in vivo halflife can be increased, but with this improvement, the potency of the protein can drop by an order of magnitude or more.13 While these tradeoffs in pharmacological performance have been
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acceptable for at least ten PEGylated protein-based drugs that have been FDA approved for systemic administration, monovalent protein-polymer conjugation has not been successfully applied for wound healing applications. In contrast, we are developing a method of multivalent protein conjugation that offers a multiple parameter approach to enhancing protein function. We generate soluble, nanoscale clusters of tethered proteins on single-chain hyaluronic acid (HyA) biopolymers with stoichiometric control over the number of proteins on each biopolymer (i.e., valency).14-15 Previous studies showed how these multivalent conjugates (MVCs) increased the potency of their cargo proteins compared to equimolar concentrations of unconjugated proteins as measured by in vitro assays.14, 16-17 The MVCs also enhanced the therapeutic function of the conjugated proteins when administered to several in vivo disease models16-19 The observed improved in vivo performance of the MVCs is due, at least in part, to their increased potency, but they also likely benefited from pharmacokinetic advantages relative to the unconjugated proteins due to their larger size. In this study, we will focus on the effect of polymer molecular weight (MW) to control MVC size and enable independent control over their in vivo bioactivity to yield greater overall therapeutic efficacy. As a model growth factor, we used Sonic hedgehog (Shh), which has been previously proposed as a drug to promote tissue regeneration and wound healing.20-22 Our overall hypothesis was that the wound healing efficacy of mvShh conjugates would be sensitive to the MW of their HyA component. To assess this parameter in isolation, we first demonstrated that the bioactivity and proteolytic protection of mvShh could be normalized for conjugates made using HyA with different MWs. Then we selected two mvShh with different MW but equivalent in vitro bioactivities to evaluate using an in vivo wound healing model. In doing so, our study
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was the first to show how the biopolymer MW parameter of a multivalent protein conjugate can be used to enhance the therapeutic performance of a protein drug.
EXPERIMENTAL: Multivalent Shh-HyA Conjugation: We prepared mvShh following a method described previously.14 We obtained HyA over a range of MWs from 150-860 kDa (LifeCore Research), which we have referenced by their weight averaged MW, as specified from the manufacturer based on their static light scattering analysis. 3 mg/mL of each HyA was dissolved by rotating overnight in MES buffer (0.1M, pH 6.5). Carbodiimide substitution was initiated by adding 10 mg/mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 0.3 mg/mL sulfoNHS, and 1.2 mg/mL N-ε-maleimidocaproic acid hydrazide (EMCH) to the solution and reacting for 2 hours at 4oC, which resulted in the addition of maleimide reactive groups on the HyA (see Fig. S1). Samples were dialyzed with 50 kDa MWCO membranes against PBS buffer (930 mg/L EDTA and 10% glycerol in DPBS) twice for 4 hours and then once for 24 hours at 4oC. Activated HyA-EMCH was then stored at -20oC. Recombinant N-terminal Shh fragment with a C-terminal cysteine residue was synthesized as previously described using an E.coli expression system.23 To obtain mvShh over a range of valencies, a fixed concentration of Shh-Cys was combined with HyA-EMCH at various defined feed ratios in a PBS and allowed to react at 4oC for four hours. The mvShh solution was then dialyzed with 100 kDa MWCO against PBS buffer twice for 4 hours and once for 24 hours at 4oC. Characterization of Shh-HyA Conjugates: The concentration of Shh protein for each mvShh conjugate was measured using the BCA assay (Thermo), and we verified that the concentration of HyA in the mvShh was ~100X lower than its detection limit by BCA.24
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Conjugation of Shh to HyA was verified by protein separation with SDS-PAGE (Mini Protean System, BioRad) using 12% acrylamide gels and staining with Bio-Safe Coomassie Stain (BioRad) and SPYRO ruby (Promega, see Supporting Information Fig. S2). mvShh conjugate MW and Rg,z was measured using size-exclusion chromatography with multi-angle light scattering paired with an in-line differential refractometer and ultraviolet spectrometer (SECMALS-RI-UV). Using a protocol previously described in detail,18, 25 the combined molecular mass distribution for HyA and Shh in the conjugates was determined based on both the distributions of light scattering and differential refraction (dn/dc). Then, based on the known dn/dc and UV extinction coefficients (εex) for HyA and Shh, we could use the conjugate dn/dc and UV absorption values to solve for the Shh and HyA mass fractions over the total MW distribution. The HyA mass fraction indicated the final MW of the HyA after the reactions, and the Shh valency was calculated by dividing the Shh mass fraction by the known MW of Shh (23 kDa). The Shh ligand spacing was calculated by dividing the nominal HyA MW by the Shh valency. Alkaline Phosphatase Bioassay: C3H10T1/2 embryonic murine fibroblasts (ATCC) were maintained in growth medium (GM) consisting of Eagle's minimum essential medium (ATCC) supplemented with 10% fetal bovine serum (FBS, Hyclone) in a 37oC humidified incubator with 5% CO2. Between passages 5 and 15, cells were plated in 96-well plates at an initial seeding density of 3,200 cells/well in GM supplemented with 20 mM HEPES (Lonza). The cells were allowed to proliferate for three days after seeding, and then the media was replaced with fresh GM supplemented with 20 mM HEPES, 2% FBS, and various treatments containing Shh or mvShh. After an additional four days, the cells were rinsed once with TBS (50 mM Tris-Cl, pH 7.6; 150 mM NaCl), and after aspirating the TBS, the cells were flash frozen at -80oC. 0.1%
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Triton-X 100 in TBS was then added to each well and then the plate was agitated for 30 minutes to thaw and lyse the cells. ALP enzyme activity in each well was detected using the pnitrophenyl phosphate (pNPP) liquid substrate system (Sigma) and normalized to the total protein concentration measured by the BCA assay. We fit the resulting dose-response curves using four-parameter logistic non-linear curves to determine the EC50 for each treatment. Enzyme Digestion Assays: Unconjugated Shh and mvShh at the indicated concentrations were incubated with various concentrations of plasmin (Sigma) at room temperature for various digestion intervals. We selected 6.25 U plasmin/mg Shh as a suitable concentration for further investigation on the effect of multivalent conjugation to prevent proteolytic deactivation of Shh (See Supporting Information Fig. S3). Proteolytic degradation was assessed using SDS-PAGE and the ALP bioactivity assays, as described previously. For analysis of digestion fragments using SDS-PAGE, the enzyme activity was stopped by adding Laemmli sample buffer followed by denaturing at 95oC for 10 minutes. This method of quantification was sufficient to measure protein fragment concentrations that were at least 7.5% of the initial protein mass loaded into each well (standardized to 1 ug/well for each sample). For bioactivity analysis of the digestion fragments using the ALP assay, the enzymes were deactivated by the 2% FBS present in the GM for C3H10T1/2 embryonic fibroblasts. The results from this assay were reported as percent bioactivity retained, which was calculated as the either the EC50 for each conjugate incubated with the enzyme divided by a matched dose response curve for a set of no-enzyme control samples. The EC50 was used for the plasmin assays, which yielded good fits (R2>0.9000) using four-parameter logistic fits. In Vivo Wound Healing Model: The IACUC at UCSF approved the animal procedures used in this study. Chronic, non-fasting hyperglycemia (>350 mg/dL) was verified in 10-week-
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old BKS.Cg-Dock7m +/+ Leprdb/J mice (Jackson Laboratory, Bar Harbor, ME) using a commercial blood glucose monitor. The mice were anesthetized with isoflurane in oxygen (13%, adjusted for adequate anesthesia), the dorsum was shaved and sterilized, and then skin tissue was excised from the dorsum using a dermal punch to make a full-thickness excisional wound. To minimize discomfort for the mouse, we used the smallest circular wound sizes needed for each experiment: two bilateral, 0.5-cm diameter wounds per animal for histology and one 1.5-cm diameter wound per animal for wound closure experiments. The animals were randomized into four treatment groups on the basis on blood glucose concentration, and one of the three treatments or vehicle control was applied to each wound via a disc of 1% (w/v) dehydrated methylcellulose (MC; Sigma Aldrich) having the same diameter as the wounds. 6.25 µg/cm2 of Shh (based on wound area) was the treatment dose for both the mvShh and unconjugated treatments. The MC discs were placed directly onto the open wound and covering the entire wound to ensure the treatment dose was applied evenly across the wound surface regardless of the wound size. The MC dissolved after contact with wound fluid, and we verified that the Shh and mvShh retained their bioactivity after release from the MC (see Supporting Information Fig. S4). Wound areas were subsequently imaged and measured every 3-4 days by tracing the wounds onto transparency paper and the calculating the pixel area of each trace with image analysis using ImageJ. Mice were sacrificed at the specified time points by CO2 asphyxiation. Following sacrifice at 24, 48, or 72 hours for histology experiments, we performed a bilateral thoracotomy to expose the heart for perfusion with PBS. Square tissue samples approximately 2 cm on each side and centered on the wound were excised, fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin. The specimens were then sectioned perpendicular to the wound surface at 10-µm intervals and transferred to slides to visualize
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wound cross-sections through the diameter of the wound. At least two sections from each wound were stained using rat anti-mouse CD31 (BD Biosciences) with biotinylated anti-rat IgG and Vectastain Elite ABC reagent (Vectorlabs). CD31+ cell quantification was performed using previously described methods.26-27 We imaged the stained cells in the tissue specimens at 10X magnification, which was sufficient to view the full thickness of epidermis, dermis and granulation tissues. For each specimen, the average cell number per field was calculated from four images taken from sections across the thickness of the wound. Statistical Analysis: All grouped data was represented as mean ± standard deviation, and the Shapiro-Wilk test was applied to all groups to verify normality. Differences between groups were assessed using one-way ANOVA with Tukey post-hoc analysis. Linear regression was used to assess strength of correlation between variables and to identify slope terms that were significantly non-zero. Statistical significance for all tests was assigned for α
