(T1530-12-69) Shear-Thinning Injectable Hyaluronic Acid Hydrogels for Diabetic Wound Healing

Purpose: Diabetic foot ulcers (DFUs) affect around 15% of the estimated 460 million global diabetes patients1-3. Approximately 5% of individuals in the US who have a DFU move on to above-ankle amputations4, meaning that of the 37 million Americans alone, around 1.85 million will require a major amputation. Reactive oxygen species (ROS) are more prevalent in DFUs, and result in irregular tissue remodeling and slower wound healing3. The only FDA-approved pharmacological therapeutic is becaplermin, which is a growth factor-loaded gel that promotes tissue remodeling. Becaplermin fails to reduce local ROS, which decreases the wound healing potential of the growth factors3,5. Besides its costliness, becaplermin, in its current NaCMC formulation, fails to offload pressure from or seal the wound, both of which increase the risk of prolonged injury or reinjury resulting from poor tissue remodeling6. To address these deficiencies, we have developed a library of ABC-triblock drug-loadable, biodegradable polymer nanoparticles (NP) and a biocompatible hyaluronic acid matrix (HA). One component of the nanoparticle contains adamantane (AD) moieties, which are physically encapsulated by the internal binding pocket of β-cyclodextrin (βCD) moieties grafted to the HA subunits. The nanoparticle-HA system creates a physically crosslinked hydrogel that can deform under shear stress or strain and regain its prior shape after removal of force. The deformation under force, or shear-thinning, allows for subcutaneous injection of the gel at the wound site, lengthening time between treatments and offloading pressure from the wound tissue by deforming under mechanical load6. Our previous research demonstrates that the polypropylene sulfide (PPS) block of our triblock nanoparticle formulation deactivates ROS, reducing local inflammation and promoting healthy tissue remodeling7 <. Additionally, ROS-mediated oxidation of PPS sulfides to sulfones and sulfoxides increases nanoparticle hydrophilicity, allowing for controlled release of hydrophobic drug from the core of the nanoparticle over an extended time period8.

Methods: Synthesis of PPS135-b-PDMAx-b-P(DMA-co-ADA)y: The following procedure, for the synthesis of PPS135-b-PDMA300-b-P(DMA160-co-ADA40) is a representative procedure for triblock synthesis. A diblock solution comprising PPS-b-PDMA grafted to a macro-chain transfer agent was mixed with DMA, N-(Adamantane-1-yl) acrylamide, and a radical initiator were mixed and heated after purging with high-purity nitrogen for 30 minutes. After reaction completion, the resulting solution was purified via precipitation into diethyl ether, and further purified via lyophilization and reconstitution. Rheological Characterization of Gels: Shear-thinning properties and mechanical strength of hydrogels were measured using an AR2000 stress-controlled rheometer (TA Instruments, New Castle, DE) fitted with a cone plate geometry (20mm diameter, 0.995°cone angle). Rheological properties were examined at 25 °C using oscillatory frequency sweeps (0.01−100 Hz; 0.5% strain) and percent strain-step based oscillatory time sweeps (0.5% low strain, 300% high strain) in 1 minute cycles at 10Hz frequency. The tan(δ) value, which is the ratio of loss modulus to bulk modulus, was calculated and plotted. Hydrogel Degradation: Polymer was dissolved in around 200µL of dichloromethane and mixed with around 5mg of Nile Red dye (Thermo Fischer, Hampton, NH). Nanoparticles were put under rotary evaporator for around 10 minutes. Samples were resuspended in 50µL of PBS, and mixed at different ratios with CD20%-HA. The resulting hydrogels were aliquoted at volumes of 50µL, and treated with 10µL of varying concentrations of hydrogen peroxide, hyaluronase (Thermo Fischer, Hampton, NH), or a 1:1 combination of the two, representative of physiological conditions. Samples were assayed using the aforementioned rheological characterization methods.

Results: We generated a library of polymers, whose resulting hydrogels produce a variety of mechanical properties (Fig. 1). The data suggest that altering the DMA block, the B-block of our triblock nanoparticle, to comprise 300 subunits (D300) of the molecule DMA rather than 200 or 150 results in higher G’ than the shorter D150 or D200 blocks (Fig. 1B-D). Further, using higher adamantane concentration increases mechanical strength. To quantify hydrogel capability of deactivating ROS, we subjected lead candidate samples (one sample per time point per concentration) to varying concentrations of H2O2 as a surrogate for ROS, then mechanically characterized each sample via rheometer. The data show that the hydrogels comprising the PPS nanoparticles maintain a lower tan(δ), meaning less chemical degradation, under equivalent ROS concentrations (particularly 100mM) than the control gels in which polycaprolactone (PCL) was substituted for the PPS A-block (Fig. 2).

Conclusion: We developed a mechanically tunable library of polymers capable of forming supramolecular guest-host interactions. Our gels address many of the present shortcomings of the existing NaCMC formulation of becaplermin. The potential further exists to dual-functionalize these polymers with the capacity to form chemical and physical crosslinks, potentially increasing tunability of mechanical properties and providing other properties favorable for wound healing, such as bioadhesion or injectability.

References: (1) Khan, M. et al. (2020), Journal of Epidemiology and Global Health, 10(1), 107–111. (2) Yazdanpanah, L. et al. (2015) World Journal of Diabetes, 6(1), 37–53. (3) Kunkemoeller, B. and T. R. Kyriakides. (2017) 27(12), 823–838. (4) Brennan, M.B. et al. (2017). “Diabetic foot ulcer severity predicts mortality among veterans with type 2 diabetes.” J. Diabetes Complications, 31(3), 556-561. (5) Fang, R. C. et al. (2008) Biologics:Targets & Therapy, 2(1), 1–12. (6) Guvendiren, L. et al. (2012) Soft Matter, 8(2), 26–272. (7) Dollinger B. R. et al. (2017) Tissue Engineering, 23(19-20), 1120-113. (8) Xiong, Y. et al. (2021) Polym. Chem., 12, 3721-3739.

Figure 1. A. Heat map of tan(δ) (loss modulus/storage modulus) values across polymer library. B-D. Storage moduli of hydrogels mixed at various ratios of HA to NP.



Figure 2. A-C. Storage moduli of PPS (radical-scavenging), PCL (NP control), and HA-HA (in vivo model) gels after treatment with varying concentrations of H2O2 as a mimic for ROS. D-F. Heat map of tan(δ) values for the corresponding hydrogels in A-C.