(316c) Antioxidant-Immobilized Backpack-Carrying Monocytes for TBI Therapy

Introduction: Traumatic brain injury (TBI) afflicts around 3 million people in the United States annually, with around 55,000 fatal cases. Furthermore, moderate or severe TBI can cause long-term disabilities including difficulty in sensorimotor function, memory issues, depression, and dementia. There are no clinically approved therapeutics to reduce TBI damage from the initial insult or secondary injury. Due to the lifelong impact of TBI, there is a strong clinical need to develop therapeutics that mitigate TBI damage. TBI damage encompasses the initial impact trauma and secondary brain injury. Elevated oxidative stress contributes a pivotal role in secondary brain injury, exacerbating excitotoxicity, blood-brain barrier (BBB) and blood-cerebrospinal fluid-barrier (BCSFB) dysfunction, neuroinflammation, and ultimately cell death in TBI. Thus, the antioxidant enzyme catalase is a promising therapeutic for mitigating TBI secondary injury. However, effective enzyme delivery is hindered by poor penetration and accumulation in diseased brain parenchyma and enzyme degradation from extracellular proteases. To overcome these obstacles, we have implemented a novel approach of immobilizing catalase into discoidal hydrogel scaffolds termed backpacks and attaching these backpacks onto monocytes to leverage chemotactically targeted therapeutic delivery to the diseased brain after TBI.

Materials and Methods: We formulated hydrogel backpacks composed of 250kDa hyaluronic acid-methacrylate (HA-MA), 1kDa 2-arm poly(ethylene glycol) methacrylate (PEGDMA), Irgacure 2959 (I2959) photoinitiator, and catalase. We converted free primary amines on catalase to sulfhydryl groups with Traut’s reagent to form immobilization reaction sites. For backpack synthesis, we spin-coated our hydrogel solution onto polydimethylsiloxane (PDMS) elastomer molds with 8 μm wells. Upon 365 nm irradiation with I2959 photo-initiation, HAMA and PEGDMA pendant MA groups covalently conjugate together via free radical polymerization to form HAMA/PEGDMA hydrogels, while MA groups also react with catalase sulfhydryl groups via the thiol-ene Michael addition-based click reaction for immobilization. Subsequent O₂ plasma etching to remove the thin hydrogel film and printing yields uniform 8 μm hydrogel backpacks. With hydrogen peroxide (H₂O₂) as the substrate for catalase, we measured catalase activity with kinetic 240nm spectrophotometric detection of H₂O₂ decomposition and evaluated catalase activity in the presence of degradative proteases over time. We isolated bone marrow cells from the tibias and femurs of C57BL/6J mice and cultured them with macrophage colony stimulating factor (MCSF)-containing media on ultra-low adhesion plates to obtain monocytes. After backpack incubation with our primary mouse monocytes, we obtained ABC-monocytes.

Results: Catalase modification with Traut’s reagent was non-denaturing, resulting in enzyme activity retention of 89.53%. Starting at 100% at time 0h, upon incubation with degradative proteases, freely suspended catalase rapidly degraded to 4.7% activity in 2h. Unmodified catalase loaded within backpacks exhibited 24.6% activity at 2h and decreased to 2.5% activity at 24h. On the other hand, Traut’s reagent-modified catalase immobilized within backpacks exhibited 42.3% activity at 2h, 39.6% activity at 24h, and 19.8% activity at 21 days before reducing to 0.0% activity at 28 days. We achieved backpack binding on 56.2% of mouse monocytes with a 3:1 catalase-immobilized backpack to mouse monocyte ratio.

Conclusions: Our work demonstrates that by immobilizing catalase within hydrogel backpacks to prevent release, the hydrogel backpack scaffold sterically protects catalase from environmental protease degradation while permitting H₂O₂ diffusion for retained enzyme activity. This antioxidant immobilization strategy extends catalase activity from less than 1 day to over 21 days. Furthermore, our hydrogel backpacks bind onto the surface of monocytes without phagocytosis, enabling continuous extracellular H₂O₂ scavenging. Antioxidant-immobilized backpack-carrying monocytes are a promising neurotherapeutic strategy for reducing oxidative stress to ultimately mitigate TBI secondary injury.