(208f) Anti-Inflammatory Backpack-Carrying Macrophages for Porcine Traumatic Brain Injury

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 inflammation contributes a pivotal role in secondary brain injury, exacerbating excitotoxicity, oxidative stress, blood-brain barrier (BBB) and blood-cerebrospinal fluid-barrier (BCSFB) dysfunction, and ultimately cell death in TBI. Thus, anti-inflammatory therapeutics are promising for mitigating TBI secondary injury. However, effective drug delivery is hindered by poor penetration and accumulation in diseased brain regions and limited duration of action. To overcome these obstacles, we have implemented a novel approach of attaching anti-inflammatory-loaded discoidal microparticle backpacks onto macrophages to leverage chemotactically targeted therapeutic delivery to the diseased brain after TBI.

Materials and Methods: We formulated anti-inflammatory polymer backpacks composed of a poly(vinyl alcohol) (PVA) layer with interleukin-4 sandwiched between layers of poly(lactic-co-glycolic acid) PLGA Resomer 502H with dexamethasone. For backpack synthesis, we spin-coated our polymer/drug solution onto polydimethylsiloxane (PDMS) elastomer molds, followed by microcontact printing to yield uniform 8 μm backpacks. We isolated bone marrow cells from the ribs of Yorkshire piglets and cultured them to obtain anti-inflammatory macrophages. We incubated macrophages with backpacks for adhesion and evaluated macrophage phenotype over time in inflammatory in vitro culturing conditions with flow cytometry. Furthermore, we assessed the therapeutic efficacy of anti-inflammatory backpack-carrying macrophages in reducing lesion size and inflammation in a cortical impact model of TBI in one-month old piglets.

Results: We achieved backpack binding onto ~35% of macrophages. In vitro, anti-inflammatory backpacks significantly reduced pro-inflammatory expression of CD80 and iNOS and increased anti-inflammatory expression of CD206 and Arg1 across 7 days compared to that of free drug in pro-inflammatory culture media. After cortical impact, backpacks specifically accumulated in the ipsilateral hemisphere. Evaluated at 7 days after injury, anti-inflammatory backpack-macrophage treatment reduced macroscopic lesion volume by 60% compared to that of saline control (p=0.0627). Furthermore, in the peri-contusion region, treated pigs exhibited a significant reduction in microglia density, microglia circularity, and CD80 expression compared to that of saline control. Pig serum and CSF had reduced concentrations of inflammatory biomarkers tumor necrosis factor alpha and glial fibrillary acidic protein.

Conclusions: Our work demonstrates that anti-inflammatory microparticle backpacks provide sustained anti-inflammatory activity that results in enhanced therapeutic effects. Therapeutic backpacks bind onto the surface of macrophages without phagocytic internalization, taking advantage of macrophage chemotaxis to injured brain regions for locally targeted extracellular therapeutic effect. Controlled release of interleukin-4 and dexamethasone from polymer backpacks extends anti-inflammatory polarization of carrier macrophages and skews the injured brain regions towards an anti-inflammatory microenvironment across 7 days. In a TBI pig model, anti-inflammatory backpack-macrophage treatment reduces lesion volume and inflammation of peri-contusion regions, resolving inflammatory expansion of secondary brain injury. Altogether, anti-inflammatory-loaded backpack-carrying macrophages are a promising neurotherapeutic strategy for treating TBI.