381884 Dendrimer Nanodevices for Pediatric Brain Injury
Introduction: Maternal-fetal inflammation (MI) and hypoxia-ischemia (HI) are major risk factors for perinatal/neonatal brain injury, and both can lead to neurological diseases including cerebral palsy (CP) and autism. Neuroinflammation, mediated by activated microglia and astrocytes, is implicated in the pathogenesis of both etiologies, resulting in diffuse white and grey matter injury. Targeting activated microglia/astrocytes may offer such an opportunity. However, this is a challenge on multiple levels because (1) transport of drugs and drug delivery vehicles across the blood-brain-barrier (BBB) is difficult to achieve, (2) inflammation and injury are often diffuse, making it difficult for therapeutics to reach target cells even if administered locally, and (3) the role of microglia and extent of BBB disruption as disease progresses in the developing brain is not well understood, especially after a therapy is administered. Recent studies from our laboratory have shown that dendrimer nanomaterials can target activated microglia in the brain upon systemic administration, producing dramatic motor function improvements and neuronal repair in a rabbit model of CP. These studies suggest that nanotechnology-based approaches provide potential platforms for CNS therapy. However, there is a major knowledge gap relating to the effect of changes in extracellular matrix, brain edema, glial cell function and BBB disruption, on the movement, interactions, and cellular uptake of nanoparticles following injury to the brain.
Materials and Methods: In this work, we use two clinically relevant, broad etiological mechanisms of neonatal brain injury (HI or MI) to study the effect of dendrimer size and surface functionality, as well as disease etiology and pathophysiology, on the ability of the dendrimer to penetrate a disrupted BBB, diffuse within the brain parenchyma, and selectively uptake in microglia cells. For efficacy, N-acetyl cysteine (NAC) conjugated to OH-dendrimer (D-NAC) was administered systemically, and animals were followed for survival or sacrificed at specified timepoints for quantitative, immunohistochem (IHC), or RT-PCR analysis. For fluorescent-dendrimer conjugates injected systemically, brains were harvested at specified timepoints and used for IHC or for quantitative analysis using spectrophotometry. Blinded neurobehavioral analysis was completed and scored to determine extent of disease and efficacy of dendrimer-drug conjugates compared to free drug (NAC), saline controls, or age-matched healthy controls.
Results and Discussion: In both etiologies, we show that systemic administration of a single dose of dendrimer-drug conjugate results in accumulation in activated microglia in the brain of newborn rabbits with MI-induced CP or newborn mice following HI, leading to sustained attenuation of neuroinflammation up to 8 days after injury. BBB breakdown is greater in HI-mediated brain disease compared to MI-induced brain disease. Following systemic administration, neutral dendrimer penetrates a disrupted BBB in both models, and rapidly accumulates in microglia within 4 hours. Cationic dendrimers do not penetrate within the brain parenchyma or localize in microglia cells, and anionic dendrimers show delayed uptake in the brain and in microglia cells. These results demonstrate the importance of dendrimer surface functionality on movement and localization within the brain. Dendrimer was not found in the brain in age-matched healthy controls. Importantly, a strong positive correlation between % injected dose of dendrimer uptake in the brain and disease score based on neurobehavioral testing was found, with sicker animals showing greater uptake, likely due to increased BBB impairment and greater microglia activation representative of a stronger inflammatory response.
Conclusions: Pediatric illnesses are often underserved by novel drug delivery technologies, which focus primarily on adults. Therefore, there is great opportunity to bring nanotherapeutic approaches to perinatal and neonatal brain injury, with implications that can be translated to adult brain injury. These findings begin to address the critical knowledge gap in identifying the physicochemical properties of nanoparticle platforms and pathological properties of disease that facilitate brain penetration, diffusion and specific cellular uptake that would promote targeted attenuation of neuroinflammation in the brain.