Introduction: Inflammation in the central nervous system, mediated by activated microglia and astrocytes, is implicated in the development of several neurologic disorders in both children and adults, including cerebral palsy (CP). Strategies to target microglia/astrocytes and treat neuroinflammation can potentially not only slow disease progression, but also promote repair and regeneration, enabling normal development and maturation of the brain. Recent findings demonstrate that intravenous administration of a dendrimer-drug nanoparticle system selectively targets and accumulates in activated microglia/astrocytes in the brain of newbown rabbits with neuroinflammation and CP, resulting in significant improvement in motor function and myelination, attenuation of activated microglia, and decrease in neuronal injury by 5 days. However, the role of dendrimer physicochemical properties and disease physiology on this selective brain uptake and localization of dendrimers that are responsible for achieving these efficacious results is not well understood. Methods: A combination of in vivo and ex vivo methods were used to study the effect of dendrimer size and surface properties on the ability to penetrate a disrupted BBB, diffuse within the brain parenchyma, and selectively uptake in microglia cells in rabbit kits exposed to endotoxin in utero. Brain slices were collected from kits exposed to endotoxin in utero as well as from healthy kits. The slices were grown on membrane insets for several days, incubated with G4-OH-Cy5 or G4-NH2-FITC, and imaged using a two-photon laser scanning microscope. For systemic administration, G4-OH-Cy5, G4-NH2-FITC, and PS-COOH nanoparticles were injected into postnatal day 1 kits exposed to endotoxin in utero. For intracranial injections, G4-OH-Cy5 dendrimer was injected 1mm superficially into the cortex, and G4-OH-FITC was injected 3mm into white matter. For all in vivo studies, at various timepoints, kits were perfused, and brains were stored in formalin followed by a sucrose gradient prior to sectioning. Sections were stained with lectin for microglia and GFAP for astrocytes, as well as DAPI to label cell nuclei. Results: In brain slices from endotoxin kits, G4-OH-Cy5 dendrimer could diffuse through the brain parenchyma, preferentially accumulating in microglia, whereas G4-NH2-FITC dendrimers did not diffuse in slices. Dendrimer uptake in microglia was minimal in age-matched healthy control slices. Following intracranial administration into the cortex of an endotoxin day 1 kit, G4-OH dendrimer was found distributed in the parenchyma at 4 hrs and 24 hrs, and localized in microglial cells at 24 hrs. When injected intracranially into white matter, dendrimer were directionally distributed along white matter tracts and localized in microglial cells. In age-matched healthy controls, minimal uptake in microglia was seen in either white matter or cortical regions; however, dendrimer was found spread throughout the parenchyma at 24 hours post-administration confirming the diffusive nature of these particles. Following systemic administration, G4-OH-Cy5 dendrimer penetrate a disrupted BBB in endotoxin kits and were found within the parenchyma at 4 hours post administration, and within microglial cells 24 hours post administration. Polystyrene nanoparticles 20 nm in size did not cross the BBB or selectively uptake into microglia cells, in ex vivo slices or in vivo endotoxin kits. G4-OH-Cy5 dendrimer was not found in the brain in age-matched healthy controls following systemic administration. Conclusions: We found that size and surface charge are not only critical to the ability of a dendrimer nanoparticle to cross an impaired BBB and diffuse within the brain parenchyma, but also are key factors in uptake into cells involved in neuroinflammation, such as microglia and astrocytes. Understanding the mechanism of uptake will allow for better design and more efficient delivery of dendrimers platforms to diseased cells, further increasing therapeutic efficacy.
1N/A, Baltimore, MD, 2Johns Hopkins University SOM, Baltimore, MD