(696d) Quantum Dot-Based Biomarkers of Neuroinflammation in the Developing Brain

Introduction: Neuroinflammation, mediated by activated microglia and astrocytes, is an important disease hallmark within the development of central nervous system (CNS) diseases, including hypoxic-ischemia encephalopathy (HIE) and autism spectrum disorders (ASD). Thus, comprehension of neuroinflammation is considered essential for both understanding CNS diseases and developing neurological disease therapeutics. However, current studies of biomarkers of neuroinflammation have been difficult due to (1) highly restricted barriers such as the blood brain barrier (BBB) and the brain parenchyma penetration barrier, and (2) the dynamic and heterogeneous neurological disease microenvironment, which is dependent on disease etiology and disease progression, and is variable from patient to patient. Relevant biomarkers and high-throughput platforms that can assay neurological disease severity representative of the in vivo environment can overcome these challenges, but are currently lacking. Our previous research showed that small fluorescent nanoparticles (e.g. 4 nm dendrimers) could target activated microglia in the brain upon systemic administration, and were able to only localize in the region of injury in newborn rabbits with cerebral palsy (CP). This suggested that nanoparticles can be targeted to the regions of the brain that contain diseased cells, as well as specific cell types within those regions, especially in the presence of inflammation. Nano-sized fluorescent semiconductor quantum dot (QD) particles come to light as potential neuroinflammation biomarkers and have several advantages over traditional biomarkers including small size (5-10 nm), tailorable excitation and emission spectra, tailorable surface functionalities, and high photoluminescence and photostability, which are ideal characteristics for in vivo imaging. In this study, we evaluated QDs as biomarkers of inflammation in the developing brain. Our overall hypotheses are that QD cell-specific uptake within the brain will be governed by (1) QD stability in the brain microenvironment, (2) QD surface functionality, and (3) the extent of neuroinflammation, evaluated by microglia and astrocyte activation.

Materials and Methods: We investigated QD stability, toxicity, and cellular uptake in both organotypic brain slices and in vivo in a neonatal rat model. QDs with different surface functionalities were incubated with saline, artificial cerebrospinal fluid (aCSF) and brain tissue homogenate to characterize their stability in relevant media. For QD toxicity, lactate dehydrogenase (LDH) assays, propidium iodide (PI) staining, and Fluoro-Jade C staining were utilized following QD incubation with 300-400 µm organotypic brain slices obtained from neonatal wide type (WT) rats. QD cellular uptake in brain slices was evaluated by using immunohistochemistry (IHC) and confocal microscopy to characterize the colocalization of QDs with cell populations in the brain, including microglia, astrocytes, and neurons. We also evaluated QD biodistribution and cellular localization following systemic administration in the presence of neuroinflammation in a newborn rat model with hallmarks of ASD (metabolic glutamate receptor 5 knockout rats (mGluR5 KO)), compared to age-matched WT controls. Biodistribution, confocal microscopy and fluorescence-activated cell sorting (FACS) were utilized for quantitative analysis of QD uptake in the brain.

Results and Discussion: We show that QDs functionalized with polyethylene glycol (PEG) chains are stable up to 24 h in all media at both room temperature and at physiological temperature. QDs without a PEG coating (e.g. QD-carboxyl) rapidly aggregate in saline and aCSF at 23ËšC and 37ËšC, independent of the composition and ion concentration of the biological fluid. This stability result correlates with QD toxicity and cellular uptake imaging, where QDs without PEG aggregate in tissue and do not localize in cells, exhibiting overall lower toxicity and lower cell internalization compared to ones with PEG. Between the two most stable QDs, QD-PEG-NH₂ (PEG-amine) is found to specifically localize into neurons, while QD-PEG-OMe (PEG-methoxy) remains in the extracellular space (ECS). This suggests that QD surface functionality can affect the stability of QDs, and therefore determine their cytotoxicity and cell co-localization. Our in vivo results demonstrate that the QD-PEG-NH₂ localizes in activated microglia in the mGluR5 KO rat brain, and does not penetrate the BBB or uptake in cells in age-matched, litter-matched WT controls. Furthermore, FACS sorted microglia cells contained QDs, demonstrating that these cells are a pro-inflammatory phenotype. Based on these results, there is an association between QD uptake in the brain and neonatal neuroinflammation severity, suggesting that QDs can be a biomarker of neuroinflammation presence and severity.

Conclusions: Biomarkers for characterizing the severity of neonatal neuroinflammation are in need for understanding developing brain injuries and furthering therapeutic development. QD-based biomarkers have unique imaging advantages over traditional organic dyes and have great potential as biomarkers in the brain. Our study systemically evaluates QD stability, toxicity and cellular uptake in the developing brain, which begins to fill the critical knowledge gap in developing ideal QD-based biomarkers for investigation of neuorinflammation etiology and progression. Exploration of QD penetration of the BBB, specific cell internalization, intracellular trafficking, and diffusion in the CNS would also bring about a detailed molecular-level understanding for the future in vivo application of QDs in the developing brain.