Design of Carbon Nanotube Neuro-Sensor Corona Phase for Improved Biocompatibility and Neuromodulator Imaging

Single-walled carbon nanotubes (SWCNT) are an emerging nanomaterial platform enabling promising advances in biomolecular imaging. Noncovalent modification of SWCNTs with amphiphilic ligands reveals intrinsic SWCNT near-infrared fluorescence and imparts molecular recognition. In particular, SWCNTs wrapped with DNA oligonucleotides develop selective fluorescent response to catecholamine neurotransmitters such as dopamine. Consequently, these DNA-SWCNT probes have been applied for recording dopamine activity in brain tissue. However, two major barriers to in vivo application of SWCNT dopamine probes exist. Firstly, DNA-SWCNTs are highly susceptible to biofouling by protein adsorption reducing efficacy of sensors in vivo. Secondly, the impact of these artificial nanomaterials on biological environments remains poorly understood. Towards the first point, we develop a novel method for the study of biomolecular adsorption to the SWCNT surface utilizing the fluorescence quenching ability of carbon nanotubes to track adsorption of fluorophore-conjugated biomolecules. We leverage multiplexing using dissimilar dyes conjugated to each molecular species in order to simultaneously monitor the competitive adsorption/desorption of proteins and single-stranded DNA, respectively. Here we show that attenuation of dopamine response is largely due to protein adsorption rather than DNA desorption. Furthermore, we identify fibrinogen as a ubiquitous protein with high affinity to the SWCNT surface./nNext, we study the effects these SWCNT probes may elicit once implanted in brain tissue. Here we utilize microglial cells as a model for neuroinflammation. Microglia are the specialized immune cells of the central nervous system which are central in maintaining the homeostasis of the neuronal environment. Microglia are activated by interaction with foreign or pathogenic material, mediating and propagating inflammatory responses throughout the brain which can elicit downstream neurotoxicity and neurodegeneration. Examining the interactions between this cell type and carbon nanotube neuro-sensors is crucial in characterizing and quantifying the net biological impact of these probes on surrounding brain tissue. We utilize high-throughput sequencing and live-cell imaging to show that exposure to SWCNT probes induces significant upregulation of inflammatory signaling pathways and cell morphology change in SIM-A9 microglia. Although inflammation response is lower in magnitude than that provoked by inflammatory simulants such as lipopolysaccharide, SWCNTs uniquely induced drastic changes to cell morphology, where cells exposed to SWCNTs progressed from round and motile states to highly ramified and stationary within several hours. These effects suggest activation of microglia into pro-inflammatory phenotypes by SWCNTs. This must be minimized in order to accurately study chemical signaling in the brain./nSubsequently, we devise strategies for noncovalent passivation of exposed SWCNT graphene lattice to improve biocompatibility. We apply protein adsorption and microglial activation as metrics by which to test noncovalently modified SWCNT catecholamine sensors. We find that passivation of DNA-SWCNTs using PEGylated phospholipid significantly decreases nonspecific protein adsorption and SWCNT-induced microglial ramification. These passivated neuro-sensors are then applied to image striatal dopamine release and reuptake events in excised mouse brain tissue. We show that brain slices labeled with passivated neuro-sensor exhibited both improved diffusivity through tissue and higher fluorescence response to dopamine release over unmodified SWCNT probes. Hence, we present three stages of development and optimization of a carbon nanotube-based neuro-sensor through design of the nanoparticle corona phase. The methodologies presented here are readily translatable to other bionanotechnologies and represent an advancement in the field of nanomaterial biosensors for molecular imaging.

Advisor: Landry, Markita