(H-311) Near-infrared fluorescent nanosensors for in vivo pharmacology and inflammation monitoring

Introduction:: Rapid, minimally-invasive in situ detection of analytes in vivo remains a goal to study inflammatory signaling molecules and pharmacology of therapeutics, as well as in the translation of personal diagnostics to the clinic. For example, local and systemic levels of inflammatory cytokines, such as IL-6 and IL-1β, are predictive of the severity and progression of various diseases, including viral infection and sepsis, as well as cardiovascular, autoimmune, and neurodegenerative diseases. Current inflammatory monitoring methods include blood draws coupled with traditional, time-consuming immunoassays, precipitating the need for more rapid and inexpensive multiplexed inflammatory diagnostics. Towards this end, we are using the near-infrared photoluminescent properties of single-walled carbon nanotubes (SWCNT) to design sensors for use in live mice and in patient fluids at the bedside. The photostable, environmental-responsive, tissue-transparent emission of SWCNT is ideal for this application.


Materials and Methods:: We have designed multiplexed inflammatory cytokine-specific fluorescent sensors using engineered SWCNT. We employed aqueous two-phase separation to separate multiple chiral species of fluorescent nanotubes, which were then separately functionalized with antibodies and/or ssDNA aptamers against specific cytokines of interest. Separately, we screened interactions with small molecule organic dyes and protein/polymer passivation agents to optimize both the sensitivity and specificity of the sensor system. We then evaluated response of the engineered sensors to the cytokines of interest in human samples and animal serum. Finally, we embedded the fluorescent nanotube sensors into an injectable methylcellulose hydrogel system, which we injected locally into mice. We measured fluorescence and sensor functionality over a several-week period using a hand-held near-infrared fluorescence probe.


Results, Conclusions, and Discussions:: Our studies have discovered several protein and polymer passivation agents which impart highly sensitive and specific cytokine sensing in human serum. Specifically, sensors pre-incubated with poly-L-lysine demonstrated detection IL-6 in human serum as low as 25 pg/mL and up to 25,000 pg/mL, encompassing a clinically-relevant range of detection for infectious and chronic inflammatory diseases. We also found a specific formulation of 3% sulfonated methylcellulose hydrogel which allows for simple injection into an animal with in situ gel formation as well as the detection of small molecule and protein analytes diffusing through the gel. We continue to apply these findings to screening patient samples ex vivo to develop low-cost bedside diagnostics. Further, we continue to deploy these sensors using rodent models of chronic inflammatory disease to understand the role of individual cytokines in these models of disease towards the development of next-generation personal diagnostics.


Acknowledgements (Optional): : We acknowledge NIH NIGMS support (R35148233).

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