(Invited) In Vitro and In Vivo Near-Infrared Imaging with Biocompatible Bottom-up and Top-Down-Synthesized Graphene Quantum Dots
Authors: Naumov Anton V , Hasan Md Tanvir , Campbell Elizabeth , Lin Ching-Wei , Belcher Angela M. 
Published: May 2020

Carbon nanomaterials offer remarkable properties including high durability, conductivity, and versatility in modification. Carbon nanotubes and graphene oxide also exhibit fluorescence in the near-infrared (NIR) making those attractive for bioimaging and drug delivery applications due to the high tissue penetration depth of the NIR emission. However, despite these remarkable properties, the biocompatibility and degradability of carbon-based platforms still rise some unfortunate controversy that hampers their clinical utilization. In order to address this, we specifically develop NIR-emissive graphene-based quantum dots with high biocompatibility. We explore both the bottom-up and the top-down synthetic approaches together with a variety of doping strategies that yield 2 – 5 nm quasi-spherical quantum dots with graphitic lattice structure observable in TEM. In the bottom-up glucosamine-based synthesis the most prominent NIR emission is observed from graphene quantum dots doped by nitrogen, sulfur or rare-earth metals exhibiting transitions in that spectral region. These GQDs decorated with oxygen-containing functional groups identified with the FTIR have high water solubility and offer efficient cell internalization in HeLa and MCF-7 cells maximized at 12 h. Few percent doping with rare earth metals or nitrogen/sulfur heteroatoms as verified by the EDX does not substantially contribute to the toxic profile of the formulation: doped GQDs exhibit high biocompatibility up to 1 – 2 mg/mL concentrations and degradation in cell culture at 36 h. Finally, these GQDs exhibit emission in the visible with quantum yields up to 60% and also in the NIR, which is utilized for in vitro fluorescence imaging in both spectral regions.
Top-down synthesized GQDs are derived from graphitic materials via UV-assisted radical-based oxidation. Unlike their parent material, these few-layered GQDs containing oxygen addends are water-soluble and exhibit fluorescence in the visible and a wavelength-independent emission in the NIR with NIR quantum yields ranging from 1 to 8%. They are also biocompatible with cell viability over 80% with up to 1 mg/mL GQD concentrations and exhibit cellular internalization within several hours tracked with their NIR fluorescence excited by the 808 nm diode laser. The NIR imaging capabilities of both bottom-up and top-down synthesized GQDs are verified in vivo in live mouse models showing detectable fluorescence in spleen with some liver and kidney signal observed with 808 laser excitation through the tissues of live animals. Excised organs show NIR GQD emission from kidneys, liver, spleen and intestine with GQDs also detected in single organ slices indicating their location within the particular organ. As a result, we suggest that NIR-emissive biocompatible GQDs synthesized both via bottom-up and top-down approaches can be developed and utilized as imaging and, potentially, drug delivery agents both in vitro and in vivo in small animal models.