Dual-Mode Fluorescence/Ultrasound Imaging with Biocompatible Metal-Doped Graphene Quantum Dots

Abstract

Sonography offers many advantages over standard methods of diagnostic imaging due to its non-invasiveness, substantial tissue penetration depth, and low cost. The benefits of ultrasound imaging call for the development of ultrasound-trackable drug delivery vehicles that can address a variety of therapeutic targets. One disadvantage of the technique is the lack of highprecision imaging, which can be circumvented by complementing ultrasound contrast agents with visible and, especially, near-infrared (NIR) fluorophores. In this work, we, for the first time, develop a variety of lightly metal-doped (iron oxide, silver, thulium, neodymium, cerium oxide, cerium chloride, and molybdenum disulfide) nitrogen-containing graphene quantum dots (NGQDs) that demonstrate high-contrast properties in the ultrasound brightness mode and exhibit visible and/or near-infrared fluorescence imaging capabilities. NGQDs synthesized from glucosamine precursors with only a few percent metal doping do not introduce additional toxicity in vitro, yielding over 80% cell viability up to 2 mg/mL doses. Their small (<50 nm) sizes warrant effective cell internalization, while oxygen-containing surface functional groups decorating their surfaces render NGQDs water soluble and allow for the attachment of therapeutics and targeting agents. Utilizing visible and/or NIR fluorescence, we demonstrate that metal-doped NGQDs experience maximum accumulation within the HEK-293 cells 6−12 h after treatment. The successful 10-fold ultrasound signal enhancement is observed at 0.5−1.6 mg/mL for most metal-doped NGQDs in the vascular phantom, agarose gel, and animal tissue. A combination of non-invasive ultrasound imaging with capabilities of high-precision fluorescence tracking makes these metal-doped NGQDs a viable agent for a variety of theragnostic applications.

Cancer Therapeutic siRNA Delivery and Imaging by Nitrogen- and Neodymium-Doped Graphene Quantum Dots

“…Due to its small amount, the metal dopant is not expected to affect the biocompatibility of the GQDs. 65 Besides Nd, EDX analysis indicates carbon, oxygen, and nitrogen, suggesting the presence of various functional groups in the GQD structure. FTIR spectra of freeze-dried GQD samples (Figure S3) indicate that hydroxyl, carboxyl, and amino groups are most likely present on the surface of the GQDs, enabling high water solubility as well as complexation with nucleic acids 50 and covalent functionalization for biological applications.…”

“…EDX spectra demonstrate ∼1 atom % of Nd in Nd-NGQDs, confirming their successful doping. Due to its small amount, the metal dopant is not expected to affect the biocompatibility of the GQDs . Besides Nd, EDX analysis indicates carbon, oxygen, and nitrogen, suggesting the presence of various functional groups in the GQD structure.…”

“…Both siEGFR and siKRAS were labeled with ROX and complexed with either NGQDs or Nd-NGQDs to form four combinations: NGQDs/siEGFR-ROX, NGQDs/siKRAS-ROX, Nd-NGQDs/siEGFR-ROX, and Nd-NGQDs/siKRAS-ROX. These complexes were left to transfect HeLa cells for 12 h, which has been previously determined as an optimal internalization time for both GQD types. , In order to separately image fluorescence from GQDs and ROX, these were spectrally separated by exciting at 480 and 540 nm, respectively, with the corresponding emission recorded at 535 ± 20 and 600 ± 20 nm (Figure ). Confocal images taken at a median plane within the cells demonstrate only fluorescence from the internalized GQDs and siRNA-ROX, disregarding those accumulated on the cell membrane.…”

Biocompatible Aluminum-Doped Graphene Quantum Dots for Dual-Modal Fluorescence and Ultrasound Imaging Applications

“…In previous work, we demonstrated that GQDs doped with various metals generate up to a 10-fold ultrasound signal enhancement compared to that of nonmetal-doped GQDs and water control in vascular phantom, agarose gel, and animal tissue. 23 In order to test the ability of Al-GQDs to enhance the ultrasound signal, they are also introduced into these simulated bioenvironments and imaged with the GE Logiq E portable ultrasound system. While nearly no ultrasound signal is detected from water control within 3% agarose gel (Figure 6a), substantial ultrasound signal enhancement is immediately observed after the injection of Al-GQDs (Figure 6b).…”

“…In such instances, neodymium and thulium-doped GQDs offer in vitro, ex vivo, and in vivo NIR imaging, enabling noninvasive image tracking in live mice, while retaining high biocompatibility. Incorporating metal dopants into the GQD platform also produces biocompatible nanostructures with additional ultrasound contrast capablities. − Such multimodality is enabled by a simple and scalable bottom-up GQD synthesis from the plethora of carbonaceous precursor materials. Moreover, GQD structures can be synthetically tailored to facilitate specific therapeutic functions, such as targeting disease sites, delivering drug and gene medicine, and assisting with photothermal ablation .…”