Multimodal Microscopy Distinguishes Extracellular Aggregation and Cellular Uptake of Single‐Walled Carbon Nanohorns

Abstract

The low toxicity, high surface area, and ease of functionalisation of carbon nanohorns (CNH) makes them attractive systems for cellular imaging, diagnostics and therapeutics. However, challenges remain for the biomedical translation of these and other nanomaterials. A significant task is tuning the surface chemistry to achieve optimal cellular interactions. Herein, we combine real-time fluorescent imaging of nanoparticle cellular uptake and real-time differential interference contrast (DIC) imaging of extracellular media to monitor a) nanoparticle/nanoparticle and b) nanoparticle/cell interactions for CNHs covalently modified with an OFF/ON near-IR dye, the fluorescence of which is switched OFF in extracellular environments and triggered upon cellular internalisation. CHN samples modified with different loadings of the hydrophobic dye are taken as a simple model of drug-loaded nanoparticle systems. The punctate fluorescence suggests the CNHs are delivered to lysosomes and other vesicles of the endocytic pathway. DIC imaging highlights the competition that exists for many particle types, between extracellular aggregation and cellular internalization, the efficiency of which would be dependent upon the amount of fluorophore loading. The results of this study illustrate how complementary real-time imaging methods together with physicochemical characterisation can be used to address the challenges involved in optimising nanoparticle/cell interactions for biomedical applications.

Investigation of the Cellular Destination of Fluorescently Labeled Carbon Nanohorns in Cultured Cells

“…Oxidized CNHs (o-CNHs) were prepared according to previously reported procedures by reacting commercially sourced pristine CNHs under reflux with nitric acid. , Scanning electron microscopy (SEM) showed they possessed a spherical shape with a core of approximately 100 nm in size, while transmission electron microscopy (TEM) revealed the conelike structure of the particles (Figure A,B). The o-CNHs were next functionalized with an amine-terminated short-chain glycol chain using carbodiimide coupling of a diamino glycol in large excess, followed by repeated centrifugal washing.…”

“…Importantly, a 10% loaded formulation was found to be uptaken by cells, which then underwent photoactivated cell death. We have also used live cell imaging to simultaneously report on intracellular uptake and extracellular aggregation as a function of surface modification with a cellular responsive fluorescent dye . However, while these experiments and others have demonstrated the uptake of CNHs into cells, their range of internal destinations and mechanisms of endocytosis and transport are only poorly described.…”

Optical Microscopy Systems for the Detection of Unlabeled Nanoparticles

“…Differential interference contrast microscopy (DICM) is frequently utilized to study the interactions between cells and NPs, particularly their cellular uptake. 66 , 67 The DIC mode of confocal microscopes can also be used to detect individual NPs. However, in contrast to DFM, DICM usually produces images with very low contrast compared to the background.…”

Carbon nanohorn modified platinum electrodes for improved immobilisation of enzyme in the design of glutamate biosensors

“…Oxidized CNHs (o-CNH) were prepared by previously reported methods. [39][40][41] Briefly, 50 mg of pristine CNHs were oxidized in 50 mL of 5 M nitric acid solution for 2 h under reflux at 120 C. Subsequent washing and centrifugation of the particles afforded aqueous dispersions of o-CNH at a concentration of 2 mg mL −1 .…”