Graphene possesses extraordinary properties that promise great potential in biomedicine. However, fully leveraging these properties requires close contact with the cell surface, raising the concern of unexpected biological consequences. Computational models have demonstrated that graphene preferentially interacts with cholesterol, a multifunctional lipid unique to eukaryotic membranes. Here we demonstrate an interaction between graphene and cholesterol. We find that graphene increases cell membrane cholesterol and potentiates neurotransmission, which is mediated by increases in the number, release probability, and recycling rate of synaptic vesicles. In fibroblasts grown on graphene, we also find an increase in cholesterol, which promotes the activation of P2Y receptors, a family of receptor regulated by cholesterol. In both cases, direct manipulation of cholesterol levels elucidates that a graphene-induced cholesterol increase underlies the observed potentiation of each cell signaling pathway. These findings identify cholesterol as a mediator of graphene’s cellular effects, providing insight into the biological impact of graphene.

Graphene, a two-dimensional carbon crystal, has emerged as a promising material for sensing and modulating neuronal activity in vitro and in vivo . In this review, we provide a primer for how manufacturing processes to produce graphene and graphene oxide result in materials properties that may be tailored for a variety of applications. We further discuss how graphene may be composited with other bio-compatible materials of interest to make novel hybrid complexes with desired characteristics for bio-interfacing. We then highlight graphene’s ever-widen utility and unique properties that may in the future be multiplexed for cross-modal modulation or interrogation of neuronal network. As the biological effects of graphene are still an area of active investigation, we discuss recent development, with special focus on how surface coatings and surface properties of graphene are relevant to its biological effects. We discuss studies conducted in both non-murine and murine systems, and emphasize the preclinical aspect of graphene’s potential without undermining its tangible clinical implementation.

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Cholesterol, a lipid molecule found ubiquitously in eukaryotic cells, plays a vital role in the integrity, dynamics, and trafficking of the lipid membrane, in addition to influencing many transmembrane proteins. However, the functionality of membrane cholesterol is far from clear, largely due to an inability to manipulate membrane cholesterol with high spatiotemporal precision. Popular tools like statins or methyl-b-cyclodextrin (MbCD) only lead to chronic and indiscriminative cholesterol reduction. Moreover, there is no selective approach to increase membrane cholesterol. Our recent work involving carbon nanomaterials provided an unexpected answer. Graphene, a one-atom thick carbon crystal, has been explored for biomedical applications because of its remarkable chemical and physical properties. Using in vitro and in vivo measurements, we have found that graphene selectively interacts with cholesterol. This enriches cholesterol at the plasma membrane, and thus enhances membrane lipid phase order, likely promoting the formation of cholesterol-rich lipid membrane nanodomains. Neurons grown on graphene exhibited presynaptic potentiation, specifically caused by a larger pool of releasable vesicles and an increase of fast recycling. By addition or depletion of membrane cholesterol, we found that the graphene-induced presynaptic enrichment of membrane cholesterol is necessary and sufficient to promote potentiation. In nonneuronal cells, graphene significantly elevates ATP-induced intracellular Ca 2þ -signaling by promoting the activation of P2Y receptors, a group of GPCRs which are selectively responsive to extracellular ATP. Furthermore, we found that graphene enhances P2Y receptor signaling on the timescale of seconds, as rapidly as its effect on membrane packing. This then reveals an intriguing interaction between graphene and cholesterol, and its impact on plasma membrane structure, trafficking, and transmembrane proteins. Given the current challenges in manipulating membrane cholesterol, this graphenecholesterol interaction will accelerate studies of membrane cholesterol function and broaden the biological application of carbon nanomaterials.

In situ imaging of biomolecular location with nanoscale resolution enables mapping of the building blocks of life throughout biological systems in normal and disease states. Expansion microscopy (ExM), by physically enlarging specimens in an isotropic fashion, enables nanoimaging on standard light microscopes. Key to ExM is the equipping of different kinds of molecule, with different kinds of anchoring moiety, so they can all be pulled apart by polymer swelling. Here we present a multifunctional anchor, an acrylate epoxide, that enables multiple kinds of molecules (e.g., proteins and RNAs) to be equipped with anchors in a single experimental step. This reagent simplifies ExM protocols and greatly reduces cost (by 2-10 fold for a typical multiplexed ExM experiment) compared to previous strategies for equipping RNAs with anchors. We show that this unified ExM (uniExM) protocol can be used to preserve and visualize RNA transcripts, proteins in biologically relevant ultrastructure, and sets of RNA transcripts in patient-derived xenograft (PDX) cancer tissues, and can support the visualization of other kinds of biomolecular species as well. Thus, uniExM may find many uses in the simple, multimodal nanoscale analysis of cells and tissues.