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Remote Nongenetic Optical Modulation of Neuronal Activity Using Fuzzy Graphene

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Remote Nongenetic Optical Modulation of Neuronal Activity Using Fuzzy Graphene Remote nongenetic optical modulation of neuronal activity using fuzzy graphene Sahil K. Rastogia,1, Raghav Gargb,1, Matteo Giuseppe Scopellitic, Bernardo I. Pintod, Jane E. Hartunge, Seokhyoung Kimf, Corban G. E. Murpheyf, Nicholas Johnsonb, Daniel San Romanb, Francisco Bezanillad, James F. Cahoonf, Michael S. Golde, Maysam Chamanzarc, and Tzahi Cohen-Karnia,b,2 aDepartment of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213; bDepartment of Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, PA 15213; cDepartment of Electrical and Computer Engineering, Carnegie Mellon University, Pittsburgh, PA 15213; dDepartment of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637; eDepartment of Neurobiology, University of Pittsburgh, Pittsburgh, PA 15213; and fDepartment of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3290 Edited by John A. Rogers, Northwestern University, Evanston, IL, and approved April 28, 2020 (received for review March 23, 2020) The ability to modulate cellular electrophysiology is fundamental absorption cross-section, and limited long-term stability when a to the investigation of development, function, and disease. stable interface is needed (15, 16). Currently, there is a need for remote, nongenetic, light-induced Two-dimensional (2D) cell culture has been foundational for control of cellular activity in two-dimensional (2D) and three- in vitro research of cellular communication (17, 18). However, its dimensional (3D) platforms. Here, we report a breakthrough use for disease modeling and designing of new therapeutic in- hybrid nanomaterial for remote, nongenetic, photothermal stimu- terventions has been limited due to its challenge in portraying a lation of 2D and 3D neural cellular systems. We combine one- native 3D tissue microenvironment (19–22). The 3D tissues, such dimensional (1D) nanowires (NWs) and 2D graphene flakes grown as spheroids and organoids, have opened new opportunities to out-of-plane for highly controlled photothermal stimulation at develop in vitro models that better mimic in vivo physiology and subcellular precision without the need for genetic modification, have enabled new ways to study disease pathology (23, 24). New with laser energies lower than a hundred nanojoules, one to technologies are needed in order to understand the mechanisms two orders of magnitude lower than Au-, C-, and Si-based nano- underlying cell−cell communication and the interplay between materials. Photothermal stimulation using NW-templated 3D fuzzy different cell populations in health and disease. Therefore, there graphene (NT-3DFG) is flexible due to its broadband absorption is a need to develop a remote, nongenetic, and minimally in- ENGINEERING and does not generate cellular stress. Therefore, it serves as a vasive modulation of neuronal activity in 3D tissues that will powerful toolset for studies of cell signaling within and between enable understanding of cellular physiology in both healthy and tissues and can enable therapeutic interventions. diseased states to develop new therapeutic platforms. Here, we resolve the limitations of the current state-of-the-art graphene | nanowire | optical modulation | dorsal root ganglia neurons | platforms with a complementary approach based on a hybrid spheroids nanomaterial composed of 3D out-of-plane fuzzy graphene grown on an intrinsic Si nanowire (i-SiNW) template. The high ell−cell communication is fundamental to the emergence of Chigher-order function in tissue. Since Hodgkin and Huxley’s Significance (1) seminal work on the electrical activity of squid giant axons, input/output interfaces with electroactive cells and tissues have Modulation of cellular electrophysiology helps develop an steadily improved through a variety of techniques and materials, understanding of cellular development and function in healthy enabling increased spatial and temporal control (2). The devel- and diseased states. We modulate the electrophysiology of opment of optogenetics (3) has revolutionized our ability to neuronal cells in two-dimensional (2D) and 3D assemblies with control the activity in excitable cells and tissues and fundamen- subcellular precision via photothermal stimulation using a tally changed the questions that we can ask and answer. Clinical multiscale fuzzy graphene nanostructure. Nanowire (NW)- translation potential of optogenetics is limited, since it requires templated 3D fuzzy graphene (NT-3DFG) nanostructures en- genetic modification of the cells or organisms to introduce the able remote, nongenetic photothermal stimulation with laser light-sensitive proteins (3, 4). Similarly, magnetogenetics, which energies as low as subhundred nanojoules without generating uses superparamagnetic nanoparticles for localized heating and cellular stress. NT-3DFG serves as a powerful toolset for studies of cell signaling within and between in vitro 3D models stimulation (5, 6), has enhanced our ability to modulate cellular (human-based organoids and spheroids) and can enable therapeutic activity, but it also requires genetic modification, that is, ex- interventions. pression of the heat-sensitive transient receptor potential ion channel (TRPV1). It also is limited by a relatively long latency of Author contributions: T.C.-K. designed research; S.K.R., R.G., M.G.S., B.I.P., J.E.H., S.K., up to 5 s between the magnetic field application and the onset of C.G.E.M., and N.J. performed research; S.K.R., R.G., M.G.S., B.I.P., J.E.H., S.K., C.G.E.M., neural activity (5). As an alternative to genetic modification, N.J., D.S.R., F.B., J.F.C., M.S.G., M.C., and T.C.-K. analyzed data and discussed results; and S.K.R., R.G., and T.C.-K. wrote the paper. direct infrared (IR) laser pulses are used to induce membrane The authors declare no competing interest. depolarization (7, 8). However, direct IR stimulation does not provide cell specificity, spatial stimulation precision, or sub- This article is a PNAS Direct Submission. This open access article is distributed under Creative Commons Attribution-NonCommercial- cellular resolution, and suffers from a narrow energy density NoDerivatives License 4.0 (CC BY-NC-ND). threshold for stimulation vs. damage to the tissue (9, 10). An Data deposition: All data related to the presented figures are available through Carnegie emerging alternative that can provide nongenetic, instantaneous, Mellon University research repository (Kilthub) at doi.org/10.1184/R1/c.4943241.v1. and spatial control is photothermal stimulation using Au- and Si- 1S.K.R. and R.G. contributed equally to this work. – based nanomaterials (11 14). Although this approach can pro- 2To whom correspondence may be addressed. Email: [email protected]. vide light-based control similar to optogenetics (with subcellular This article contains supporting information online at https://www.pnas.org/lookup/suppl/ precision), it has limitations, including the need for relatively doi:10.1073/pnas.1919921117/-/DCSupplemental. high laser power, low photothermal conversion efficiency, low www.pnas.org/cgi/doi/10.1073/pnas.1919921117 PNAS Latest Articles | 1of11 Downloaded by guest on September 28, 2021 photothermal efficiency and subcellular dimensions of nanowire- membrane. To create stable aqueous suspensions and increase templated 3D fuzzy graphene (NT-3DFG) (25) enable in- cell membrane adhesion, graphene flakes were treated with stantaneous, remote, and nongenetic photothermal stimulation HNO3 and functionalized with lysine-phenylalanine based peptide of cells at subcellular spatial resolution using significantly lower (KF)4 (30, 31), as confirmed by X-ray photoelectron spectroscopy laser energies, that is, one to two orders of magnitude lower than (XPS) characterization (for detailed XPS characterization, see SI other optical platforms. NT-3DFG’s high broadband absorbance Appendix). spectrum provides flexibility to modulate neuronal behavior with different excitation wavelengths. The ease with which the surface High Photothermal Efficiency of NT-3DFG. Bottom-up synthesis of chemistry of NT-3DFG can be tuned via chemical or peptide- NT-3DFG allows us to tailor the 3DFG flake size and density by based functionalization enables stable biointerfaces. This allows changing the synthesis time (SI Appendix, Fig. S5A). Changing us to modulate the neuronal activity in both 2D neuronal cul- the physical architecture of NT-3DFG by increasing the 3DFG tures and 3D neural spheroids, toward an understanding of flake size and density leads to an increase in the absorbance in cellular physiology, with the long-term goal of applications in the ultraviolet visible (UV-Vis) regime (SI Appendix, Fig. S5 B intact tissues. and C). The UV-Vis spectrum for i-SiNW agrees well with previously reported results (32–34). The enhanced broadband Results absorption by NT-3DFG can be attributed to the increased light Highly Controlled Out-of-Plane Growth of Single- to Few-Layer 3D trapping by the densely packed out-of-plane 3DFG flakes, as this Graphene on an SiNW. Previously, we demonstrated synthesis of structure is synonymous to that of nanostructured silicon and NT-3DFG mesh composed of 3D out-of-plane single- to few- carbon metamaterials (35–37). Due to its high broadband ab- layer graphene flakes grown on an interconnected SiNW mesh sorption, that is, UV to near-IR (NIR), of ca. 95%, NT-3DFG template (25). Here, we isolate individual NT-3DFG to achieve synthesized for 90 min was selected for subsequent character- subcellular
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