Effect of Hydrophobicity on Nano-Bio Interactions of Zwitterionic

Communication Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Effect of Hydrophobicity on Nano-Bio Interactions of Zwitterionic Luminescent Gold Nanoparticles at the Cellular Level Shasha Sun, Yingyu Huang, Chen Zhou, Sishan Chen, Mengxiao Yu, Jinbin Liu, and Jie Zheng* Department of Chemistry, The University of Texas at Dallas, Richardson, Texas 75080, United States S Supporting Information *

ABSTRACT: Fundamental understanding of how the hydrophobicity impacts cellular interactions of engineered nanoparticles is critical to their future success in healthcare. Herein, we report that inserting hydrophobic octanethiol onto the surface of zwitterionic luminescent glutathione coated gold nanoparticles (GS-AuNPs) of 2 nm enhanced their affinity to the cellular membrane and increased cellular uptake kinetics by more than one order of magnitude, rather than inducing the accumulation of the AuNPs in the bilayer core or enhancing their passive diffusion. These studies highlight the diversity and heterogeneity in the hydrophobicity-induced nano−bio interactions at the cellular level and offer a new pathway to expediting cellular uptake of engineered nanoparticles. In addition, the amphiphilic luminescent AuNPs with high affinity to cell membrane and rapid endocytosis potentially serve as dual-modality imaging probes to correlate fluorescence and electron microscopies at the cellular level.

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To address these challenges, in this study, we used luminescent AuNPs with an intrinsic emission from the gold core as a model.20,21 Unlike previous studies that AuNPs are often not luminescent and require conjugation of organic dyes for visualizing their cellular uptake,22 luminescent gold nanoparticles with intrinsic emission help us to minimize the potential interference from organic dyes on the nano−bio interactions as well as potential artifacts resulting from the dissociation of organic dyes from the particle surface. By finely controlling their surface chemistries with hydrophobic alkyl thiols,23 we were able to unravel how the hydrophobicity impacts the interactions of the zwitterionic AuNPs of 2 nm with the cell membranes and their cellular uptake kinetics. Unlike the previous report that insertion of hydrophobic ligands enabled the AuNPs to escape the cell membrane barrier and rapidly diffuse into the cytoplasm and nucleus,24 our results showed that the increased hydrophobicity of AuNPs significantly enhanced the affinity of the AuNPs to the cell membrane and induced rapid endocytosis. Although these AuNPs were monodispersed on the cell surface, a large number of AuNPs were retained in individual endosomes, suggesting that the nanoparticles were engulfed through cell membrane wrapping. The binding of amphiphilic-AuNPs to cell membrane also induced more than 12 times faster endocytosis compared to that of zwitterionic AuNPs. These amphiphilic luminescent AuNPs with high affinity to cell membrane and rapid endocytosis potentially serve as multimodality imaging probes for unraveling nano−bio interactions in vitro at high temporal and spatial resolution.

undamental understanding of in vitro nano−bio interactions are important to advance applications of engineered nanoparticles in healthcare.1−9 Among all the interactions, how the nanoparticles interact with the cell membrane is particularly critical since the cell membrane is the first barrier that controls the entrance of engineered nanoparticles into the intracellular environment. Many structural factors of engineered nanoparticles have been found to affect these interactions. For example, Chan et al. studied the cellular uptake of 14, 50, and 70 nm AuNPs and found the 50-nm-sized particles showed the highest cellular uptake efficiency, indicating an optimal size in the efficient cellular uptake.10 Surface chemistries of engineered nanoparticles also play a key role in the interactions.11 Rotello et al. found that AuNPs with neutral surfaces are less readily taken up by the cells than cationic AuNPs.12 The positively charged particles were found to more readily penetrate cell membrane into the cytoplasma because they can bind to the negatively charged cell membrane (e.g., sialic acid).13 Moreover, recent studies showed that positively charged nanoparticles depolarized the cell membrane, which results in increased intracellular [Ca2+] and inhibits the proliferation of normal cells.14 In addition to the charge, the hydrophobicity and surface structure might also affect the nanoparticle−cell interaction.15 Stellacci et al. found that amphiphilic AuNPs coated with ordered surface structures can penetrate the cell membrane through passive diffusion.16 However, a very recent study showed that the translocation of hydrophobic NPs through the cell membrane does not need any specific nanopatterning.17 These contradictory observations suggest that it remains highly desired to further unravel how the hydrophobicity impacts on the nano−bio interactions at the cellular level.18,19 © XXXX American Chemical Society

Received: March 19, 2018 Revised: May 14, 2018

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DOI: 10.1021/acs.bioconjchem.8b00202 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Communication

Bioconjugate Chemistry

Figure 1. (A) HR-TEM image and size distribution of OG-AuNPs. (Scale bar: 5 nm). (B) Zeta potentials of OG-AuNPs and GS-AuNPs at pH 7.4. (C) Excitation and emission spectra of GS-AuNPs and OG-AuNPs, respectively. (D) FT-IR spectra of OG-AuNPs (red curve) and GS-AuNPs (black curve).

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RESULTS AND DISCUSSION Hydrophilic glutathione coated AuNPs (GS-AuNPs) with maximum emission at 600 nm were synthesized as our group previously reported.20 Amphiphilic octanethiol/glutathione coated AuNPs (OG-AuNPs) were synthesized by inserting alkyl thiol ligands-octanethiol to the GS-AuNPs through the ligand exchange reaction.25,26 The reaction ratio between octanethiol and glutathione on AuNPs is 2:1. The size of GSAuNPs and OG-AuNPs was measured using transmission electron microscope. The average diameter of OG-AuNPs is 2.0 ± 0.5 nm (Figure 1A), consistent with the core size of GSAuNPs (1.8 ± 0.3 nm), which indicated that the ligand replacement reaction did not etch the nanoparticle gold core. The surface charge of OG-AuNPs (zeta potential: −15.3 mV, pH 7.4) dramatically decreased in comparison with that of GSAuNPs (−46.3 mV, pH 7.4), suggesting the replacement of negatively charged glutathione by neutral octanethiol (Figure 1B). The OG-AuNPs showed almost the same emission spectra as GS-AuNPs with the maximum emission intensity at 600 nm (Figure 1C), suggesting the emission is a result from the charge transfer between ligand−metal (S−Au),27 and the hydrophobic octanethiol ligands did not alter the emission of the AuNPs. To confirm the successful synthesis of amphiphilic OG-AuNPs, we analyzed the FT-IR spectra of OG-AuNPs and GS-AuNPs. Compared to GS-AuNPs, the OG-AuNPs showed transmittance at 2917 and 2850 cm−1, indicating methylene bonding groups of 1-octanethiol ligands (Figure 1D). To determine the average ratio between gold, glutathione, and 1-octanethiol, we conducted element analysis of carbon and nitrogen content in a dried OG-AuNPs sample. The result showed that OG-AuNPs were composed of 17.85% carbon and 2.07% nitrogen; thus, the

ratio of gold and glutathione and 1-octanethiol turned out to be 14:2:5. Therefore, the OG-AuNPs are amphiphilic due to the coating of both hydrophilic glutathione and hydrophobic 1octanethiol molecules. To determine how the hydrophobicity of these NPs influences their interaction with cell membrane, we used human cervical carcinoma cell lines (HeLa cells) as a model and imaged the binding of AuNPs with live HeLa cells fluorescently. Briefly, live HeLa cells were incubated with OGAuNPs or GS-AuNPs in PBS at pH 7.4 at 25 °C for 3 min and then washed with PBS once. The live cells incubated in PBS were then imaged under a fluorescence microscope with a 100× objective in epifluorescence geometry. The highly fluorescent cell membrane after OG-AuNP incubation and PBS wash indicated a strong binding of the OG-AuNPs to the cell membrane (Figure 2A,B). The interaction between OG-AuNPs and cell membrane was much stronger than the membrane adsorption of glutathione/cysteamine-AuNPs at acidic pH, which was driven by local electrostatic attraction but can be easily removed by PBS wash.21 On the other hand, for the cells incubated with GS-AuNPs, there was almost no fluorescence detected on the cell surface, suggesting that GS-AuNPs has almost no affinity to the cell membrane because both cell membrane and GS-AuNPs are highly negatively charged (Figure 2C,D).5,28 This agrees with previous results that AuNPs coated by hydrophilic materials can hardly passively transport artificial phospholipid membranes.29 Quantitatively, the fluorescence intensity of the cell membrane incubated with OG-AuNPs was around 18 times higher than that of GS-AuNPs (Figure 2E). The high affinity of the amphiphilic AuNPs to the cell membrane indicated that increasing hydrophobicity enhanced the cell−nanoparticle interactions rather than B

DOI: 10.1021/acs.bioconjchem.8b00202 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Communication

Bioconjugate Chemistry

Figure 3. (A) TEM images of HeLa cells incubated with OG-AuNPs. (Scale bar: 100 nm). (B) OG-AuNPs on the cell membrane. (C) Cell membrane wrapping of OG-AuNPs. (Scale bar: 200 nm). (D) OGAuNPs inside the wrapped cell membrane. (E) Illustration of cellular endocytosis process of AuNPs.

Figure 2. (A,B) Bright field and fluorescence images of live HeLa cells incubated with OG-AuNPs for 3 min. (C,D) Bright field and fluorescence images of live HeLa cells incubated with GS-AuNPs for 3 min. (Scale bar: 20 μm). (E) Cell membrane fluorescence intensity of OG-AuNPs and GS-AuNPs.

encapsulated into the individual endosomes (Figure 3C,D). These results further confirmed that endosomes were formed because of the wrapping of the cell membrane (Figure 3E). It should be noted that the AuNPs remained monodispersed even in the individual endosomes, suggesting that the AuNPs were highly stable and tightly bind to the cell membrane during the endocytosis.11 To gain a more quantitative understanding of how the hydrophobicity impacts the endocytic kinetics, we used fluorescence microscopy to quantify the average number of endosomes at different incubation time points. We incubated the HeLa cells with AuNPs in MEM at 37 °C and took fluorescence imaging at different time points. As time progressed, a lot of bright spots appeared inside the cells (Figure 4B,E), which indicated that the nanoparticles were taken up by cells through the endocytosis process rather than being translocated passively through the cell membrane.17 Interestingly, the uptake of OG-AuNPs was fairly rapid in the first 2 h and then reached the maximum amount with 42 ± 8 endosomes at 3 h (Figure 4C); however, the uptake process of GS-AuNPs was much slower: after 6 h, there were only 11 ± 3 endosomes. With the increase of incubation time, the number of endosomes continued to increase and reached 31 ± 6 at 24 h

enabling them to passively diffuse into the cytoplasm.14,30 Since there are two kinds of forces between the NPs and the cell membrane, the hydrophobic interaction between the octanethiol and hydrophobic fatty acids of the cell membrane, and the electrostatic repulsion between glutathione and the negatively charged phosphate groups of cell membrane, these results also indicated that the van de Waals force resulting from the hydrophobic interactions could overcome the electrostatic repulsion between the AuNPs and the cell membrane. As a result, the negatively charged amphiphilic AuNPs can still bind to the cell membrane. To gain a deeper understanding of the binding of the amphiphilic AuNPs to the cell membrane, we used biological EM to image the AuNPs on the cell membrane. As shown in Figure 3A, AuNPs were found on the outer layer of the cell membrane rather than being embedded in the middle of cellular membrane.17 These nanoparticles were still monodispersed on the cell membrane (Figure 3B), distinct from the previous reports that hydrophobic nanoparticles usually form aggregates on the cell membrane.31 Interestingly, after 1 h, the monodispersed OG-AuNPs on the cell membrane were C

DOI: 10.1021/acs.bioconjchem.8b00202 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Communication

Bioconjugate Chemistry

Figure 4. (A,B) Bright field and fluorescence images of live HeLa cells incubated with OG-AuNPs in MEM at 37 °C for 24 h. (C) Cellular uptake kinetics of OG-AuNPs in 5 h. (D,E) Bright field and fluorescence images of live HeLa cells incubated with GS-AuNPs in MEM at 37 °C for 24 h. (Scale bar: 20 μm). (F) Cellular uptake kinetics of GS-AuNPs in 24 h.

(Figure 4F), which was still fewer than the number of endosomes induced by OG-AuNPs. The much faster cellular endocytosis of OG-AuNPs than GS-AuNPs might be attributed to the decrease of localized Gibbs free energy induced by nanoparticle−cell interactions.1,32 As a result, the membrane around the NPs quickly wrapped and formed endosomes (Figure S1A,B). The high affinity of amphiphilic luminescent AuNPs to the cell membrane also makes them useful as a membrane staining probe. We studied a potential application of the OG-AuNPs in neuron imaging. The conventional method for the detection and characterization of living neuron cells depends on immunodetection of marker molecules, which involves the use of xenogenic antibodies and is limited to the expression of cell surface molecules.33 Since OG-AuNPs can bind to cell membrane through hydrophobic interactions, we investigated whether the OG-AuNPs could bind to neuron cells nonspecifically. Live neuron cells have very low fluorescence intensity and the dendritic branching pattern could not be observed under fluorescence microscope (Figure 5A,B). After being incubated with OG-AuNPs, the live neuron cells became highly fluorescent and even the dendrites were clearly observed under the microscope (Figure 5C,D), which opens an opportunity for the future investigation of the interactions of the nanoparticles with neuron.34 In conclusion, by using 2 nm luminescent AuNPs with intrinsic emissions as probes, we found that increasing hydrophobicity of zwitterionic nanoparticles did not enable the passive diffusion of the particles into the cytoplasm or being trapped in the middle of cell membrane, but enhanced their affinity to the cell membrane and greatly increased their

Figure 5. (A,B) Bright field and fluorescence images of neuron incubated with PBS at pH 7.4, 25 °C for 3 min. (C,D) Bright field and fluorescence images of neuron incubated with OG-AuNPs at pH 7.4, 25 °C for 3 min, and then washed with PBS once. (Scale bar: 20 μm).

endocytosis kinetics by more than one order of magnitude. These results also indicate that hydrophobicity could play a more important role than electrostatic repulsion in the cellular uptake of the engineered nanoparticles. These findings are D

DOI: 10.1021/acs.bioconjchem.8b00202 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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(9) Nicol, J. R., Dixon, D., and Coulter, J. A. (2015) Gold nanoparticle surface functionalization: a necessary requirement in the development of novel nanotherapeutics. Nanomedicine 10, 1315−1326. (10) Chithrani, B. D., Ghazani, A. A., and Chan, W. C. W. (2006) Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett. 6, 662−668. (11) Ding, H. M., Tian, W. D., and Ma, Y. Q. (2012) Designing Nanoparticle Translocation through Membranes by Computer Simulations. ACS Nano 6, 1230−1238. (12) Zhu, Z. J., Ghosh, P. S., Miranda, O. R., Vachet, R. W., and Rotello, V. M. (2008) Multiplexed Screening of Cellular Uptake of Gold Nanoparticles Using Laser Desorption/Ionization Mass Spectrometry. J. Am. Chem. Soc. 130, 14139−14143. (13) Verma, A., and Stellacci, F. (2010) Effect of Surface Properties on Nanoparticle-Cell Interactions. Small 6, 12−21. (14) McNamee, C. E., Pyo, N., and Higashitani, K. (2006) Atomic force microscopy study of the specific adhesion between a colloid particle and a living melanoma cell: Effect of the charge and the hydrophobicity of the particle surface. Biophys. J. 91, 1960−1969. (15) Han, H. S., Martin, J. D., Lee, J., Harris, D. K., Fukumura, D., Jain, R. K., and Bawendi, M. (2013) Spatial Charge Configuration Regulates Nanoparticle Transport and Binding Behavior In Vivo. Angew. Chem., Int. Ed. 52, 1414−1419. (16) Verma, A., Uzun, O., Hu, Y. H., Hu, Y., Han, H. S., Watson, N., Chen, S. L., Irvine, D. J., and Stellacci, F. (2008) Surface-structureregulated cell-membrane penetration by monolayer-protected nanoparticles. Nat. Mater. 7, 588−595. (17) Guo, Y. C., Terazzi, E., Seemann, R., Fleury, J. B., and Baulin, V. A. (2016) Direct proof of spontaneous translocation of lipid-covered hydrophobic nanoparticles through a phospholipid bilayer. Sci. Adv. 2, e1600261. (18) Cesbron, Y., Shaw, C. P., Birchall, J. P., Free, P., and Levy, R. (2012) Stripy Nanoparticles Revisited. Small 8, 3714−3719. (19) Yu, M., and Stellacci, F. (2012) Response to ″Stripy Nanoparticles Revisited″. Small 8, 3720−3726. (20) Zhou, C., Sun, C., Yu, M. X., Qin, Y. P., Wang, J. G., Kim, M., and Zheng, J. (2010) Luminescent Gold Nanoparticles with Mixed Valence States Generated from Dissociation of Polymeric Au(I) Thiolates. J. Phys. Chem. C 114, 7727−7732. (21) Yu, M. X., Zhou, C., Liu, J. B., Hankins, J. D., and Zheng, J. (2011) Luminescent Gold Nanoparticles with pH-Dependent Membrane Adsorption. J. Am. Chem. Soc. 133, 11014−11017. (22) Xia, T., Rome, L., and Nel, A. (2008) Nanobiology - Particles slip cell security. Nat. Mater. 7, 519−520. (23) Clapp, C. H., Grandizio, A. M., Yang, Y., Kagey, M., Turner, D., Bicker, A., and Muskardin, D. (2002) Irreversible inactivation of soybean lipoxygenase-1 by hydrophobic thiols. Biochemistry 41, 11504−11511. (24) Conroy, C. V., Jiang, J., Zhang, C., Ahuja, T., Tang, Z., Prickett, C. A., Yang, J. J., and Wang, G. (2014) Enhancing near IR luminescence of thiolate Au nanoclusters by thermo treatments and heterogeneous subcellular distributions. Nanoscale 6, 7416−7423. (25) AbdulHalim, L. G., Kothalawala, N., Sinatra, L., Dass, A., and Bakr, O. M. (2014) Neat and Complete: Thiolate-Ligand Exchange on a Silver Molecular Nanoparticle. J. Am. Chem. Soc. 136, 15865−15868. (26) Ojea-Jimenez, I., Garcia-Fernandez, L., Lorenzo, J., and Puntes, V. F. (2012) Facile Preparation of Cationic Gold NanoparticleBioconjugates for Cell Penetration and Nuclear Targeting. ACS Nano 6, 7692−7702. (27) Zheng, J., Zhou, C., Yu, M. X., and Liu, J. B. (2012) Different sized luminescent gold nanoparticles. Nanoscale 4, 4073−4083. (28) Sun, S. S., Ning, X. H., Zhang, G., Wang, Y. C., Peng, C. Q., and Zheng, J. (2016) Dimerization of Organic Dyes on Luminescent Gold Nanoparticles for Ratiometric pH Sensing. Angew. Chem., Int. Ed. 55, 2421−2424. (29) Banerji, S. K., and Hayes, M. A. (2007) Examination of nonendocytotic bulk transport of nanoparticles across phospholipid membranes. Langmuir 23, 13244−13244.

expected to help further precisely control nano−bio interactions in vitro, broadening their applications in disease detection and treatment.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00202. Materials and equipment, experimental procedures, supplementary figures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 972-883-5768. Fax: 972883-2925. ORCID

Jie Zheng: 0000-0001-8546-1882 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the Electron Microscopy Core of UT Southwestern Medical Center for the help with the TEM imaging of cells. This work was supported in part by the NIH (1R21EB011762) and the start-up fund from The University of Texas at Dallas (J.Z.).

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ABBREVIATIONS AuNPs, gold nanoparticles; GS-AuNPs, glutathione coated gold nanoparticles; OG-AuNPs, octanethiol/glutathione coated gold nanoparticles; TEM, transmission electron microscopy; PBS, phosphate buffer saline; MEM, minimum essential media

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REFERENCES

(1) Nel, A. E., Madler, L., Velegol, D., Xia, T., Hoek, E. M. V., Somasundaran, P., Klaessig, F., Castranova, V., and Thompson, M. (2009) Understanding biophysicochemical interactions at the nanobio interface. Nat. Mater. 8, 543−557. (2) Liu, J. B., Yu, M. X., Zhou, C., and Zheng, J. (2013) Renal clearable inorganic nanoparticles: a new frontier of bionanotechnology. Mater. Today 16, 477−486. (3) Arvizo, R. R., Miranda, O. R., Thompson, M. A., Pabelick, C. M., Bhattacharya, R., Robertson, J. D., Rotello, V. M., Prakash, Y. S., and Mukherjee, P. (2010) Effect of Nanoparticle Surface Charge at the Plasma Membrane and Beyond. Nano Lett. 10, 2543−2548. (4) Kim, C. S., Le, N. D. B., Xing, Y. Q., Yan, B., Tonga, G. Y., Kim, C., Vachet, R. W., and Rotello, V. M. (2014) The Role of Surface Functionality in Nanoparticle Exocytosis. Adv. Healthcare Mater. 3, 1200−1202. (5) Su, G. X., Zhou, H. Y., Mu, Q. X., Zhang, Y., Li, L. W., Jiao, P. F., Jiang, G. B., and Yan, B. (2012) Effective Surface Charge Density Determines the Electrostatic Attraction between Nanoparticles and Cells. J. Phys. Chem. C 116, 4993−4998. (6) Huynh, N. T., Roger, E., Lautram, N., Benoit, J. P., and Passirani, C. (2010) The rise and rise of stealth nanocarriers for cancer therapy: passive versus active targeting. Nanomedicine 5, 1415−1433. (7) Jing, B. X., and Zhu, Y. X. (2011) Disruption of Supported Lipid Bilayers by Semihydrophobic Nanoparticles. J. Am. Chem. Soc. 133, 10983−10989. (8) Pelaz, B., Alexiou, C. H., Alvarez -Puebla, R. A., Alves, F., Andrews, A. M., Ashraf, S., Balogh, L. P., Ballerini, L., Bestetti, A., Brendel, C., et al. (2017) Diverse Applications of Nanomedicine. ACS Nano 11, 2313−2381. E

DOI: 10.1021/acs.bioconjchem.8b00202 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry (30) Li, Y., Chen, X., and Gu, N. (2008) Computational Investigation of Interaction between Nanoparticles and Membranes: Hydrophobic/ Hydrophilic Effect. J. Phys. Chem. B 112, 16647−16653. (31) Tian, F. L., Zhang, X. R., and Dong, W. (2014) How hydrophobic nanoparticles aggregate in the interior of membranes: A computer simulation. Phys. Rev. E 90, 052701. (32) Ginzburg, V. V., and Balijepailli, S. (2007) Modeling the thermodynamics of the interaction of nanoparticles with cell membranes. Nano Lett. 7, 3716−3722. (33) Yun, S. W., Leong, C., Zhai, D. T., Tan, Y. L., Lim, L., Bi, X. Z., Lee, J. J., Kim, H. J., Kang, N. Y., Ng, S. H., et al. (2012) Neural stem cell specific fluorescent chemical probe binding to FABP7. Proc. Natl. Acad. Sci. U. S. A. 109, 10214−10217. (34) Er, J. C., Leong, C., Teoh, C. L., Yuan, Q., Merchant, P., Dunn, M., Sulzer, D., Sames, D., Bhinge, A., Kim, D., et al. (2015) NeuO: a Fluorescent Chemical Probe for Live Neuron Labeling. Angew. Chem., Int. Ed. 54, 2442−2446.

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DOI: 10.1021/acs.bioconjchem.8b00202 Bioconjugate Chem. XXXX, XXX, XXX−XXX