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A Bioorthogonally Deciphered Binary Nanoemitters for Tumor Diagnostics Hong-Wei An, Sheng-Lin Qiao, Li-Li Li, Chao Yang, YaoXin Lin, Yi Wang, Zeng-Ying Qiao, Lei Wang, and Hao Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07497 • Publication Date (Web): 19 Jul 2016 Downloaded from http://pubs.acs.org on July 20, 2016

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ACS Applied Materials & Interfaces

A Bioorthogonally Deciphered Binary Nanoemitters for Tumor Diagnostics Hong-Wei An,†,‡ Sheng-Lin Qiao,†,‡ Li-Li Li,† Chao Yang,† Yao-Xin Lin,†,‡Yi Wang,†,‡ Zeng-Ying Qiao,† Lei Wang,*,† and Hao Wang*,† †

CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biological Effects of

Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), No. 11 Beiyitiao, Zhongguancun, Beijing, China ‡

University of Chinese Academy of Science (UCAS), No. 19A Yuquan Road, Beijing, China.

KEYWORDS: supramolecular, nanoparticles, bioorthogonal, self-assembly, tumor

ABSTRACT: Bio-inspired design concept has been recognized as one of the most promising strategies for discovering new biomaterials. However, smart biomaterials that are of growing interests in biomedical field need biological processability to meet their emergent applications in vivo. Herein, a new bioorthogonally deciphered approach has been demonstrated for modulating optical properties of nanomaterials in living systems. The self-assembled nanoemitters based on cyanine-pyrene molecule 1 with inert optical property are designed and prepared. The structure and optical feature of the nanoemitters 1 can be efficiently and reliably modulated by a unique bioorthogonal mechanism with abundant glutathione (GSH) as an activator. As a result, the self-

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assembled nanoemitters 1 spontaneously exhibits binary emissions for high-performance tumor imaging in vivo. We believe that this bioorthogonally deciphered strategy open a new avenue for designing variable smart biomaterials or devices in biomedical applications.

Molecular self-assembly has been considered as a powerful approach1,2 to fabricate artificial materials for biomedical applications3, such as drug delivery4,5, bioimaging6-8 and tissue engineering9-11 etc.. The unique dynamic characteristics of self-assembly systems are endowed with interesting structural changes and signal on/off switches during the reassembly or transformation process upon environmental stimuli.12-14 However, for application in biological systems, the interference of complicated environment commonly leads to unpredictable structure transformation. Besides the traditional stimuli-factors, such as pH15, enzymes16, reactive oxygen species (ROS)17 etc., the more specific and highly efficient chemistry strategy has been paid extensive attention, e.g., bioorthogonal reactions. Some bioorthogonal reactions have been developed, such as the Staudinger ligation of azides18 and copper catalyzed azide-alkyne 1,3dipolar cycloaddition (CuAAC)19, which are successfully applied for bio-labeling in vitro and in vivo. However, the bioorthogonal reaction as a trigger factor for construction of nanomaterials or molecular recognition in biological systems is rarely reported. A typical example using bioorthogonal reaction for stimuli-construction of nanoaggregates has been introduced by Rao’s group, which is a biocompatible reaction between free cysteine and cyanobenzothiazole (CBT).8, 20

Inspired by GSH distribution under a pathologic condition, i. e. cancer,21 we first develop a

new bioorthogonal reaction, which can specifically trigger binary nanoemitters with dualwavelength fluorescence for tumor-imaging in vivo.


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Previously, we reported the stimuli-construction of nano or micro-sized assemblies in situ showed extraordinary assembly-induced retention (AIR) effect16 compared to small molecules in the tumor that greatly enhanced the performance of cancer imaging and therapy.22 In this study, a new GSH-based bioorthogonal reaction as an activator for converting the nanoemitters from "inert" to "active" states with binary fluorescence was developed for tumor diagnostics in vivo. Two emissive cyanine (Cy) and bis(pyrene) (BP) motifs were covalently linked to form molecule 1, which self-assembled into nanoemitters in “inert” states with quenched fluorescence due to aggregation-caused quenching (ACQ).23, 24 In the presence of GSH, the Cy motif was covalently tethered with GSH and the hydrophobic BP residue further gathered to form larger aggregates accompanying activated dual-color fluorescence signals (Scheme 1). Especially, the enhancement of fluorescence exhibited 30-fold for GSH labeled Cy at NIR region (λem = 820 nm) and 36-fold for BP aggregates at 520 nm. The bioorthogonal triggered approach could be utilized to prepare highly sensitive and specific bioimaging system for diagnostics of diseases in vivo. The molecule 1 was obtained by coupling amine-derived BP with chloro-substituted Cy (detailed synthesis process in Supporting Information and Scheme S1) in DMF at room temperature for 3 h with a yield of 80% (Figure 1a). The molecule 1 was purified by column chromatography and characterized by 1HNMR and MALDI-TOF (See Supporting Information). In monomeric state, the molecule 1 exhibited dual-color fluorescence at 440 and 770 nm, which was ascribed to characteristic monomer fluorescence of BP and Cy, respectively. Accordingly, the absorbance of the molecule 1 was centered at 346 nm (for BP) and 645 nm (for Cy). Upon gradually addition of H2O to DMSO solution, the molecule 1 transformed from the monomeric states to aggregated states and led to a fluorescence quenching (Figure S1). As the addition of


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H2O up to 99% (v/v), the fluorescence was completely quenched due to ACQ effect (Figure 1b). Moreover, the morphology of the nanoemitters 1 was studied by transmission electron microscope (TEM) (Figure 1c). The results indicated that the nanoemitters 1 showed particulate structures with a size (32.8 ± 6.3 nm), which well corresponded to the results obtained by dynamic light scattering (DLS) measurement (43.3 ± 11.6 nm, Figure S2). Treatment the molecule 1 with GSH (500 equiv.) generated two major products compounds 2 and 3, which were confirmed by MALDI-TOF (Figure 2a). Visually, the remarkable color and fluorescence changes of the solution occurred at the same time. Upon treatment with GSH, the absorption of the nanoemitters 1 at 645 nm decreased, and a new peak simultaneously appeared at 788 nm with significant colorimetric changes from blue to green (Figure 2b). Correspondingly, the dual-color fluorescence emission at λmax = 820 nm and λmax = 520 nm displayed a remarkable increase (Figure 2c and 2d). The large red-shift in absorption (143 nm) and strong fluorescence at 820 nm were attributed to the formation of S-substituted cyanine compound25, which was identified with the molecule 3 synthesized as a control (Scheme S2 and Figure S3). Meanwhile, the increased green emission at 520 nm was ascribed to J-type aggregates of the molecule 2 corresponding to the results reported previously.26-28 For further confirmation of the bioorthogonal reaction between the nanoemitters 1 and GSH, the 1H NMR measurements were carried out (Figure 3a). GSH was firstly added into the nanoemitters 1 solution and the resultant supernatant solutions at different intervals (0, 0.5 and 2 h) were collected and dried, respectively. The 1H NMR of each dried samples in a mixture of solution (CD3OD/D2O = 9:1, v/v) showed that the conversion from the molecule 1 to 3 was developed gradually during the addition of GSH (Figure 3b). In the presence of 500 equiv. GSH,


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the vinylic protons of the molecule 1 were shifted to low-field regions (Ha 7.81 ppm to 8.75 ppm, Hb 5.79 ppm to 6.25 ppm) as a result of substitution of thiol groups (GSH) after the “S-S” cleaved. The noticeable 1HNMR spectral changes indicated that the released cyanine dye was easily substituted by the thiol group of GSH. This assumption was confirmed by a dependable molecule 3 synthesized as a control (Figure 3b). Meanwhile, after treatment with GSH, the precipitate was also carefully separated by centrifugation. The isolated aggregates were redissolved in chloroform-d and the chemical structure was confirmed by 1H NMR analysis and MALDI-TOF (Figure 3c and S4). The results explicitly confirmed the proposed product (molecule 2) of the bioorthogonal reaction, which had enhanced hydrophobic properties and selfaggregated into large aggregates. To further confirm the generation of larger nanoaggregates, the confocal laser scanning microscopy (CLSM) was presented to observe the GSH treated the nanoemitters 1 in buffer solutions (Figure S5). Upon treatment with GSH (500 equiv.) at 37 °C for 3 h in DMSO/PBS solution (v/v = 1/99, pH = 7.4), the green fluorescence dots and red background were observed in two different channels, respectively. Besides, the morphology and size were also studied by TEM and DLS measurements (Figure S6a and b). The sizes of nanostructures increased from 32.8 ± 6.3 nm to 67.8 ± 12.3 nm upon treatment the nanoemitters 1 (100 µM) with GSH (500 equiv.) in aqueous solution for 12 h. As a contrast, no size changes were observed over the same time period in the absence of GSH (Figure S6c and d). To verify the mechanism of this bioorthogonal reaction, parallel experiments of the nanoemitters 1 treated with DL-Dithiothreitol (DTT) and control molecule of n-butylamine labeled chloro-substituted cyanine (Cy-CC, without a disulfide linker “S-S”) were carried out. As expected, the DTT also realized the substitution after cleavage of disulfide bond (Figure S7). However, the absorption of Cy-CC treated with GSH had almost no changes with the increase of


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GSH concentration (0-500 equiv.), implying the cleavage of disulfide linker is necessary for Cy release and GSH substitution (Figure S8). Moreover, we observed that the reaction rate of the molecule 1 with GSH was severely affected by the solvents. The rate was dramatically accelerated in protic solvent (methanol) than aprotic solvent (DMSO) (Figure S9). Based on the above experimental results, the mechanism of this bioorthogonal reaction was proposed to be a nucleophilic addition and an elimination reaction in the protic solvent (Figure S10). As a bioorthogonal reaction for in vitro and in vivo application, the parameters, i.e. concentration, pH, cytotoxicity and specificity should be considered. Upon treatment with different equivalent of GSH to the nanoemitters 1, the absorption exhibited a gradually red shift from 645 nm to 788 nm and reached a plateau at 500 equiv. of GSH, which implied that the nanoemitters 1 totally reacted (Figure 4a). According to literature, the concentration of GSH inside cell was about 10 mM, which was sufficiently for intracellular bioorthogonal reaction.21 Then the reactions in several of pH buffer were tested. The results displayed that at pH > 6.0, both absorption (788 nm) and fluorescence intensities (λem 520 and 820 nm) of the nanoemitters 1 increased upon the addition of GSH (500 equiv.). Meanwhile, without GSH addition, the nanoemitters 1 remained stable (Figure 4b and Figure S11). Next, the cell viability of the nanoemitters 1 on breast cancer cells (MCF-7 cells) were tested and negligible cytotoxicity was detected as expected with the concentration of the nanoemitters 1 up to 100 µΜ (Figure S12). Besides GSH, five small molecules i.e., cysteine (Cys), glutamic (Glu), threonine (Thr), tyrosine (Tyr) did not trigger the reaction under the physiological concentrations (the concentration of cysteine acid varied between 4-16 µM),31 which was confirmed by the absorption spectra. Only GSH induced the absorption exhibited a red shift from 645 nm to 788 nm (Figure 4c). All above results suggested that this reaction could be well controlled under physiological environment.


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Overall, the GSH-based bioorthogonal reaction enabled for bioimaging the activity of GSH in intricate environment in vitro and in vivo with good biocompatibility. For validation of the bioorthogonal reaction on a cellular level, the MCF-7 cell line was chosen for further experiments. Cells were pre-treated with LPA (a lipoic acid, a GSH enhancer) and NEM (N-ethylmalemide a GSH scavenger) to enhance and decrease the express of GSH, respectively.25,29 Both in green and red fluorescence channels the fluorescence intensity increased almost 1.5 fold in the positive LPA group. As a sharp contrast, the fluorescence intensity in negative NEM group was almost non-detectable (Figure 5a). The quantification analysis based on CLSM images indicated that the bioorthogonal reaction enabled to decipher the nanoemitters 1 with binary turn-on fluorescence for sensitive and specific GSH imaging inside cells (Figure 5b and Figure S13). As expected, the molecular weight of molecule 2 and 3 were detected in cell lysates by MOLDI-TOF (Figure 5c). The fluorescence spectra at λem = 520 nm and 820 nm of cell lysates (Figure S14) were obtained, corresponding to the aggregates 2 and molecule 3, respectively. These cellular experiments illustrated that the endogenous GSH could activate the nanoemitters 1 in complicated cellular environment and that the fluorescence intensity was related to the level of intracellular GSH. The feasibility of the bioorthogonal reaction in the tumor tissue for bioimaging application was examined using MCF-7 xenograft BALB/c nude mice model. All animal experiments were performed according to the NIH guidelines for the care and use of laboratory animals of Peking University Animal Study Committee’s requirements and using protocol approved by the Institutional Animal Care. After the subcutaneous tumors developed to approximately 0.6-0.8 mm, the mice were randomly divided into two groups (N = 6). The mice were intravenously injected with PBS or the nanoemitters 1 (200 µL with a concentration of 60 µM) separately. The


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green (from 500 nm to 650 nm) and red (from 780 nm to 950 nm) fluorescence channels were detected 12 h post administration with a multispectral imaging system. As shown in Figure 6a, the tumor was distinguishable from other tissues with good green and red fluorescence contrast in the mice, indicating the highly efficient accumulation in tumor due to EPR effect and highly specific tumor response of the bioorthogonal reaction. The biodistribution of the nanoemitters 1 in mice was further evaluated by ex vivo experiments. As shown in Figure 6b and c, the tumor exhibited a stronger signal in green and red channels than other harvested organs, i.e., lung, heart, kidney and spleen except for liver at 12 h after injection. Therefore, the tumor sections were taken from mice at 12 h after injection of the nanoemitters 1 for CLSM imaging (Figure 6d). The accumulated structures within the tumor, resulting from the bioorthogonal reaction between the injected nanoemitters 1 and GSH, were clearly observed (the green emission from the aggregates of molecule 2 and the red emission from the formation of S-substituted Cy compound 3). The results demonstrated that the bioorthogonally deciphered nanoemitters 1 could be applied for tumor-specific diagnostics in vivo. In conclusion, we designed and prepared a building block molecule 1 with two emissive motifs (Cy and BP), which can self-assemble into nanoemitters 1. These nanoemitter 1 could be deciphered by a GSH-based bioorthogonal reaction, resulting in turn-on binary fluorescence signals with a large red shift excitation/emission profiles. The hydrophobic BP residue 2 selfassembled into larger nanoaggregates with almost 36-fold enhancement in fluorescence intensity at 520 nm. Interestingly, the newly formed GSH labeled Cy 3 exhibited 30-fold fluorescence enhancement at NIR region (λem = 820 nm). This bioorthogonal reaction exhibited high specificity and good biocompatibility for GSH imaging in vitro and in vivo, which could be further unitized for tumor diagnostics. We believe that the bioorthogonally deciphered approach


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will open a new avenue for in situ construction of self-assembled biomaterials for diagnostic of diseases in vivo. ASSOCIATED CONTENT Supporting Information. Materials, experimental methods, and additional measurements. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by National Basic Research Program of China (973 Program, 2013CB932701), National Natural Science Foundation of China (21374026, 51573031, 21304023, and 51303036). REFERENCES (1) Würthner, F., Supramolecular polymerization: Living it up. Nat. Chem. 2014, 6, 171-173. (2) Aida, T.; Meijer, E. W.; Stupp, S. I., Functional supramolecular polymers. Science 2012, 335, 813-817.


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(3) Guo, D.-S.; Liu, Y., Supramolecular Chemistry of p-Sulfonatocalix[n]arenes and Its Biological Applications. Acc. Chem. Res. 2014, 47, 1925-1934. (4) Lee, J. H.; Chen, K. J.; Noh, S. H.; Garcia, M. A.; Wang, H.; Lin, W. Y.; Jeong, H.; Kong, B. J.; Stout, D. B.; Cheon, J.; Tseng, H. R., On-demand drug release system for in vivo cancer treatment through self-assembled magnetic nanoparticles. Angew. Chem. Int. Ed. 2013, 52, 43844388. (5) Yao, Y.; Xue, M.; Chen, J.; Zhang, M.; Huang, F., An amphiphilic pillar[5]arene: synthesis, controllable self-assembly in water, and application in calcein release and TNT adsorption. J. Am. Chem. Soc. 2012, 134, 15712-15715. (6) Yang, Q.; Dong, Y.; Wu, W.; Zhu, C.; Chong, H.; Lu, J.; Yu, D.; Liu, L.; Lv, F.; Wang, S., Detection and differential diagnosis of colon cancer by a cumulative analysis of promoter methylation. Nat. Commun. 2012, 3, 1206. (7) Wang, H.; Wang, S.; Su, H.; Chen, K. J.; Armijo, A. L.; Lin, W. Y.; Wang, Y.; Sun, J.; Kamei, K.; Czernin, J.; Radu, C. G.; Tseng, H. R., A supramolecular approach for preparation of size-controlled nanoparticles. Angew. Chem. Int. Ed. 2009, 48, 4344-4348. (8) Ye, D.; Liang, G.; Ma, M. L.; Rao, J., Controlling intracellular macrocyclization for the imaging of protease activity. Angew. Chem. Int. Ed. 2011, 50, 2275-2279. (9) Williams, R. J.; Smith, A. M.; Collins, R.; Hodson, N.; Das, A. K.; Ulijn, R. V., Enzymeassisted self-assembly under thermodynamic control. Nat. Nanotech. 2009, 4, 19-24. (10) Li, L. L.; Qi, G. B.; Yu, F.; Liu, S. J.; Wang, H., An adaptive biointerface from selfassembled functional peptides for tissue engineering. Adv. Mater. 2015, 27, 3181-3188. (11) Zhang, S., Fabrication of novel biomaterials through molecular self-assembly. Nat. Biotech. 2003, 21, 1171-1178. (12) Huang, Z.; Yang, L.; Liu, Y.; Wang, Z.; Scherman, O. A.; Zhang, X., Supramolecular polymerization promoted and controlled through self-sorting. Angew. Chem. Int. Ed. 2014, 53, 5351-5355. (13) Gao, Y.; Shi, J.; Yuan, D.; Xu, B., Imaging enzyme-triggered self-assembly of small molecules inside live cells. Nat. Commun. 2012, 3, 1033. (14) Zhang, X.; Gorl, D.; Stepanenko, V.; Würthner, F., Hierarchical growth of fluorescent dye aggregates in water by fusion of segmented nanostructures. Angew. Chem. Int. Ed. 2014, 53, 1270-1274. (15) Wang, Y.; Zhou, K.; Huang, G.; Hensley, C.; Huang, X.; Ma, X.; Zhao, T.; Sumer, B. D.; DeBerardinis, R. J.; Gao, J., A nanoparticle-based strategy for the imaging of a broad range of tumours by nonlinear amplification of microenvironment signals. Nat. Mater. 2014, 13, 204-212. (16) Zhang, D.; Qi, G.-B.;Zhao, Y.-Q.; Qiao, S.-L.; Yang, C.; Wang, H., A In Situ Formation of Nanofibers from Purpurin18-Peptide Conjugates and the Assembly Induced Retention Effect in Tumor Sites. Adv. Mater. 2015, 27, 6125-6130. (17) Zhang, D.; Wei, Y.; Chen, K.; Zhang, X.; Xu, X.; Shi, Q.; Han, S.; Chen, X.; Gong, H.; Li, X.; Zhang, J., Biocompatible reactive oxygen species (ROS)-responsive nanoparticles as superior drug delivery vehicles. Adv. Healthc. Mater. 2015, 4, 69-76. (18) Eliana Saxon; Bertozzi, C. R., Cell Surface Engineering by a ModiÞed Staudinger Reaction. science 2000, 287, 2007-2010. (19) Vsevolod V. Rostovtsev; Luke G. Green; Valery V. Fokin; Sharpless, K. B., A Stepwise Huisgen Cycloaddition Process:Copper(I)-Catalyzed Regioselective "Ligation" of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. 2002, 114, 2708-2711.


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Scheme 1. Schematic illustration of a new GSH-based bioorthogonal reaction that enable to decipher the nanometters of cyanine-pyrene dye (1), resulting in turn-on binary fluorescence signals with a large shift excitation/emission profiles in living systems.


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Figure 1 The synthesis and characterization of molecule 1. a) Synthesis of a cyanine-pyrene dye (1). b) The UV-Vis (the blue line) and fluorescence spectra of the molecule 1 in monomeric (dot lines, DMSO solution) and aggregated state (solid lines, aqueous solution). The green line was the fluorescence spectra of BP motifs and the red line was the fluorescence spectra of Cy motifs c) The TEM image of the nanoemitters 1 (100 µM) in DMSO/PBS solution (v/v = 1/99, pH = 7.4).


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Figure 2 The confirmation of the reaction product by optical and mass spectra. a) A proposed GSH-based bioorthogonal reaction and the MALDI-TOF spectrum of reaction products (molecule 2 and 3). Time-dependent UV-Vis b) and fluorescence (c, d) spectra of the nanoemitters 1 (10 µM) upon addition of GSH (500 equiv.). Inset: Photographs of the color changing b), and the emission λem=820 nm c) and λem=520 nm d) of the nanoemitters 1 before/after the addition of GSH (500 equiv.). The buffer is DMSO/PBS solution (v/v = 1/99, pH = 7.4).


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Figure 3 The confirmation of the reaction product by NMR. a) The procedure of separation the major products of GSH (500 equiv.) treated the nanoemitters 1 (10 mM). b) Partial 1H NMR spectra of supernatants collected during the reaction of the nanoemitters 1 (10 mM) with GSH (500 equiv.) in 0 h (i), 0.5 h (ii) and 2 h (iii) comparing to the synthesized molecule 3 (iv). All supernatants were dried and dissolved by a mixture of solution (CD3OD/D2O = 9:1, v/v) before examination. c) The 1H NMR spectrum of collecting precipitate of nanoemitters 1 (10 mM) upon addition of GSH (500 equiv.). Inset: The molecular structure of molecule 1, 3 b) and 2 c). The buffer is DMSO/PBS solution (v/v = 1/99, pH = 7.4).


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Figure 4 The exploration of the reaction conditions. a) UV-Vis spectra of the nanoemitters 1 (10 µM) in the presence of various content of GSH at 37 °C. Arrows indicate the increase of GSH ratios. b) The response of the nanoemitters 1 (10 µM) to the pH variations (3.5–10.0) in presence/absence of GSH (500 equiv.). c) UV-Vis response of the nanoemitters 1 (10 µM) upon addition of GSH (10 mM), Cys (100 µM), and Glu, Thr, Tyr (500 µM). The buffer is DMSO/PBS solution (v/v = 1/99, pH = 7.4).


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Figure 5 The GSH-based bioorthogonal reaction for intracellular application. a) CLSM images of the nanoemitters 1 (30 µM) in MCF-7 cells (control), LPA pre-treated cells (1.8 mM, 24 h, positive) and NEM treated cells (500 µM, 2 h, negative). b) The representative line plot of MCF-7 cells and the corresponding fluorescence signal distribution based on the white line in images. c) The MALDI-TOF spectrum of MCF-7 cell lysates after incubation with the nanoemitters 1 (50 µM) for 3 h.


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Figure 6 The GSH-based bioorthogonal reaction for in vivo application. a) In vivo optical imaging (green channel and red channel) of nude mice bearing MCF-7 tumor at 12 h post tailvein injection of the nanoemitters 1 or PBS. (The arrows show the location of subcutaneous tumors) b) Ex vivo optical imaging (green channel and red channel) of tissues and tumors at 12 h post tail-vein injection of the nanoemitters 1 or PBS; 1. Tumor; 2. Lung; 3. Heart; 4. Kidney; 5. Spleen; 6. Liver. c) Quantitative analysis of the biodistribution of the nanoemitters 1 in major organs 12 h post–injection to mice in green and red channel. (d) Confocal fluorescence microscopy images of tumor resected from mice treated with the nanoemitters 1 at 12 h post– injection to mice in green and red channel. Each column represented the mean value (n=6). Data were represented as means ± SD. Mice treated with PBS were regarded as the control group.


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TOC

Bioorthogonal-Reaction Deciphered Nanoemitters: We prepare a cyanine-pyrene building block (1) that can be efficiently replaced by abundant Glutathione through a new bioorthogonal reaction. This reaction activates the nanoemitters 1 and results in turn-on binary fluorescence signals for tumor imaging in vitro and in vivo.


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Bio-orthogonally Deciphered Binary Nanoemitters ... - ACS Publications

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