Article pubs.acs.org/bc
Activatable Water-Soluble Probes Enhance Tumor Imaging by Responding to Dysregulated pH and Exhibiting High Tumor-to-Liver Fluorescence Emission Contrast Hu Xiong, Petra Kos, Yunfeng Yan, Kejin Zhou, Jason B. Miller, Sussana Elkassih, and Daniel J. Siegwart* Simmons Comprehensive Cancer Center, Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States S Supporting Information *
ABSTRACT: Dysregulated pH has been recognized as a universal tumor microenvironment signature that can delineate tumors from normal tissues. Existing fluorescent probes that activate in response to pH are hindered by either fast clearance (in the case of small molecules) or high liver background emission (in the case of large particles). There remains a need to design watersoluble, long circulating, pH-responsive nanoprobes with high tumor-to-liver contrast. Herein, we report a modular chemical strategy to create acidic pH-sensitive and water-soluble fluorescent probes for high in vivo tumor detection and minimal liver activation. A combination of a modified Knoevenagel reaction and PEGylation yielded a series of NIR BODIPY fluorophores with tunable pKas, high quantum yield, and optimal orbital energies to enable photoinduced electron transfer (PeT) activation in response to pH. After intravenous administration, Probe 5c localized to tumors and provided excellent tumor-to-liver contrast (apparent T/L = 3) because it minimally activates in the liver. This phenomenon was further confirmed by direct ex vivo imaging experiments on harvested organs. Because no targeting ligands were required, we believe that this report introduces a versatile strategy to directly synthesize soluble probes with broad potential utility including fluorescence-based image-guided surgery, cancer diagnosis, and theranostic nanomedicine.
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INTRODUCTION Tumor-targeted fluorescence imaging is a promising technique for in situ cancer diagnosis and tracking, image-guided surgery, and theranostic nanomedicine due to excellent spatiotemporal resolution with chemically tunable emission colors.1−7 Probes are commonly designed to respond to specific molecular signatures of cancer by attachment of antibodies, peptides, and other targeting groups,5,7−17 but these “always on” probes are limited in their ability to universally image tumors of different types in different locations. Alternatively, probes can be designed to respond to general hallmarks of cancer, such as the slightly lower pH of the tumor microenvironment.18 This has emerged as a targetable strategy to universally image tumors of different types.7,18,19 Ideal probes maximize signal contrast through mediation of “off” and “on” states in normal and cancerous tissues. Despite advances, existing pH-responsive probes suffer from either fast clearance (in the case of small molecules) or high liver © 2016 American Chemical Society
background emission (in the case of nanoparticles). Moreover, simultaneous control of pH activation and near-infrared (NIR) emission (650−900 nm)9,16 remains synthetically challenging, due in part to inherent photosensitivity of unsaturated bonds within typical NIR dyes (e.g., cyanine) that preclude pH responsiveness by mechanisms such as photoinduced electron transfer (PeT).8,9,20−23 In this work, we report comprehensive, water-soluble probes that exhibit high tumor-to-background contrast, tunable pH response, extended blood circulation, and high targeting ligand-free tumor uptake to improve the application of tumor imaging (Figure 1). Notably, Probe 5c localized to tumors after i.v. administration and provided excellent tumor-to-liver contrast because it minimally activates in the liver (Figures 1b, 6e, and S15). Received: May 12, 2016 Revised: May 30, 2016 Published: June 10, 2016 1737
DOI: 10.1021/acs.bioconjchem.6b00242 Bioconjugate Chem. 2016, 27, 1737−1744
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Figure 1. pH-responsive, water-soluble probes can detect tumors with minimal liver activation and without attachment of targeting ligands. (a) Water-soluble PEGylated NIR BODIPYs activate in response to a decrease in pH. Electron flow is involved in a PeT process from the aniline group to the excited BODIPY core. (b) Responsive Probe 5c turns on in MDA-MB-231 tumors and not in livers after ex vivo injection due to the low pH of the tumor microenvironment. Normalized emission is presented to show contrast. Imaging was performed 10 min after direct injection of probes (1 μM, 100 μL).
Figure 2. Series of pH-responsive, water-soluble probes were synthesized using indole and aldehyde condensation reactions. Chemical modifications rendered typically green BODIPY scaffolds as NIR far red emitting dyes. PEGylation provided water-solubility, and anilines tuned the pKa. A modified Knoevenagel condensation reaction, Fischer aldehydes, and modular linker regions (indole, ester, ether) were key to tuning the HOMO/ LUMO electronic properties.
Low microenvironment pH is a tumor hallmark18,19 that can be exploited by pH-sensitive probes11,24−32 to image tumors. Multiple parameters including chemical composition, molecular weight, solubility/dispersion, size, and shape alter pharmacokinetic (PK) profiles and organ localiazation.33 Water-soluble, small molecule probes5,9,11,16,34 have required attachment of targeting moieties to increase signal-to-noise and reduce rapid renal clearance.11,35,36 Nanoparticle-based probes7,37−46 can provide ultrasharp pH transitions, but result in high liver emission because of unavoidable liver accumulation. High liver background emission from NPs (>20 nm) is particularly adverse for detecting liver cancer and liver metastasis.33 Probes that are water-soluble (low liver uptake and retention), emit in
the NIR region (low background), and have good PK profiles (i.e., not fast clearing), have been elusive. Herein, we unified molecular weight and chemical design approaches to synthesize water-soluble, long circulating, pHresponsive probes, with high tumor-to-liver contrast. We defined chemical rules to increase pKa and emission wavelength by chemical modification, and demonstrate advantageous tumor imaging in vivo in multiple tumor types. Because no targeting ligands were required, we believe that this report introduces a versatile method to directly synthesize modular probes with broad potential utility including image-guided surgery, cancer diagnosis, and theranostic nanomedicine. 1738
DOI: 10.1021/acs.bioconjchem.6b00242 Bioconjugate Chem. 2016, 27, 1737−1744
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RESULTS AND DISCUSSION
pH-Responsive Fluorescent Probes Were Designed to Provide High Tumor Response and Minimal Activation in the Liver. To rationally design pH-responsive probes, we selected tetramethyl-BODIPY (4,4′-difluoro-4-bora-3a,4adiaza-s-indacene)47,48 as a core scaffold and installed anilines to tune the pKa, linkers (indole, ester, ether) to enable NIR (>650 nm9,16) emission, and poly(ethylene glycol) (PEG) chains to provide water solubility and slow kidney clearance (Figure 2). We hypothesized that N,N-dialkylated anilines would provide sufficiently high highest occupied molecular orbital (HOMO) energy levels to quench the NIR BODIPY core at neutral or basic pH by the PeT mechanism (Figure S1). Inspired by previous reports, we aimed to overcome the green emission 11 and targeting ligand attachment requirement5,7,11,15,30,45 that can limit in vivo utility. We utilized Knoevenagel-type49 condensation of the methyl groups adjacent to BODIPY nitrogens with aromatic aldehydes to install ester, ether, and indole groups through a unique position of conjugation. This enabled NIR emission, which is optimal for in vivo imaging. The cumulative PEGylated design alleviated the need to attach targeting ligands. As the first step, 1a−e were synthesized by acid-catalyzed condensation of 2,4-dimethylpyrrole with N,N-dialkylaminobenzaldehydes, followed by oxidation with tetrachlorobenzoquinone (TCQ) and treatment with BF3·OEt2.11,50 Subsequently, the BODIPY core 1b was condensed with 1,3dimethyl-1H-indole-2-carbaldehyde or a Fisher aldehyde to obtain NIR chromophores (Figure 2). This reaction typically reaches low conversions using classic Knoevenagel conditions. Through optimization, we found that the use of acetonitrile as solvent at modest temperature (85 °C) could afford the desired product in high conversion (>90%) (Table S1). The resulting dyes with the NIR units 2b and 3b showed strong emission at 746 and 730 nm, respectively, but were unfortunately not pHsensitive in DMSO and methanol (Figure S2). This implied that the HOMO energy level of the NIR core may be too high to accept electrons donated from the N,N-dimethylaniline moiety, thus inhibiting the fluorescence quenching in the PeT process. Using this understanding, the NIR dye 4b-1 was rationally synthesized by a new synthetic route as shown in Figure 2 by inclusion of two ester groups to reduce the HOMO energy level of the NIR core. As expected, 4b-1 was pH-responsive in DMSO and methanol (Figure S3). To render the dye watersoluble, two PEG chains were attached via click chemistry to obtain water-soluble 5b-1. Importantly, the pH-activatable fluorescence property of 5b-1 was not affected by PEGylation, which was confirmed by pH titration experiments. However, owing to the weakened basicity of N,N-dimethylaniline by the ester groups, the pKa of 5b-1 was only about 2.9 (Figure 3a), which rendered it unsuitable for in vivo imaging. To increase the pKa, 5b-2 was synthesized by replacing the ester groups with electron-donating oxygen atoms to increase the basicity of amine moiety. This subtle change increased the pKa to 3.1 (Figure 3a). To further develop probes to cover a larger pH range, the pKa and pH-transition points were finetuned by changing the alkyl substituents (hydrophobicity) on the aniline nitrogen. In this way, a series of pH-responsive Probes 5c−e and always-on Probe 5a were developed by the modified synthetic pathway. The pKa values were 4.5 and 4.0 for p-diethylanilino 5c and p-piperidinylphenyl 5e, respectively.
Figure 3. pH transition of water-soluble NIR probes is highly tunable. (a) Normalized pH titration profiles of the 5 series of probes. 5a is the always-on probe and Fmax/Fmin is expressed. pKa: 5b-1, 2.9; 5b-2, 3.1; 5c, 4.5; 5d, 1.75; 5e, 4.0. (b) pH-dependent fluorescent images of 10 μM aq. solutions of probes. (c) NIR fluorescence spectra of 5c (10 μM, λex = 640 nm). (d) Relationship between concentration and dependent fluorescence intensity of 5c in pH 4.5 buffer.
Interestingly, the pKa value was only 1.75 for 5d, probably due to the rigid conformation of the pyrrolidinyl moiety, which inhibits protonation of the amine group (Figure 3a). Fluorescent images of aqueous solutions of 5a−e at different pH values illustrate exquisite control of pH transitions for each probe (Figure 3b). Notably, 5c (emission at 670 nm) exhibits enhanced fluorescence at pH 4.5−5.0 (Figure 3c), good quantum yield (Φ = 0.22, Table S2), and excellent water solubility at low concentration (Figure 3d). Because the probes exhibited a linear increase in fluorescence intensity as a function of concentration from 0 to ∼10 μM, the probe concentration was kept below 10 μM for all in vitro and in vivo experiments (0.5 mg/kg). Accordingly, liver uptake is likely minimized because the water-soluble probes do not form micelles or nanoparticles at these concentrations. pH-Responsive Probes Activate Inside of Cancer Cells. We first examined utility for imaging cancer cells in vitro. To determine the performance of pH-dependent intracellular signal activation, we applied Probes 5a−e at the same concentration (5.0 μM) to HeLa cells in vitro and stained for lysosomes and cell nuclei. All pH-responsive probes were off in cell culture medium (pH ∼ 7.4). After 30 min, NIR fluorescence was observed most strongly for Probes 5c (pKa 4.5) and 5e (pKa 4.0) (Figure S5). This indicates that probes with higher pH transition are activated faster than those with a lower pH transition, likely within early endosomes. After 4 h, most of the probes (especially 5c) produced bright NIR emission (Figures S6−S8), suggesting that all of the probes (except 5b-1) could eventually activate as the pH in maturing endosomes decreased. Eventual colocalization with Lysotracker Green confirmed that the probes were endocytosed and progressed to the lysosomes. Among the probes, 5c exhibited the brightest emission and possessed an optimal pKa of 4.5. Furthermore, no cell cytotoxicity was observed even at high concentration (116 μM, cell viability >90%, Figure S9). 1739
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Figure 4. NIR Probe 5c activates in a variety of cancer cell types. Probe 5c (5.0 μM) was incubated with 6 different cancer cell lines for 4 h at 37 °C. Confocal fluorescence microscope images (20×) of fixed cells indicate that probes rapidly activate inside of a variety of cancer cell lines. Probe 5c (red), cell nuclei (blue), and lysosomes (green) are shown. Eventual colocalization of probes and lysosomes is visualized as yellow in the merged images. Scale bar = 20 μm.
To investigate the potential universality of Probe 5c activation, we incubated 5c with 6 different cancer cell lines (MDA-MB-231, HCC4017, A549, HeLa, SKOV3, and Huh7). All 6 cancer cell types showed accumulation and activation of 5c within 4 h (Figure 4). It is worth noting that these in vitro experiments examined uptake into cancer cells, where the activation occurred intracellularly. Next, we aimed to examine extracellular activation in vivo, where the difference between blood pH and the tumor extracellular microenvironment (pH ∼ 6.8) can activate pH-responsive probes.7 Based on these cancer cell imaging results, Probe 5c was selected as a promising probe for in vivo tumor imaging. Probe 5c Can Localize to Tumors in Mice and Turn “On” After Intravenous Administration. The acidic cancer tissue microenvironment (∼6.8) is recognized as a general hallmark of cancer due to metabolically accumulated lactic acid,18,51 providing an actionable target for tumor therapy52−54 and imaging.7,55−58 To begin investigating 5c, we directly injected it into MDA-MB-231 breast cancer xenograft tumors. Intense, bright NIR emission was visible, which persisted for >6 days (Figure S10). Next, we evaluated the ability of 5c to activate in tumors after intravenous (i.v.) injection. Mice bearing subcutaneous MDA-MB-231 tumors were injected i.v. with either the pH-activatable Probe 5c or the always-on Probe 5a at a dose of 0.5 mg/kg. Strong and sustained emission was observed (Figure 5a). Thus, the fast clearance drawback typical of small molecule probes could be avoided by increasing the molecular weight of the probe. Importantly, for 5c, the tumor tissue could be clearly distinguished from the surrounding normal tissues 2 h (h) after injection. 5c NIR emission became
considerably brighter in the tumor after 6 h (Figure 5a) and was still detectable 24 h post-injection (Figure S11). In contrast, the always-on control 5a displayed normal tissue interference and exhibited low tumor-to-background contrast (Figure 5a). Probe 5c is brighter than 5a due to its higher quantum yield (Table S2). To provide further evidence of improved PK, the water-soluble sulfo-Cyanine5 carboxylic acid (sulfo-Cy5) and indocyanine green (ICG) dyes were injected i.v. for comparison. After only 15 min, sulfo-Cy5 and ICG dyes were visible in the liver and kidneys due to rapid renal clearance (Figure S12) and were almost totally cleared within 2 h. In addition, no tumor emission was observed at any time point (Figure 5a). These results indicate that 5c possesses a high ability to accumulate and activate in tumors and that the clearance rate of 5c in tumor-bearing mice was much slower than sulfo-Cy5 and ICG due to the prolonged blood-circulation time provided by PEGylation.59,60 To further examine biodistribution, harvested organs were analyzed 8 h post-injection. 5c exhibited strong NIR emission in the tumor and low emission in other organs (Figure 5b). In contrast, the always-on 5a showed nearly the same fluorescence intensity in the liver, tumor, and kidneys (Figure 5b). Sulfo-Cy5 and ICG had no detectable emission 6 h after injection, which verified that they were rapidly cleared. The essential pHactivatable response of 5c was confirmed by normalizing the relative fluorescence signal of each organ/tissue to muscle (Figure 6a). To demonstrate that 5c responds to dysregulated pH as a general tumor hallmark, we examined imaging capability in two additional cancer types. We selected HCC4017 because it was derived from a lung cancer patient 1740
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Figure 5. Probe 5c activates in the low pH tumor microenvironment. (a) Time-dependent in vivo fluorescence images of mice bearing subcutaneous MDA-MB-231 tumors after i.v. injection of 0.5 mg/kg pH-activatable probe 5c, always-on probe 5a, sulfo-Cyanine5 carboxylic acid (Sulfo-Cy5), and ICG. (b) Representative fluorescence images of harvested tumors and organs from MDA-MB-231 tumor-bearing mice sacrificed at 6 h (Sulfo-Cy5 or ICG) or 8 h (5a or 5c) post-injection.
∼0.5). This indicates that although 5c localized to the tumor over the liver by a factor of only 0.5, it activated selectively in the tumor microenvironment due to lower pH, which provided an apparent T/L ratio of ∼3. Confocal fluorescence imaging of tissue cryosections further verified stronger emission in the tumor over the liver (Figure 6d). Because apparent contrast is most relevant for practical application, these results demonstrate successful utility. Finally, to further examine this effect, we harvested MDAMB-231 tumors and livers from noninjected mice and directly injected probes ex vivo. When 5c was injected into the harvested tissues (1 μM, 100 μL), bright NIR signal was observed only in the tumor with no signal being detected in the liver (Figure 6e). This is due to the different extracellular pH in harvested tumors and livers. We then repeated this study using harvested HCC4017 lung tumors. Similarly, 5c activates only in the lung tumor and not in the liver (Figure S15a). In contrast,
at our medical center, thereby capturing the genetic NSCLC features.61 Time-dependent imaging of HCC4017 (Figure S13) and HeLa (common cancer line) (Figure S14) xenografts indicates that 5c effectively accumulates in the tumors within 2 h after injection with negligible background fluorescence. Probe 5c Provides Remarkably High Tumor-to-Liver Contrast. To further understand why PEGylated 5c can balance size and pH activation to overcome the issues of small molecules (rapid clearance) and nanoparticles (high liver accumulation), we closely examined T/L ratio. It is clear that 5c produces effectively high T/L contrast (ratio ∼3, Figure 6b), even though the liver can collect PEGylated materials.62−66 To help understand the origin of the increased T/L ratio, the tumor and liver were homogenized and 5c was extracted into methanol. The total amount of 5c was quantified by adding TFA (Figure S4). As shown in Figure 6b,c, the liver actually took up a significant fraction of the total probe dose (T/L ratio 1741
DOI: 10.1021/acs.bioconjchem.6b00242 Bioconjugate Chem. 2016, 27, 1737−1744
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Figure 6. Probe 5c improves tumor-to-liver contrast by minimally activating in the liver. (a) Relative 5c fluorescence intensity of organs in MDAMB-231 tumor-bearing mice (8 h post-injection of 0.5 mg/kg i.v.). Emission was normalized to the fluorescence signal in the muscle. (b) T/L (Tumor/Liver) ratio of 5c fluorescence intensity at 8 h post-injection. T/La ratio was normalized by ROI analysis. T/Lb ratio was normalized to the fluorescent signal per gram tissue after homogenization in MeOH with 1% TFA (to turn on all 5c molecules). Mean ± s.d. (n = 3). (c) NIR fluorescence spectrum of homogenized liver and tumor in MeOH with 1% (v/v) TFA. (d) Fluorescence microscopy images of tumor and liver tissue cryosections 8 h post-injection of 5c (0.5 mg/kg i.v.). Scale bar = 100 μm. (e) Probe 5c was directly injected into the harvested tumor and liver ex vivo to evaluate tumor specific pH-activation. Imaging was performed 10 min after direct injection of 5c (1 μM, 100 μL).
groups. Therefore, these rationally designed and carefully engineered probes may provide a robust platform for cancer imaging in vivo in a variety of modalities and applications. We are currently exploring the effects of alternative routes of administration on PK and tumor uptake. We are also examining the effect of PEG length on tumor imaging. These efforts will be published in future reports. Finally, we envision future utility in theranostic drug delivery and imaging materials due to the modular design capability.
the always-on 5a is bright in both the tumor and liver after injection (Figure S15b). These results further prove that 5c can turn on in acidic tumor microenvironment (low extracellular pH). Cumulatively, these results suggest that the acidic tumor microenvironment and intracellular pH dynamics activate 5c in cancer cells in vivo. 5c ultimately results in high tumor-tonormal and tumor-to-liver contrast to improve imaging resolution and cancer detection.
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CONCLUSIONS In summary, we have successfully developed a series of watersoluble, pH-tunable, and NIR fluorescence probes based on rational chemical design by harnessing the PeT effect. The pHresponsive Probe 5c can localize to cancer cells in vivo and be activated by acidic pH to produce high tumor-to-normal signal contrast and high tumor-to-liver signal contract in mice bearing breast, lung, and cervical tumors after i.v. injection. This new class of small molecule probes is a promising step for detecting different cancer cells without having to attach tumor-targeting
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00242. Synthetic descriptions of NIR probes, photophysical properties of NIR BODIPYs, cell culture and confocal microscopy, cell viability studies, in vivo imaging and biodistribution of tumor-bearing mice, ex vivo analysis of 1742
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liver and tumor, 1H NMR and 13C NMR spectra of the probes, and additional supplemental figures (PDF)
of epidermal growth factor receptor in breast cancer xenografts. Cancer Res. 63, 7870−7875. (13) Luo, S. L., Zhang, E. L., Su, Y. P., Cheng, T. M., and Shi, C. M. (2011) A review of NIR dyes in cancer targeting and imaging. Biomaterials 32, 7127−7138. (14) Bagalkot, V., Zhang, L., Levy-Nissenbaum, E., Jon, S., Kantoff, P. W., Langer, R., and Farokhzad, O. C. (2007) Quantum dot - Aptamer conjugates for synchronous cancer imaging, therapy, and sensing of drug delivery based on Bi-fluorescence resonance energy transfer. Nano Lett. 7, 3065−3070. (15) Urano, Y., Sakabe, M., Kosaka, N., Ogawa, M., Mitsunaga, M., Asanuma, D., Kamiya, M., Young, M. R., Nagano, T., Choyke, P. L., et al. (2011) Rapid cancer detection by topically spraying a gammaglutamyltranspeptidase-activated fluorescent probe. Sci. Transl. Med. 3, 110ra119. (16) Guo, Z. Q., Park, S., Yoon, J., and Shin, I. (2014) Recent progress in the development of near-infrared fluorescent probes for bioimaging applications. Chem. Soc. Rev. 43, 16−29. (17) Wu, X. J., Yu, M. Z., Lin, B. J., Xing, H. J., Han, J. H., and Han, S. F. (2015) A sialic acid-targeted near-infrared theranostic for signal activation based intraoperative tumor ablation. Chem. Sci. 6, 798−803. (18) Webb, B. A., Chimenti, M., Jacobson, M. P., and Barber, D. L. (2011) Dysregulated pH: A perfect storm for cancer progression. Nat. Rev. Cancer 11, 671−677. (19) Hanahan, D., and Weinberg, R. A. (2011) Hallmarks of cancer: The next generation. Cell 144, 646−674. (20) Tanaka, K., Miura, T., Umezawa, N., Urano, Y., Kikuchi, K., Higuchi, T., and Nagano, T. (2001) Rational design of fluoresceinbased fluorescence probes, mechanism-based design of a maximum fluorescence probe for singlet oxygen. J. Am. Chem. Soc. 123, 2530− 2536. (21) Sasaki, E., Kojima, H., Nishimatsu, H., Urano, Y., Kikuchi, K., Hirata, Y., and Nagano, T. (2005) Highly sensitive near-infrared fluorescent probes for nitric oxide and their application to isolated organs. J. Am. Chem. Soc. 127, 3684−3685. (22) Yu, F. B. A., Li, P., Li, G. Y., Zhao, G. J., Chu, T. S., and Han, K. L. (2011) A near-IR reversible fluorescent probe modulated by selenium for monitoring peroxynitrite and imaging in living cells. J. Am. Chem. Soc. 133, 11030−11033. (23) Yu, F. B., Li, P., Wang, B. S., and Han, K. L. (2013) Reversible near-infrared fluorescent probe introducing tellurium to mimetic glutathione peroxidase for monitoring the redox cycles between peroxynitrite and glutathione in vivo. J. Am. Chem. Soc. 135, 7674− 7680. (24) Han, J. Y., and Burgess, K. (2010) Fluorescent indicators for intracellular pH. Chem. Rev. 110, 2709−2728. (25) Lee, H., Akers, W., Bhushan, K., Bloch, S., Sudlow, G., Tang, R., and Achilefu, S. (2011) Near-infrared pH-activatable fluorescent probes for imaging primary and metastatic breast tumors. Bioconjugate Chem. 22, 777−784. (26) Myochin, T., Kiyose, K., Hanaoka, K., Kojima, H., Terai, T., and Nagano, T. (2011) Rational design of ratiometric near-infrared fluorescent pH probes with various pK(a) values, based on aminocyanine. J. Am. Chem. Soc. 133, 3401−3409. (27) Hoogendoorn, S., Blom, A. E. M., Willems, L. I., van der Marel, G. A., and Overkleeft, H. S. (2011) Synthesis of pH-activatable red fluorescent BODIPY dyes with distinct functionalities. Org. Lett. 13, 5656−5659. (28) Asanuma, D., Takaoka, Y., Namiki, S., Takikawa, K., Kamiya, M., Nagano, T., Urano, Y., and Hirose, K. (2014) Acidic-pH-activatable fluorescence probes for visualizing exocytosis dynamics. Angew. Chem., Int. Ed. 53, 6085−6089. (29) Lee, M. H., Park, N., Yi, C., Han, J. H., Hong, J. H., Kim, K. P., Kang, D. H., Sessler, J. L., Kang, C., and Kim, J. S. (2014) Mitochondria-immobilized pH-sensitive off-on fluorescent probe. J. Am. Chem. Soc. 136, 14136−14142. (30) Asanuma, D., Sakabe, M., Kamiya, M., Yamamoto, K., Hiratake, J., Ogawa, M., Kosaka, N., Choyke, P. L., Nagano, T., Kobayashi, H., et al. (2015) Sensitive beta-galactosidase-targeting fluorescence probe
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS D.J.S. gratefully acknowledges financial support from the Welch Foundation (I-1855) and the Cancer Prevention and Research Institute of Texas (CPRIT) (R1212). J.B.M. acknowledges CPRIT (RP140110) for fellowship support. S.E. acknowledges the National Science Foundation (NSF) (GRFP 1000198224) for fellowship support. We also acknowledge the UT Southwestern Live Cell Imaging Facility, a Shared Cancer Center Resource supported by an NCI Cancer Center Support Grant (1P30 CA142543-05). We thank Professor Jinming Gao for scientific inspiration and helpful discussions. The content is solely the responsibility of the authors and does not necessarily represent the official views of any of the above-mentioned funding agencies.
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REFERENCES
(1) Weissleder, R. (2001) A clearer vision for in vivo imaging. Nat. Biotechnol. 19, 316−317. (2) Weissleder, R., and Pittet, M. J. (2008) Imaging in the era of molecular oncology. Nature 452, 580−589. (3) Fernandez-Suarez, M., and Ting, A. Y. (2008) Fluorescent probes for super-resolution imaging in living cells. Nat. Rev. Mol. Cell Biol. 9, 929−943. (4) Vahrmeijer, A. L., Hutteman, M., van der Vorst, J. R., van de Velde, C. J. H., and Frangioni, J. V. (2013) Image-guided cancer surgery using near-infrared fluorescence. Nat. Rev. Clin. Oncol. 10, 507−518. (5) van Dam, G. M., Themelis, G., Crane, L. M. A., Harlaar, N. J., Pleijhuis, R. G., Kelder, W., Sarantopoulos, A., de Jong, J. S., Arts, H. J. G., van der Zee, A. G. J., et al. (2011) Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-alpha targeting: first in-human results. Nat. Med. 17, 1315−1319. (6) Weichert, J. P., Clark, P. A., Kandela, I. K., Vaccaro, A. M., Clarke, W., Longino, M. A., Pinchuk, A. N., Farhoud, M., Swanson, K. I., Floberg, J. M., et al. (2014) Alkylphosphocholine analogs for broadspectrum cancer imaging and therapy. Sci. Transl. Med. 6, 240ra275. (7) Wang, Y. G., Zhou, K. J., Huang, G., Hensley, C., Huang, X. N., Ma, X. P., Zhao, T., Sumer, B. D., DeBerardinis, R. J., and Gao, J. M. (2013) A nanoparticle-based strategy for the imaging of a broad range of tumours by nonlinear amplification of microenvironment signals. Nat. Mater. 13, 204−212. (8) Kobayashi, H., and Choyke, P. L. (2011) Target-cancer-cellspecific activatable fluorescence imaging probes: rational design and in vivo applications. Acc. Chem. Res. 44, 83−90. (9) Kobayashi, H., Ogawa, M., Alford, R., Choyke, P. L., and Urano, Y. (2010) New strategies for fluorescent probe design in medical diagnostic imaging. Chem. Rev. 110, 2620−2640. (10) Becker, A., Hessenius, C., Licha, K., Ebert, B., Sukowski, U., Semmler, W., Wiedenmann, B., and Grotzinger, C. (2001) Receptortargeted optical imaging of tumors with near-infrared fluorescent ligands. Nat. Biotechnol. 19, 327−331. (11) Urano, Y., Asanuma, D., Hama, Y., Koyama, Y., Barrett, T., Kamiya, M., Nagano, T., Watanabe, T., Hasegawa, A., Choyke, P. L., et al. (2009) Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes. Nat. Med. 15, 104−109. (12) Ke, S., Wen, X. X., Gurfinkel, M., Charnsangavej, C., Wallace, S., Sevick-Muraca, E. M., and Li, C. (2003) Near-infrared optical imaging 1743
DOI: 10.1021/acs.bioconjchem.6b00242 Bioconjugate Chem. 2016, 27, 1737−1744
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Bioconjugate Chemistry for visualizing small peritoneal metastatic tumours in vivo. Nat. Commun. 6, 6463. (31) Wang, L., Fan, Z., Zhang, J., Changyi, Y., Huang, C., Gu, Y., Xu, Z., Tang, Z., Lu, W., Wei, X., et al. (2015) Evaluating tumor metastatic potential by imaging intratumoral acidosis via pH-activatable nearinfrared fluorescent probe. Int. J. Cancer 136, 107−116. (32) Yin, J., Hu, Y., and Yoon, J. (2015) Fluorescent probes and bioimaging: alkali metals, alkaline earth metals and pH. Chem. Soc. Rev. 44, 4619−4644. (33) Blanco, E., Shen, H., and Ferrari, M. (2015) Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 33, 941−951. (34) Vendrell, M., Zhai, D. T., Er, J. C., and Chang, Y. T. (2012) Combinatorial strategies in fluorescent probe development. Chem. Rev. 112, 4391−4420. (35) Lee, S., Xie, J., and Chen, X. Y. (2010) Peptide-based probes for targeted molecular imaging. Biochemistry 49, 1364−1376. (36) Liu, J. B., Yu, M. X., Zhou, C., Yang, S. Y., Ning, X. H., and Zheng, J. (2013) Passive tumor targeting of renal-clearable luminescent gold nanoparticles: Long tumor retention and fast normal tissue clearance. J. Am. Chem. Soc. 135, 4978−4981. (37) Lee, E. S., Na, K., and Bae, Y. H. (2003) Polymeric micelle for tumor pH and folate-mediated targeting. J. Controlled Release 91, 103− 113. (38) Zhou, K. J., Wang, Y. G., Huang, X. N., Luby-Phelps, K., Sumer, B. D., and Gao, J. M. (2011) Tunable, ultrasensitive pH-responsive nanoparticles targeting Specific endocytic organelles in living cells. Angew. Chem., Int. Ed. 50, 6109−6114. (39) Wang, L., and Li, C. (2011) pH responsive fluorescence nanoprobe imaging of tumors by sensing the acidic microenvironment. J. Mater. Chem. 21, 15862−15871. (40) Gao, G. H., Lee, J. W., Nguyen, M. K., Im, G. H., Yang, J., Heo, H., Jeon, P., Park, T. G., Lee, J. H., and Lee, D. S. (2011) pHresponsive polymeric micelle based on PEG-poly(beta-amino ester)/ (amido amine) as intelligent vehicle for magnetic resonance imaging in detection of cerebral ischemic area. J. Controlled Release 155, 11−17. (41) Zhou, K. J., Liu, H. M., Zhang, S. R., Huang, X. N., Wang, Y. G., Huang, G., Sumer, B. D., and Gao, J. M. (2012) Multicolored pHtunable and activatable fluorescence nanoplatform responsive to physiologic pH stimuli. J. Am. Chem. Soc. 134, 7803−7811. (42) Kim, K. S., Park, W., Hu, J., Bae, Y. H., and Na, K. (2014) A cancer-recognizable MRI contrast agents using pH-responsive polymeric micelle. Biomaterials 35, 337−343. (43) Si, Z., Huang, C. Y., Gao, X. H., and Li, C. (2014) pHresponsive near-infrared nanoprobe imaging metastases by sensing acidic microenvironment. RSC Adv. 4, 55548−55555. (44) Zhao, Y., Ji, T. J., Wang, H., Li, S. P., Zhao, Y. L., and Nie, G. J. (2014) Self-assembled peptide nanoparticles as tumor microenvironment activatable probes for tumor targeting and imaging. J. Controlled Release 177, 11−19. (45) Wu, X. J., Tian, Y. P., Yu, M. Z., Han, J. H., and Han, S. F. (2014) A targetable acid-responsive micellar system for signal activation based high performance surgical resolution of tumors. Biomater. Sci. 2, 972−979. (46) Nagai, A., Miller, J. B., Kos, P., Elkassih, S., Xiong, H., and Siegwart, D. J. (2015) Tumor imaging based on photon upconversion of Pt(II) porphyrin rhodamine co-modified NIR excitable cellulose enhanced by aggregation. ACS Biomater. Sci. Eng. 1, 1206−1210. (47) Loudet, A., and Burgess, K. (2007) BODIPY dyes and their derivatives: Syntheses and spectroscopic properties. Chem. Rev. 107, 4891−4932. (48) Kowada, T., Maeda, H., and Kikuchi, K. (2015) BODIPY-based probes for the fluorescence imaging of biomolecules in living cells. Chem. Soc. Rev. 44, 4953−4972. (49) Bura, T., Retailleau, P., Ulrich, G., and Ziessel, R. (2011) Highly substituted bodipy dyes with spectroscopic features sensitive to the environment. J. Org. Chem. 76, 1109−1117. (50) Shie, J. J., Liu, Y. C., Lee, Y. M., Lim, C., Fang, J. M., and Wong, C. H. (2014) An azido-BODIPY probe for glycosylation: Initiation of
strong fluorescence upon triazole formation. J. Am. Chem. Soc. 136, 9953−9961. (51) Gatenby, R. A., and Gillies, R. J. (2004) Why do cancers have high aerobic glycolysis? Nat. Rev. Cancer 4, 891−899. (52) Tannock, I. F., and Rotin, D. (1989) Acid pH in tumors and its potential for therapeutic exploitation. Cancer Res. 49, 4373−4384. (53) Ahmed, F., Pakunlu, R. I., Srinivas, G., Brannan, A., Bates, F., Klein, M. L., Minko, T., and Discher, D. E. (2006) Shrinkage of a rapidly growing tumor by drug-loaded polymersomes: pH-triggered release through copolymer degradation. Mol. Pharmaceutics 3, 340− 350. (54) Ling, D., Park, W., Park, S. J., Lu, Y., Kim, K. S., Hackett, M. J., Kim, B. H., Yim, H., Jeon, Y. S., Na, K., et al. (2014) Multifunctional tumor pH-sensitive self-assembled nanoparticles for bimodal imaging and treatment of resistant heterogeneous tumors. J. Am. Chem. Soc. 136, 5647−5655. (55) Li, C., Xia, J. A., Wei, X. B., Yan, H. H., Si, Z., and Ju, S. H. (2010) pH-activated near-infrared fluorescence nanoprobe imaging tumors by sensing the acidic microenvironment. Adv. Funct. Mater. 20, 2222−2230. (56) Wang, L., Zhu, X., Xie, C., Ding, N., Weng, X. F., Lu, W. Y., Wei, X. B., and Li, C. (2012) Imaging acidosis in tumors using a pHactivated near-infrared fluorescence probe. Chem. Commun. 48, 11677−11679. (57) Weerakkody, D., Moshnikova, A., Thakur, M. S., Moshnikova, V., Daniels, J., Engelman, D. M., Andreev, O. A., and Reshetnyak, Y. K. (2013) Family of pH (low) insertion peptides for tumor targeting. Proc. Natl. Acad. Sci. U. S. A. 110, 5834−5839. (58) Tung, C. H., Qi, J., Hu, L., Han, M. S., and Kim, Y. (2015) A quick responsive fluorogenic pH probe for ovarian tumor imaging. Theranostics 5, 1166−1174. (59) Weissleder, R., Tung, C. H., Mahmood, U., and Bogdanov, A., Jr. (1999) In vivo imaging of tumors with protease-activated nearinfrared fluorescent probes. Nat. Biotechnol. 17, 375−378. (60) Zhu, L., Xie, J., Swierczewska, M., Zhang, F., Quan, Q., Ma, Y., Fang, X., Kim, K., Lee, S., and Chen, X. (2011) Real-time video imaging of protease expression in vivo. Theranostics 1, 18−27. (61) Whitehurst, A. W., Bodemann, B. O., Cardenas, J., Ferguson, D., Girard, L., Peyton, M., Minna, J. D., Michnoff, C., Hao, W., Roth, M. G., et al. (2007) Synthetic lethal screen identification of chemosensitizer loci in cancer cells. Nature 446, 815−819. (62) Nagai, A., Miller, J. B., Du, J., Kos, P., Stefan, M. C., and Siegwart, D. J. (2015) Biocompatible organic charge transfer complex nanoparticles based on a semi-crystalline cellulose template. Chem. Commun. 51, 11868−11871. (63) Hao, J., Kos, P., Zhou, K. J., Miller, J. B., Xue, L., Yan, Y. F., Xiong, H., Elkassih, S., and Siegwart, D. J. (2015) Rapid synthesis of a lipocationic polyester library via ring-opening polymerization of functional valerolactones for efficacious siRNA delivery. J. Am. Chem. Soc. 137, 9206−9209. (64) Zhou, K., Nguyen, L. H., Miller, J. B., Yan, Y., Kos, P., Xiong, H., Li, L., Hao, J., Minnig, J. T., Zhu, H., et al. (2016) Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model. Proc. Natl. Acad. Sci. U. S. A. 113, 520−525. (65) Venditto, V. J., and Szoka, F. C. (2013) Cancer nanomedicines: So many papers and so few drugs! Adv. Drug Delivery Rev. 65, 80−88. (66) Bertrand, N., and Leroux, J. C. (2012) The journey of a drugcarrier in the body: An anatomo-physiological perspective. J. Controlled Release 161, 152−163.
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DOI: 10.1021/acs.bioconjchem.6b00242 Bioconjugate Chem. 2016, 27, 1737−1744
