High‐Contrast Fluorescence Detection of Metastatic Breast ... · 2017 WILEY-VCH Verlag GmbH & Co....

CommuniCation

1700131 (1 of 10) © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advmat.de

High-Contrast Fluorescence Detection of Metastatic Breast Cancer Including Bone and Liver Micrometastases via Size-Controlled pH-Activatable Water-Soluble Probes

Hu Xiong, Hao Zuo, Yunfeng Yan, Gino Occhialini, Kejin Zhou, Yihong Wan, and Daniel J. Siegwart*

Dr. H. Xiong, Dr. Y. Yan, G. Occhialini, Dr. K. Zhou, Prof. D. J. SiegwartSimmons Comprehensive Cancer CenterDepartment of BiochemistryUniversity of Texas Southwestern Medical CenterDallas, TX 75390, USAE-mail: [email protected]. H. Zuo, Prof. Y. WanDepartment of PharmacologySimmons Comprehensive Cancer CenterUniversity of Texas Southwestern Medical CenterDallas, TX 75390, USA

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adma.201700131.

DOI: 10.1002/adma.201700131

from the primary tumor and colonize in distant sites. Tracking the spread of cancer is a major challenge, particularly when it disseminates to regions such as bones and liver where there is frequently high background contrast that can prevent detection by existing imaging modalities.[2] In the United States (U.S.), ≈11% of U.S. women will develop invasive breast cancer over the course of their lifetime, and about 40 000 women die of breast cancer every year.[1b] The unique clinical challenges for detecting metastases include their small size, low vascularization, high multiplicity, dispersed distribution nature, and limited sensitivity and spatial resolution of current clinical imaging technologies.[2] Despite successful development of various molecular imaging probes that can visualize breast cancer, fluorescent probes that can detect the various different micrometastases of breast cancer, including migration of cancer cells to bone and liver, have not been realized.[3] There remains a pressing demand to develop highly sensitive imaging techniques to precisely detect cancer metastasis. Such detection would offer more timely treatment options, and

greatly increase survival rates of breast cancer patients.Nanomaterials can effectively target tumors by the enhanced

permeability and retention (EPR) effect,[4] which suggests potential for metastasis imaging.[5] However, metastatic tumors have poor vasculature and angiogenic dormancy when the diameter of micrometastatic lesions is less than 1–2 mm.[6] Unfortunately, the EPR effect is limited to vascularized tumors larger than ≈4.6 mm in diameter, which prevents the use of nanomaterials for imaging small or unvascularized metastases.[2,7] Accordingly, there remains a need to design watersoluble materials for imaging that do not rely on the EPR effect for detecting breast cancer metastasis. Dysregulated pH has been recognized as a universal hallmark of the tumor microenvironment that can promote dormant metastasis by increasing the migration of cancer cells.[8] pHsensitive probes can exploit this low pH to enhance metastases imaging while minimizing background fluorescence. A number of exciting pHsensitive probes have been developed for imaging tumors.[9] However, true

Breast cancer metastasis is the major cause of cancer death in women worldwide. Early detection would save many lives, but current fluorescence imaging probes are limited in their detection ability, particularly of bone and liver micrometastases. Herein, probes that are capable of imaging tiny (<1 mm) micrometastases in the liver, lung, pancreas, kidneys, and bone, that have disseminated from the primary site, are reported. The influence of the poly(ethylene glycol) (PEG) chain length on the performance of water-soluble, pH-responsive, near-infrared 4,4′-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) probes is systematically investigated to demonstrate that PEG tuning can provide control over micrometastasis tracking with high tumor-to-background contrast (up to 12/1). Optimized probes can effectively visualize tumor boundaries and successfully detect micrometastases with diameters <1 mm. The bone-metastasis-targeting ability of these probes is further enhanced by covalent functionalization with bisphosphonate. This improved detection of both bone and liver micrometastases (<2 mm) with excellent tumor-to-normal contrast (5.2/1). A versatile method is thus introduced to directly synthesize modular water-soluble probes with broad potential utility. Through a single intravenous injection, these materials can image micro-metastases in multiple organs with spatiotemporal resolution. They thus hold promise for metastasis diagnosis, image-guided surgery, and theranostic PEGylated drug therapies.

Cancer Detection

Breast cancer is the most frequently diagnosed cancer in women worldwide.[1] Metastatic disease is the main underlying clinical cause of death, occurring when cancer cells migrate

Adv. Mater. 2017, 1700131

© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim1700131 (2 of 10)

www.advmat.dewww.advancedsciencenews.com

delineation of deep micrometastases (especially in the bone and liver) requires a unique combination of deep tissue penetrating nearinfrared (NIR) emission (>650 nm),[10] activation at low pH to enhance contrast, and a proper molecular weight balance to avoid the fast clearance issue of small molecules and the high dependence on the EPR effect of nanoparticles.

We previously developed watersoluble PEGylated NIR 4,4′difluoro4bora3a,4adiazasindacene (BODIPY) probes that activate (“turn on”) in tumors by responding to low pH and provide high tumortoliver (T/L) contrast.[11] The NIR probes are totally dark at physiological pH and only turn on in acidic microenvironment with strong fluorescence emission by harnessing the photoinduced electron transfer (PeT) effect. In our prior work, we showed that diethylaminefunctionalized probe 5c was capable of imaging various xenograft tumors with high contrast. However, solid tumors are not what kill breast cancer patients. Rather, metastasis is the major cause of death and clinical problem. To detect and image breast cancer metastasis, including bone and liver micrometastases, further development of this class of probes was required. As a core part of this challenge, the relationship between physicochemical parameters of PEGylated probes and their cellular interactions remains largely unexplored, further hindering the design of effective watersoluble probes that possess controllable and consistent metastatic cancer interactions.

Herein, we systematically investigated the effect of poly(ethylene glycol) (PEG) length on pHresponsive NIR BODIPY probes for tumor imaging, and found that PEG length dramatically affects metastasis detection and presents an opportunity to finetune detection of small tumors. Our experimental results first demonstrate that a high level of control over the micrometastasis tracking can be achieved through modulation of the PEG length (Figure 1, top). PEGylated NIR BODIPY probes with a PEG length between 1000 and 5000 g mol−1 could effectively image not only primary tumors but also universally detect breast cancer metastases (including liver, lung, pancreas, and kidney metastases of breast cancer) without having to attach tumortargeting groups. Interestingly, probes with

PEG molecular weight (MW) less than 1000 or greater than 5000 were not effective, suggesting a design guide for future PEGylated probes and drug conjugates for passive tumor targeting. Optimized PEG2K5c could effectively visualize the boundary of breast cancer metastasis and successfully detect micrometastasis with diameter less than 1 mm. Notably, covalent functionalization with bisphosphonate (PEG3.5K5c-PO3H2) could further enhance bone metastasis imaging after i.v. administration and provide high signaltonoise ratio (Figure 1, bottom). Moreover, PEG3.5K5cPO3H2 localized to a micrometastasis (<2 mm) in the liver with excellent tumortonormal contrast. To the best of our knowledge, PEG3.5K5cPO3H2 is the first pHactivatable NIR fluorescent probe to successfully visualize bone and liver metastases of breast cancer in vivo. We believe that this report introduces a versatile method to directly synthesize modular watersoluble probes with broad potential utility including imageguided surgery, metastasis diagnosis, and theranostic PEGylated drug therapies.

The molecular weight of PEG drug conjugates is known to affect both pharmacokinetics and ultimate efficacy in tumors,[12] but has not been systematically examined for tumor imaging probes. To better understand the effects of PEG length on NIR BODIPY probes for metastasis imaging, we synthesized a series of PEGylated 5cbased probes (Scheme S1, Supporting Information) by expanding our previously reported protocols.[11] Building from the NIR core 4c, click chemistry was employed to attach two PEG chains, resulting in five new probes: PEG4005c, PEG1K5c, PEG2K5c, PEG5K5c, and PEG10K5c. Interestingly, the five PEGylated probes exhibited similar absorbance and emission spectrum in acidic buffer (Figure S1, Supporting Information). To further investigate the solubility of all PEGylated 5c probes in water, we examined the concentration dependent properties. Owing to the linear increase in fluorescence intensity as a function of concentration from 0 to 5 × 10−6 m for PEG4005c, to 10 × 10−6 m for PEG1K5c and PEG2K5c, and to 20 × 10−6 m for PEG5K5c and PEG10K5c, all the probes exhibited excellent water solubility at relevant concentrations for in vitro and in vivo imaging (Figure 2a).

Adv. Mater. 2017, 1700131

Figure 1. Schematic illustration of PEGylated NIR BODIPYs optimized for effectively tracking of different breast cancer metastases to distant sites. Optimization of the probe design indicated that the PEG length defined tumor tropism and that covalent functionalization with bisphosphonate enhanced bone metastasis imaging.



Next, we examined the pharmacokinetics of these PEGylated 5c probes, because a long circulation halflife is essential for tumor accumulation of the probes. Five 5cbased probes with different PEG lengths were intravenously (i.v.) injected into separate healthy mice at the same dose of 2.0 nmol (10 × 10−6 m, 200 µL), and blood was withdrawn at indicated time points for the measurement of NIR fluorescence intensity. The blood circulation halflives were 30 min, 2.5 h, 3.0 h, 9.0 h, and 16.0 h for PEG400, PEG1K, PEG2K, PEG5K, and PEG10K5c, respectively, which indicated the PEGylated 5c probes with higher MW were cleared more slowly than probes with lower PEG length (Figure 2b). Furthermore, the absence of any weight loss indicated no appreciable toxicity following multiple i.v. injections of 2 nmol PEGylated 5c (Figure 2c).

To assess the capabilities of PEGylated 5c to preferentially accumulate and be retained in the breast tumors in vivo, mice bearing subcutaneous MDAMB231 breast cancer xenografts were injected i.v. separately with each of the five pHactivatable PEGylated 5c at the same dose of 2.0 nmol (10 × 10−6 m, 200 µL) and timedependent wholebody NIR fluorescence images were collected (Figure 2d). Strong and sustained NIR emission (Cy5.5 filter was used) was observed for PEG1K, PEG2K, and PEG5K5c. Importantly, for PEG1K, PEG2K, and PEG5K5c, the tumor tissues could be clearly distinguished from surrounding normal tissues 2 h after tail vein injection and still remained considerably bright at 24 h postinjection with negligible background fluorescence (Figure 2d). In contrast, PEG4005c (fastest cellular uptake; see the the text and Figure S2–S4 in

Adv. Mater. 2017, 1700131

Figure 2. Systematic evaluation of PEG length effects on NIR BODIPY probes in breast cancer xenograft tumors. a) Relationship between concentration and dependent fluorescence intensity of PEGylated 5c in pH 2.0 buffer. b) Pharmacokinetic profile of PEGylated 5c. Plasma concentrations of PEGylated 5c were normalized to relative fluorescence signal after i.v. injection of 2.0 nmol (10 × 10−6 m, 200 µL) of each probe into the tail vein (mean ± s.d., n = 3). c) Healthy BL6 mice (day 30) were injected i.v. with each PEGylated 5c (2.0 nmol) to evaluate the toxicity in vivo (mean ± s.d., n = 3). Time course of mice weight were measured. The arrows indicate the i.v. administration of each PEGylated 5c at days 30, 32, and 34 postbirth. d) Time-dependent in vivo fluorescence images of mice bearing subcutaneous MDA-MB-231 breast cancer xenograft tumors after i.v. injection of 2.0 nmol (10 × 10−6 m, 200 µL) PEGylated 5c. Representative fluorescence images of harvested tumors and organs from MDA-MB-231 tumor-bearing mice sacrificed at 24 h postinjection. e) In vivo time-dependent NIR fluorescence intensities on the subcutaneous MDA-MB-231 tumors were measured by ROI analysis (mean ± s.d., n = 3). f) T/L (tumor/liver) ratios of relative PEGylated 5c fluorescence intensity at 24 h postinjection. T/L ratio was normalized by ROI analysis (mean ± s.d., n = 3).



the Supporting Information) could not effectively accumulate in the tumor and was visible in the liver and kidneys due to rapid renal clearance (Figure 2d). Thus, the typical fast clearance drawback of small molecular probes could be avoided by increasing the PEG length of the probes. However, PEG10K5c with longer PEG length exhibited low tumortobackground contrast and displayed normal tissue interference (Figure 2d). Interestingly, if we normalized timedependent NIR fluorescence intensities on the subcutaneous MDAMB231 tumors, maximal NIR fluorescence intensities were observed at 4, 6, 6, 10, and 24 h postinjection for PEG400, PEG1K, PEG2K, PEG5K, and PEG10K5c, respectively, which correlated well with cellular interactions (Figure 2e). These results indicate that PEGylated 5c probes with PEG length between 1000 and 5000 g mol−1 possess a high ability to accumulate and activate in tumors, demonstrating that the in vitro and in vivo PEG length effects (cellular uptake and tumor clearance) should be balanced to design effective primary and metastatic tumortargeting watersoluble probes.

To further examine biodistribution, harvested tumors and organs were analyzed 24 h postinjection. PEG1K, PEG2K, and PEG5K5c exhibited remarkable NIR emission in the tumors and low emission in all other organs (Figure 2d), which validated that PEGylated 5c with PEG length between 1000 and 5000 g mol−1 could effectively accumulate and be retained in the tumors where they were activated to “turnon” state. In contrast, PEG4005c had no detectable NIR emission in the tumor and only had (low) NIR emission in the liver and kidneys, which verified that it was rapidly cleared. In addition, PEG10K5c had low tumor accumulation even though it circulated the longest in the blood stream (Figure 2d). Next, we normalized T/L ratios of PEGylated 5c fluorescence intensities at

24 h postinjection. Significantly, both PEG1K5c and PEG2K5c provided high apparent T/L ratio of ≈3, probably due to comparable cellular interactions and pharmacokinetics (Figure 2f). PEG5K5c showed a slightly lower T/L ratio of ≈1.7. Nevertheless, the apparent T/L ratios of PEG4005c and PEG10K5c were less than 1 (Figure 2f). These results indicate that PEGylated 5c with PEG length between 1000 and 5000 g mol−1 can balance size and pH activation to overcome the issues of small molecule probes (fast clearance) and nanoparticlebased probes (high liver background emission).

To further investigate the efficacy of PEGylated 5c with PEG length between 1000 and 5000 g mol−1 for cancer detection, we examined imaging capabilities of PEGylated 5c in an orthotopic lung tumor because lung is a common site of the metastatic spread of breast cancer.[1] Luciferaseexpressing cells (A549Luc) were employed to be able to confirm localization of the probe to the tumor via overlap of luciferase and fluorescence. Impressively, ex vivo fluorescence images of harvested tumors and organs from orthotopic A549 lung tumorbearing mice demonstrate that PEG1K, PEG2K, and PEG5K5c can not only effectively detect orthotopic lung tumors but also provide high T/L contrast (Figure 3a,c). As was observed in the xenograft tumor study, PEG4005c and PEG10K5c exhibited weak NIR emission in orthotopic lung tumors and low T/L contrast (Figure 3a,c). In the control group, PEG2K5c could not efficiently accumulate and activate in the normal lung (Figure 3a,b), implying that activation of requires the presence of cancer. Moreover, pHresponsive PEGylated 5c with appropriate PEG length can specifically target and turn on in orthotopic lung tumors. Haemotoxylin and eosin (H&E) staining and confocal images of tissue cryosections confirmed that PEG1K, PEG2K, and PEG5K5c were able to efficiently accumulate and light up in orthotopic lung tumors

Adv. Mater. 2017, 1700131

Figure 3. Evaluation of PEG length effect of PEGylated 5c in orthotopic lung tumor model, a common site of breast cancer metastasis. a) Representative ex vivo fluorescence images of harvested tumors and organs from orthotopic A549-Luc lung tumor-bearing mice or normal NOD-SCID mice sacrificed at 24 h postinjection. Orthotopic A549 lung tumor-bearing mice were injected i.v. separately with the series of five pH-activatable PEGylated 5c at the same dose of 2.0 nmol (10 × 10−6 m, 200 µL) and, 24 h later, organs were collected for NIR imaging. In the control group, 2 nmol of PEG2K-5c was injected i.v. into normal NOD-SCID mice. b) Comparison of orthotopic lung tumor and normal lung after i.v. injection of 2.0 nmol PEG2K-5c. i) White light, ii) bioluminescence (signal from luciferase-expressing A549 tumor), iii) fluorescence images of orthotopic lung tumor and normal lung (coming from PEG2K-5c). c) Relative T/L (lung tumor/liver) ratios of PEGylated 5c fluorescence intensity at 24 h postinjection. In the control group, normal lung-to-liver ratio was used. T/L ratio was normalized by ROI analysis (mean ± s.d., n = 3). Statistical significance was determined using a two-tailed Student’s t test (***, P ≤ 0.001). d) H&E images of orthotopic lung tumor and normal lung tissue cryosections. Scale bar = 50 µm.



(Figure 3d and Figure S5 (Supporting Information)). Through these studies in xenograft and orthotopic tumors, PEGylated 5c probes with PEG length between 1000 and 5000 g mol−1 were determined to hold the most potential for detecting breast cancer metastasis based on quantitative evaluation of water solubility, pharmacokinetics, cellular interactions, tumor accumulation, and biodistribution.

Having optimized PEG length, we moved on to a challenging model of breast cancer metastasis derived from polyoma middle T antigen (PyMT) oncogene overexpression driven by the mouse mammary tumor virus (MMTV) promoter.[13] Py8119 metastasisprone mammary tumor cells derived from the C57BL/6 backgroundcompatible MMTVPyMT mice were employed. Upon intracardiac injection, Py8119 cells accurately capture metastatic breast cancer diseases by forming metastasis in multiple organs including bone and liver, which can be confirmed by the engineered luciferase and green fluorescent protein (GFP) reporters in the Py8119LucGFP cells.[14] Nanomaterials can passively accumulate into tumor site due to EPR effect,[4] but they distribute only in the perivascular space of tumor area,[9h] hindering the use of nanomaterials for imaging small or unvascularized micrometastases (0.2–2 mm in size).[2] In contrast, watersoluble materials with optimized molecular weight to favor biodistribution to tumors do not completely rely on the EPR effect to localize to tumors. Thus, we envisioned that PEGylated 5c with PEG length between 1000 and 5000 g mol−1 may effectively detect micrometastases of breast cancer. To evaluate the feasibility of imaging metastasis with pHactivatable PEGylated 5c, we selected PEG2K5c as a representative imaging beacon in the middle of the ideal molecular weight range for tracking breast cancer metastasis. We injected Py8119 cells intracardially, which formed metastatic tumors in distant organs (Figure 4a).[15] Mice bearing pancreatic and tibia metastases of breast cancer were injected i.v. with pHactivatable PEG2K5c at a dose of 0.5 mg kg−1 and organs were harvested for bioluminescence and fluorescence imaging at 24 h postinjection. Pancreatic metastases displayed considerably bright NIR emission that correlated well with bioluminescence signals and high tumortospleen (T/S) contrast up to 12/1 (Figure 4b,c), indicating that PEG2K5c can effectively detect pancreatic metastasis of breast cancer without having to attach tumortargeting ligands. Because liver is a common site of metastatic dissemination of breast cancer,[1] we use T/S ratio instead of T/L ratio to evaluate fluorescence intensity of PEG2K5c. To confirm that the generation of pancreatic and tibia metastases results from Py8119 cell line, tissues of interest were homogenized and analyzed for the cancer specific PyMT gene of Py8119 cells by qPCR. High tumor burdens were quantified in the pancreas and tibia (Figure 4d), demonstrating that PEG2K5c is able to detect breast cancer metastasis. H&E staining further confirmed that pancreases have metastases (Figure 4e). Moreover, the same slides of metastasis tissues were imaged using confocal imaging and captured in three channels including green for GFP, far red for PEG2K5c, and blue for cell nuclei, which verified that PEG2K5c efficiently accumulated and activated in the pancreatic metastases (Figure 4e). More importantly, PEG2K5c that respond to acidic tumor microenvironment can also differentiate healthy cells from cancer cells within the metastasis tissue (Figure 4eii), supporting that areas of

low pH often observed at tumor boundaries correspond to the high proteolytic activity.[16] Because small, low volume tumors are acidic,[17] the previously demonstrated PeT mechanism of action is likely responsible for tumor activation.[11] In addition, although boneimaging is challenging, PEG2K5c exhibited high NIR emission in some tibia metastases of breast cancer (Figure 4b) and clearly colocalized with GFP signals (Figure 4eiii). Moreover, a tibia micrometastasis less than 1 mm was also visualized by PEG2K5c (Figure S6, Supporting Information). In contrast, PEG2K5c did not exhibit any NIR fluorescence in the normal pancreas and tibia (Figure 4biii).

To further investigate the potential universality of PEG2K5c for metastasis imaging, we evaluated PEG2K5c to detect other sites of breast cancer metastasis including liver, lung, and kidney. Liver is a common organ for metastasis of breast cancer.[1] However, imaging liver metastasis is challenging due to the fact that nanoparticles readily accumulate in the liver because of the discontinuous features of hepatic vasculature,[18] typical 80–100 nm diameters,[19] adsorption of apolipoproteins,[20] and are cleared by the Kupffer cells in the liver, which creates a high background signal and potential false positive diagnoses. Meanwhile, the reticuloendothelial system (liver and spleen) is responsive for the uptake of most of the nanomaterials due to EPR effect.[21] Therefore, liver metastasis imaging via the EPR effect is largely compromised. The accurate detection of relatively large liver metastatic tumors (>1 cm in size) has been significantly improved by the development of computed tomography (CT), fluorodeoxyglucose–positron emission tomography (FDGPET), and magnetic resonance imaging (MRI).[22] Nevertheless, small liver metastases can rarely be detected by current imaging technologies. Although it was recently reported that a small liver metastatic tumor (≈1 mm) could be visualized by MRI using polymeric micelles, the T/N contrast ratio was only 1.7, hindering the application for precisely detecting other micrometastases.[23] Herein, we show that PEG2K5c holds promise for tracking liver metastasis of breast cancer due to its water solubility and pHresponsive property that can suppress normal liver uptake and activation of the probes. We next applied PEG2K5c to liver metastasisbearing mice at a dose of 0.5 mg kg−1 and organs were harvested for bioluminescence and fluorescence imaging at 24 h postinjection. Liver metastasis was clearly visualized (Figure 5ai) and displayed high T/S contrast up to 9/1 (Figure 5b). H&E staining confirmed that the livers had metastatic tumors (Figure 5di and Figure S7a (Supporting Information)). Furthermore, confocal images of cryosections verified that PEG2K5c efficiently activated in the liver metastases (Figure 5di and Figure S7a and S8 (Supporting Information)) due to low pH in tumor microenvironment and not in the normal liver (Figure S7b, Supporting Information). In addition, a micrometastasis (1.0 mm × 0.75 mm by histology) in the liver was also visualized by PEG2K5c, which was further confirmed by histological and confocal imaging analysis (Figure S9, Supporting Information). Lung is also a common site for metastatic spread of breast cancer.[1] Similarly, PEG2K5c exhibited strong NIR emission in lung metastases (Figure 5aii) and Figure S10a (Supporting Information)) and high T/S contrast up to 7.4/1 (Figure 5b). H&E and confocal images confirmed that PEG2K5c could efficiently accumulate and activate in the

Adv. Mater. 2017, 1700131



Adv. Mater. 2017, 1700131

Figure 4. pH-responsive, water-soluble PEG2K-5c can detect pancreatic and bone metastases of breast cancer without attachment of targeting ligands. a) Illustration mouse model for breast cancer metastasis to distant sites. b) Representative ex vivo bioluminescence and fluorescence images of harvested organs from Py8119 breast cancer metastasis-bearing mice (i,ii) and normal BL6 mice (iii) sacrificed at 24 h postinjection. Mice were intravenously administered with pH-activatable PEG2K-5c at a dose of 0.5 mg kg−1 (10 × 10−6 m, 200 µL) and, 24 h later, organs were collected for bioluminescence and NIR imaging. c) T/S (tumor/spleen) ratios of PEG2K-5c fluorescence intensity at 24 h postinjection. T/S ratio was normalized by ROI analysis (mean ± s.d., n = 5). Statistical significance was determined using a two-tailed Student’s t test (***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05). d) Tumor burden of relative metastasis analyzed by qPCR (****, P ≤ 0.0001). e) Histology and fluorescence microscopy analysis of PEG2K-5c accu-mulation and activation in pancreatic and tibia metastases (10× magnification). PEG2K-5c, GFP, and cell nuclei are shown in red, green, and blue colors, respectively. Yellow is the merged color of green and red signals. White is the merged color of green, red, and blue signals. Scale bar = 100 µm.



lung metastasis (Figure 5dii and Figure S8 (Supporting Information)). Moreover, several lung micrometastases were also detected by PEG2K5c (Figure S10, Supporting Information), which was further verified by histological and confocal imaging analysis (Figure S11, Supporting Information). Remarkably, a kidney micrometastasis (1.0 mm × 0.65 mm) that lost bioluminescence signal was also illuminated by PEG2K5c (Figure 5aiii) and displayed a 5.2/1 of tumortospleen contrast (Figure 5b). The kidney metastasis was confirmed by H&E staining (Figure 5diii). To further confirm that the kidney had metastasis of breast cancer, tissues of kidney were analyzed for presence of the tumor specific PyMT gene of Py8119 cells by qPCR and indeed the tumor burden was detected in the suspected kidney of interest (Figure 5c). Cumulatively, these results suggest that PEGylated 5c with PEG length between 1000 and 5000 g mol−1 may provide a robust platform for detecting different metastases of breast cancer in vivo without having to attach tumortargeting ligands.

Bone is the most common site of distant metastases from breast cancer.[1] Metastases to the bone are observed in up to 70% of all cancer patients and the growth of metastases causes the major mortality.[24] Despite the development of various molecular imaging probes that can visualize bone cancer, fluorescent probes that can consistently detect bone metastases with high signaltonoise ratio are elusive.[25] A few fluorescent imaging probes with bonetargeting groups have been reported to illuminate bone tissues,[26] but none of these “alwayson” probes were applied for in vivo bone metastasis imaging. In

addition, Kikuchi and coworkers reported pHresponsive fluorescent probes that imaged normal boneresorbing osteoclasts after subcutaneous administration.[27] However, owing to the limited tissue penetration ability (green emission) and fast clearance for i.v. administration, the application of these probes in bone metastasis imaging in vivo is not yet possible. To the best of our knowledge, there have been no reports using pHactivatable NIR probes on the detection of bone metastasis of breast cancer. Therefore, there remains a great demand for developing novel pHactivatable NIR probes to precisely detect bone metastasis and improve treatment outcomes. As shown above, the PEGylated 5c with PEG length between 1000 and 5000 g mol−1 showed great potential for imaging bone metastasis of breast cancer in vivo. To further enhance bone metastasis imaging of PEGylated 5c, we decided to combine PEGylated 5c with carboxylate or bisphosphonate groups due to their high affinity to hydroxyapatite (HA) of bone matrix.[28] These functional groups can bind to Ca2+ in bone. With this idea in mind, we first hydrolyzed a commercially available bifunctionalized PEG (N3PEG78NHS) into N3PEG78CO2H. We chose the MW of 3500 because it was in the middle of the optimal 1000–5000 range. Then, PEG3.5K5cCO2H was synthesized via click chemistry (Scheme S2, Supporting Information). By engineering bisphosphonate groups into PEG3.5K5cCO2H, we obtained PEG3.5K5cPO3H2 in high yield (Scheme S2 (Supporting Information) and Figure 6a). To investigate their potential for binding to HA, we directly mixed the suspension of HA (1 mg mL−1) with 10 × 10−6 m solutions of PEG2K5c,

Adv. Mater. 2017, 1700131

Figure 5. pH-responsive, water-soluble PEG2K-5c can clearly illuminate liver, lung, and kidney metastases of breast cancer with minimal background signal. a) Representative ex vivo bioluminescence and fluorescence images of harvested organs from Py8119 breast cancer metastasis-bearing mice sacrificed at 24 h postinjection. Metastasis-bearing mice were injected i.v. with pH-activatable PEG2K-5c at a dose of 0.5 mg kg−1 (10 × 10−6 m, 200 µL) and, 24 h later, organs were collected for bioluminescence and NIR imaging. b) T/S ratios of PEG2K-5c fluorescence intensity at 24 h postinjection. T/S ratio was normalized by ROI analysis (mean ± s.d., n = 4). c) Tumor burden of relative metastasis analyzed by qPCR. d) Histology and fluorescence microscopy analysis of PEG2K-5c accumulation and activation in liver, lung, and kidney metastases (10× magnification). From histological analysis, the metastatic tumor in the kidney had dimensions of 1.0 mm × 0.65 mm. PEG2K-5c, GFP, and cell nucleus are shown in red, green, and blue colors, respectively. Yellow is the merged color of green and red signals. White is the merged color of green, red, and blue signals. Scale bar = 100 µm.



Adv. Mater. 2017, 1700131

PEG3.5K5cCO2H, and PEG3.5K5cPO3H2. After physicochemical adsorption and rinsing after two days, only PEG3.5K5cPO3H2 displayed excellent binding to HA (Figure 6b and Figure S12 (Supporting Information)). The binding to HA ratios of PEG2K5c, PEG3.5K5cCO2H, and PEG3.5K5cPO3H2 were 0, 0.19, and 0.81, respectively (Figure 6c). Furthermore, the probes were still pHresponsive after binding to HA (Figure 6b). To further confirm the pHactivatable property of PEG3.5K5cPO3H2 after binding to HA, confocal microscopy was performed. Intense NIR fluorescence was only observed at acidic pH and not at physiological pH (Figure S13, Supporting Information). These results indicated that PEG3.5K5cPO3H2 is pHsensitive not only in aqueous solution but also in the solid state after binding to HA. Moreover, PEG3.5K5cPO3H2 exhibits excellent water solubility at low concentration (low liver uptake and retention) and good pKa (4.85) that is suitable for detecting the bone resorptive microenvironment with pH ≈ 4.5 (Figure S14, Supporting Information).[29] Based on these results, PEG3.5K5cPO3H2 was selected as a promising probe for enhancing in vivo bone metastasis imaging. To examine if PEG3.5K5cPO3H2 could enhance bone metastasis imaging in vivo, bone metastasisbearing mice were injected i.v. with pHactivatable PEG3.5K5cPO3H2 at a dose of 0.77 mg kg−1 and organs were collected for bioluminescence and NIR imaging. Because of the advantage of bisphosphonate binding to bone matrix, PEG3.5K5cPO3H2 clearly illuminated tibia metastases of breast cancer (Figure 6d and Figure S15 (Supporting

Information)) and exhibited a higher T/S ratio up to 5.2/1 in contrast with the T/S ratio (2.7/1) of the parent PEG2K5c probe (Figure 6e). Moreover, a micrometastasis (<2 mm) in the liver was simultaneously detected by PEG3.5K5cPO3H2 with excellent tumortonormal contrast, increasing 350% over background (Figure 6d and Figure S16 and S17 (Supporting Information)). Therefore, we believe that PEG3.5K5cPO3H2 will provide a powerful tool for detecting many different micrometastases of breast cancer in vivo, including bone and liver.

In summary, we have systematically investigated PEG length effects on the performance of the watersoluble NIR BODIPY probes and demonstrated a high level of controllability over the tracking of breast cancer metastases through the modulation of the PEG length and the functional group on the tail of PEG. Our data indicates that highcontrast tumor imaging is controlled by fast accumulation into the tumors coupled to slow clearance of the probes from the tumor. The pHresponsive PEGylated 5c with PEG length between 1000 and 5000 g mol−1 could localize to different metastases of breast cancer in vivo and be activated by acidic pH to produce high tumortonormal signal contrast in mice bearing pancreas, liver, lung, and kidney metastases after i.v. injection. Notably, by engineering bisphosphonate groups into PEGylated probes, PEG3.5K5cPO3H2 successfully enhanced bone metastasis imaging and simultaneously illuminated micrometastasis in the liver in vivo with high signaltonoise ratio. Therefore, these pHactivatable NIR probes may provide a robust platform for cancer metastasis imaging in a

Figure 6. Covalent attachment of bisphosphonate groups onto pH-activatable, water-soluble 5c (PEG3.5K-5c-PO3H2) can further enhance bone meta-stasis imaging. a) Illustration the structures of PEG3.5K-5c-CO2H and PEG3.5K-5c-PO3H2. b) PEG3.5K-5c-PO3H2 can specifically bind to hydroxyapatite and remain pH-responsive. i) The suspensions of hydroxyapatite with PEG2K-5c (α), PEG3.5K-5c-CO2H (β), and PEG3.5K-5c-PO3H2 (γ) after physico-chemical adsorption. ii,iiii) Fluorescent images of corresponding diluted suspension in pH 7.4 buffer (ii) and pH 5.0 buffer (iii). c) Quantitative analysis of the probes binding to hydroxyapatite. Statistical significance was determined using a two-tailed Student’s t test (***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05). d) Representative ex vivo bioluminescence and fluorescence images of harvested organs from Py8119 breast cancer metastasis-bearing mice sacrificed at 24 h postinjection. Bone metastasis-bearing mice were injected i.v. with pH-activatable PEG3.5K-5c-PO3H2 at a dose of 0.77 mg kg−1 (10 × 10−6 m, 200 µL) and, 24 h later, organs were collected for bioluminescence and NIR imaging. A white light image of a micrometastasis (<2 mm) in the liver clearly showed the position of the tumor, which matched the luminescence and fluorescence images. e) PEG3.5K-5c-PO3H2 exhibited enhanced tumor-to-spleen contrast. T/S ratios of PEG3.5K-5c-PO3H2 and PEG2K-5c fluorescence intensity at 24 h postinjection. T/S ratio was normalized by ROI analysis (mean ± s.d., n = 4; **, P ≤ 0.01).



Adv. Mater. 2017, 1700131

variety of modalities and applications. The modular nature suggests potential to include other targeting groups and use other nonfouling polymers.[30] Finally, we envision future utility in theranostic PEGylated drugs delivery and imaging materials due to the modular design capability.

Supporting InformationSupporting Information is available from the Wiley Online Library or from the author.

AcknowledgmentsThe authors thank Dr. Jean Jiang (University of Texas Health Science Center at San Antonio) and Dr. Lesley G. Ellies (University of California at San Diego) for providing Py8119 cell line. This work was supported in part by the CPRIT (R1212, D.J.S.; RP130145, Y.W.), the Mary Kay Foundation (049-15, D.J.S.; 073.14, Y.W.), and the Welch Foundation (I-1855 and 1-1855-20170325, D.J.S.; I-1751, Y.W.). All animal experiments were approved by the Institutional Animal Care and Use Committees of The University of Texas Southwestern Medical Center and were consistent with local, state and federal regulations as applicable.

Conflict of InterestThe authors declare no conflict of interest.

Keywordsfluorescence, imaging, metastasis, pH, water-soluble probes

Received: January 6, 2017Revised: April 19, 2017

Published online:

[1] a) B. Weigelt, J. L. Peterse, L. J. Van’t Veer, Nat. Rev. Cancer 2005, 5, 591; b) R. L. Siegel, K. D. Miller, A. Jemal, Ca-Cancer J. Clin. 2016, 66, 7.

[2] A. Schroeder, D. A. Heller, M. M. Winslow, J. E. Dahlman, G. W. Pratt, R. Langer, T. Jacks, D. G. Anderson, Nat. Rev. Cancer 2012, 12, 39.

[3] a) R. Weissleder, Science 2006, 312, 1168; b) Q. Meng, Z. Li, Int. J. Biomed. Imaging 2013, 2013, 230487.

[4] a) K. H. Bae, H. J. Chung, T. G. Park, Mol. Cells 2011, 31, 295; b) H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, J. Controlled Release 2000, 65, 271; c) J. Fang, H. Nakamura, H. Maeda, Adv. Drug Delivery Rev. 2011, 63, 136.

[5] a) X. Y. He, X. Y. Bao, H. Q. Cao, Z. W. Zhang, Q. Yin, W. W. Gu, L. L. Chen, H. J. Yu, Y. P. Li, Adv. Funct. Mater. 2015, 25, 2831; b) P. M. Peiris, R. Toy, E. Doolittle, J. Pansky, A. Abramowski, M. Tam, P. Vicente, E. Tran, E. Hayden, A. Camann, A. Mayer, B. O. Erokwu, Z. Berman, D. Wilson, H. Baskaran, C. A. Flask, R. A. Keri, E. Karathanasis, ACS Nano 2012, 6, 8783; c) E. Doolittle, P. M. Peiris, G. Doron, A. Goldberg, S. Tucci, S. Rao, S. Shah, M. Sylvestre, P. Govender, O. Turan, Z. Lee, W. R. Schiemann, E. Karathanasis, ACS Nano 2015, 9, 8012; d) P. Rychahou, F. Haque, Y. Shu, Y. Zaytseva, H. L. Weiss, E. Y. Lee, W. Mustain, J. Valentino, P. X. Guo, B. M. Evers, ACS Nano 2015, 9, 1108.

[6] D. F. Quail, J. A. Joyce, Nat. Med. 2013, 19, 1423.[7] J. W. Nichols, Y. H. Bae, J. Controlled Release 2014, 190, 451.[8] a) B. A. Webb, M. Chimenti, M. P. Jacobson, D. L. Barber, Nat. Rev.

Cancer 2011, 11, 671; b) D. Hanahan, R. A. Weinberg, Cell 2011, 144, 646.

[9] a) H. Kobayashi, M. Ogawa, R. Alford, P. L. Choyke, Y. Urano, Chem. Rev. 2010, 110, 2620; b) Z. Q. Guo, S. Park, J. Yoon, I. Shin, Chem. Soc. Rev. 2014, 43, 16; c) Y. Urano, D. Asanuma, Y. Hama, Y. Koyama, T. Barrett, M. Kamiya, T. Nagano, T. Watanabe, A. Hasegawa, P. L. Choyke, H. Kobayashi, Nat. Med. 2009, 15, 104; d) G. M. van Dam, G. Themelis, L. M. A. Crane, N. J. Harlaar, R. G. Pleijhuis, W. Kelder, A. Sarantopoulos, J. S. de Jong, H. J. G. Arts, A. G. J. van der Zee, J. Bart, P. S. Low, V. Ntziachristos, Nat. Med. 2011, 17, 1315; e) Y. Urano, M. Sakabe, N. Kosaka, M. Ogawa, M. Mitsunaga, D. Asanuma, M. Kamiya, M. R. Young, T. Nagano, P. L. Choyke, H. Kobayashi, Sci. Transl. Med. 2011, 3, 110ra119; f) K. J. Zhou, H. M. Liu, S. R. Zhang, X. N. Huang, Y. G. Wang, G. Huang, B. D. Sumer, J. M. Gao, J. Am. Chem. Soc. 2012, 134, 7803; g) J. P. Weichert, P. A. Clark, I. K. Kandela, A. M. Vaccaro, W. Clarke, M. A. Longino, A. N. Pinchuk, M. Farhoud, K. I. Swanson, J. M. Floberg, J. Grudzinski, B. Titz, A. M. Traynor, H. E. Chen, L. T. Hall, C. J. Pazoles, P. J. Pickhardt, J. S. Kuo, Sci. Transl. Med. 2014, 6, 240ra75; h) Y. G. Wang, K. J. Zhou, G. Huang, C. Hensley, X. N. Huang, X. P. Ma, T. Zhao, B. D. Sumer, R. J. DeBerardinis, J. M. Gao, Nat. Mater. 2014, 13, 204; i) Z. Si, C. Y. Huang, X. H. Gao, C. Li, RSC Adv. 2014, 4, 55548; j) L. Wang, Z. Fan, J. Zhang, Y. Changyi, C. Huang, Y. Gu, Z. Xu, Z. Tang, W. Lu, X. Wei, C. Li, Int. J. Cancer 2015, 136, 107; k) A. Nagai, J. B. Miller, P. Kos, S. Elkassih, H. Xiong, D. J. Siegwart, ACS Biomater. Sci. Eng. 2015, 1, 1206; l) A. Nagai, J. B. Miller, J. Du, P. Kos, M. C. Stefan, D. J. Siegwart, Chem. Commun. 2015, 51, 11868; m) L. Wang, C. Li, J. Mater. Chem. 2011, 21, 15862; n) C. Li, J. A. Xia, X. B. Wei, H. H. Yan, Z. Si, S. H. Ju, Adv. Funct. Mater. 2010, 20, 2222; o) X. J. Wu, B. J. Lin, M. Z. Yu, L. Yang, J. H. Han, S. F. Han, Chem. Sci. 2015, 6, 2002.

[10] S. L. Luo, E. L. Zhang, Y. P. Su, T. M. Cheng, C. M. Shi, Biomaterials 2011, 32, 7127.

[11] H. Xiong, P. Kos, Y. Yan, K. Zhou, J. B. Miller, S. Elkassih, D. J. Siegwart, Bioconjugate Chem. 2016, 27, 1737.

[12] a) Y. Murakami, Y. Tabata, Y. Ikada, Drug Delivery 1997, 4, 23; b) J. M. Harris, R. B. Chess, Nat. Rev. Drug Discovery 2003, 2, 214; c) J. Khandare, T. Minko, Prog. Polym. Sci. 2006, 31, 359; d) N. Bertrand, J. C. Leroux, J. Controlled Release 2012, 161, 152.

[13] A. Fantozzi, G. Christofori, Breast Cancer Res. 2006, 8, 212.[14] a) T. Biswas, X. Gu, J. H. Yang, L. G. Ellies, L. Z. Sun, Cancer Lett.

2014, 346, 129; b) J. Z. Zhou, M. A. Riquelme, X. Gao, L. G. Ellies, L. Z. Sun, J. X. Jiang, Oncogene 2015, 34, 1831.

[15] a) K. Gibby, W. K. You, K. Kadoya, H. Helgadottir, L. J. T. Young, L. G. Ellies, Y. C. Chang, R. D. Cardiff, W. B. Stallcup, Breast Cancer Res. 2012, 14; b) J. Rautela, N. Baschuk, C. Y. Slaney, K. M. Jayatilleke, K. Xiao, B. N. Bidwell, E. C. Lucas, E. D. Hawkins, P. Lock, C. S. Wong, W. Chen, R. L. Anderson, P. J. Hertzog, D. M. Andrews, A. Moller, B. S. Parker, Cancer Immunol. Res. 2015, 3, 1207.

[16] G. Busco, R. A. Cardone, M. R. Greco, A. Bellizzi, M. Colella, E. Antelmi, M. T. Mancini, M. E. Dell’Aquila, V. Casavola, A. Paradiso, S. J. Reshkin, FASEB J. 2010, 24, 3903.

[17] L. Q. Chen, E. A. Randtke, K. M. Jones, B. F. Moon, C. M. Howison, M. D. Pagel, Mol. Imaging Biol. 2015, 17, 488.

[18] C. Lorenzer, M. Dirin, A. M. Winkler, V. Baumann, J. Winkler, J. Controlled Release 2015, 203, 1.

[19] R. Kanasty, J. R. Dorkin, A. Vegas, D. Anderson, Nat. Mater. 2013, 12, 967.

[20] a) A. Akinc, W. Querbes, S. De, J. Qin, M. Frank-Kamenetsky, K. N. Jayaprakash, M. Jayaraman, K. G. Rajeev, W. L. Cantley,



Adv. Mater. 2017, 1700131

J. R. Dorkin, J. S. Butler, L. Qin, T. Racie, A. Sprague, E. Fava, A. Zeigerer, M. J. Hope, M. Zerial, D. W. Sah, K. Fitzgerald, M. A. Tracy, M. Manoharan, V. Koteliansky, A. Fougerolles, M. A. Maier, Mol. Ther. 2010, 18, 1357; b) Y. Dong, K. T. Love, J. R. Dorkin, S. Sirirungruang, Y. Zhang, D. Chen, R. L. Bogorad, H. Yin, Y. Chen, A. J. Vegas, C. A. Alabi, G. Sahay, K. T. Olejnik, W. Wang, A. Schroeder, A. K. R. Lytton-Jean, D. J. Siegwart, A. Akinc, C. Barnes, S. A. Barros, M. Carioto, K. Fitzgerald, J. Hettinger, V. Kumar, T. I. Novobrantseva, J. Qin, W. Querbes, V. Koteliansky, R. Langer, D. G. Anderson, Proc. Natl. Acad. Sci. USA 2014, 111, 3955.

[21] a) J. Hao, P. Kos, K. Zhou, J. B. Miller, L. Xue, Y. Yan, H. Xiong, S. Elkassih, D. J. Siegwart, J. Am. Chem. Soc. 2015, 137, 9206; b) K. Zhou, L. H. Nguyen, J. B. Miller, Y. Yan, P. Kos, H. Xiong, L. Li, J. Hao, J. T. Minnig, H. Zhu, D. J. Siegwart, Proc. Natl. Acad. Sci. USA 2016, 113, 520; c) Y. Yan, K. Zhou, H. Xiong, J. B. Miller, E. A. Motea, D. A. Boothman, L. Liu, D. J. Siegwart, Biomaterials 2017, 118, 84; d) Y. F. Yan, L. Liu, H. Xiong, J. B. Miller, K. J. Zhou, P. Kos, K. E. Huffman, S. Elkassih, J. W. Norman, R. Carstens, J. Kim, J. D. Minna, D. J. Siegwart, Proc. Natl. Acad. Sci. USA 2016, 113, E5702; e) J. B. Miller, S. Zhang, P. Kos, H. Xiong, K. Zhou, S. S. Perelman, H. Zhu, D. J. Siegwart, Angew. Chem., Int. Ed. 2017, 56, 1059.

[22] a) K. Kinkel, Y. Lu, M. Both, R. S. Warren, R. F. Thoeni, Radiology 2002, 224, 748; b) S. Bipat, M. S. van Leeuwen, E. F. I. Comans,

M. E. J. Pijl, P. M. M. Bossuyt, A. H. Zwinderman, J. Stoker, Radiology 2005, 237, 123; c) M. C. Niekel, S. Bipat, J. Stoker, Radi-ology 2010, 257, 674.

[23] P. Mi, D. Kokuryo, H. Cabral, H. L. Wu, Y. Terada, T. Saga, I. Aoki, N. Nishiyama, K. Kataoka, Nat. Nanotechnol. 2016, 11, 724.

[24] R. E. Coleman, Clin. Cancer Res. 2006, 12, 6243s.[25] R. S. Fernandes, D. Dos Santos Ferreira, C. de Aguiar Ferreira,

F. Giammarile, D. Rubello, A. L. de Barros, Biomed. Pharmacother. 2016, 83, 1253.

[26] a) A. Zaheer, R. E. Lenkinski, A. Mahmood, A. G. Jones, L. C. Cantley, J. V. Frangioni, Nat. Biotechnol. 2001, 19, 1148; b) K. M. Harmatys, E. L. Cole, B. D. Smith, Mol. Pharmaceutics 2013, 10, 4263; c) E. M. Peck, P. M. Battles, D. R. Rice, F. M. Roland, K. A. Norquest, B. D. Smith, Bioconjugate Chem. 2016, 27, 1400; d) S. T. Sun, K. M. Blaiewska, A. P. Kadina, B. A. Kashemirov, X. C. Duan, J. T. Triffitt, J. E. Dunford, R. G. G. Russell, F. H. Ebetino, A. J. Roelofs, F. P. Coxon, M. W. Lundy, C. E. McKenna, Bioconjugate Chem. 2016, 27, 329.

[27] T. Kowada, J. Kikuta, A. Kubo, M. Ishii, H. Maeda, S. Mizukami, K. Kikuchi, J. Am. Chem. Soc. 2011, 133, 17772.

[28] D. Wang, S. C. Miller, P. Kopeckova, J. Kopecek, Adv. Drug Delivery Rev. 2005, 57, 1049.

[29] S. L. Teitelbaum, Science 2000, 289, 1504.[30] S. Y. Jiang, Z. Q. Cao, Adv. Mater. 2010, 22, 920.

High‐Contrast Fluorescence Detection of Metastatic Breast ... · 2017 WILEY-VCH Verlag GmbH & Co....

Documents

Transcript of High‐Contrast Fluorescence Detection of Metastatic Breast ... · 2017 WILEY-VCH Verlag GmbH & Co....