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Cite This: ACS Appl. Mater. Interfaces 2018, 10, 16335−16343
Tumor-Activated Water-Soluble Photosensitizers for Near-Infrared Photodynamic Cancer Therapy Hu Xiong, Kejin Zhou, Yunfeng Yan, Jason B. Miller, and Daniel J. Siegwart* Department of Biochemistry, Simmons Comprehensive Cancer Center, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States S Supporting Information *
ABSTRACT: Current photosensitizers (PSs) for photodynamic therapy (PDT) are limited by their low water solubility and tendency to aggregate, low near-infrared (NIR) absorption, and low cancer selectivity. Here, we designed iodinated, water-soluble NIR boron dipyrromethene-based PSs to achieve image-guided and efficient PDT against cancer in vivo that is enhanced by leveraging tumor-specific pHresponsive activation. PEG2k5c-I and PEG2k5c-OMe-I localized to tumors and were activated by acidic pH in the tumor microenvironment to produce 1O2 and fluorescence for efficient PDT and effective cancer detection after intravenous administration. Upon NIR irradiation, these PSs exhibited strong NIR absorption at 660 and 690 nm, stable NIR emission at 692 and 742 nm, and high 1O2 quantum yields of 0.78 and 0.72 in acidic pH. PEG2k5c-I and PEG2k5c-OMe-I killed cancer cells upon irradiation of NIR light and were nontoxic without irradiation. Light-activated PDT treatment of breast cancer tumors in mice resulted in suppression of tumor growth, DNA damage, and necrosis selectively in tumors. This work thus introduces a versatile method to directly synthesize modular pHresponsive water-soluble PSs and provides a versatile strategy for activatable PDT against cancer. KEYWORDS: photodynamic therapy, photosensitizer, activatable nanoprobes, near-infrared, pH-responsive fluorescence imaging
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INTRODUCTION Photodynamic therapy (PDT) has emerged as a minimally invasive cancer treatment that can kill cancer cells and improve clinical outcomes.1 The most important element of PDT is the chemical design of the photosensitizer (PS). Most PSs, including those approved for use in humans, contain aromatic porphyrin and chlorin motifs that can aggregate in aqueous media.2 There is a clear need for improved PS designs that are water-soluble, absorb in the near-infrared (NIR) region to improve the penetration depth, and are selective to cancer to reduce the damage to normal tissues. Here, we report acidic pH-activatable boron dipyrromethene (BODIPY)-based PSs that were rationally designed to possess the following features: (1) iodine was introduced into the BODIPY core to enhance 1O2 generation efficiency, (2) a diethylaminophenyl moiety was appended to achieve pHcontrollable activity via photoinduced electron transfer (PeT), (3) a conjugated distyryl ether core was designed to absorb and emit in the NIR region, and (4) poly(ethylene glycol) (PEG) chains were attached to obtain excellent water solubility (Figures 1a and 2a). The 1O2 release is “turned off” by exploiting the PeT process at physiological pH, whereas upon the protonation of the amino groups at acidic pH, the quenching process is inhibited and the 1O2 release is “turned on” (Figure 1a). Because an established pH-responsive activation mechanism was leveraged, these new molecules can generate 1O2 in cancer cells and induce cancer cell death via © 2018 American Chemical Society
apoptosis in a cancer-selective manner (Figure 1b). Compared with a Food and Drug Administration (FDA)-approved PDT drug protoporphyrin (PpIX),2 PEG2k5c-I and PEG2k5cOMe-I exhibit better water solubility, stronger NIR absorption, and higher 1O2 quantum yield. Notably, they are localized to tumors on their own without the need to encapsulate in nano/ microparticle carriers3−6 and inhibited tumor growth in vivo after intravenous (iv) administration. To the best of our knowledge, PEG2k5c-I and PEG2k5c-OMe-I are the first pHactivatable water-soluble NIR PSs to successfully image MDAMB-231 breast cancer and inhibit tumor growth in vivo via PDT. We believe that this report introduces a versatile method to directly synthesize modular pH-responsive water-soluble PSs and provides a versatile strategy for activatable PDT against cancer. PDT utilizes a combination of a PS, light, and oxygen to generate a singlet oxygen (1O2) or other reactive oxygen species (ROS) during photochemical reactions. Antitumor efficacy relies on the chemical design of the PS to mediate ROS that causes apoptosis and necrosis in cells and damage to tumor microvasculature and stimulates antitumor immunity.2,7 PDT is ideally minimally invasive and offers negligible drug resistance, repeatable administration without significant side effects, and Received: March 22, 2018 Accepted: April 25, 2018 Published: April 26, 2018 16335
DOI: 10.1021/acsami.8b04710 ACS Appl. Mater. Interfaces 2018, 10, 16335−16343
Research Article
ACS Applied Materials & Interfaces
Figure 1. Illustration of pH-activatable PEGylated NIR PS-mediated PDT. (a) Structure and function of pH-activatable BODIPY-based PSs for 1O2 generation. (b) pH-activatable BODIPY-based PSs turn on in cancer tissues and cells for selective PDT against cancer.
Figure 2. (a) Structures of PEG2k5c-I and PEG2k5c-OMe-I. (b) Normalized absorption and emission spectra of PEG2k5c-I (10 μM) in phosphate-buffered saline (PBS). (c) Normalized absorption and emission spectra of PEG2k5c-OMe-I (10 μM) in PBS. (d) NIR fluorescence spectra of PEG2k5c-I and PEG2k5c-OMe-I (10 μM) in pH 5.0 and 7.4 citrate buffer. (e) pH-dependent fluorescent images of 10 μM aq solutions of PSs. (f) Confocal NIR fluorescence images of MDA-MB-231 cells incubated with different PSs (5 μM) for 8 h. The NIR PS is red, and the cell nuclei are blue. Scale bar = 50 μm.
activation of an immune response against targeted cells.8−11 PSs with both efficient 1O2 generation and bright fluorescence emission have also been used for image-guided PDT.12−15 Certain ideal criteria for effective image-guided PDT against cancer have been established: (1) high absorption and emission in the NIR region (650−900 nm) for deep tissue penetration and cancer imaging, (2) sufficient 1O2 production under NIR light irradiation, and (3) selective toxicity in cancer tissues not normal tissues.16
Unfortunately, most clinically approved PSs based on porphyrin and chlorin derivatives have weak absorption and low 1O2 quantum yield upon NIR irradiation.2 Moreover, they can easily aggregate in aqueous media through π−π stacking because of their hydrophobic and rigid planar structures, which can dramatically decrease their 1O2 production and result in aggregation-caused fluorescence quenching.17 To address this issue, PSs with aggregation-induced emission characteristics (AIEgen) were recently developed by Tang and others.18−20 16336
DOI: 10.1021/acsami.8b04710 ACS Appl. Mater. Interfaces 2018, 10, 16335−16343
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ACS Applied Materials & Interfaces
Figure 3. Concentration-dependent absorption spectra of PEG2k5c-I in PBS (a), PEG2k5c-OMe-I in PBS (b), and PpIX in DMSO (c). The insets show a correlation between concentration and dependent absorbance intensity of PEG2k5c-I (a), PEG2k5c-OMe-I (b), and PpIX (c). Photosensitized 1O2 generation by PEG2k5c-I, PEG2k5c-OMe-I, and PpIX. Time-dependent absorption spectrum changes seen for 100 μM DMSO solutions of DPBF and 2 μM PEG2k5c-I or PpIX with (d) or without (e, f) 0.1% trifluoroacetic acid (TFA) (v/v) upon irradiation at 600− 700 nm (0.2 W/cm2).
This work provides a chemical framework for rational design of cancer-selective PSs.
Nevertheless, AIEgen PSs usually show absorption in the UV and visible range because of the low conjugation of rotor structures, resulting in limited biological applications at the tissue level. In addition, most existing PSs are “always-on” small molecules without activatable functionality, resulting in unwanted phototoxicity and background fluorescence in normal tissues. Apart from conventional PSs, BODIPY-based PSs have recently attracted great attention owing to their high 1O2 quantum yield, good photostability, biocompatibility, and tolerance to chemical modification.21−26 Despite the successful development of BODIPY-based PSs, activatable PSs with good water solubility and high NIR absorption have not been realized.27−30 pH is a logical stimulus to achieve tumor activation because the tumor microenvironment (pH 6.5− 6.8) is acidic31,32 and the pH in lysosomes (pH 4.5−5.0) further promotes 1O2 generation and release.15,27,30,33 Recently, several pH-activatable BODIPY-based PSs were reported to produce 1O2 upon NIR irradiation27,30,34 but these PSs were not water-soluble and were confined to aza-BODIPY structures that intrinsically absorb in the NIR region. Phototherapeutic effects of non-aza-BODIPYs have not been fully tested in vivo. Thus, the simultaneous control of good water solubility, high NIR absorption, activation by cancer-associated stimuli,15,33,35−39 and effective 1O2 generation with low dark toxicity in a single PS remains challenging. In this article, we leveraged our experience with watersoluble, stimuli-responsive40,41 probe designs42,43 to create PSs capable of localizing to tumors and activating in response to tumor pH to both emit fluorescence and generate ROS to kill cancer cells selectively. PEG2k5c-I and PEG2k5c-OMe-I successfully imaged and inhibited tumor growth in vivo via PDT, whereas a FDA-approved PDT agent PpIX could not.
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RESULTS AND DISCUSSION pH-Activatable NIR BODIPY-Based PSs Were Chemically Synthesized To Enable Effective PDT. Our goal was to design an efficient PS that is inactive at physiological pH but emits 1O2 with high quantum yield at acidic tumor pH under NIR light (650−900 nm).16 To rationally design pH-activatable NIR PSs, we utilized tetramethyl-BODIPY as a core scaffold, installed a diethylaminophenyl moiety that allows response to tumor pH via PeT,40,41 conjugated distyryl ether groups to enable NIR absorption and emission over 650 nm, and attached PEG chains to provide water solubility and slow kidney clearance. To covert an imaging probe to a PS, we added iodine to enhance 1O2 generation44 due to the heavy atom effect (Figure 2a and Scheme S1). As the first step, BODIPY 1 was treated with a mixture of iodine and iodic acid in acetonitrile, giving iodinated BODIPY 2 with enhanced 1O2 generation. Subsequently, BODIPY 2 was condensed with 4-(propargyloxy)-benzaldehyde to obtain NIR BODIPY 3a by our previously reported protocol.40,41 To render 3a water-soluble, two PEG chains with a molecular weight of 2000 g/mol (MW 2k) were attached via a coppercatalyzed click reaction to obtain water-soluble PEG2k5c-I (Scheme S1). This MW was selected on the basis of our prior report showing enhancement of metastatic imaging with MW between 1000 and 5000 g/mol.41 We previously found that lower MW chains (e.g., 400 g/mol) resulted in low water solubility and minimal tumor uptake, whereas longer PEG chains (e.g., 10 000 g/mol) increased blood circulation time but did not enhance tumor uptake.41 Therefore, we focused on PEG2000 in this work. PEG2k5c-I could easily dissolve in 16337
DOI: 10.1021/acsami.8b04710 ACS Appl. Mater. Interfaces 2018, 10, 16335−16343
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Figure 4. Photocytotoxicity of PEG2k5c-I and PEG2k5c-OMe-I in cancer cells. Cell viability of MDA-MB-231 after treatment with BODIPY-based PSs (1 μM) with or without irradiation at 600−700 nm for different times: (a) PS: PEG2k5c-I, control: PEG2k5c; (b) PS: PEG2k5c-OMe-I, control: PEG2k5c-OMe. (c) Confocal microscopy images of MDA-MB-231 cells with PEG2k5c-I or PEG2k5c-OMe-I (1 μM) after 600−700 nm laser irradiation (0.2 W/cm2, 10 min). Live/dead cells are green/red (calcein AM/PI staining), respectively.
ability of PEG2k5c-I and PEG2k5c-OMe-I to produce 1O2 upon NIR light irradiation, which was studied in DMSO using 1,3-diphenylisobenzofuran (DPBF) as the 1O2 indicator (Figures 3d,e and S4).45 A dramatic quenching of the DPBF absorption band at 418 nm over a time period of 0−150 s was observed only in the acidic pH solution not in the neutral pH solution (Figure 3d,e), indicating that the 1O2 production efficiency of PEG2k5c-I is significantly dependent on solution pH. Meanwhile, the NIR absorption of PEG2k5c-I remained same at 660 nm upon irradiation (Figure 3d,e), which suggests that PEG2k5c-I is stable under light. Similarly, PEG2k5cOMe-I exhibited pH-controllable 1O2 generation with NIR absorption at 690 nm (Figure S4). Moreover, PEG2k5c-I and PEG2k5c-OMe-I could generate 1O2 in pH 6.0 citrate buffer that is suitable for in vivo application (Figure S5). In addition, they both displayed a faster 1O2 production ratio and stronger NIR absorption in comparison with PpIX (Figure 3f). According to an established protocol,45 1O2 quantum yields (Φ Δ) of PEG2k5c-I and PEG2k5c-OMe-I were then calculated using methylene blue (MB) as the standard (ΦΔ = 0.52 in DMSO). ΦΔ values of PEG2k5c-I and PEG2k5c-OMeI are 0.78 and 0.72, respectively, which are higher than that of PpIX (ΦΔ = 0.54). We also measured the fluorescence quantum yields (Φfl) of PEG2k5c-I and PEG2k5c-OMe-I using our previously reported method.40 The Φfl of PEG2k5c-I and PEG2k5c-OMe-I are 0.01 and 0.016 in 0.001 M aqueous HCl, respectively. Therefore, these results suggest that PEG2k5c-I and PEG2k5c-OMe-I possess an ability to work as promising PSs for pH-activatable PDT against cancer in vitro and in vivo. PEG2k5c-I and PEG2k5c-OMe-I Generate Photocytotoxicity Selectively upon NIR Irradiation in Cancer Cells. Successful PDT PSs exhibit low cytotoxicity in the dark but
water and exhibited a high NIR absorption at 660 nm and emission at 692 nm (Figure 2b). To make the BODIPY-based PS more red-shifted, PEG2k5c-OMe-I with four methoxyl groups was synthesized via the similar protocols (Scheme S1), which exhibited a strong absorption at 690 nm and emission at 742 nm (Figure 2c), thereby facilitating utility for deeper tissues. Notably, both molecules are “off” at neutral pH but exhibit NIR emission at acidic pH (Figure 2d,e), indicating that the diethylaminophenyl moiety can quench the excited iodinated NIR BODIPY core by harnessing the PeT effect. Moreover, they were taken up by MDA-MB-231 breast cancer cells and activated by the low pH in lysosomes to give NIR emission (Figures 2f and S1), demonstrating that PEG2k5c-I and PEG2k5c-OMe-I are able to respond to acidic pH for activatable PDT in vitro. In addition, PEG2k5c and PEG2k5cOMe without iodine were also synthesized as the control molecules (Scheme S2), which exhibited fast acidic pH response and suitable pKa’s at pH 4.5 and 4.76, respectively (Figure S2). PEGylated BODIPY-Based PSs Are Water-Soluble and Can Generate Singlet Oxygen. To investigate the solubility of PEGylated BODIPY-based PSs in water, we examined the concentration-dependent properties. Owing to the linear increase in the absorption intensity as a function of concentration from 10 to 100 μM for PEG2k5c-I and PEG2k5c-OMe-I, they both exhibited excellent water solubility for in vitro and in vivo applications (Figure 3a,b). In contrast, a FDA-approved PDT agent (PpIX) exhibited low water solubility and weak absorption in the NIR region (Figure S3). Although PpIX has higher solubility in dimethyl sulfoxide (DMSO), it still aggregated at high concentration, as indicated by the nonlinear relationship between the absorption intensity and the concentration (Figure 3c). Next, we examined the 16338
DOI: 10.1021/acsami.8b04710 ACS Appl. Mater. Interfaces 2018, 10, 16335−16343
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Figure 5. In vivo antitumor efficacy of PEG2k5c-I and PEG2k5c-OMe-I on athymic nude mice bearing subcutaneous MDA-MB-231 xenograft tumors. (a) Tumor volume is plotted. PDT was performed 6, 24, and 48 h after iv injection of 1 mg/kg PS (10 μM, 400 μL) or PBS (400 μL) (mean ± sd, n = 4). The tumors were irradiated with a 600−700 nm laser at 0.2 W/cm2 for 30 min. Statistical significance was determined using a two-tailed Student’s t test (**P = 0.0012). (b) Mouse body weight curves with relevant treatments. (c) Photos of the harvested tumors from each group on the last day of the experiment. (d) In vivo NIR fluorescence images of subcutaneous MDA-MB-231 tumor-bearing mice 6 h after iv injection of 1 mg/kg PEG2k5c-OMe-I. (e) Representative ex vivo fluorescence images of harvested tumors and organs on the last day of the experiment. (f) Histology and fluorescence microscopic analysis of the PSs. PSs, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and cell nuclei are shown in red, green, and blue, respectively. Scale bar = 50 μm.
considerable cancer cell death when exposed to light. To examine cytotoxicity, we added PEG2k5c-I and PEG2k5cOMe-I to MDA-MB-231 breast cancer cells in the presence and absence of irradiation with 600−700 nm light (0.2 W/cm2) (Figure 4a,b). In the absence of light, they were negligibly cytotoxic. In contrast, PEG2k5c-I exhibited high cytotoxicity upon irradiation with 600−700 nm light for 5 min, which could cause cancer cell death over 90% (Figure 4a). Similarly, PEG2k5c-OMe-I exhibited moderate and high cytotoxicity under irradiation for 5 and 10 min, respectively (Figure 4b). There was no cytotoxicity for untreated cells under light or dark conditions, indicating that photocytotoxicity is caused by PS under NIR light. To further confirm the lack of background
cytotoxicity, we conducted a dose response experiment under dark conditions that showed fully viable cells (Figure S6). To prove that the attachment of iodine was responsible for successful PDT, we examined control molecules that lack the iodine modification (PEG2k5c and PEG2c5c-OMe). No cytotoxicity was observed under both light and dark conditions in MDA-MB-231, demonstrating that the iodine on the BODIPY-based PS enhances 1O2 production and release (Figure 4a,b). Furthermore, they exhibited excellent inhibition of growth of IGROV1 cells (ovarian cancer), HeLa cells (cervical cancer), and B16F10 cells (melanoma cancer) under NIR light (Figure S7). To further verify photocytotoxicity of PEG2k5c-I and PEG2k5c-OMe-I, a calcein acetoxymethyl 16339
DOI: 10.1021/acsami.8b04710 ACS Appl. Mater. Interfaces 2018, 10, 16335−16343
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tumors treated with PEG2k5c-I or PEG2k5c-OMe-I combined with NIR irradiation. In contrast, negligible apoptotic tumor cells were observed in the PBS group. Confocal fluorescence imaging of tumor tissue cyrosections further verified NIR emission in the groups of mice treated with PEG2k5c-I and PEG2k5c-OMe-I (Figure 5f), which confirmed that PEG2k5cI and PEG2k5c-OMe-I efficiently accumulated and activated in the tumors after iv administration. These results therefore demonstrate that PEG2k5c-I and PEG2k5c-OMe-I can act as therapeutic drugs for activatable PDT against cancer in vivo.
(AM) and propidium iodide (PI) assay was performed after PDT treatment for labeling live and dead cells, respectively. As shown in Figure 4c, the cells kept largely protected from light showed green calcein AM fluorescence in the control group, whereas red PI fluorescence signals were seen in the cells treated with PS and photoirradiation. Therefore, these results indicate that PEG2k5c-I and PEG2k5c-OMe-I can achieve activatable NIR PDT. PEG2k5c-I and PEG2k5c-OMe-I Inhibit the Growth of Subcutaneous MDA-MB-231 Tumors Better than FDAApproved PplX. To further investigate the in vivo anticancer efficacy of PEG2k5c-I and PEG2k5c-OMe-I via PDT, mice bearing subcutaneous MDA-MB-231 breast cancer xenograft tumors were injected iv with pH-activatable PEG2k5c-I and PEG2k5c-OMe-I at a dose of 1 mg/kg (administrated five times within 2 weeks). PDT was performed 6, 24, and 48 h after iv injection by irradiation (600−700 nm, 0.2 W/cm2) on the tumors for 30 min. The MDA-MB-231 tumor-bearing mice were randomly divided into six groups with different treatments. The therapeutic effects after treatment were assessed by monitoring the change of relative tumor volumes during the subsequent 13 days (Figure 5a). No tumor growth inhibition was observed in the groups of mice treated with PBS regardless of irradiation, which indicated that irradiation with such a lowpower-intensity NIR light had little photothermal effect. PEG2k5c-I and PEG2k5c-OMe-I in the absence of irradiation also exhibited similar tumor growth behaviors to those of PBS, suggesting that they have negligible dark toxicity. In contrast, the tumor growth was remarkably suppressed for the mice treated with PEG2k5c-I or PEG2k5c-OMe-I under NIR light irradiation (Figure 5a,c), demonstrating that both molecules could be effectively activated upon NIR irradiation to produce a strong phototoxicity to the tumors. In addition, the absence of any significant mice body weight loss indicated no off-target toxicity or side effects following multiple iv injections and PDT treatments (Figure 5b). Moreover, the tumor tissues in the PEG2k5c-I- or PEG2k5c-OMe-I-treated mice could be imaged and distinguished from the surrounding normal tissues (Figures 5d and S8), indicating that they are suitable for image-guided PDT in vivo because of the NIR emission at 692 and 742 nm, respectively. To further examine biodistribution, harvested tumors and organs were analyzed on the last day of PDT treatment (Figures 5e and S9). PEG2k5c-I and PEG2k5cOMe-I both exhibited remarkable NIR fluorescence emission in the tumors and low emission in all other organs, except liver, which validated that they could effectively accumulate and be retained in the tumors where they were activated to the “turnon” state. As an additional control, the MDA-MB-231 tumorbearing mice were injected iv with PpIX, followed by the same PDT treatment. However, PpIX without encapsulation into nanocarriers had no tumor accumulation and negligible tumor growth inhibition after iv administration (Figures S8 and S10), indicating that the water-soluble PEG2k5c-I and PEG2k5cOMe-I can achieve PDT against cancer similar to nanocarrierPS.8,9 To further confirm in vivo phototherapeutic efficacy, tumor sections were examined using hematoxylin and eosin (H&E) staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Prominent necrosis was observed in the PDT-treated tumor tissue, whereas necrosis was indiscernible in the group of mice treated with PBS and irradiation (Figure 5f). Moreover, the TUNEL assay results revealed a significant increase in the amount of apoptosis in the
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CONCLUSIONS In summary, we developed pH-activatable, water-soluble, and NIR BODIPY-based PSs that achieve effective, image-guided PDT against cancer in vivo. The pH-activatable PEG2k5c-I and PEG2k5c-OMe-I molecules can localize to tumors without encapsulation into nanocarriers, where they are activated by acidic pH to produce 1O2 and fluorescence for efficient PDT and effective cancer detection after iv administration. They exhibit excellent water solubility, strong NIR absorption, and high 1O2 quantum yield. Thus, our two-in-one NIR PDT/ imaging BODIPY-based PSs are promising therapeutic and diagnostic candidates for cancer treatment. Overall, these carefully engineered PSs represent a robust platform for cancer imaging and treatment with potential use in a variety of modalities and applications. The successful synthesis via rational design suggests methods to develop further pHactivatable PDT molecules for safer and more attractive clinical application.
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METHODS
Synthesis of Iodinated BODIPY 2. Iodic acid (2 mmol, 352 mg) was dissolved in 1.5 mL of deionized (DI) water and added dropwise to a solution of BODIPY 1 (1 mmol, 395 mg) and iodine (2 mmol, 508 mg) in 30 mL of CH3CN under a N2 atmosphere. The resulting mixture was stirred at room temperature for 1 h, and a red precipitate was formed. The reaction mixture was extracted with dichloromethane (DCM), washed with brine, and dried over anhydrous sodium sulfate. The solvent was then evaporated under vacuum, and the resulting residue was purified by silica gel column chromatography using hexanes/DCM (1:0, 4:1, and 2:1) for elution, affording 2 as a red solid with 39% yield. Synthesis of Iodinated NIR BODIPY 3. Iodinated BODIPY 2 (258.8 mg, 0.4 mmol) and the corresponding propargyl benzaldehyde (2.0 mmol) were refluxed in a mixture of dry acetonitrile (20 mL), piperidine (800 μL), glacial acetic acid (480 μL), and 4 Å molecular sieves (∼50 pellets) under a N2 atmosphere. The reaction mixture was stirred at 85 °C for 8−12 h. Then, the solvent was removed under reduced pressure. The crude product was first purified by flash column chromatography over silica gel using ethyl acetate/hexanes as the eluent to remove the excess propargyl benzaldehyde. Subsequently, the eluent was changed to DCM/hexanes to obtain the desired product. The collected product was then recrystallized in CH2Cl2/ hexanes (10:90), yielding 3a and 3b as a wine-colored solid. Synthesis of pH-Activatable NIR BODIPY-Based PSs. NIR BODIPY 3 (0.04 mmol), methoxy-poly(ethylene glycol)-azide (PEGN3, average Mn = 2000, 176 mg, 0.088 mmol), CuSO4 (1.3 mg, 0.008 mmol), and sodium ascorbate (VcNa, 15.8 mg, 0.08 mmol) were added to a 10 mL Schlenk flask. The vessel was evacuated and backfilled with N2. Then, the flask was cooled to 0 °C for 20 min and 4 mL of tetrahydrofuran (THF) (degassed) was added via a syringe, followed by 1 mL of DI water (degassed). The reaction mixture was slowly warmed to room temperature and stirred overnight. After evaporation of THF, the residue was dialyzed in DI water for 8 h and lyophilized to obtain a blue solid. The collected product was then purified by column chromatography through a Sephadex LH-20 resin 16340
DOI: 10.1021/acsami.8b04710 ACS Appl. Mater. Interfaces 2018, 10, 16335−16343
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ACS Applied Materials & Interfaces using MeOH as the eluent, affording PEG2k5c-I and PEG2k5c-OMeI as a blue solid. Cell Culture and Confocal Microscopy Imaging. All cancer cell lines were cultured in a high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) or Roswell Park Memorial Institute (RPMI)-1640 medium containing 5% fetal bovine serum (FBS). Cells were maintained at 37 °C under a humidified atmosphere with 5% CO2. (DMEM for B16F10 and HeLa cell lines; RPMI for MDA-MB-231 and IGROV1 cell lines.) Prior to confocal imaging studies, cells were seeded at a density of 25 000 cells per well in four-chambered cover glass slides (Nunc) in 600 μL of media and allowed to attach for 24 h. On the following day, the media were removed and replaced with fresh DMEM or RPMI (600 μL) with 5% FBS. The NIR BODIPY-based PS (5 μM) was added, and the cells were incubated for 8 h. Then, the cells were washed twice with phosphate-buffered saline (PBS) and incubated with the LysoTracker Green DND-26 (0.5 μM, 500 μL) for 30 min. The cells were fixed by 4% paraformaldehyde (400 μL) and stained with 4′,6-diamidino-2-phenylindole (DAPI) (0.3 μM, 500 μL) for 10 min. After labeling, cells were washed twice with PBS and incubated with fresh PBS for fluorescence imaging. Confocal microscopy imaging was performed using a Zeiss LSM 700 Confocal microscope. Images were analyzed using ImageJ (NIH). Calcein AM/PI Staining Assay. MDA-MB-231 cells were seeded at a density of 25 000 cells per well in four-chambered cover glass slides (Nunc) in 600 μL of media and allowed to attach for 24 h. On the following day, the media were removed and replaced with fresh RPMI (600 μL). The NIR BODIPY-based PS (1 μM) was added, and the cells were incubated for 8 h. Then, the cells were irradiated with a 600−700 nm laser at 0.2 W/cm2 for 10 min. Next, the cells were washed once with PBS and incubated with calcein AM (4 μM) and PI (4 μM) for 30 min. After labeling, cells were washed twice with PBS and incubated with fresh PBS for fluorescence imaging. Confocal microscopy imaging was performed using a Zeiss LSM 700 Confocal microscope. Images were analyzed using ImageJ (NIH). Photocytotoxicity Assay. Cancer cells were seeded at a density of 10 000 cells per well in a 96-well plate in 100 μL of media and incubated for 24 h at 37 °C. On the following day, the media were removed and replaced with fresh DMEM or RPMI (200 μL per well) containing 5% FBS. The NIR BODIPY-based PS or control (1 μM) was added, and the cells were incubated for 8 h at 37 °C. Then, the cells were irradiated with a 600−700 nm laser at 0.2 W/cm2 for different times. Afterward, the media were removed and replaced with fresh DMEM or RPMI (200 μL per well), followed by incubation at 37 °C for 12 h. After this time, cell viability was evaluated using the CellTiter-Glo assay (Promega) using a Tecan InfiniTe F/M200 Pro microplate reader (n = 3). In Vivo Phototoxicity Assay on a Subcutaneous Tumor Model. Female athymic nude Foxn1nu mice (∼20 g, 6−8 weeks) were purchased from Harlan Laboratories. All 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. MDA-MB-231 tumor cells (5 × 106) in 100 μL of HBSS/Matrigel (v/v, 50:50) were injected subcutaneously into each flank of 6−8 week old nude mice. After 10 days, when the tumors reached adequate size (∼5−6 mm in diameter), mice were randomly divided into six groups with different treatments: group 1, PBS with laser exposure; group 2, PBS without laser exposure; group 3, iv injection of PEG2k5c-I with laser exposure; group 4, iv injection of PEG2k5c-I without laser exposure; group 5, iv injection of PEG2k5c-OMe-I with laser exposure; group 6, iv injection of PEG2k5c-OMe-I without laser exposure. Each group contained four mice. PDT was performed 6, 24, and 48 h after iv injection of 1 mg/kg NIR BODIPY-based PS (10 μM, 400 μL) or PBS (400 μL) into the tail vein at day 1, day 3, day 6, day 9, and day 12. The tumors were irradiated with a 600−700 nm laser at 0.2 W/cm2 for 30 min. Then, the tumor volume was measured by a Vernier Caliper and calculated on the basis of 0.5 × length × width2. In Vivo Fluorescence Imaging and Biodistribution of BreastTumor-Bearing Mice. When mice bearing subcutaneous MDA-MB231 xenograft tumors were injected iv with NIR BODIPY-based PS,
PBS, or PpIX, mice were anesthetized with 2.5% isofluorane in oxygen at selected time points and the whole-body NIR fluorescence images (Cy5.5 filter was used) were captured using an IVIS Lumina imaging system (Caliper Life Sciences). At the end point of PDT treatment (day 13), mice were euthanized and their tumors, livers, lungs, hearts, kidneys, spleens, and pancreases were collected. Ex vivo fluorescence imaging of these organs was immediately performed on the IVIS Lumina system. Histology and Fluorescence Microscopy Analysis of Breast Tumors. Representative tumors were isolated from MDA-MB-231 tumor-bearing mice and immediately fixed in 10% formalin buffered with PBS for 20 h. The fixed tumor tissues were washed with fresh PBS and processed to make paraffin sections (8 μm). The paraffin sections were stained with H&E, labeled with TUNEL reagents (Thermo Fisher Scientific), or mounted with DAPI medium (Vector Laboratories) after deparaffinizing and rehydrating using the following steps: 1 × 2 min xylene (blot excess xylene before going into ethanol), 1 × 30 s 100% ethanol, 1 × 30 s 95% ethanol, 1 × 30 s 70% ethanol, 1 × 15 s DI water, and 1 × 15 s PBS. The images of H&E staining were captured on a Keyence BZ-X710 microscope (40× magnification). Fluorescence images of the sections were captured on a Zeiss LSM 700 Confocal microscope (20× magnification) and analyzed using ImageJ (NIH).
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b04710. Additional general information about instrumentation and reagent sources, Schemes S1 and S2, Figures S1− S10, and 1H NMR and 13C NMR of the PSs (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Daniel J. Siegwart: 0000-0003-3823-1931 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Support from Cancer Prevention and Research Institute of Texas (CPRIT) (R1212), Welch Foundation (I-1855), and American Cancer Society (RSG-17-012-01) is acknowledged.
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REFERENCES
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