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In vivo near-infrared imaging and phototherapy of tumors using a cathepsin B-activated fluorescent probe
Biomaterials 122 (2017) 130e140
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In vivo near-infrared imaging and phototherapy of tumors using a cathepsin B-activated fluorescent probe Xiaoqiang Chen a, b, 1, Dayoung Lee a, 1, Sungsook Yu c, 1, Gyoungmi Kim a, Songyi Lee a, Yejin Cho c, Haengdueng Jeong c, Ki Taek Nam c, **, Juyoung Yoon a, * a
Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, South Korea State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemical Engineering, Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing 210009, China c Severance Biomedical Science Institute, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, South Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2016 Received in revised form 13 January 2017 Accepted 14 January 2017 Available online 16 January 2017
The development of multifunctional reagents for simultaneous specific near-infrared (NIR) imaging and phototherapy of tumors is of great significance. This work describes the design of a cathepsin B-activated fluorescent probe (CyA-P-CyB) and its applications as an NIR imaging probe for tumor cells and as a phototherapy reagent for tumors. In vitro experiments demonstrated that CyA-P-CyB was activated via the cleavage of a peptide linker by cathepsin B in tumor cells to produce fluorescence in the NIR region based on a FRET mechanism. MTT assays showed that the phototoxicity of CyA-P-CyB toward cells depended on the activity of cathepsin B, and the probe exhibited specific phototoxicity toward tumor cells. CyA-P-CyB was also successfully applied to the in vivo imaging and phototherapy of tumors. Histological analysis indicated that CyA-P-CyB had no cytotoxic effects on seven mouse tissues (lung, liver, heart, kidney, pancreas, spleen and brain) after the CyA-P-CyB treatment and laser irradiation. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Specific phototherapy NIR bioimaging Enzyme-activated fluorescence Multiple functional probe
1. Introduction Fluorescent probes have become powerful tools that allow biologists to study biological processes because they can provide visual information in real time with high spatial resolution [1]. In particular, probes with near-infrared (NIR) emission (650e900 nm) are preferable for in vivo imaging. Compared to the conventional probes with shorter emission wavelengths, the NIR fluorescence probes have unique advantages, such as that the NIR emission can avoid the interference from auto-fluorescence of indigenous biomolecules, achieving a higher signal-to-noise ratio; NIR fluorescent probes can achieve imaging in deeper tissues because of their longer excitation wavelength; and NIR excitation and emission cause less damage to biological samples than the shorter wavelengths of light. In the past two decades, a number of NIR fluorescence probes have been developed to monitor various biological species and drug release in vivo and in vitro [2,3].
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (K.T. Nam), [email protected] (J. Yoon). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2017.01.020 0142-9612/© 2017 Elsevier Ltd. All rights reserved.
The development of multifunctional reagents that can enable both specific imaging and tumor therapy is of great significance due to that they provide the information on where (W), when (W), and how (H) drugs are delivered and activated in vivo [4,5]. Phototherapy is a mild and noninvasive approach for cancer treatment [6,7]. In this treatment modality, with the assistance of the photosensitizer or photothermal agent, light is absorbed and transferred to reactive oxygen species (ROS) or causes local hyperthermia, inducing tumor cell death [8,9]. The NIR light-induced PDT is preferable to shorter-wavelength light due to its deeper tissue penetration [10e12]. To date, the most suitable NIRphotosensitizable molecule is indocyanine green dye (ICG) [13,14]. ICG is an NIR dye that has been approved for clinical use by the U.S. Food and Drug Administration (FDA). However, ICG itself is not specific for tumor cells, indicating that phototherapy with ICG would damage normal cells or tissues upon NIR laser irradiation. Therefore, phototherapy agents that cause specific and efficient damage to the targeted cancer cells rather than the normal cells are preferable. Cathepsin B, one of lysosomal proteases, plays an important role in the regulation of angiogenesis and invasion during cancer progression [15,16]. Some antitumor prodrugs, cleaved by cathepsin B, which is highly upregulated in malignant
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tumors and premalignant lesions, has been developed to achieve a higher drug concentration in tumor tissue and a lower concentration in normal tissue, resulting in enhance the efficacy and reduce toxicity to normal cells [17,18]. Though some excellent cathepsin Bactivated imaging probes have been reported for the evaluation of enzyme activity and the diagnosis of cancer [19e27], few of them based on cathepsin B-activated reaction were explored as multifunctional reagents that can enable both specific imaging and targeted tumor therapy [26,27]. Herein, we describe a new NIR fluorescent probe (CyA-P-CyB) that possesses multiple properties, including specific NIR imaging, specific phototoxic effects on tumor cells, and a phototherapeutic effect in mice bearing tumors. Compared to the protease-trigged antitumor PDT agents [26,27], the selective antitumor activity of the present probe come from the effective diffusion of cleavage product (PDT agent) in tumor cells. The probe is designed based on a fluorescence resonance energy transfer (FRET) mechanism. As shown in Fig. 1, the probe (CyA-P-CyB) features two cyanine moieties (CyA and CyB) linked via a cathepsin B-activated peptide(GlyPhe-Leu-Gly), which has been used in design of cathepsin Bcleavable prodrugs for targeted cancer therapy [28,29]. CyA acts as a fluorescent donor, and CyB acts as a quencher. The initial form of the probe does not emit fluorescence due to the efficient FRET from CyA to CyB. After the peptide linker is cleaved by cathepsin B, the
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separation between the fluorescence donor CyA and acceptor CyB leads to strong NIR fluorescence. This probe can be used to visualize the activity of cathepsin B and lysosome membrane permeabilization in cells. Following the cleavage of the peptide linker, a PDT reagent, Cy-S-Ph-NH2, is released; this reagent has a maximum absorption peak at approximately 800 nm and has strong cytotoxicity to cells upon NIR laser irradiation. Given the much higher activity of cathepsin B in tumor cells than normal cells, the probe is expected to exhibit greater phototoxicity to tumor cells. The in vivo bioimaging application and antitumor efficacy of CyA-P-CyB are also evaluated by injecting CyA-P-CyB into tumorbearing mice. 2. Materials and experimental section 2.1. Materials and methods IR-780, 4-aminothiophenol, 3-azidopropan-1-amine and cathepsin B from human liver were obtained from Sigma-Aldrich. Alkyne-Peptide-PABC was purchased from DgPeptides Co.,Ltd (Hangzhou, China), and PEG5000-PLA3000 was purchased from Daigang Biomaterial Co., Ltd (Jinan, China). A detailed description of the synthesis of the probe and intermediates is provided in the Supplementary Methods section. The probe and intermediates
Fig. 1. Illustration of near-infrared imaging and phototherapy of tumor cells triggered by cathepsin B using the probe CyA-P-CyB. The CyA-P-CyB probe is composed of CyA (FRET donor) and CyB (FRET acceptor, quencher). After cleavage of the peptide linker by cathepsin B, the separation between CyA and CyB results in the NIR emission of CyA moiety. The final cleavage product Cy-S-Ph-NH2 has PDT effect and causes strong phototoxicity to tumor cells upon laser irradiation at 808 nm.
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were characterized by 1H NMR, 13C NMR and ESI mass spectrometry.1H NMR and 13C NMR spectra were record using Bruker 300 MHz. Fluorescence emission spectra were obtained using RF5301/PC spectrophotometer (Shimadzu). Cell images were acquired by using a confocal laser scanning microscope (Fluoview 1200, Olympus, Japan). 2.2. Enzymatic activity toward CyA-P-CyB CyA-P-CyB was dissolved in DMSO at an initial concentration of 1 mM. Cathepsin B was first activated in 60 mM DL-dithiothreitol (DTT)/30 mM EDTA (5 mL) for 60 min at room temperature and then incubated with CyA-P-CyB in reaction buffer (25 mM acetate, 1.0 mM EDTA, pH 5.0, and 500 mM GSH) for different time intervals; the final concentrations of CyA-P-CyB and cathepsin B were 10 mM and 10 mg/mL, respectively. The enzymatic reaction was monitored by measuring emission with a fluorescence spectrometer at an excitation wavelength of 650 nm. The inhibition experiment was performed by adding 100 mM calpain inhibitor I under the same conditions. 2.3. Cell imaging by confocal microscopy Cells were seeded in a 35-mm glass bottomed dishes at a density of 3 105 cells per dish in culture media. After overnight culture, cells were treated with 150 mM of H2O2 for 24 h, 10 mM of camptothecin for 24 h and 30 nM of Calpain inhibitor I for 30 min, respectively. Then incubated with 10 mM of CyA-P-CyB for 2 h and washed with DPBS. 200 nM of Lysotracker (Life Technologies) was used for costaining and the incubation time is 30 min. Fluorescence images were acquired by confocal microscopy. 2.4. Singlet oxygen generation from Cy-S-Ph-NH2 by 808 laser irradiation The fluorescence of DPBF(10 mM) in the aqueous solution containing Cy-S-Ph-NH2 (10 mM) was measured before irradiation with an 808 nm laser under excitation at 410 nm, which is denoted as the fluorescence at time 0 h (t ¼ 0). The solution was then irradiated with 808 nm NIR light at a power of 0.5 W/cm2 for up to 5 min, and the emission spectra (430 nm-600 nm) were collected at 10 s, 30 s, 1 min, 2 min, and 5 min. The control experiment was performed without laser irradiation under the same conditions. 2.5. Cytotoxicity test Cells were seeded in a 96-well plate with culture media. After overnight culture, cells were incubated with samples for 16 h and washed with DPBS. After irradiation with 808 nm NIR laser (2 W/ cm2) for 10 min, cells were incubated another 24 h. To identify cell viability, 0.5 mg/ml of MTT (Sigma) media was added to the cells for 4 h, and the produced formazan was dissolved in 0.1 ml of dimethylsulfoxide (DMSO) and read at OD 650 nm with a Spectramax Microwell plate reader. 2.6. Encapsulation of CyA-P-CyB into PEG-PLA CyA-P-CyB (5 mg) and PEG5000-PLA3000 (45 mg) (weight ratio ¼ 1:9) were completely dissolved in 0.5 mL of CH2Cl2 with stirring and dried by rotary evaporation (2 h) to obtain a thin film. Then, deionized water (10 mL) was added dropwise to form a micellar solution. The size of the PEG-PLA nanoparticles loaded with CyA-P-CyB was measured using a dynamic laser scattering spectrometer.
2.7. Determination of loading efficiency and in vitro release study PEG-PLA (45.0 mg) and CyA-P-CyB (5.0 mg) were dissolved in 3.0 mL of CH2Cl2. After the solution was ultrasonicated for 5 min, CH2Cl2 was removed by rotary evaporation. Then, 10.0 mL of PBS (pH ¼ 7.4) was added dropwise to form a micelles solution. After further ultrasonication for 5 min, the drug-loaded micelles were centrifuged (7000 g, 10 min), and the supernatant was collected. The supernatant sample was used to measure drug loading by collecting UVevis absorption value at 790 nm. The loading efficiency (LE) was calculated from the total concentration of the added amount of CyA-P-CyB present in the PEG-PLA and the concentration of that in the supernatant using the following equation [30]: LE (%) ¼ ([CyA-P-CyB]T e [CyA-P-CyB]s)/[CyA-P-CyB]T 100 The in vitro drug release profiles were studied in 50 mM PBS (containing 30% DMSO) at pH 7.4. The CyA-P-CyB-loaded micelles (containing 0.47 mg CyA-P-CyB) were dissolved in 4.0 mL of PBS and enclosed with a dialysis bag (MWCO 3500 Da). The dialysis bag was placed in 40.0 mL of PBS (containing 30% DMSO) at 37 C under constant shaking at 100 rpm. 1.5 mL of external release medium was withdrawn at chosen time intervals, and then 1.5 mL of fresh PBS (containing 30% DMSO) was added. The amount of released CyA-P-CyB was quantified using absorption at 790 nm. The percent cumulative release was calculated using the following formula [31]:
Present cumulative release ðQ %Þ ¼
! P Cn V þ Vi n1 i¼0 Ci 100 weight of drug
where Cn is the sample concentration at Tn, V the total volume of release medium, Vi the sampling volume at Ti, Ci the sample concentration at Ti (both V0 and C0 are equal to zero). 2.8. Analysis of the NIR fluorescence of CyA-P-CyB in vivo All animal procedures were approved by the Institutional Animal Care Committee at Yonsei University. H640 lung cancer cells (1.0 107 cells per mouse) were subcutaneously injected into male BALB/c nude mice. When the tumor volumes reached approximately 100 mm3, PEG-PLA/CyA-P-CyB (2 mg/kg) was intravenously injected into the BALB/c nude mice. The NIR images were captured at 0, 1, 2, 4, 8, 11, 24, 32 and 48 h after the injection of PEG-PLA/CyAP-CyB using an animal optical imaging system (IVIS, Caliper Life Sciences). 2.9. Phototherapeutic efficacy of CyA-P-CyB in vivo H640 lung cancer cells (1.0 107 cells per mouse) were subcutaneously injected into male BALB/c nude mice. When the tumor volumes reached 100e200 mm3, the mice were intravenously injected with PEG-PLA/CyA-P-CyB (5 mg/kg, n ¼ 3e6) daily for 4 days as the test group. Mice treated with an equal volume of PEGPLA (n ¼ 3) were used as the control group. Twenty-four hours after the PEG-PLA/CyA-P-CyB injection, the mice were irradiated with an 808 nm laser (1 W/cm2 for 5 min). Mice bearing A549 lung adenocarcinoma tumor (injection of 1.0 107 cells per mice) at 100e200 mm3 were intravenously injected by PEG-PLA/CyA-P-CyB (5 mg/kg, n ¼ 4) daily for 2 days as the test group. Then the mice were irradiated with an 808 nm laser (1 W/cm2 for 5 min) at an interval of 11 h after each injection. Tumor diameters were measured using a Vernier caliper. The volume (V) of the tumor was estimated using the following formula, V ¼ p x (W x H x D)/6
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(where W is the shortest diameter of the tumor, H is the longest diameter of the tumor and D is the depth of the tumor). The relative tumor volumes were calculated as V/V0 (V0 is the initial tumor volume when the treatment began). The data are represented as the mean ± SEM. Multiple groups were compared using ANOVA. Statistical significance was set at P < 0.05. 2.10. Tests of CyA-P-CyB toxicity in vivo Male BALB/c nude mice were intravenously injected with equal volumes of PEG-PLA (n ¼ 3) or PEG-PLA/CyA-P-CyB (20 mg/kg, n ¼ 3e6). Male BALB/c nude mice (n ¼ 3e6) were injected with PEG-PLA/CyA-P-CyB (50 mg/kg, 200 mL per mouse) daily for 4 days in the tail vein. BALB/c nude mice (n ¼ 3) injected with an equal volume of PEG-PLA were used as the control group. After 14 days, the mice were euthanized. The lung, liver, heart, kidney, pancreas, spleen, and brain were collected, fixed in formalin, and cell proliferation and apoptosis were analyzed histopathologically using H&E staining and immunohistochemistry. For the proliferation assay, sections from the paraffin-embedded tissues were deparaffinized, rehydrated, and then antigen-retrieved. After blocking endogenous peroxidases and proteins, the tissues were incubated with an antiKi-67 antibody (Abcam, USA), followed by a secondary rabbit IgG (Dako, Denmark), and detected with DAKO Envision þ System-HRP DAB (Dako, Denmark). The sections were counterstained with hematoxylin (Sigma-Aldrich, USA). A TUNEL assay kit (Life Technologies, USA) was used for the apoptosis assay. The nuclei were visualized by DAPI staining. 3. Results and discussion CyA bearing an azido group was obtained, and then CyB was linked to an alkyne-bearing peptide at its terminus (Alkyne-GlyPhe-Leu-Gly) via a p-aminobenzyloxycarbonyl bridge to synthesize the CyA-P-CyB probe. After a “click chemistry” reaction catalyzed by Cuþ, the CyA moiety was conjugated to CyB with a cathepsin Bactivated peptide spacer to afford the target product CyA-P-CyB (Scheme S1). CyA-P-CyB and its intermediates were purified by silica gel column chromatography, and their structures were identified by 1H NMR, 13C NMR and ESI-MS (Fig. S1eS12). The large spectral overlap between Cy-N3 emission and Cy-SPh-NH2 absorption (Fig. 2a) suggested that the CyB moiety was an effective acceptor for the CyA donor. CyB does not emit
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fluorescence, thus acting as a dark quencher for the FRET system. The changes in fluorescence of a mixed solution containing CyA-PCyB and activated cathepsin B were monitored to confirm the specific cleavage of CyA-P-CyB by cathepsin B. An obvious increase in fluorescence intensity at 720 nm attributed to the emission of CyA was detected after the mixed solution was incubated at 37 C (Fig. 2b). The fluorescence assay exhibited increased NIR fluorescence in a time-dependent manner. There was a 21-fold increase in the fluorescence signal from the enzyme-activated probe compared with the unactivated probe after an 8 h incubation. The increase in fluorescence was not observed in the reaction solution containing a cathepsin B inhibitor (Fig. S13), indicating that cathepsin B is needed to cleave peptide linker (Gly-Phe-Leu-Gly). In some previous reports, the short peptide (Gly-Phe-Leu-Gly) has been used as a cleavable linker to design of cathepsin B-cleavable prodrugs for targeted cancer therapy [28,29]. We investigated the enzyme cleavage product of CyA-P-CyB by MALDI-TOF, and the result showed that the product Cy-S-Ph-NH2 (628.3641 m/z) generated (Fig. S14a) after the enzyme cleavage reaction was carried out for 4 h. Similarly, we also investigated the enzyme cleavage product of Alkyne-Peptide-PABC-CyB (the structure shown in Scheme S1) by MALDI-TOF. The peak of the cleavage product Cy-S-Ph-NH2 can also be observed (Fig. S14b). These results indicated that cathepsin B catalyzed the cleavage of peptide (Gly-Phe-Leu-Gly) linker of the probe. Given the marked increase in the NIR fluorescence of CyA-P-CyB in the presence of cathepsin B, the probe was further used to monitor the activity and location of cathepsin B. Because cathepsin B is primarily located in lysosomes in tumor cells, when HeLa cells were pretreated with 10 mM CyA-P-CyB for 2 h, we can observe the released NIR emission in intact lysosomes, where LysoTracker also emits blue fluorescence, which showed that CyA-P-CyB can image cathepsin B in lysosomes in cells (Fig. 3a). Lysosomes are cytoplasmic, membrane-enclosed organelles that contain hydrolytic enzymes, including proteases, lipases, nucleases, and glycosidases. Studies have shown that lysosomal rupture is an early event in various apoptotic processes, including apoptosis initiated by reactive oxygen species, lysosomotropic compounds with detergent activity, and some endogenous cell death effectors [32e34]. Upon lysosome membrane permeabilization (LMP), a large amount of hydrolytic enzymes are released from the lysosomal lumen to the cytosol, leading to rapid cell death. After the lysosome membrane is destroyed by ROS and antitumor drugs, a large amount of
Fig. 2. The overlap between the FRET donor and acceptor and cathepsin B-induced increase in CyA-P-CyB emission. (a) The absorption spectrum of Cy-S-Ph-NH2 (red) and the emission spectrum of Cy-N3 (black) are shown (lex ¼ 650 nm). (b) Changes in the emission spectra of CyA-P-CyB (10 mM) after incubation with cathepsin B in buffered solution (25 mM acetate, 1.0 mM EDTA, pH 5.0, and 500 mM GSH) for different times (lex ¼ 650 nm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Fluorescence imaging of CyA-P-CyB in the cell. HeLa cells were pretreated with a blank (a), 150 mM of H2O2 for 24 h (b), 10 mM of camptothecin for 24 h (c) or 30 nM of calpain inhibitor I for 30 min (d) and then were incubated with 10 mM of CyA-P-CyB for 2 h. The cells were washed with DPBS, and fluorescence images were acquired by confocal microscopy. LysoTracker was used to co-stain the cells. Top: CyA-P-CyB (ex. 635 nm/em. 655e755 nm), middle: LysoTracker (ex. 405 nm/em. 430e455 nm), bottom: merged with DIC. Scale bar: 10 mm.
hydrolytic enzymes will be released from the lysosomal lumen to the cytosol. When the cells were treated with 150 mM H2O2 (ROS) or camptothecin (CPT), which acted as a DNA topoisomerase I inhibitor to cause DNA damage, a slight increase in fluorescence and diffused fluorescence in the cytosol were observed (Fig. 3b and c),
indicating that LMP was induced by H2O2 or CPT, and cathepsin moved from the lysosomal lumen to the cytosol. Thus, the present CyA-P-CyB probe is capable of identifying the location of cathepsin B and monitoring the LMP process. In addition, when HeLa cells were pre-treated with calpain inhibitor I before incubation with
Fig. 4. Singlet oxygen generation and assay of Cy-S-Ph-NH2 following 808 laser irradiation. (a) The fluorescence spectra of DPBF (10 mM) in the presence of Cy-S-Ph-NH2 (10 mM) at different irradiation times using an 808 nm laser (0.5 w cm2) (lex ¼ 410 nm). (b) The fluorescence spectra of DPBF (10 mM) in the presence of Cy-S-Ph-NH2 (10 mM) without laser irradiation.
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CyA-P-CyB, NIR fluorescence was not observed (Fig. 3d), indicating that the NIR emission resulted from the cleavage of CyA-P-CyB by cathepsin B. As described above, the CyB moiety was cleaved to liberate CyS-Ph-NH2 in the presence of cathepsin B (Fig. 1). Moreover, Cy-SPh-NH2 can act as an effective PDT agent, leading to the apoptosis of tumor cells following NIR laser irradiation. 1,3Diphenylisobenzofuran (DPBF) was used as a singlet oxygen trap to verify the generation of singlet oxygen. As shown in Fig. 4a, after the solution containing Cy-S-Ph-NH2 and DPBF was irradiated with an 808 nm laser, the fluorescence of DPBF at 480 nm gradually decreased as the irradiation time increased. In contrast, if the mixture of Cy-S-Ph-NH2 and DPBF did not undergo laser
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irradiation, no obvious change was observed (Fig. 4b). These results demonstrated the generation of singlet oxygen in the solution of Cy-S-Ph-NH2 upon NIR laser irradiation. MTT assays showed that CyA-P-CyB was phototoxic to tumor cells, including U87-MG and A549 cells, in a concentrationdependent manner (Fig. 5a and b). A 10 min irradiation with the 808 nm laser caused a significant increase in cytotoxicity. In the presence of 10 mM CyA-P-CyB, the viability of the U87-MG cells decreased from more than 60% to less than 20% upon laser irradiation (808 nm) (Fig. 5a). A similar result was observed in A549 cells (Fig. 5b). The laser-enhanced cytotoxicity depended on the cathepsin B activity in tumor cells. When U87 and A549 cells were pretreated with 30 nM calpain inhibitor I (CI) for 30 min and then
Fig. 5. MTT assay of cells grown in the presence of CyA-P-CyB with or without laser irradiation and the cathepsin B inhibitor I (CI). a and b: Cytotoxic effect of CyA-P-CyB with or without 808 nm laser irradiation. U87 cells (a) and A549 cells (b) were incubated with different concentrations of CyA-P-CyB for 16 h, then CyA-P-CyB was removed. The cells were then irradiated with an 808 nm laser (2 W cm2, 10 min) and cultured for another 24 h c and d: Cytotoxicity was inhibited by the cathepsin B inhibitor. U87 cells (c) and A549 cells (d) were pretreated with 30 nM calpain inhibitor I for 30 min, incubated with 5 mM CyA-P-CyB for 16 h, and then CyA-P-CyB was removed. The cells were then irradiated with an 808 nm laser (2 W/cm2, 10 min) and cultured for another 24 h e: Comparisons of the cytotoxicity toward normal cells and cancer cells. The cells were incubated with 5 mM CyA-PCyB for 16 h, and the CyA-P-CyB was removed. The cells were irradiated with an 808 nm laser (2 W cm2, 10 min) and cultured for another 24 h. The results are expressed as the mean ± standard deviation of three independent experiments.
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Fig. 6. HeLa Cells were incubated with 10 mM alkyne-Peptide-PABC-CyB without (a, b, e, f, I, j) or with (c, d, g, h, k, l) 30 nM calpain inhibitor I for 2 h. After washing with DPBS, cells were irradiated with 808 nm laser (2 W cm2, 10 min) or not and acquired fluorescence images. Calpain inhibitor I was pretreated for 30 min and costained with Lysotracker blue. Blue: ex. 405 nm/em. 430e455 nm (a, b, c, d), green: ex. 473 nm/em 490e590 nm (e, f, g, h), merged with DIC (i, j, k, l). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
incubated with 5 mM CyA-P-CyB for 16 h, the viability of U87 cells increased from 20.6% to 48.5% (Fig. 5c) and that of A549 cells increased from 30.2% to 57.9% upon laser irradiation (Fig. 5d), indicating that the phototoxicity of CyA-P-CyB toward tumor cells depends on the activity of cathepsin B. In other words, cathepsin B may trigger tumor cell death in response to NIR laser irradiation. The cathepsin B-triggered phototoxicity indicated that CyA-P-CyB had significantly different cytotoxicity toward normal cells and tumor cells. As shown in Fig. 5e, when normal cells, including IMR90, NIH-3T3 and W38-VA13 cells, were incubated with 5 mM CyAP-CyB for 16 h and irradiated with an 808 nm laser for 10 min, the cell survival rate was over 50%. However, for the tumor cells, such as HeLa, MCF-7, U87-MG, SK-Hep1 and A549 cells, subjected to the same treatments, the cell viability decreased to 18.8% for HeLa cells, 12.2% for MCF-7 cells, 27.9% for U87-MG cells, 22.1% for SK-HEP1 cells and 31.4% for A549 cells. The specific phototoxicity of CyA-PCyB toward tumor cells can be attributed to the fact that the cathepsin B levels are higher in tumor cells than in normal cells [35e37]. Based on the singlet oxygen generation assay and photothermal assay, CyA-P-CyB itself can also produce ROS (Fig. S15). However,
increased phototoxicity was only observed when CyA-P-CyB was cleaved by cathepsin B to form Cy-S-Ph-NH2. We speculated that the initial CyA-P-CyB probe was restricted to the lysosome, mostly due to the peptide linker, leading to a high concentration of CyA-PCyB in a local space. Local high concentrations of PDT agents will lead to the inner filter effect and the quenching of photo-excited species by collision with non-excited PDT agents or the degradation products from the laser irradiation. The inner filter effect and the quenching of photo-excited species further inhibits the generation of singlet oxygen, thus improving the viability of tumor cells [26,38]. After CyA-P-CyB was cleaved by cathepsin B, the product Cy-S-Ph-NH2 diffused, and the local concentration of the cyanine dye decreased, which is in favor of the generation of singlet oxygen, and caused more serious toxicity to tumor cells. This hypothesis was supported by the subsequent verification experiments. The CyB moiety can be activated by irradiation with an 808 nm laser to emit green fluorescence from the degradation products, which is related to the activity of singlet oxygen. When Alkyne-PeptidePABC-CyB was used as a tracer, strong and diffused green fluorescence was observed in both the cytoplasm and lysosome after irradiation with an 808 nm laser (Fig. 6). In contrast, when
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cathepsin B activity was inhibited with calpain inhibitor I, much weaker fluorescence was observed after irradiation with an 808 nm laser, indicating that the uncleaved Alkyne-Peptide-PABC-CyB was maintained in lysosomes, thus producing less singlet oxygen and less damage to the cell. Therefore, the increased phototoxicity of CyA-P-CyB in tumor cells is related to the diffusion of the cleavage product Cy-S-Ph-NH2. The specific phototoxicity toward tumor cells inspired us to further investigate the phototherapeutic efficacy of CyA-P-CyB in vivo. In vivo NIR fluorescence imaging was performed on H640 tumor-bearing mice. To improve the water solubility and slow the blood clearance, CyA-P-CyB was encapsulated into PEG-PLA micelles (PEG-PLA/CyA-P-CyB). The PEG-PLA micelles loaded with CyA-P-CyB had an average size of 230 nm and a polydispersion index (PDI) of 0.461 (Fig. S16). The loading efficiency of CyA-P-CyB to PEG-PLA was calculated to be 46.7%, and the in vitro release behavior was studied by dialysis in 40.0 mL of PBS (containing 30% DMSO) at 37 C. The results showed that 70% of CyA-P-CyB was released in the first 5 h (Fig. S17). The strong fluorescent in the tumor region was observed at 4 h after the intravenous injection of
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PEG-PLA/CyA-P-CyB, and remained high fluorescent intensity until 32 h, indicating that CyA-P-CyB can accumulate in tumors (Fig. 7a, lower panels). In contrast, treatment with PEG-PLA did not produce any meaningful fluorescence signals (Fig. 7a, upper panels). When the tumor volume reached approximately 100e200 mm3, the mice bearing H640 tumors were intravenously four times injected with PEG-PLA/CyA-P-CyB (5 mg/kg) in 4 days (once injection per day) as the test group. Mice treated with an equal volume of PEG-PLA were used as the control group. Twenty-four hours after the last injection, the mice were irradiated with an 808 nm laser (1 W/cm2 for 5 min). The tumors in the mice treated with PEG-PLA/CyA-P-CyB and laser irradiation were significantly smaller than the tumors in the mice treated with PEG-PLA injection alone, PEG-PLA injection with laser irradiation, or PEG-PLA/CyA-P-CyB injection without laser irradiation (Fig. 7b). The tumor volumes in mice treated with PEG-PLA/CyA-P-CyB injection and laser irradiation were decreased to one-third of the volumes of the other three groups on the 14th day (Fig. 7c). We also investigated the phototherapeutic efficacy of CyA-P-CyB in vivo to A549 tumor-bearing mice, and similar tumorsuppression effect was observed (Fig. S18). These results suggest
Fig. 7. NIR fluorescence imaging of CyA-P-CyB and phototherapeutic efficacy of CyA-P-CyB in vivo. (a) NIR images of tumor-bearing mice were collected over 48 h after the intravenous injections of PEG-PLA/CyA-P-CyB. The tumors are circled with a dotted line. (b) Tumor growth in mice bearing H640 tumors after administration of the indicated treatments. The tumor volumes were normalized to their initial values. (The red arrows represent the day of the injection of PEG-PLA/CyA-P-CyB, and the blue arrow represents the day of laser irradiation. The animals were treated with 5 mg/kg PEG-PLA/CyA-P-CyB daily for 4 days (20 mg kg1)). The data are expressed as the mean ± SEM (n ¼ 3e6). *P < 0.05, compared to the PEG-PLA with laser group. (c) Photos of mice bearing H640 tumors on day 14 after administration of the indicated treatments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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that CyA-P-CyB has notable phototherapeutic efficacy in suppressing tumor growth. We checked the tumor site temperature after injection of PEG-PLA or PEG-PLA/CyA-P-CyB followed by laser irradiation (808 nm), and the results showed temperature increase by 3 C and 7 C for the cases with the injection of PEG-PLA and PEG-PLA/CyA-P-CyB, respectively (Fig. S19). The temperature increase of tumor with the treatment of PEG-PLA/CyA-P-CyB during laser irradiation (5 min) was not enough for photothermal ablation. Therefore, the major therapeutic effect should be due to photodynamic effect rather than photothermal ablation. The toxicity of CyA-P-CyB was assessed in mice that were treated with PEG-PLA/CyA-P-CyB or PEG-PLA via the tail vein. Mice
in the PEG-PLA/CyA-P-CyB group (20 mg kg1) did not die and survived for over 16 days. Seven organs (lung, liver, heart, kidney, pancreas, spleen and brain) were acquired from the mice on the 14th day following injection with PEG-PLA or PEG-PLA/CyA-P-CyB (20 mg kg1) and assessed using H&E staining (Fig. 8a). There were no distinct lesions (no necrosis, edema, inflammatory infiltration, or hyperplasia) in the sections of the seven tissues. These histological results indicate that at the dose used here, CyA-P-CyB does not have cytotoxic effects on mice. We also analyzed cell proliferation and apoptosis in the tumor masses treated with PEG-PLA with or without laser and PEG-PLA/CyA-P-CyB with or without laser (Fig. 8b). Cell proliferation was tested by anti-Ki-67 staining and
Fig. 8. Toxicity of CyA-P-CyB in vivo and the effects of CyA-P-CyB on proliferation and apoptosis in vivo. (a) Histological analysis of the organs (lung, liver, heart, kidney, pancreas, spleen and brain) acquired from mice on the 14th day after the injection with PEG-PLA or PEG-PLA/CyA-P-CyB (20 mg/kg). (b) Proliferation and apoptosis of the tumors. Proliferation was determined by staining the tissues with the Ki-67 antibody and is shown as brown signals. The nuclei were counterstained with hematoxylin. Apoptosis was determined using the TUNEL assay. Green represents a positive signal. The nuclei were visualized by DAPI staining (blue). (c) Quantification of the TUNEL assay. Positive signals were counted in five frames per slide. The data are expressed as the means ± SEM (n ¼ 3e6). **P < 0.01, compared to the control (PEG-PLA), control (PEG-PLA) with laser or PEG-PLA/CyAP-Cy B groups, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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was dramatically decreased in the group treated with PEG-PLA/ CyA-P-CyB and the laser, whereas cell proliferation was comparatively strong in the other three groups (Fig. 8b, left panels). In the apoptosis assay using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, the group treated with PEGPLA/CyA-P-CyB and the laser showed an increase in the apoptotic, green signal (Fig. 8b, right panels) and a significant increase in the number of apoptotic cells compared with the other three groups in the quantitative analysis (Fig. 8c). These results suggest that the treatment with PEG-PLA/CyA-P-CyB and the laser substantially affected tumor cell apoptosis. Together, our results indicate that the treatment with PEG-PLA/CyA-P-CyB and the laser affected cell proliferation and apoptosis in the tumor mass. 4. Conclusions In conclusion, a cathepsin B-activated fluorescent probe, CyA-PCyB, was designed and constructed based on FRET between a fluorescent cyanine unit and a non-fluorescent cyanine acceptor that were covalently coupled through a specific peptide substrate linker. As the key moiety, CyB based on Cy-S-Ph-NH2 serves as two key functions owing to the great absorption in NIR region (650e800 nm), weak emission and high phototoxicity. One is as a quencher, which may tune the NIR fluorescence of CyA based on the FRET effect. The other role is as a PDT agent to realize the phototherapy with NIR irradiation. In vitro cell-based studies show that the probe can be used to visualize the location of cathepsin B and monitor the LMP process during apoptosis. Furthermore, the probe exhibited specific phototoxicity toward tumor cells. In vivo imaging of tumors and the antitumor efficacy of the probe in mice bearing tumors indicated the present probe was capable of being used for NIR imaging in mice and phototherapy for tumors in vivo. Most importantly, the deposition of CyA-P-CyB in the tumor can be directly visualized by the release of NIR fluorescence; thus, we can choose the optimum irradiation time using the fluorescence intensity in the tumor. As expected, the tumor growth in mice was markedly inhibited upon treatment with CyA-P-CyB encapsulated in PEG-PLA and laser irradiation. CyA-P-CyB had no cytotoxic effects on mice, based on the histological assessment, and the treatment with PEG-PLA/CyA-P-CyB and laser irradiation inhibited cell proliferation and induced apoptosis in the tumor mass. All these results indicate that CyA-P-CyB is a truly efficient phototherapy reagent. Acknowledgments This research was supported financially by grants from the National Creative Research Initiative Program (2012R1A3A2048814). It was also supported by the Korea Mouse Phenotyping Center (2016M3A9D5A01952416), and the Brain Korea 21 PLUS Project for Medical Science, Yonsei University, the Bio and Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF2013M3A9D5072551). X. Chen acknowledges funding from the National Natural Science Foundation of China (21376117), the Jiangsu Natural Science Funds for Distinguished Young Scholars (BK20140043), and the Natural Science Foundation of Jiangsu Higher Education Institutions of China (14KJA150005). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2017.01.020.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
In vivo near-infrared imaging and phototherapy of tumors using a cathepsin B-activated fluorescent probe Xiaoqiang Chen a, b, 1, Dayoung Lee a, 1, Sungsook Yu c, 1, Gyoungmi Kim a, Songyi Lee a, Yejin Cho c, Haengdueng Jeong c, Ki Taek Nam c, **, Juyoung Yoon a, * a
Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, South Korea State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemical Engineering, Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing 210009, China c Severance Biomedical Science Institute, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, South Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2016 Received in revised form 13 January 2017 Accepted 14 January 2017 Available online 16 January 2017
The development of multifunctional reagents for simultaneous specific near-infrared (NIR) imaging and phototherapy of tumors is of great significance. This work describes the design of a cathepsin B-activated fluorescent probe (CyA-P-CyB) and its applications as an NIR imaging probe for tumor cells and as a phototherapy reagent for tumors. In vitro experiments demonstrated that CyA-P-CyB was activated via the cleavage of a peptide linker by cathepsin B in tumor cells to produce fluorescence in the NIR region based on a FRET mechanism. MTT assays showed that the phototoxicity of CyA-P-CyB toward cells depended on the activity of cathepsin B, and the probe exhibited specific phototoxicity toward tumor cells. CyA-P-CyB was also successfully applied to the in vivo imaging and phototherapy of tumors. Histological analysis indicated that CyA-P-CyB had no cytotoxic effects on seven mouse tissues (lung, liver, heart, kidney, pancreas, spleen and brain) after the CyA-P-CyB treatment and laser irradiation. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Specific phototherapy NIR bioimaging Enzyme-activated fluorescence Multiple functional probe
1. Introduction Fluorescent probes have become powerful tools that allow biologists to study biological processes because they can provide visual information in real time with high spatial resolution [1]. In particular, probes with near-infrared (NIR) emission (650e900 nm) are preferable for in vivo imaging. Compared to the conventional probes with shorter emission wavelengths, the NIR fluorescence probes have unique advantages, such as that the NIR emission can avoid the interference from auto-fluorescence of indigenous biomolecules, achieving a higher signal-to-noise ratio; NIR fluorescent probes can achieve imaging in deeper tissues because of their longer excitation wavelength; and NIR excitation and emission cause less damage to biological samples than the shorter wavelengths of light. In the past two decades, a number of NIR fluorescence probes have been developed to monitor various biological species and drug release in vivo and in vitro [2,3].
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (K.T. Nam), [email protected] (J. Yoon). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2017.01.020 0142-9612/© 2017 Elsevier Ltd. All rights reserved.
The development of multifunctional reagents that can enable both specific imaging and tumor therapy is of great significance due to that they provide the information on where (W), when (W), and how (H) drugs are delivered and activated in vivo [4,5]. Phototherapy is a mild and noninvasive approach for cancer treatment [6,7]. In this treatment modality, with the assistance of the photosensitizer or photothermal agent, light is absorbed and transferred to reactive oxygen species (ROS) or causes local hyperthermia, inducing tumor cell death [8,9]. The NIR light-induced PDT is preferable to shorter-wavelength light due to its deeper tissue penetration [10e12]. To date, the most suitable NIRphotosensitizable molecule is indocyanine green dye (ICG) [13,14]. ICG is an NIR dye that has been approved for clinical use by the U.S. Food and Drug Administration (FDA). However, ICG itself is not specific for tumor cells, indicating that phototherapy with ICG would damage normal cells or tissues upon NIR laser irradiation. Therefore, phototherapy agents that cause specific and efficient damage to the targeted cancer cells rather than the normal cells are preferable. Cathepsin B, one of lysosomal proteases, plays an important role in the regulation of angiogenesis and invasion during cancer progression [15,16]. Some antitumor prodrugs, cleaved by cathepsin B, which is highly upregulated in malignant
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tumors and premalignant lesions, has been developed to achieve a higher drug concentration in tumor tissue and a lower concentration in normal tissue, resulting in enhance the efficacy and reduce toxicity to normal cells [17,18]. Though some excellent cathepsin Bactivated imaging probes have been reported for the evaluation of enzyme activity and the diagnosis of cancer [19e27], few of them based on cathepsin B-activated reaction were explored as multifunctional reagents that can enable both specific imaging and targeted tumor therapy [26,27]. Herein, we describe a new NIR fluorescent probe (CyA-P-CyB) that possesses multiple properties, including specific NIR imaging, specific phototoxic effects on tumor cells, and a phototherapeutic effect in mice bearing tumors. Compared to the protease-trigged antitumor PDT agents [26,27], the selective antitumor activity of the present probe come from the effective diffusion of cleavage product (PDT agent) in tumor cells. The probe is designed based on a fluorescence resonance energy transfer (FRET) mechanism. As shown in Fig. 1, the probe (CyA-P-CyB) features two cyanine moieties (CyA and CyB) linked via a cathepsin B-activated peptide(GlyPhe-Leu-Gly), which has been used in design of cathepsin Bcleavable prodrugs for targeted cancer therapy [28,29]. CyA acts as a fluorescent donor, and CyB acts as a quencher. The initial form of the probe does not emit fluorescence due to the efficient FRET from CyA to CyB. After the peptide linker is cleaved by cathepsin B, the
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separation between the fluorescence donor CyA and acceptor CyB leads to strong NIR fluorescence. This probe can be used to visualize the activity of cathepsin B and lysosome membrane permeabilization in cells. Following the cleavage of the peptide linker, a PDT reagent, Cy-S-Ph-NH2, is released; this reagent has a maximum absorption peak at approximately 800 nm and has strong cytotoxicity to cells upon NIR laser irradiation. Given the much higher activity of cathepsin B in tumor cells than normal cells, the probe is expected to exhibit greater phototoxicity to tumor cells. The in vivo bioimaging application and antitumor efficacy of CyA-P-CyB are also evaluated by injecting CyA-P-CyB into tumorbearing mice. 2. Materials and experimental section 2.1. Materials and methods IR-780, 4-aminothiophenol, 3-azidopropan-1-amine and cathepsin B from human liver were obtained from Sigma-Aldrich. Alkyne-Peptide-PABC was purchased from DgPeptides Co.,Ltd (Hangzhou, China), and PEG5000-PLA3000 was purchased from Daigang Biomaterial Co., Ltd (Jinan, China). A detailed description of the synthesis of the probe and intermediates is provided in the Supplementary Methods section. The probe and intermediates
Fig. 1. Illustration of near-infrared imaging and phototherapy of tumor cells triggered by cathepsin B using the probe CyA-P-CyB. The CyA-P-CyB probe is composed of CyA (FRET donor) and CyB (FRET acceptor, quencher). After cleavage of the peptide linker by cathepsin B, the separation between CyA and CyB results in the NIR emission of CyA moiety. The final cleavage product Cy-S-Ph-NH2 has PDT effect and causes strong phototoxicity to tumor cells upon laser irradiation at 808 nm.
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were characterized by 1H NMR, 13C NMR and ESI mass spectrometry.1H NMR and 13C NMR spectra were record using Bruker 300 MHz. Fluorescence emission spectra were obtained using RF5301/PC spectrophotometer (Shimadzu). Cell images were acquired by using a confocal laser scanning microscope (Fluoview 1200, Olympus, Japan). 2.2. Enzymatic activity toward CyA-P-CyB CyA-P-CyB was dissolved in DMSO at an initial concentration of 1 mM. Cathepsin B was first activated in 60 mM DL-dithiothreitol (DTT)/30 mM EDTA (5 mL) for 60 min at room temperature and then incubated with CyA-P-CyB in reaction buffer (25 mM acetate, 1.0 mM EDTA, pH 5.0, and 500 mM GSH) for different time intervals; the final concentrations of CyA-P-CyB and cathepsin B were 10 mM and 10 mg/mL, respectively. The enzymatic reaction was monitored by measuring emission with a fluorescence spectrometer at an excitation wavelength of 650 nm. The inhibition experiment was performed by adding 100 mM calpain inhibitor I under the same conditions. 2.3. Cell imaging by confocal microscopy Cells were seeded in a 35-mm glass bottomed dishes at a density of 3 105 cells per dish in culture media. After overnight culture, cells were treated with 150 mM of H2O2 for 24 h, 10 mM of camptothecin for 24 h and 30 nM of Calpain inhibitor I for 30 min, respectively. Then incubated with 10 mM of CyA-P-CyB for 2 h and washed with DPBS. 200 nM of Lysotracker (Life Technologies) was used for costaining and the incubation time is 30 min. Fluorescence images were acquired by confocal microscopy. 2.4. Singlet oxygen generation from Cy-S-Ph-NH2 by 808 laser irradiation The fluorescence of DPBF(10 mM) in the aqueous solution containing Cy-S-Ph-NH2 (10 mM) was measured before irradiation with an 808 nm laser under excitation at 410 nm, which is denoted as the fluorescence at time 0 h (t ¼ 0). The solution was then irradiated with 808 nm NIR light at a power of 0.5 W/cm2 for up to 5 min, and the emission spectra (430 nm-600 nm) were collected at 10 s, 30 s, 1 min, 2 min, and 5 min. The control experiment was performed without laser irradiation under the same conditions. 2.5. Cytotoxicity test Cells were seeded in a 96-well plate with culture media. After overnight culture, cells were incubated with samples for 16 h and washed with DPBS. After irradiation with 808 nm NIR laser (2 W/ cm2) for 10 min, cells were incubated another 24 h. To identify cell viability, 0.5 mg/ml of MTT (Sigma) media was added to the cells for 4 h, and the produced formazan was dissolved in 0.1 ml of dimethylsulfoxide (DMSO) and read at OD 650 nm with a Spectramax Microwell plate reader. 2.6. Encapsulation of CyA-P-CyB into PEG-PLA CyA-P-CyB (5 mg) and PEG5000-PLA3000 (45 mg) (weight ratio ¼ 1:9) were completely dissolved in 0.5 mL of CH2Cl2 with stirring and dried by rotary evaporation (2 h) to obtain a thin film. Then, deionized water (10 mL) was added dropwise to form a micellar solution. The size of the PEG-PLA nanoparticles loaded with CyA-P-CyB was measured using a dynamic laser scattering spectrometer.
2.7. Determination of loading efficiency and in vitro release study PEG-PLA (45.0 mg) and CyA-P-CyB (5.0 mg) were dissolved in 3.0 mL of CH2Cl2. After the solution was ultrasonicated for 5 min, CH2Cl2 was removed by rotary evaporation. Then, 10.0 mL of PBS (pH ¼ 7.4) was added dropwise to form a micelles solution. After further ultrasonication for 5 min, the drug-loaded micelles were centrifuged (7000 g, 10 min), and the supernatant was collected. The supernatant sample was used to measure drug loading by collecting UVevis absorption value at 790 nm. The loading efficiency (LE) was calculated from the total concentration of the added amount of CyA-P-CyB present in the PEG-PLA and the concentration of that in the supernatant using the following equation [30]: LE (%) ¼ ([CyA-P-CyB]T e [CyA-P-CyB]s)/[CyA-P-CyB]T 100 The in vitro drug release profiles were studied in 50 mM PBS (containing 30% DMSO) at pH 7.4. The CyA-P-CyB-loaded micelles (containing 0.47 mg CyA-P-CyB) were dissolved in 4.0 mL of PBS and enclosed with a dialysis bag (MWCO 3500 Da). The dialysis bag was placed in 40.0 mL of PBS (containing 30% DMSO) at 37 C under constant shaking at 100 rpm. 1.5 mL of external release medium was withdrawn at chosen time intervals, and then 1.5 mL of fresh PBS (containing 30% DMSO) was added. The amount of released CyA-P-CyB was quantified using absorption at 790 nm. The percent cumulative release was calculated using the following formula [31]:
Present cumulative release ðQ %Þ ¼
! P Cn V þ Vi n1 i¼0 Ci 100 weight of drug
where Cn is the sample concentration at Tn, V the total volume of release medium, Vi the sampling volume at Ti, Ci the sample concentration at Ti (both V0 and C0 are equal to zero). 2.8. Analysis of the NIR fluorescence of CyA-P-CyB in vivo All animal procedures were approved by the Institutional Animal Care Committee at Yonsei University. H640 lung cancer cells (1.0 107 cells per mouse) were subcutaneously injected into male BALB/c nude mice. When the tumor volumes reached approximately 100 mm3, PEG-PLA/CyA-P-CyB (2 mg/kg) was intravenously injected into the BALB/c nude mice. The NIR images were captured at 0, 1, 2, 4, 8, 11, 24, 32 and 48 h after the injection of PEG-PLA/CyAP-CyB using an animal optical imaging system (IVIS, Caliper Life Sciences). 2.9. Phototherapeutic efficacy of CyA-P-CyB in vivo H640 lung cancer cells (1.0 107 cells per mouse) were subcutaneously injected into male BALB/c nude mice. When the tumor volumes reached 100e200 mm3, the mice were intravenously injected with PEG-PLA/CyA-P-CyB (5 mg/kg, n ¼ 3e6) daily for 4 days as the test group. Mice treated with an equal volume of PEGPLA (n ¼ 3) were used as the control group. Twenty-four hours after the PEG-PLA/CyA-P-CyB injection, the mice were irradiated with an 808 nm laser (1 W/cm2 for 5 min). Mice bearing A549 lung adenocarcinoma tumor (injection of 1.0 107 cells per mice) at 100e200 mm3 were intravenously injected by PEG-PLA/CyA-P-CyB (5 mg/kg, n ¼ 4) daily for 2 days as the test group. Then the mice were irradiated with an 808 nm laser (1 W/cm2 for 5 min) at an interval of 11 h after each injection. Tumor diameters were measured using a Vernier caliper. The volume (V) of the tumor was estimated using the following formula, V ¼ p x (W x H x D)/6
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(where W is the shortest diameter of the tumor, H is the longest diameter of the tumor and D is the depth of the tumor). The relative tumor volumes were calculated as V/V0 (V0 is the initial tumor volume when the treatment began). The data are represented as the mean ± SEM. Multiple groups were compared using ANOVA. Statistical significance was set at P < 0.05. 2.10. Tests of CyA-P-CyB toxicity in vivo Male BALB/c nude mice were intravenously injected with equal volumes of PEG-PLA (n ¼ 3) or PEG-PLA/CyA-P-CyB (20 mg/kg, n ¼ 3e6). Male BALB/c nude mice (n ¼ 3e6) were injected with PEG-PLA/CyA-P-CyB (50 mg/kg, 200 mL per mouse) daily for 4 days in the tail vein. BALB/c nude mice (n ¼ 3) injected with an equal volume of PEG-PLA were used as the control group. After 14 days, the mice were euthanized. The lung, liver, heart, kidney, pancreas, spleen, and brain were collected, fixed in formalin, and cell proliferation and apoptosis were analyzed histopathologically using H&E staining and immunohistochemistry. For the proliferation assay, sections from the paraffin-embedded tissues were deparaffinized, rehydrated, and then antigen-retrieved. After blocking endogenous peroxidases and proteins, the tissues were incubated with an antiKi-67 antibody (Abcam, USA), followed by a secondary rabbit IgG (Dako, Denmark), and detected with DAKO Envision þ System-HRP DAB (Dako, Denmark). The sections were counterstained with hematoxylin (Sigma-Aldrich, USA). A TUNEL assay kit (Life Technologies, USA) was used for the apoptosis assay. The nuclei were visualized by DAPI staining. 3. Results and discussion CyA bearing an azido group was obtained, and then CyB was linked to an alkyne-bearing peptide at its terminus (Alkyne-GlyPhe-Leu-Gly) via a p-aminobenzyloxycarbonyl bridge to synthesize the CyA-P-CyB probe. After a “click chemistry” reaction catalyzed by Cuþ, the CyA moiety was conjugated to CyB with a cathepsin Bactivated peptide spacer to afford the target product CyA-P-CyB (Scheme S1). CyA-P-CyB and its intermediates were purified by silica gel column chromatography, and their structures were identified by 1H NMR, 13C NMR and ESI-MS (Fig. S1eS12). The large spectral overlap between Cy-N3 emission and Cy-SPh-NH2 absorption (Fig. 2a) suggested that the CyB moiety was an effective acceptor for the CyA donor. CyB does not emit
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fluorescence, thus acting as a dark quencher for the FRET system. The changes in fluorescence of a mixed solution containing CyA-PCyB and activated cathepsin B were monitored to confirm the specific cleavage of CyA-P-CyB by cathepsin B. An obvious increase in fluorescence intensity at 720 nm attributed to the emission of CyA was detected after the mixed solution was incubated at 37 C (Fig. 2b). The fluorescence assay exhibited increased NIR fluorescence in a time-dependent manner. There was a 21-fold increase in the fluorescence signal from the enzyme-activated probe compared with the unactivated probe after an 8 h incubation. The increase in fluorescence was not observed in the reaction solution containing a cathepsin B inhibitor (Fig. S13), indicating that cathepsin B is needed to cleave peptide linker (Gly-Phe-Leu-Gly). In some previous reports, the short peptide (Gly-Phe-Leu-Gly) has been used as a cleavable linker to design of cathepsin B-cleavable prodrugs for targeted cancer therapy [28,29]. We investigated the enzyme cleavage product of CyA-P-CyB by MALDI-TOF, and the result showed that the product Cy-S-Ph-NH2 (628.3641 m/z) generated (Fig. S14a) after the enzyme cleavage reaction was carried out for 4 h. Similarly, we also investigated the enzyme cleavage product of Alkyne-Peptide-PABC-CyB (the structure shown in Scheme S1) by MALDI-TOF. The peak of the cleavage product Cy-S-Ph-NH2 can also be observed (Fig. S14b). These results indicated that cathepsin B catalyzed the cleavage of peptide (Gly-Phe-Leu-Gly) linker of the probe. Given the marked increase in the NIR fluorescence of CyA-P-CyB in the presence of cathepsin B, the probe was further used to monitor the activity and location of cathepsin B. Because cathepsin B is primarily located in lysosomes in tumor cells, when HeLa cells were pretreated with 10 mM CyA-P-CyB for 2 h, we can observe the released NIR emission in intact lysosomes, where LysoTracker also emits blue fluorescence, which showed that CyA-P-CyB can image cathepsin B in lysosomes in cells (Fig. 3a). Lysosomes are cytoplasmic, membrane-enclosed organelles that contain hydrolytic enzymes, including proteases, lipases, nucleases, and glycosidases. Studies have shown that lysosomal rupture is an early event in various apoptotic processes, including apoptosis initiated by reactive oxygen species, lysosomotropic compounds with detergent activity, and some endogenous cell death effectors [32e34]. Upon lysosome membrane permeabilization (LMP), a large amount of hydrolytic enzymes are released from the lysosomal lumen to the cytosol, leading to rapid cell death. After the lysosome membrane is destroyed by ROS and antitumor drugs, a large amount of
Fig. 2. The overlap between the FRET donor and acceptor and cathepsin B-induced increase in CyA-P-CyB emission. (a) The absorption spectrum of Cy-S-Ph-NH2 (red) and the emission spectrum of Cy-N3 (black) are shown (lex ¼ 650 nm). (b) Changes in the emission spectra of CyA-P-CyB (10 mM) after incubation with cathepsin B in buffered solution (25 mM acetate, 1.0 mM EDTA, pH 5.0, and 500 mM GSH) for different times (lex ¼ 650 nm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Fluorescence imaging of CyA-P-CyB in the cell. HeLa cells were pretreated with a blank (a), 150 mM of H2O2 for 24 h (b), 10 mM of camptothecin for 24 h (c) or 30 nM of calpain inhibitor I for 30 min (d) and then were incubated with 10 mM of CyA-P-CyB for 2 h. The cells were washed with DPBS, and fluorescence images were acquired by confocal microscopy. LysoTracker was used to co-stain the cells. Top: CyA-P-CyB (ex. 635 nm/em. 655e755 nm), middle: LysoTracker (ex. 405 nm/em. 430e455 nm), bottom: merged with DIC. Scale bar: 10 mm.
hydrolytic enzymes will be released from the lysosomal lumen to the cytosol. When the cells were treated with 150 mM H2O2 (ROS) or camptothecin (CPT), which acted as a DNA topoisomerase I inhibitor to cause DNA damage, a slight increase in fluorescence and diffused fluorescence in the cytosol were observed (Fig. 3b and c),
indicating that LMP was induced by H2O2 or CPT, and cathepsin moved from the lysosomal lumen to the cytosol. Thus, the present CyA-P-CyB probe is capable of identifying the location of cathepsin B and monitoring the LMP process. In addition, when HeLa cells were pre-treated with calpain inhibitor I before incubation with
Fig. 4. Singlet oxygen generation and assay of Cy-S-Ph-NH2 following 808 laser irradiation. (a) The fluorescence spectra of DPBF (10 mM) in the presence of Cy-S-Ph-NH2 (10 mM) at different irradiation times using an 808 nm laser (0.5 w cm2) (lex ¼ 410 nm). (b) The fluorescence spectra of DPBF (10 mM) in the presence of Cy-S-Ph-NH2 (10 mM) without laser irradiation.
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CyA-P-CyB, NIR fluorescence was not observed (Fig. 3d), indicating that the NIR emission resulted from the cleavage of CyA-P-CyB by cathepsin B. As described above, the CyB moiety was cleaved to liberate CyS-Ph-NH2 in the presence of cathepsin B (Fig. 1). Moreover, Cy-SPh-NH2 can act as an effective PDT agent, leading to the apoptosis of tumor cells following NIR laser irradiation. 1,3Diphenylisobenzofuran (DPBF) was used as a singlet oxygen trap to verify the generation of singlet oxygen. As shown in Fig. 4a, after the solution containing Cy-S-Ph-NH2 and DPBF was irradiated with an 808 nm laser, the fluorescence of DPBF at 480 nm gradually decreased as the irradiation time increased. In contrast, if the mixture of Cy-S-Ph-NH2 and DPBF did not undergo laser
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irradiation, no obvious change was observed (Fig. 4b). These results demonstrated the generation of singlet oxygen in the solution of Cy-S-Ph-NH2 upon NIR laser irradiation. MTT assays showed that CyA-P-CyB was phototoxic to tumor cells, including U87-MG and A549 cells, in a concentrationdependent manner (Fig. 5a and b). A 10 min irradiation with the 808 nm laser caused a significant increase in cytotoxicity. In the presence of 10 mM CyA-P-CyB, the viability of the U87-MG cells decreased from more than 60% to less than 20% upon laser irradiation (808 nm) (Fig. 5a). A similar result was observed in A549 cells (Fig. 5b). The laser-enhanced cytotoxicity depended on the cathepsin B activity in tumor cells. When U87 and A549 cells were pretreated with 30 nM calpain inhibitor I (CI) for 30 min and then
Fig. 5. MTT assay of cells grown in the presence of CyA-P-CyB with or without laser irradiation and the cathepsin B inhibitor I (CI). a and b: Cytotoxic effect of CyA-P-CyB with or without 808 nm laser irradiation. U87 cells (a) and A549 cells (b) were incubated with different concentrations of CyA-P-CyB for 16 h, then CyA-P-CyB was removed. The cells were then irradiated with an 808 nm laser (2 W cm2, 10 min) and cultured for another 24 h c and d: Cytotoxicity was inhibited by the cathepsin B inhibitor. U87 cells (c) and A549 cells (d) were pretreated with 30 nM calpain inhibitor I for 30 min, incubated with 5 mM CyA-P-CyB for 16 h, and then CyA-P-CyB was removed. The cells were then irradiated with an 808 nm laser (2 W/cm2, 10 min) and cultured for another 24 h e: Comparisons of the cytotoxicity toward normal cells and cancer cells. The cells were incubated with 5 mM CyA-PCyB for 16 h, and the CyA-P-CyB was removed. The cells were irradiated with an 808 nm laser (2 W cm2, 10 min) and cultured for another 24 h. The results are expressed as the mean ± standard deviation of three independent experiments.
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Fig. 6. HeLa Cells were incubated with 10 mM alkyne-Peptide-PABC-CyB without (a, b, e, f, I, j) or with (c, d, g, h, k, l) 30 nM calpain inhibitor I for 2 h. After washing with DPBS, cells were irradiated with 808 nm laser (2 W cm2, 10 min) or not and acquired fluorescence images. Calpain inhibitor I was pretreated for 30 min and costained with Lysotracker blue. Blue: ex. 405 nm/em. 430e455 nm (a, b, c, d), green: ex. 473 nm/em 490e590 nm (e, f, g, h), merged with DIC (i, j, k, l). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
incubated with 5 mM CyA-P-CyB for 16 h, the viability of U87 cells increased from 20.6% to 48.5% (Fig. 5c) and that of A549 cells increased from 30.2% to 57.9% upon laser irradiation (Fig. 5d), indicating that the phototoxicity of CyA-P-CyB toward tumor cells depends on the activity of cathepsin B. In other words, cathepsin B may trigger tumor cell death in response to NIR laser irradiation. The cathepsin B-triggered phototoxicity indicated that CyA-P-CyB had significantly different cytotoxicity toward normal cells and tumor cells. As shown in Fig. 5e, when normal cells, including IMR90, NIH-3T3 and W38-VA13 cells, were incubated with 5 mM CyAP-CyB for 16 h and irradiated with an 808 nm laser for 10 min, the cell survival rate was over 50%. However, for the tumor cells, such as HeLa, MCF-7, U87-MG, SK-Hep1 and A549 cells, subjected to the same treatments, the cell viability decreased to 18.8% for HeLa cells, 12.2% for MCF-7 cells, 27.9% for U87-MG cells, 22.1% for SK-HEP1 cells and 31.4% for A549 cells. The specific phototoxicity of CyA-PCyB toward tumor cells can be attributed to the fact that the cathepsin B levels are higher in tumor cells than in normal cells [35e37]. Based on the singlet oxygen generation assay and photothermal assay, CyA-P-CyB itself can also produce ROS (Fig. S15). However,
increased phototoxicity was only observed when CyA-P-CyB was cleaved by cathepsin B to form Cy-S-Ph-NH2. We speculated that the initial CyA-P-CyB probe was restricted to the lysosome, mostly due to the peptide linker, leading to a high concentration of CyA-PCyB in a local space. Local high concentrations of PDT agents will lead to the inner filter effect and the quenching of photo-excited species by collision with non-excited PDT agents or the degradation products from the laser irradiation. The inner filter effect and the quenching of photo-excited species further inhibits the generation of singlet oxygen, thus improving the viability of tumor cells [26,38]. After CyA-P-CyB was cleaved by cathepsin B, the product Cy-S-Ph-NH2 diffused, and the local concentration of the cyanine dye decreased, which is in favor of the generation of singlet oxygen, and caused more serious toxicity to tumor cells. This hypothesis was supported by the subsequent verification experiments. The CyB moiety can be activated by irradiation with an 808 nm laser to emit green fluorescence from the degradation products, which is related to the activity of singlet oxygen. When Alkyne-PeptidePABC-CyB was used as a tracer, strong and diffused green fluorescence was observed in both the cytoplasm and lysosome after irradiation with an 808 nm laser (Fig. 6). In contrast, when
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cathepsin B activity was inhibited with calpain inhibitor I, much weaker fluorescence was observed after irradiation with an 808 nm laser, indicating that the uncleaved Alkyne-Peptide-PABC-CyB was maintained in lysosomes, thus producing less singlet oxygen and less damage to the cell. Therefore, the increased phototoxicity of CyA-P-CyB in tumor cells is related to the diffusion of the cleavage product Cy-S-Ph-NH2. The specific phototoxicity toward tumor cells inspired us to further investigate the phototherapeutic efficacy of CyA-P-CyB in vivo. In vivo NIR fluorescence imaging was performed on H640 tumor-bearing mice. To improve the water solubility and slow the blood clearance, CyA-P-CyB was encapsulated into PEG-PLA micelles (PEG-PLA/CyA-P-CyB). The PEG-PLA micelles loaded with CyA-P-CyB had an average size of 230 nm and a polydispersion index (PDI) of 0.461 (Fig. S16). The loading efficiency of CyA-P-CyB to PEG-PLA was calculated to be 46.7%, and the in vitro release behavior was studied by dialysis in 40.0 mL of PBS (containing 30% DMSO) at 37 C. The results showed that 70% of CyA-P-CyB was released in the first 5 h (Fig. S17). The strong fluorescent in the tumor region was observed at 4 h after the intravenous injection of
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PEG-PLA/CyA-P-CyB, and remained high fluorescent intensity until 32 h, indicating that CyA-P-CyB can accumulate in tumors (Fig. 7a, lower panels). In contrast, treatment with PEG-PLA did not produce any meaningful fluorescence signals (Fig. 7a, upper panels). When the tumor volume reached approximately 100e200 mm3, the mice bearing H640 tumors were intravenously four times injected with PEG-PLA/CyA-P-CyB (5 mg/kg) in 4 days (once injection per day) as the test group. Mice treated with an equal volume of PEG-PLA were used as the control group. Twenty-four hours after the last injection, the mice were irradiated with an 808 nm laser (1 W/cm2 for 5 min). The tumors in the mice treated with PEG-PLA/CyA-P-CyB and laser irradiation were significantly smaller than the tumors in the mice treated with PEG-PLA injection alone, PEG-PLA injection with laser irradiation, or PEG-PLA/CyA-P-CyB injection without laser irradiation (Fig. 7b). The tumor volumes in mice treated with PEG-PLA/CyA-P-CyB injection and laser irradiation were decreased to one-third of the volumes of the other three groups on the 14th day (Fig. 7c). We also investigated the phototherapeutic efficacy of CyA-P-CyB in vivo to A549 tumor-bearing mice, and similar tumorsuppression effect was observed (Fig. S18). These results suggest
Fig. 7. NIR fluorescence imaging of CyA-P-CyB and phototherapeutic efficacy of CyA-P-CyB in vivo. (a) NIR images of tumor-bearing mice were collected over 48 h after the intravenous injections of PEG-PLA/CyA-P-CyB. The tumors are circled with a dotted line. (b) Tumor growth in mice bearing H640 tumors after administration of the indicated treatments. The tumor volumes were normalized to their initial values. (The red arrows represent the day of the injection of PEG-PLA/CyA-P-CyB, and the blue arrow represents the day of laser irradiation. The animals were treated with 5 mg/kg PEG-PLA/CyA-P-CyB daily for 4 days (20 mg kg1)). The data are expressed as the mean ± SEM (n ¼ 3e6). *P < 0.05, compared to the PEG-PLA with laser group. (c) Photos of mice bearing H640 tumors on day 14 after administration of the indicated treatments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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that CyA-P-CyB has notable phototherapeutic efficacy in suppressing tumor growth. We checked the tumor site temperature after injection of PEG-PLA or PEG-PLA/CyA-P-CyB followed by laser irradiation (808 nm), and the results showed temperature increase by 3 C and 7 C for the cases with the injection of PEG-PLA and PEG-PLA/CyA-P-CyB, respectively (Fig. S19). The temperature increase of tumor with the treatment of PEG-PLA/CyA-P-CyB during laser irradiation (5 min) was not enough for photothermal ablation. Therefore, the major therapeutic effect should be due to photodynamic effect rather than photothermal ablation. The toxicity of CyA-P-CyB was assessed in mice that were treated with PEG-PLA/CyA-P-CyB or PEG-PLA via the tail vein. Mice
in the PEG-PLA/CyA-P-CyB group (20 mg kg1) did not die and survived for over 16 days. Seven organs (lung, liver, heart, kidney, pancreas, spleen and brain) were acquired from the mice on the 14th day following injection with PEG-PLA or PEG-PLA/CyA-P-CyB (20 mg kg1) and assessed using H&E staining (Fig. 8a). There were no distinct lesions (no necrosis, edema, inflammatory infiltration, or hyperplasia) in the sections of the seven tissues. These histological results indicate that at the dose used here, CyA-P-CyB does not have cytotoxic effects on mice. We also analyzed cell proliferation and apoptosis in the tumor masses treated with PEG-PLA with or without laser and PEG-PLA/CyA-P-CyB with or without laser (Fig. 8b). Cell proliferation was tested by anti-Ki-67 staining and
Fig. 8. Toxicity of CyA-P-CyB in vivo and the effects of CyA-P-CyB on proliferation and apoptosis in vivo. (a) Histological analysis of the organs (lung, liver, heart, kidney, pancreas, spleen and brain) acquired from mice on the 14th day after the injection with PEG-PLA or PEG-PLA/CyA-P-CyB (20 mg/kg). (b) Proliferation and apoptosis of the tumors. Proliferation was determined by staining the tissues with the Ki-67 antibody and is shown as brown signals. The nuclei were counterstained with hematoxylin. Apoptosis was determined using the TUNEL assay. Green represents a positive signal. The nuclei were visualized by DAPI staining (blue). (c) Quantification of the TUNEL assay. Positive signals were counted in five frames per slide. The data are expressed as the means ± SEM (n ¼ 3e6). **P < 0.01, compared to the control (PEG-PLA), control (PEG-PLA) with laser or PEG-PLA/CyAP-Cy B groups, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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was dramatically decreased in the group treated with PEG-PLA/ CyA-P-CyB and the laser, whereas cell proliferation was comparatively strong in the other three groups (Fig. 8b, left panels). In the apoptosis assay using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, the group treated with PEGPLA/CyA-P-CyB and the laser showed an increase in the apoptotic, green signal (Fig. 8b, right panels) and a significant increase in the number of apoptotic cells compared with the other three groups in the quantitative analysis (Fig. 8c). These results suggest that the treatment with PEG-PLA/CyA-P-CyB and the laser substantially affected tumor cell apoptosis. Together, our results indicate that the treatment with PEG-PLA/CyA-P-CyB and the laser affected cell proliferation and apoptosis in the tumor mass. 4. Conclusions In conclusion, a cathepsin B-activated fluorescent probe, CyA-PCyB, was designed and constructed based on FRET between a fluorescent cyanine unit and a non-fluorescent cyanine acceptor that were covalently coupled through a specific peptide substrate linker. As the key moiety, CyB based on Cy-S-Ph-NH2 serves as two key functions owing to the great absorption in NIR region (650e800 nm), weak emission and high phototoxicity. One is as a quencher, which may tune the NIR fluorescence of CyA based on the FRET effect. The other role is as a PDT agent to realize the phototherapy with NIR irradiation. In vitro cell-based studies show that the probe can be used to visualize the location of cathepsin B and monitor the LMP process during apoptosis. Furthermore, the probe exhibited specific phototoxicity toward tumor cells. In vivo imaging of tumors and the antitumor efficacy of the probe in mice bearing tumors indicated the present probe was capable of being used for NIR imaging in mice and phototherapy for tumors in vivo. Most importantly, the deposition of CyA-P-CyB in the tumor can be directly visualized by the release of NIR fluorescence; thus, we can choose the optimum irradiation time using the fluorescence intensity in the tumor. As expected, the tumor growth in mice was markedly inhibited upon treatment with CyA-P-CyB encapsulated in PEG-PLA and laser irradiation. CyA-P-CyB had no cytotoxic effects on mice, based on the histological assessment, and the treatment with PEG-PLA/CyA-P-CyB and laser irradiation inhibited cell proliferation and induced apoptosis in the tumor mass. All these results indicate that CyA-P-CyB is a truly efficient phototherapy reagent. Acknowledgments This research was supported financially by grants from the National Creative Research Initiative Program (2012R1A3A2048814). It was also supported by the Korea Mouse Phenotyping Center (2016M3A9D5A01952416), and the Brain Korea 21 PLUS Project for Medical Science, Yonsei University, the Bio and Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF2013M3A9D5072551). X. Chen acknowledges funding from the National Natural Science Foundation of China (21376117), the Jiangsu Natural Science Funds for Distinguished Young Scholars (BK20140043), and the Natural Science Foundation of Jiangsu Higher Education Institutions of China (14KJA150005). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2017.01.020.
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