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Contents lists available at ScienceDirect
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Zinc phthalocyanine conjugated with the amino-terminal fragment of urokinase for tumor-targeting photodynamic therapy
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Zhuo Chen a,⇑, Peng Xu a, Jincan Chen a, Hongwei Chen a, Ping Hu a, Xueyuan Chen b, Lin Lin c, Yunmei Huang d, Ke Zheng e, Shanyong Zhou a, Rui Li a, Song Chen a, Jianyong Liu e, Jinping Xue e, Mingdong Huang a,e,⇑ a State Key Laboratory of Structural Chemistry, Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, People’s Republic of China b Key Laboratory of Optoelectronic Materials Chemistry and Physics, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, People’s Republic of China c Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA d Fujian Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350108, People’s Republic of China e College of Chemistry and Chemical Engineering, Fuzhou University, Fujian 350108, People’s Republic of China
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Article history: Received 1 March 2014 Received in revised form 10 June 2014 Accepted 17 June 2014 Available online xxxx
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Keywords: Tumor-targeting Tumor-imaging Photosensitizer Zinc phthalocyanine
a b s t r a c t Photodynamic therapy (PDT) has attracted much interest for the treatment of cancer due to the increased incidence of multidrug resistance and systemic toxicity in conventional chemotherapy. Phthalocyanine (Pc) is one of main classes of photosensitizers for PDT and possesses optimal photophysical and photochemical properties. A higher specificity can ideally be achieved when Pcs are targeted towards tumor-specific receptors, which may also facilitate specific drug delivery. Herein, we develop a simple and unique strategy to prepare a hydrophilic tumor-targeting photosensitizer ATF-ZnPc by covalently coupling zinc phthalocyanine (ZnPc) to the amino-terminal fragment (ATF) of urokinase-type plasminogen activator (uPA), a fragment responsible for uPA receptor (uPAR, a biomarker overexpressed in cancer cells), through the carboxyl groups of ATF. We demonstrate the high efficacy of this tumor-targeting PDT agent for the inhibition of tumor growth both in vitro and in vivo. Our in vivo optical imaging results using H22 tumor-bearing mice show clearly the selective accumulation of ATF-ZnPc in tumor region, thereby revealing the great potential of ATF-ZnPc for clinical applications such as cancer detection and guidance of tumor resection in addition to photodynamic treatment. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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1. Introduction
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Cancer is one of the deadliest diseases of our time and throughout the world. Billions of dollars are spent annually on research in order to cure cancer or improve the quality of life of cancer patients. Traditional cancer treatments, including surgery, radiation therapy and chemotherapy, result in serious side effects caused by the loss of normal organ function. In contrast, photodynamic therapy (PDT) is more controllable and has the potential to selectively destroy malignant cells while sparing the normal
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⇑ Corresponding authors. Tel.: +86 591 83704996; fax: +86 591 83714946 (Z. Chen). Address: State Key Laboratory of Structural Chemistry, Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, People’s Republic of China. Tel.: +86 591 83704996; fax: +86 591 83714946 (M. Huang). E-mail addresses: [email protected] (Z. Chen), [email protected] (M. Huang).
tissues, and thus is recognized as a treatment strategy that is both minimally invasive and minimally toxic. It is a form of phototherapy using nontoxic light-sensitive compounds (i.e. photosensitizers) that are exposed selectively to light, whereupon they become toxic to targeted malignant and other diseased cells [1–3]. In addition, some photosensitizers are able to provide intense fluorescence signals in tumor tissues that allow their photodynamic imaging. Among many types of photosensitizers for PDT, phthalocyanine (Pc) is one of the main classes of photosensitizers with advantageous photophysical properties [4]. Its stronger absorption at 670 nm where the depth of light penetration in tissue is twice that obtained at 630 nm with porfimer sodium (Photofrin), which has been used as an effective photosensitizer in clinical cancer treatment or in cancer clinical trials [5]. However, Pcs tend to have low tumor-targeting efficacy, which limits their use in clinical applications. A number of strategies have been used to enhance
http://dx.doi.org/10.1016/j.actbio.2014.06.026 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Chen Z et al. Zinc phthalocyanine conjugated with the amino-terminal fragment of urokinase for tumor-targeting photodynamic therapy. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.06.026
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the tumor-targeting specificity of Pc compounds [3,6,7]. In this study, we report a strategy to improve the tumor-targeting properties of zinc phthalocyanine (ZnPc) by coupling ZnPc to the aminoterminal fragment (ATF) of urokinase. Urokinase, or urokinase-type plasminogen activator (uPA), is recommended as a prognostic marker for breast cancer by the American Society of Clinical Oncology (ASCO) [8] and the European Organization for Research and Treatment of Cancer (EORTC) [9]. Biochemical and structural studies from our laboratory [10,11] and others [12–14] have demonstrated that the ATF of uPA (uPA1-143, molecular weight 16 kDa) is solely responsible for uPA binding to the receptor of uPA (uPAR). uPAR has a low expression level in most quiescent cells, but greatly increases in several cancers including breast, colorectal and gastric cancers cells [15]. Several independent studies have correlated uPAR expression in vivo to various pathological conditions, especially cancer invasion and metastasis [16]. Moreover, a high level of soluble uPAR in plasma correlates with poor patient prognosis in many different human cancers [17]. Due to the importance of uPAR in cancer invasion and metastasis, several types of uPAR antagonists have been developed during the last decades, and used as targeting agents for imaging of uPAR via various imaging modalities such as magnetic resonance imaging, single-photon-emission computer tomography and positron emission topography [18]. A multifunctional nanoscale uPAR-targeted delivery vehicle was engineered to enhance the selective and specific delivery of the cargo (noscapine) to prostate cancer cells [19]. In another study, an uPAR-targeted dual-modality nanoparticle probe was generated and shown to selectively accumulate into primary and metastatic pancreatic cancer lesions in pancreatic cancer in mice by molecular imaging [20]. Recently, a 64Cu-labeled peptidyl uPAR inhibitor was demonstrated to accumulate specifically on a xenografted tumor in mice 2- to 3-fold more than the non-inhibitory 64Cu-labeled peptide [21]. Taken together, uPAR has attracted considerable attention as a promising molecular target for intervention and/or cytotoxin-based cancer therapies. The ATF of uPA is an often used targeting agent for uPAR and has been used to conjugate to different nanoparticles [20,22]. However, recent studies have raised concern that chemical modification of ATF may alter its uPAR binding capability. Small molecular antagonists were developed in the 1990s by several pharmaceutical companies to intervene in the uPAR–uPA interaction. These inhibitors were reported to have high potencies, and some of them had IC50s in the nanomolar range. However, two inhibitors were later resynthesized by an independent group and found to have much weaker potencies (IC50s were of the order of micromoles) [23]. This discrepancy was traced to a problem in the first competitive assay using ATF with 125I labeling, which modified the ATF residues important for uPAR binding and greatly reduced the affinity for uPAR of the labeled fragment [24], thus making the labeled fragment an invalid probe to measure uPAR binding. This example highlights that caution should be taken in the modification of ATF protein in order to preserve its uPAR-binding capability. In this study, we conjugate ZnPc to the carboxyl groups of ATF, which was shown not to interfere with uPAR binding [10], and thus preserve the binding activity of conjugated ATF to uPAR. Besides the increased tumor-targeting efficacy, this conjugation of ATF with ZnPc has an added benefit of enhancing the water solubility of ZnPc. ZnPc is not soluble in aqueous solution, and various formulation approaches have been used in order to solve this solubility issue, which include the use of detergents (castor oil) or nanoparticles [25,26]. Derivatization of the Pc ring is also commonly used to enhance the solubility [27]. Both in vitro and in vivo PDT studies demonstrate that ATF-ZnPc preferentially accumulates onto uPAR-positive cells, internalizes into cells and localizes in the lysosomes, and thus exhibits selective
PDT effects on tumor cells (as shown in Fig. 1). The selectivity of ATF-ZnPc towards uPAR-positive cells is further identified by fluorescent imaging of a tumor model in mice.
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2. Experimental
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2.1. Materials and cell lines
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All the starting materials were obtained from commercial suppliers and used as received without further purification. Common chemicals were purchased from Shanghai Chemical, Inc. Sepharose fast flow (SPFF) and Superdex75 HR 10/30 size exclusion columns were obtained from GE Life Sciences. All enzymes were from Takara Bio Inc., except that Pfu DNA polymerase was from Sangon Biotech. Synthetic DNA oligonucleotides were from Beijing Sunbiotech Co. Ltd. Zeocin, Top10F’ and eukaryotic Pichia pastoris cells were purchased from Invitrogen. Deionized water was used throughout the experiments. The mammalian cell lines used in the studies were histiocytic lymphoma cell line U937, non-small cell lung carcinoma cell line H1299, mouse hepatocellular carcinoma cell line H22, and human embryo lung fibroblast cell line HELF. All these cell lines were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences and grown in RPMI-1640 supplemented with 10% fetal calf serum and antibiotics. Cells were kept at 37 °C in a humidified incubator with 5% CO2 atmosphere. The viability of cells was determined by the dye Trypan blue. Cells were maintained in logarithmic phase with viability >95%.
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2.2. Preparation of ATF protein
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The cloning, expression and purification of human ATF (residues 1–143) were carried out according to our previously published protocols [28]. ATF was expressed in eukaryotic P. pastoris to ensure proper protein folding and disulfide bond formation, and was captured from expression medium by an SPFF cation exchange column. The protein was further purified by a preparative C4 reversed-phase column (VYDACÒ, 250 10 mm, 5 lm) on a high-performance liquid chromatography (HPLC) system (Dalian Elite Analytical Instruments Co. Ltd., Dalian, China), eluted with a linear gradient of 20–70% acetonitrile. The final product ATF was
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Fig. 1. Conjugation of ATF with zinc phthalocyanine (ZnPc) renders ZnPc soluble in water and generates a high degree of specificity to uPAR-abundant tumor cells. ATFZnPc preferentially accumulates in lysosome organelle, mainly through uPA receptor-dependent endocytosis, and induces cell death upon light illumination.
Please cite this article in press as: Chen Z et al. Zinc phthalocyanine conjugated with the amino-terminal fragment of urokinase for tumor-targeting photodynamic therapy. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.06.026
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lyophilized and stored as lyophilized powder. Several batches of ATF have been produced on a regular basis and been tested without losing uPAR binding activity over a year.
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2.3. Synthesis of ATF-ZnPc conjugate
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2.3.1. Synthesis of mono-substituted b-carboxyphthalocyanine zinc (ZnPc-COOH 3) Mono-substituted b-carboxyphthalocyanine zinc (ZnPc-COOH 3) was prepared and purified as described before [29] (Scheme 1). Typically, a mixture of trimellitic anhydride 1 (2.40 g, 0.0125 mol) and phthalic anhydride 2 (12.96 g, 0.0875 mol) was added into a flask together with anhydrous zinc acetate (22.0 g, 0.1002 mol), urea (60.0 g, 1.0 mol), ammonium molybdate (0.5 g, 0.4 mmol) and ammonium chloride (2.0 g, 0.0374 mol). After 4 h of reaction at 170 °C, the mixture was hydrolyzed under an alkaline condition with 100 ml of 1 M potassium hydroxide (KOH) for 24 h. The hydrolysis product was evaporated at 80 °C, and the green residual b-carboxyphthalocyanine zinc (ZnPc-COOH, 3) was then purified on a self-packed silica gel column (30 cm 3 cm, 100 mesh) with a mixed elution solvent (N0 ,N-dimethylformamide (DMF):acetone, 3:1 ratio) to be separated from the other two products (ZnPc-CO-NH2 and ZnPc) due to its larger polarity. After being purified twice over a silica gel column, analytical HPLC at 670 nm was performed with a gradient elution from water to DMF (5–100%) at a flow rate of 1 ml min1, yielding 10.5%.
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2.3.2. Synthesis of ZnPc-CO-NH-(CH2)2-NH-Boc 4 Typically, ZnPc-COOH 3 (9.3 mg, 0.015 mmol) was dissolved in 1.5 ml DMF followed by addition of hexafluorophosphate tetramethylurea (HBTU, 23.1 mg, 0.06 mmol) and a base N,N-diisopropylethylamine (DIEA, 0.093 ml). After stirring for 30 min at room temperature, tert-butyl-2-aminoethylcarbamate (H2N-(CH2)2-NHBoc, 2.89 mg, 0.018 mmol) in DMF (0.5 ml) was added dropwise to the solution under stirring. 24 h later, the mixture was purified by silica column chromatography with a mixed elution solvent (hexanol:tetrahydrofuran, 1:2 ratio) to yield the dark blue product ZnPc-CO-NH-(CH2)2-NH-Boc 4. The final yield was 71.4% based on
3
compound 3 (ZnPc-COOH). This product was characterized by 1H nuclear magnetic resonance (NMR) spectrometry (Fig. S1A, Bruker AV-400, 400 MHz, [d6] dimethyl sulfoxide (DMSO): d = 9.5 (s, 1H), 9.1 (m, 1H), 8.9 (m, 2H), 8.8 (t, 5H), 8.6 (d, 1H), 8.0 (m, 6H), 7.2 (m, 1H), 3.8 (m, 2H), 3.5 (m, 2H), 1.6 (s, 9H) ppm); electrospray ionization mass spectrometry (ESI-MS) (Fig. S2, DECAX-30000 LCQ Deca XP: calcd m/z for C40H30N10O3Zn [M+2H2O-H] = 799.1; found m/z [M+2H2O-H] = 800.2); and Fourier transform infrared (FT-IR) spectrometry (Fig. S3A, t = 3360 cm1 (N–H stretch), 2932 cm1 (aliphatic C–H, stretch), 1686 cm1 (amide I), 1547 cm1 (amide II)). These results confirmed the molecular structure of 4 we obtained.
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2.3.3. Synthesis of ZnPc-CO-NH-(CH2)2-NH2 5 ZnPc-CO-NH-(CH2)2-NH-Boc 4 (5 mg, 6.54 lmol) obtained was dissolved in 95% trifluoroacetic acid (TFA) in deionized water. After 3 h of stirring, the solution was precipitated by dried ethylether. The precipitate was dried under vacuum and further purified by a C18 column (SinoChrom ODS-BP column, 250 20 mm, 10 lm) on a HPLC system (Dalian Elite Analytical Instruments Co. Ltd., Dalian, China), eluted with a linear gradient of a 50–100% mixture of acetonitrile and methanol (volume ratio 1:3) and 0.1% TFA. The final yield was 55.5% based on compound 3 (ZnPc-COOH). ZnPc-CO-NH-(CH2)2-NH2 5 was characterized by 1H-NMR (Fig. S1B, Bruker AV-400, 400 MHz); FT-IR (Fig. S3B, t = 3400 cm1 (N–H2 stretch), 2941 cm1 (aliphatic C–H, stretch), 1647 cm–1 (amide I), 1543 cm–1 (amide II)); and ESI-MS (Fig. S4, DECAX-30000 LCQ Deca XP: C35H22N10OZn, calcd m/z [M+H]+ = 663.1; found m/z [M+H]+ = 662.9). Both results confirmed the molecular structure of 5. The UV–vis absorption spectrum of 5 in DMSO (Fig. S5) was recorded from 250 to 800 nm using quartz cuvettes with 1 cm path length on a Lambda-35 UV–vis spectrometer (PerkinElmer, MA, USA) in DMSO [kmax (log e): 284 (4.17), 348 (4.66), 610 (4.36), 678 (5.09) nm].
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2.3.4. Synthesis of ATF-ZnPc 6 ATF (10.91 mg, 0.71 lmol), ZnPc-CO-NH-(CH2)2-NH2 (2.83 mg, 4.26 lmol) and DIEA (50 ll) were mixed together with DMF
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Scheme 1. Synthetic route of ATF-ZnPc conjugate.
Please cite this article in press as: Chen Z et al. Zinc phthalocyanine conjugated with the amino-terminal fragment of urokinase for tumor-targeting photodynamic therapy. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.06.026
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(2 ml) at room temperature. HBTU (5 mg ml1, 320 ll, 5.0 lmol) was then added into the solution dropwise, and the whole mixture was stirred for 2 h followed by a centrifugation. The supernatant was purified through a C4 reverse-phase column (VYDACÒ, 250 10 mm, 5 lm) on a HPLC system (Dalian Elite Analytical Instruments Co. Ltd., Dalian, China), eluted with a linear gradient of 20–70% acetonitrile at a flow rate of 2 ml min1. As shown in Fig. S6, HPLC chromatogram of ATF-ZnPc exhibited three well-separated peaks (I–III). The fraction from peak II (ATF-ZnPc 6) was separated from the reactant ZnPc-CO-NH-(CH2)2-NH2 (peak III, blue) using acetonitrile as mobile phase and dried in vacuum, yielding 5.85 mg of the deep blue powder. The final yield was 53.6% based on compound 3 (ZnPc-COOH). ATF-ZnPc 6 was characterized by matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry (Bruker REFLEX III, Bruker-Franzen, Bremen, Germany, in reflex and positive modes with an accumulation of 300 times (10 times/spot)), showing a dominant peak at 16746 Da (Fig. S7). This value is higher than the MALDI MW of ATF (16088 Da), which indicates the attachment of one molecule of ZnPc to each ATF molecule in our preparation. The molecular weight of 6 was further estimated by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE, Fig. S8), showing its molecular weight to be 20 kDa. Meanwhile, the band of ATF-ZnPc showed an obvious green color before the application of Coomassie brilliant blue (CBB) staining, suggesting the presence of ZnPc. The UV–vis absorption spectrum of 6 in DMSO (Fig. S5) was recorded from 250 to 800 nm using quartz cuvettes with 1 cm path length on a Lambda-35 UV–vis spectrometer (PerkinElmer, MA, USA) in DMSO [kmax (log e): 286 (4.58), 349 (4.78), 611 (4.46), 682 (5.16) nm]. The wavelength with the maximum absorbance of 6 was located at 682 nm, which was close to that of 5 (kmax = 678 nm). The absorption band at 282 nm was mainly due to the tryptophan residues from 6. Furthermore, the absolute fluorescence quantum yield of ATFZnPc measured at room temperature on a FLS920 spectrometer (Edinburgh Instruments, Edinburgh, UK) was 3.39%. ATF-ZnPc’s fluorescence emission and excitation spectra and decay time were recorded on a spectrometer equipped with both continuous (450 W) xenon and pulsed flash lamps (FLS920, Edinburgh Instruments) at room temperature. The fluorescence decay time (Fig. S9) of ATF-ZnPc is 5.49 ns, which is >50% longer than that of ZnPc-CONH-(CH2)2-NH2 (3.50 ns), indicating the great potential for using ATF-ZnPc as an imaging probe for cancer detection and guidance of tumor resection in addition to photodynamic treatment.
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2.4. Degree of labeling for ATF-ZnPc
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We use the following equation to calculate the number of ZnPc molecules that were covalently coupled to each ATF molecule (degree of labeling, DOL):
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2.5. Singlet oxygen quantum yield of ATF-ZnPc
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Singlet oxygen (1O2) is believed to be the major cytotoxic agent involved in PDT. The quantum yield of 1O2 generated by ATF-ZnPc 6 was measured using the unsubstituted ZnPc as a reference [30]. Briefly, ATF-ZnPc (0.5 lM) and 1,3-diphenylisobenzofuran (DPBF, a scavenger for 1O2) were mixed in DMSO (2 ml) and the absorbance of DPBF was monitored at 417 nm at intervals of 5 s with a light illumination (670 nm at 80 mW cm2) in the presence of air (without degassing). The concentration of DPBF was kept
No. of Pages 12, Model 5G
5 July 2014 Acta Biomaterialia xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat 5 6
Zinc phthalocyanine conjugated with the amino-terminal fragment of urokinase for tumor-targeting photodynamic therapy
3 4 7
Q1
8 9 10 11 12 13 14 15 16
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Zhuo Chen a,⇑, Peng Xu a, Jincan Chen a, Hongwei Chen a, Ping Hu a, Xueyuan Chen b, Lin Lin c, Yunmei Huang d, Ke Zheng e, Shanyong Zhou a, Rui Li a, Song Chen a, Jianyong Liu e, Jinping Xue e, Mingdong Huang a,e,⇑ a State Key Laboratory of Structural Chemistry, Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, People’s Republic of China b Key Laboratory of Optoelectronic Materials Chemistry and Physics, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, People’s Republic of China c Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA d Fujian Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350108, People’s Republic of China e College of Chemistry and Chemical Engineering, Fuzhou University, Fujian 350108, People’s Republic of China
a r t i c l e
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i n f o
Article history: Received 1 March 2014 Received in revised form 10 June 2014 Accepted 17 June 2014 Available online xxxx
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Keywords: Tumor-targeting Tumor-imaging Photosensitizer Zinc phthalocyanine
a b s t r a c t Photodynamic therapy (PDT) has attracted much interest for the treatment of cancer due to the increased incidence of multidrug resistance and systemic toxicity in conventional chemotherapy. Phthalocyanine (Pc) is one of main classes of photosensitizers for PDT and possesses optimal photophysical and photochemical properties. A higher specificity can ideally be achieved when Pcs are targeted towards tumor-specific receptors, which may also facilitate specific drug delivery. Herein, we develop a simple and unique strategy to prepare a hydrophilic tumor-targeting photosensitizer ATF-ZnPc by covalently coupling zinc phthalocyanine (ZnPc) to the amino-terminal fragment (ATF) of urokinase-type plasminogen activator (uPA), a fragment responsible for uPA receptor (uPAR, a biomarker overexpressed in cancer cells), through the carboxyl groups of ATF. We demonstrate the high efficacy of this tumor-targeting PDT agent for the inhibition of tumor growth both in vitro and in vivo. Our in vivo optical imaging results using H22 tumor-bearing mice show clearly the selective accumulation of ATF-ZnPc in tumor region, thereby revealing the great potential of ATF-ZnPc for clinical applications such as cancer detection and guidance of tumor resection in addition to photodynamic treatment. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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1. Introduction
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Cancer is one of the deadliest diseases of our time and throughout the world. Billions of dollars are spent annually on research in order to cure cancer or improve the quality of life of cancer patients. Traditional cancer treatments, including surgery, radiation therapy and chemotherapy, result in serious side effects caused by the loss of normal organ function. In contrast, photodynamic therapy (PDT) is more controllable and has the potential to selectively destroy malignant cells while sparing the normal
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Q3
⇑ Corresponding authors. Tel.: +86 591 83704996; fax: +86 591 83714946 (Z. Chen). Address: State Key Laboratory of Structural Chemistry, Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, People’s Republic of China. Tel.: +86 591 83704996; fax: +86 591 83714946 (M. Huang). E-mail addresses: [email protected] (Z. Chen), [email protected] (M. Huang).
tissues, and thus is recognized as a treatment strategy that is both minimally invasive and minimally toxic. It is a form of phototherapy using nontoxic light-sensitive compounds (i.e. photosensitizers) that are exposed selectively to light, whereupon they become toxic to targeted malignant and other diseased cells [1–3]. In addition, some photosensitizers are able to provide intense fluorescence signals in tumor tissues that allow their photodynamic imaging. Among many types of photosensitizers for PDT, phthalocyanine (Pc) is one of the main classes of photosensitizers with advantageous photophysical properties [4]. Its stronger absorption at 670 nm where the depth of light penetration in tissue is twice that obtained at 630 nm with porfimer sodium (Photofrin), which has been used as an effective photosensitizer in clinical cancer treatment or in cancer clinical trials [5]. However, Pcs tend to have low tumor-targeting efficacy, which limits their use in clinical applications. A number of strategies have been used to enhance
http://dx.doi.org/10.1016/j.actbio.2014.06.026 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Chen Z et al. Zinc phthalocyanine conjugated with the amino-terminal fragment of urokinase for tumor-targeting photodynamic therapy. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.06.026
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the tumor-targeting specificity of Pc compounds [3,6,7]. In this study, we report a strategy to improve the tumor-targeting properties of zinc phthalocyanine (ZnPc) by coupling ZnPc to the aminoterminal fragment (ATF) of urokinase. Urokinase, or urokinase-type plasminogen activator (uPA), is recommended as a prognostic marker for breast cancer by the American Society of Clinical Oncology (ASCO) [8] and the European Organization for Research and Treatment of Cancer (EORTC) [9]. Biochemical and structural studies from our laboratory [10,11] and others [12–14] have demonstrated that the ATF of uPA (uPA1-143, molecular weight 16 kDa) is solely responsible for uPA binding to the receptor of uPA (uPAR). uPAR has a low expression level in most quiescent cells, but greatly increases in several cancers including breast, colorectal and gastric cancers cells [15]. Several independent studies have correlated uPAR expression in vivo to various pathological conditions, especially cancer invasion and metastasis [16]. Moreover, a high level of soluble uPAR in plasma correlates with poor patient prognosis in many different human cancers [17]. Due to the importance of uPAR in cancer invasion and metastasis, several types of uPAR antagonists have been developed during the last decades, and used as targeting agents for imaging of uPAR via various imaging modalities such as magnetic resonance imaging, single-photon-emission computer tomography and positron emission topography [18]. A multifunctional nanoscale uPAR-targeted delivery vehicle was engineered to enhance the selective and specific delivery of the cargo (noscapine) to prostate cancer cells [19]. In another study, an uPAR-targeted dual-modality nanoparticle probe was generated and shown to selectively accumulate into primary and metastatic pancreatic cancer lesions in pancreatic cancer in mice by molecular imaging [20]. Recently, a 64Cu-labeled peptidyl uPAR inhibitor was demonstrated to accumulate specifically on a xenografted tumor in mice 2- to 3-fold more than the non-inhibitory 64Cu-labeled peptide [21]. Taken together, uPAR has attracted considerable attention as a promising molecular target for intervention and/or cytotoxin-based cancer therapies. The ATF of uPA is an often used targeting agent for uPAR and has been used to conjugate to different nanoparticles [20,22]. However, recent studies have raised concern that chemical modification of ATF may alter its uPAR binding capability. Small molecular antagonists were developed in the 1990s by several pharmaceutical companies to intervene in the uPAR–uPA interaction. These inhibitors were reported to have high potencies, and some of them had IC50s in the nanomolar range. However, two inhibitors were later resynthesized by an independent group and found to have much weaker potencies (IC50s were of the order of micromoles) [23]. This discrepancy was traced to a problem in the first competitive assay using ATF with 125I labeling, which modified the ATF residues important for uPAR binding and greatly reduced the affinity for uPAR of the labeled fragment [24], thus making the labeled fragment an invalid probe to measure uPAR binding. This example highlights that caution should be taken in the modification of ATF protein in order to preserve its uPAR-binding capability. In this study, we conjugate ZnPc to the carboxyl groups of ATF, which was shown not to interfere with uPAR binding [10], and thus preserve the binding activity of conjugated ATF to uPAR. Besides the increased tumor-targeting efficacy, this conjugation of ATF with ZnPc has an added benefit of enhancing the water solubility of ZnPc. ZnPc is not soluble in aqueous solution, and various formulation approaches have been used in order to solve this solubility issue, which include the use of detergents (castor oil) or nanoparticles [25,26]. Derivatization of the Pc ring is also commonly used to enhance the solubility [27]. Both in vitro and in vivo PDT studies demonstrate that ATF-ZnPc preferentially accumulates onto uPAR-positive cells, internalizes into cells and localizes in the lysosomes, and thus exhibits selective
PDT effects on tumor cells (as shown in Fig. 1). The selectivity of ATF-ZnPc towards uPAR-positive cells is further identified by fluorescent imaging of a tumor model in mice.
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2. Experimental
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2.1. Materials and cell lines
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All the starting materials were obtained from commercial suppliers and used as received without further purification. Common chemicals were purchased from Shanghai Chemical, Inc. Sepharose fast flow (SPFF) and Superdex75 HR 10/30 size exclusion columns were obtained from GE Life Sciences. All enzymes were from Takara Bio Inc., except that Pfu DNA polymerase was from Sangon Biotech. Synthetic DNA oligonucleotides were from Beijing Sunbiotech Co. Ltd. Zeocin, Top10F’ and eukaryotic Pichia pastoris cells were purchased from Invitrogen. Deionized water was used throughout the experiments. The mammalian cell lines used in the studies were histiocytic lymphoma cell line U937, non-small cell lung carcinoma cell line H1299, mouse hepatocellular carcinoma cell line H22, and human embryo lung fibroblast cell line HELF. All these cell lines were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences and grown in RPMI-1640 supplemented with 10% fetal calf serum and antibiotics. Cells were kept at 37 °C in a humidified incubator with 5% CO2 atmosphere. The viability of cells was determined by the dye Trypan blue. Cells were maintained in logarithmic phase with viability >95%.
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2.2. Preparation of ATF protein
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The cloning, expression and purification of human ATF (residues 1–143) were carried out according to our previously published protocols [28]. ATF was expressed in eukaryotic P. pastoris to ensure proper protein folding and disulfide bond formation, and was captured from expression medium by an SPFF cation exchange column. The protein was further purified by a preparative C4 reversed-phase column (VYDACÒ, 250 10 mm, 5 lm) on a high-performance liquid chromatography (HPLC) system (Dalian Elite Analytical Instruments Co. Ltd., Dalian, China), eluted with a linear gradient of 20–70% acetonitrile. The final product ATF was
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Fig. 1. Conjugation of ATF with zinc phthalocyanine (ZnPc) renders ZnPc soluble in water and generates a high degree of specificity to uPAR-abundant tumor cells. ATFZnPc preferentially accumulates in lysosome organelle, mainly through uPA receptor-dependent endocytosis, and induces cell death upon light illumination.
Please cite this article in press as: Chen Z et al. Zinc phthalocyanine conjugated with the amino-terminal fragment of urokinase for tumor-targeting photodynamic therapy. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.06.026
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lyophilized and stored as lyophilized powder. Several batches of ATF have been produced on a regular basis and been tested without losing uPAR binding activity over a year.
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2.3. Synthesis of ATF-ZnPc conjugate
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2.3.1. Synthesis of mono-substituted b-carboxyphthalocyanine zinc (ZnPc-COOH 3) Mono-substituted b-carboxyphthalocyanine zinc (ZnPc-COOH 3) was prepared and purified as described before [29] (Scheme 1). Typically, a mixture of trimellitic anhydride 1 (2.40 g, 0.0125 mol) and phthalic anhydride 2 (12.96 g, 0.0875 mol) was added into a flask together with anhydrous zinc acetate (22.0 g, 0.1002 mol), urea (60.0 g, 1.0 mol), ammonium molybdate (0.5 g, 0.4 mmol) and ammonium chloride (2.0 g, 0.0374 mol). After 4 h of reaction at 170 °C, the mixture was hydrolyzed under an alkaline condition with 100 ml of 1 M potassium hydroxide (KOH) for 24 h. The hydrolysis product was evaporated at 80 °C, and the green residual b-carboxyphthalocyanine zinc (ZnPc-COOH, 3) was then purified on a self-packed silica gel column (30 cm 3 cm, 100 mesh) with a mixed elution solvent (N0 ,N-dimethylformamide (DMF):acetone, 3:1 ratio) to be separated from the other two products (ZnPc-CO-NH2 and ZnPc) due to its larger polarity. After being purified twice over a silica gel column, analytical HPLC at 670 nm was performed with a gradient elution from water to DMF (5–100%) at a flow rate of 1 ml min1, yielding 10.5%.
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2.3.2. Synthesis of ZnPc-CO-NH-(CH2)2-NH-Boc 4 Typically, ZnPc-COOH 3 (9.3 mg, 0.015 mmol) was dissolved in 1.5 ml DMF followed by addition of hexafluorophosphate tetramethylurea (HBTU, 23.1 mg, 0.06 mmol) and a base N,N-diisopropylethylamine (DIEA, 0.093 ml). After stirring for 30 min at room temperature, tert-butyl-2-aminoethylcarbamate (H2N-(CH2)2-NHBoc, 2.89 mg, 0.018 mmol) in DMF (0.5 ml) was added dropwise to the solution under stirring. 24 h later, the mixture was purified by silica column chromatography with a mixed elution solvent (hexanol:tetrahydrofuran, 1:2 ratio) to yield the dark blue product ZnPc-CO-NH-(CH2)2-NH-Boc 4. The final yield was 71.4% based on
3
compound 3 (ZnPc-COOH). This product was characterized by 1H nuclear magnetic resonance (NMR) spectrometry (Fig. S1A, Bruker AV-400, 400 MHz, [d6] dimethyl sulfoxide (DMSO): d = 9.5 (s, 1H), 9.1 (m, 1H), 8.9 (m, 2H), 8.8 (t, 5H), 8.6 (d, 1H), 8.0 (m, 6H), 7.2 (m, 1H), 3.8 (m, 2H), 3.5 (m, 2H), 1.6 (s, 9H) ppm); electrospray ionization mass spectrometry (ESI-MS) (Fig. S2, DECAX-30000 LCQ Deca XP: calcd m/z for C40H30N10O3Zn [M+2H2O-H] = 799.1; found m/z [M+2H2O-H] = 800.2); and Fourier transform infrared (FT-IR) spectrometry (Fig. S3A, t = 3360 cm1 (N–H stretch), 2932 cm1 (aliphatic C–H, stretch), 1686 cm1 (amide I), 1547 cm1 (amide II)). These results confirmed the molecular structure of 4 we obtained.
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2.3.3. Synthesis of ZnPc-CO-NH-(CH2)2-NH2 5 ZnPc-CO-NH-(CH2)2-NH-Boc 4 (5 mg, 6.54 lmol) obtained was dissolved in 95% trifluoroacetic acid (TFA) in deionized water. After 3 h of stirring, the solution was precipitated by dried ethylether. The precipitate was dried under vacuum and further purified by a C18 column (SinoChrom ODS-BP column, 250 20 mm, 10 lm) on a HPLC system (Dalian Elite Analytical Instruments Co. Ltd., Dalian, China), eluted with a linear gradient of a 50–100% mixture of acetonitrile and methanol (volume ratio 1:3) and 0.1% TFA. The final yield was 55.5% based on compound 3 (ZnPc-COOH). ZnPc-CO-NH-(CH2)2-NH2 5 was characterized by 1H-NMR (Fig. S1B, Bruker AV-400, 400 MHz); FT-IR (Fig. S3B, t = 3400 cm1 (N–H2 stretch), 2941 cm1 (aliphatic C–H, stretch), 1647 cm–1 (amide I), 1543 cm–1 (amide II)); and ESI-MS (Fig. S4, DECAX-30000 LCQ Deca XP: C35H22N10OZn, calcd m/z [M+H]+ = 663.1; found m/z [M+H]+ = 662.9). Both results confirmed the molecular structure of 5. The UV–vis absorption spectrum of 5 in DMSO (Fig. S5) was recorded from 250 to 800 nm using quartz cuvettes with 1 cm path length on a Lambda-35 UV–vis spectrometer (PerkinElmer, MA, USA) in DMSO [kmax (log e): 284 (4.17), 348 (4.66), 610 (4.36), 678 (5.09) nm].
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2.3.4. Synthesis of ATF-ZnPc 6 ATF (10.91 mg, 0.71 lmol), ZnPc-CO-NH-(CH2)2-NH2 (2.83 mg, 4.26 lmol) and DIEA (50 ll) were mixed together with DMF
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Scheme 1. Synthetic route of ATF-ZnPc conjugate.
Please cite this article in press as: Chen Z et al. Zinc phthalocyanine conjugated with the amino-terminal fragment of urokinase for tumor-targeting photodynamic therapy. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.06.026
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(2 ml) at room temperature. HBTU (5 mg ml1, 320 ll, 5.0 lmol) was then added into the solution dropwise, and the whole mixture was stirred for 2 h followed by a centrifugation. The supernatant was purified through a C4 reverse-phase column (VYDACÒ, 250 10 mm, 5 lm) on a HPLC system (Dalian Elite Analytical Instruments Co. Ltd., Dalian, China), eluted with a linear gradient of 20–70% acetonitrile at a flow rate of 2 ml min1. As shown in Fig. S6, HPLC chromatogram of ATF-ZnPc exhibited three well-separated peaks (I–III). The fraction from peak II (ATF-ZnPc 6) was separated from the reactant ZnPc-CO-NH-(CH2)2-NH2 (peak III, blue) using acetonitrile as mobile phase and dried in vacuum, yielding 5.85 mg of the deep blue powder. The final yield was 53.6% based on compound 3 (ZnPc-COOH). ATF-ZnPc 6 was characterized by matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry (Bruker REFLEX III, Bruker-Franzen, Bremen, Germany, in reflex and positive modes with an accumulation of 300 times (10 times/spot)), showing a dominant peak at 16746 Da (Fig. S7). This value is higher than the MALDI MW of ATF (16088 Da), which indicates the attachment of one molecule of ZnPc to each ATF molecule in our preparation. The molecular weight of 6 was further estimated by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE, Fig. S8), showing its molecular weight to be 20 kDa. Meanwhile, the band of ATF-ZnPc showed an obvious green color before the application of Coomassie brilliant blue (CBB) staining, suggesting the presence of ZnPc. The UV–vis absorption spectrum of 6 in DMSO (Fig. S5) was recorded from 250 to 800 nm using quartz cuvettes with 1 cm path length on a Lambda-35 UV–vis spectrometer (PerkinElmer, MA, USA) in DMSO [kmax (log e): 286 (4.58), 349 (4.78), 611 (4.46), 682 (5.16) nm]. The wavelength with the maximum absorbance of 6 was located at 682 nm, which was close to that of 5 (kmax = 678 nm). The absorption band at 282 nm was mainly due to the tryptophan residues from 6. Furthermore, the absolute fluorescence quantum yield of ATFZnPc measured at room temperature on a FLS920 spectrometer (Edinburgh Instruments, Edinburgh, UK) was 3.39%. ATF-ZnPc’s fluorescence emission and excitation spectra and decay time were recorded on a spectrometer equipped with both continuous (450 W) xenon and pulsed flash lamps (FLS920, Edinburgh Instruments) at room temperature. The fluorescence decay time (Fig. S9) of ATF-ZnPc is 5.49 ns, which is >50% longer than that of ZnPc-CONH-(CH2)2-NH2 (3.50 ns), indicating the great potential for using ATF-ZnPc as an imaging probe for cancer detection and guidance of tumor resection in addition to photodynamic treatment.
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2.4. Degree of labeling for ATF-ZnPc
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We use the following equation to calculate the number of ZnPc molecules that were covalently coupled to each ATF molecule (degree of labeling, DOL):
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2.5. Singlet oxygen quantum yield of ATF-ZnPc
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Singlet oxygen (1O2) is believed to be the major cytotoxic agent involved in PDT. The quantum yield of 1O2 generated by ATF-ZnPc 6 was measured using the unsubstituted ZnPc as a reference [30]. Briefly, ATF-ZnPc (0.5 lM) and 1,3-diphenylisobenzofuran (DPBF, a scavenger for 1O2) were mixed in DMSO (2 ml) and the absorbance of DPBF was monitored at 417 nm at intervals of 5 s with a light illumination (670 nm at 80 mW cm2) in the presence of air (without degassing). The concentration of DPBF was kept
