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Photosensitizer-Conjugated Gold Nanorods for Enzyme-Activatable Fluo- Rescence Imaging and Photodynamic Therapy Boseung Jang and Yongdoo Choi 

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Photosensitizer-Conjugated Gold Nanorods for Enzyme-Activatable Fluo- Rescence Imaging and Photodynamic Therapy Boseung Jang and Yongdoo Choi  Theranostics 2012, 2(2) 190 Ivyspring International Publisher Theranostics 2012; 2(2):190-197. doi: 10.7150/thno.3478 Research Paper Photosensitizer-Conjugated Gold Nanorods for Enzyme-Activatable Fluo- rescence Imaging and Photodynamic Therapy Boseung Jang and Yongdoo Choi Molecular Imaging & Therapy Branch, Division of Convergence Technology, National Cancer Center, 111 Jungbalsan-ro, Ilsandong-gu, Goyang, Gyeonggi-do 410-769, Republic of Korea Corresponding author: Yongdoo Choi, PhD, Molecular Imaging & Therapy Branch, Division of Convergence Technology, National Cancer Center, 111 Jungbalsan-ro, Ilsandong-gu, Goyang, Gyeonggi-do 410-769, Republic of Korea. Tel/Fax: +82-31-920-2512. E-mail: [email protected] © Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/ licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Received: 2011.09.05; Accepted: 2011.10.07; Published: 2012.02.11 Abstract We report on the development of photosensitizer-conjugated gold nanorods (MMP2P-GNR) in which photosensitizers were conjugated onto the surface of gold nanorods (GNR) via a protease-cleavable peptide linker. We hypothesized that fluorescence and phototoxicity of the conjugated photosensitizers would be suppressed in their native state, becoming activated only after cleavage by the target protease matrix metalloprotease-2 (MMP2). Quantitative analysis of the fluorescence and singlet oxygen generation (SOG) demonstrated that the MMP2P-GNR conjugate emitted fluorescence intensity corresponding to 0.4% 0.01% and an SOG efficiency of 0.89% 1.04% compared to free pyropheophorbide-a. From the in vitro cell studies using HT1080 cells that overexpress MMP2 and BT20 cells that lack MMP2, we ob- served that fluorescence and SOG was mediated by the presence or absence of MMP2 in these cell lines. This novel activatable photosensitizing system may be useful for prote- ase-mediated fluorescence imaging and subsequent photodynamic therapy for various can- cers. Key words: Photodynamic therapy, Gold nanorod, Resonance energy transfer, Quenching, En- zyme activatable Introduction Photodynamic therapy (PDT) describes the use which selectively damage tumor tissues in situ. of chemical photosensitizers combined with specific However, current photosensitizers can also nonspe- wavelengths of light. This form of therapy has been cifically accumulate in the skin or the eyes of patients. used successfully to treat cancers and is capable of If, following PDT, these patients are exposed to light, overcoming side effects associated with conventional such as sunlight or bright indoor light, side effects cancer treatments such as surgery, radiation therapy, caused by damage to normal cells in these tissues can and chemotherapy [1-2]. Photosensitizers accumulate occur. Limited tumor selectivity and unfavorable bi- in tumor tissues and are typically nontoxic to cells in odistribution of conventional PDT agents have re- the absence of light. When photosensitizers are ex- mained major obstacles to their clinical applications. cited by light radiation of a specific wavelength, they “Activatable PDT” agents have recently attracted a react with the surrounding substrates or with molec- great deal of attention as a strategy to overcome ular oxygen to produce reactive oxygen species (ROS), nonspecific activation of phototoxicity. In an ideal http://www.thno.org Theranostics 2012, 2(2) 191 PDT agent, generation of both fluorescence signal and (GNRs) via a protease-cleavable peptide linker. GNRs cytotoxic singlet oxygen would be completely inhib- have immense absorption coefficients, 104- to 106-fold ited in its native state, even upon light exposure. Their higher than those of conventional organic dyes, and optical properties would be selectively activated by therefore, may serve as ultraefficient energy quench- stimuli such as enzymes [3-6] and pH [7-8]. Choi et al. ers of excited photosensitizers through their sur- reported the first successful in vivo results demon- face-energy-transfer properties [13-14]. In addition, strating the potential use of an activatable PDT agent gold nanoparticles, including GNRs, can quench the in near-infrared fluorescence imaging and subsequent excited energy of fluorochromes even at a distance of PDT of cancers. [3] Various mechanisms for control- ~40 nm [15]. Therefore, we expected the fluorescence ling optical properties of photosensitizers have been and phototoxicity of the photosensitizers conjugated tested, including self-quenching between the same on the surface of GNRs to be suppressed in their na- type of photosensitizers [3, 5-6, 9], quenching by or- tive state, and activated following release from the ganic quenchers [4, 10], and quenching by carbon GNR surface by the action of a target protease. This nanotubes [11] and gold nanorods [12]. strategy may allow a photodynamic reaction to take Herein, we report on photosensitizer-conjugated place in a target area with greater selectivity and effi- gold nanorods (Figure 1), in which photosensitizers ciency. were conjugated onto the surface of gold nanorods Figure 1. A concept of enzyme activatable fluorescence imaging and photodynamic therapy using a photosensitizer-conjugated gold nanorod (MMP2P-GNR). Box: Structure of the MMP2P containing MMP2-cleavable peptide sequence and photosensitizer. Arrow indicates cleavable site by MMP2 activity. http://www.thno.org Theranostics 2012, 2(2) 192 In this study, the sequence GPLGVRG trifuged at 15000 × g at 25 °C for 15 min, thereby ob- (-Gly-Pro-Leu-Gly-Val-Arg-Gly-), which is specific to taining a CTAB-coated gold nanorod. the matrix metalloproteinase-2 (MMP2) substrate Preparation of photosensitizer-peptide conju- (Figure 1), was introduced as the target for enzymatic gate (MMP2P) hydrolysis by MMP2 [16]. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases and are MMP2-cleavable peptide substrate coupled with known to play key roles in tumor growth, progres- PPa (MMP2P) was purchased from Peptron Inc. sion, and metastasis as well as in dysregulated angi- (Daejeon, Republic of Korea). Supplementary Materi- ogenesis [17-20]. Therefore, MMPs, including MMP2, al: Figure S1 briefly summarizes the procedure of are considered important therapeutic and diagnostic MMP2P synthesis. An HPLC and mass analyses of targets for the treatment and detection of human MMP2P shows a purity of about 98%, and a molecular cancers. Cysteine was added to the peptide sequence weight of 2039 g/mol (Supplementary Material: Fig- in order to conjugate MMP2P on the surface of the ure S2 and S3). GNRs via thiol chemistry. Preparation of MMP2P-gold nanorod (MMP2P-GNR) conjugate Materials and methods The centrifuged CTAB-coated GNRs were re- Materials suspended in 1 mL of DW to remove excess CTAB. Gold ( Ⅲ ) chloride trihydrate (HAuCl43H2O, The concentration of the GNR solution was 100 nM 99.9%), sodium borohydride (NaBH4, 99%), silver after resuspension. An aqueous solution was pre- nitrate (AgNO3, 99%) and L-ascorbic acid (AA, 99%), pared by dissolving the MMP2P to a concentration of and hexadecyltrimethylammonium bromide (CTAB, 1 mM, of which 200 µL was added to the dispersion in 99%) were purchased from Sigma-Aldrich (St. Louis, addition to 100 L of 2 mM K2CO3 aqueous solution MO). Pyropheophorbide-a (PPa) was purchased from and allowed to react at room temperature for 5 days. Frontier Scientific, Inc. (Logan, Utah). Singlet oxygen The resulting solution was dialyzed in DW using an sensor green was purchased from Molecular Probes. ultrafiltration membrane with a molecular weight Ultrapure deionized water was used in all experi- cut-off (MWCO) of 50,000 Da to remove MMP2P that ments. HT1080 cells (human fibrosarcoma cell line) did not participate in the reaction with the GNRs. The overexpressing MMP2 and BT20 cells (human mam- remaining solution represented MMP2P-GNR conju- mary adenocarcinoma cell line), which lack detectable gates in which the photosensitizer-oligopeptide com- MMP2 expression [16], were obtained from American bination was linked with the GNRs. Type Culture Collection (ATCC, Rockville, MD). Cell To calculate the number of conjugated MMP2P lines were maintained in Dulbecco’s modified Eagle’s per GNR, the absorption spectrum of the purified medium (DMEM) supplemented with 10% fetal bo- MMP2P-GNR was measured in both DW (Figure 2B) vine serum (FBS), (GIBCO, Invitrogen) in a humidi- and dimethylformamide (DMF) using a UV/Vis fied 5% CO2 incubator at 37°C. scanning spectrophotometer (DU730, Beckman). The Preparation of gold nanorod solution CTAB-coated GNR has a molar absorption coefficient of 4.6 × 109 M-1cm-1 at 785 nm in DW [22]. PPa is Gold nanorods were prepared using a known to have a molar absorption coefficient of 9.98 × seed-mediated method as described previously [12, 104 M-1cm-1 at 413 nm in DMF [23]. These values were 21]. In brief, CTAB was dissolved in deionized water used to calculate the average number of MMP2P (DW) to a molarity of 100 mM; 7.5 ml of this CTAB conjugated per GNR in the MMP2P-GNR conjugates. aqueous solution was mixed with 250 L of 10mM Analysis of fluorescence and singlet oxygen gen- HAuCl4 aqueous solution, followed by the addition of eration (SOG) from the MMP2P-GNR conjugate 600 L of 10mM NaBH4 aqueous solution. The mix- ture was stirred for 2 min, and maintained at 25 °C for To observe fluorescence quenching characteris- 2 h to prepare a seed solution. tics, the MMP2P-GNR conjugates were dissolved in Meanwhile, 1.7 mL of 10mM HAuCl4 aqueous phosphate-buffered saline ([PBS], 6.7 mM; pH 7.4; solution was added to 40 mL of 100mM CTAB aque- NaCl, 154 mM), and their fluorescence spectra were ous solution, followed by the sequential addition of measured (Ex. 410 nm). In this study, a concentration 250 L of 10 mM AgNO3 aqueous solution and 270 L equivalent to 1 μM PPa was used for each solution. of 100 mM ascorbic acid. At this point, 420 L of the For comparison, free PPa was dissolved in PBS con- seed solution was added, and then maintained to re- taining 1% (v/v) Tween 20 to prevent fluorescence act for 12 h.
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