Int. J. Radiation Oncology Biol. Phys., Vol. 72, No. 3, pp. 633–635, 2008 Published by Elsevier Inc. Printed in the USA 0360-3016/08/$–see front matter
doi:10.1016/j.ijrobp.2008.06.1916
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SEMICONDUCTOR NANOPARTICLES AS ENERGY MEDIATORS FOR PHOTOSENSITIZER-ENHANCED RADIOTHERAPY WENSHA YANG, PH.D.,* PAUL W. READ, PH.D., M.D.,* JUN MI, M.D.,* JOSEPH M. BAISDEN, PH.D., M.D.,* KELLI A. REARDON, M.D.,* JAMES M. LARNER, M.D.,* BRIAN P. HELMKE, PH.D.,y AND KE SHENG, PH.D.* Departments of *Radiation Oncology and y Biomedical Engineering, University of Virginia, Charlottesville, VA Purpose: It has been proposed that quantum dots (QDs) can be used to excite conjugated photosensitizers and produce cytotoxic singlet oxygen. To study the potential of using such a conjugate synergistically with radiotherapy to enhance cell killing, we investigated the energy transfer from megavoltage (MV) X-rays to a photosensitizer using QDs as the mediator and quantitated the enhancement in cell killing. Methods and Materials: The photon emission efficiency of QDs on excitation by 6-MV X-rays was measured using dose rates of 100–600 cGy/min. A QD-Photofrin conjugate was synthesized by formation of an amide bond. The role of Fo¨rster resonance energy transfer in the energy transferred to the Photofrin was determined by measuring the degree of quenching at different QD/Photofrin molar ratios. The enhancement of H460 human lung carcinoma cell killing by radiation in the presence of the conjugates was studied using a clonogenic survival assay. Results: The number of visible photons generated from QDs excited by 6-MV X-rays was linearly proportional to the radiation dose rate. The Fo¨rster resonance energy transfer efficiency approached 100% as the number of Photofrin molecules conjugated to the QDs increased. The combination of the conjugate with radiation resulted in significantly lower H460 cell survival in clonogenic assays compared with radiation alone. Conclusion: The novel QD-Photofrin conjugate shows promise as a mediator for enhanced cell killing through a linear and highly efficient energy transfer from X-rays to Photofrin. Published by Elsevier Inc. Radioenhancer, Nanoparticle, Quantum dot.
labeling (3–6), and a nano-iron oxide particle has shown promise as a contrast agent in magnetic resonance imaging (7, 8). In these applications, the success of nanoparticles was largely a result of their versatility in binding with various molecules to provide biologic targeting. The idea of developing a nanoparticle-facilitated radioenhancer was initially proposed by Chen et al. (9), whose approach was clearly advantageous compared with traditional sensitizers, because the toxicity was ‘‘switched on’’ only by the radiation to achieve high geometric specificity when working with conformal radiotherapy. In their work, semiconductive nanoparticles were used to convert kilovoltage X-ray energy to visible light that could be used to excite conjugated photosensitizers. However, it was not reported whether the particles were excited by megavolatage (MV) X-rays, which are normally used in conformal external radiotherapy, or whether the energy transfer to the conjugated photosensitizer was efficient enough to significantly enhance cell killing. To
INTRODUCTION The delivery of conformal radiation by X-ray has been researched extensively with techniques such as intensity-modulated radiotherapy. However, in many cases, the conformality offered by advanced radiotherapy is still not sufficient to sterilize the tumor without causing severe complications. Increased geometric specificity might require methods in addition to pure physics and engineering. One such method uses chemical radiosensitizers, which were developed to increase tumor cells’ sensitivity to radiation. Although many types of radiosensitizers have been developed for clinical applications, they are generally toxic to normal tissues, resulting in unsatisfactory geometrically specific enhancement (1). Nanoparticles, because of their small size and unique biologic, chemical, and physical properties, are gaining wide application in biotechnology (2). In oncologic imaging, a semiconductor nanocrystal has been used for fluorescent
Conflict of interest: none. Acknowledgments—We thank Melissa Stauffer, Ph.D., of Scientific Editing Solutions, for editorial assistance. Received April 16, 2008, and in revised form April 24, 2008. Accepted for publication June 18, 2008.
Reprint requests to: Ke Sheng, Ph.D., Department of Radiation Oncology, University of Virginia, P.O. Box 800383, Charlottesville, VA 22908. Tel: (434) 872-0427; Fax: (434) 982-3520; E-mail:
[email protected] Supported by the University of Virginia Cancer Center Grant and the Coulter Foundation. 633
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address these critical questions, we conducted more detailed studies of the energy transfer and performed a cell survival assay to measure the cell killing enhancement. METHODS AND MATERIALS Polyethylene glycol-coated, amine-functionalized quantum dots (QDs) were purchased from Evident Technologies (Troy, NY). The QD has a CdSe core with a ZnS shell. The crystal diameter of the QDs is 2.1 nm. The hydrodynamic diameter of the coated QDs is approximately 25 nm, and the molar mass is distributed between 1 105 g/mol and 3 105 g/mol, as provided by the vendor. Light emission after irradiation was measured by a photomultiplier tube (Hamamatsu, H7467, Japan). The lens of the collimator is 5 cm from the center of the vial with the QD solution. The dark box containing the QD solution was irradiated using 6-MV X-rays from a clinical linear accelerator (Varian 2300, Varian, Palo Alto, CA), with dose rates of 100–600 cGy/min, at a 100-cm source-to-surface distance, with 1.5 cm of solid water for dose buildup. The dose was calibrated by an ion chamber at the measurement point. A photosensitizer was covalently linked to the QDs using previously described methods (10). In brief, pharmaceutical grade Photofrin (Wyeth-Ayerst Lederle, Collegeville, PA) was dissolved at 1.6 mmol/mL in deionized water. The carboxylic group of Photofrin was activated by 32 mmol/mL of 1-ethyl-3-(3-dimethylaminopropyl)carbodimide and stabilized by 30 mmol/mL N-hydroxysulfosuccinimide sodium (Solution A). We mixed 42 mL of Solution A with 20 mL of a 12 nmol/mL suspension of amine-terminated QDs with a known 520-nm emission (Evident Technologies). The mixture was stirred using a vortex mixer overnight at room temperature and then diluted to 48 pmol/mL for the clonogenic cell survival studies. Previous studies have shown that the energy transfer between conjugated QDs and photosensitizers can be Fo¨rster resonance energy transfer (FRET), which uses a dipole–dipole coupling mechanism as a competing mechanism to the fluorescence emissionabsorption process (11, 12). To determine the role of FRET in the QD-Photofrin conjugate, we excited conjugate suspensions with 6-MV X-rays at a known dose rate and compared the photon emis-
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Fig. 2. Conjugate emission quenching by comparing emission of quantum dots (QDs) alone with that of conjugates when irradiated by 6-MV X-rays. PS = photosensitizer. sion of the conjugate, synthesized with a range of reactant stoichiometries, to that of a simple, unconjugated QD-Photofrin mixture. By comparing the light emission, the amount of energy being transferred by FRET can be derived based on the conservation of energy. In vitro cell culture clonogenic survival studies were performed using H460 cells according to the protocol described by Franken et al. (13). In this assay, the cells were harvested from a donor culture by trypsinization. The cells were resuspended in complete growth medium consisting of Roswell Park Memorial Institute1640 supplemented with 10% fetal bovine serum (Invitrogen, San Diego, CA), and the number of cells was counted in a hemacytometer. The cell suspension was diluted to the desired seeding concentration and plated in a 100-mm 20-mm cell culture dish. The plated cells were incubated at 37 C and 5% carbon dioxide for 24 h before they were treated with the drug (conjugate, QD, or Photofrin). To avoid the phototoxicity from Photofrin, the dishes were wrapped in aluminum foil and incubated with conjugate for 4 h before irradiation. The aluminum foil was kept on throughout the radiation. The radiation doses were 2–8 Gy in 2-Gy increments. The cells were incubated for an additional 2 days before the drug was removed from the medium and for another 8 days to allow for large colony formation. The cells were fixed with 10% methanol/10% acetic acid for 5 min at room temperature and stained with 1 mL of crystal violet solution (1% wt/vol in methanol). The stained cells were scanned by an Epson V700 flatbed scanner with a 1,200 dpi resolution and automatically counted by the particle analysis module of ImageJ (National Institutes of Health, Bethesda, MD) with a threshold of 220 pixels and a circularity of 0.3–1.0.
RESULTS The QD light emission on excitation by 6-MV X-rays is shown in Fig. 1 as the emitted photon flux on exposure to X-rays at clinically relevant dose rates. A linear relationship between the dose rate and photon flux was observed (R2 = 0.9999). Thus, the number of visible photons generated from the QDs at the concentrations tested depended linearly on the radiation dose rate, and the dosimetry of the QD-Photofrin conjugate could be correlated with the radiation dose through a simple linear calibration equation. The photon emission comparison is shown in Fig. 2. Compared with QDs at the same concentration, the conjugate
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Fig. 3. Semilog plot of cell survival curves #8 Gy, at 2-Gy increments, delivered in single fraction. Cell survival fraction of radiation-only group normalized to control group unexposed to radiation, and drug survival fraction of cells exposed to both radiation and drug normalized to control group exposed to drug only. Concentration of quantum dots (QDs) and Photofrin was consistent in either conjugate or single chemical group.
emitted systematically fewer photons than QDs alone, and the difference increased significantly with the molar ratio of Photofrin and QDs in the conjugate. The result indicates energy transfer predominantly by FRET. The emission of the QDs was completely quenched when the molar ratio reached 20:1. We used a Photofrin/QD ratio of 291:1 in the synthesis of the conjugate for the cell culture study. The
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FRET efficiency at that molar ratio was therefore close to 100%. Figure 3 shows the dose dependence of H460 cell survival fraction with and without exposure to the drug. Significantly enhanced cell killing in the presence of conjugates was observed for all dose levels but not for the groups with only one of the compounds. Furthermore, the enhancement increased as the dose increased. The accelerated enhancement, in conjunction with conformal radiotherapy, would lead to a greater multiplier factor in the target volume receiving a greater dose and a lower increase in the toxicity to the surrounding volume receiving the lower dose. Moderate toxicity was observed with the conjugate alone without radiation, resulting in 24% 6% cell death. This might have been caused by a small, but unavoidable, amount of light leakage during the experiment. CONCLUSION A novel conjugate of a semiconductor nanoparticle, a QD, and a photosensitizer, Photofrin, were synthesized and tested for its ability to enhance radiotherapy. High efficiency energy transfer from MV X-rays to the photosensitizer by a mechanism known as FRET was measured. Significantly enhanced cell killing was observed with combined treatment of 6-MV radiation and QD-Photofrin conjugates compared with radiation alone. The linear relationship between the radiation dose rate and QD excitation predicted for uncomplicated dosimetry in the clinical use of this novel conjugate.
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