Quantum dot-aluminum phthalocyanine conjugates perform photodynamic reactions to kill cancer cells via fluorescence resonance energy transfer
© Li et al.; licensee Springer. 2012
Received: 25 May 2012
Accepted: 22 June 2012
Published: 12 July 2012
Sulfonated aluminum phthalocyanines (AlPcSs), commonly used photosensitizers for photodynamic therapy of cancers (PDT), were conjugated with amine-dihydrolipoic acid-coated quantum dots (QDs) by electrostatic binding, achieving 70 AlPcSs per QD. The AlPcS-QD conjugates can utilize the intense light absorptions of conjugated QDs to indirectly excite AlPcSs producing singlet oxygen via fluorescence resonance energy transfer (FRET), demonstrating a new excitation model for PDT. The AlPcS-QD conjugates easily penetrated into human nasopharyngeal carcinoma cells and carried out the FRET in cells, with efficiency around 80%. Under the irradiation of a 532-nm laser, which is at the absorption region of QDs but not fit for the absorption of AlPcSs, the cellular AlPcS-QD conjugates can destroy most cancer cells via FRET-mediated PDT, showing the potential of this new strategy for PDT.
KeywordsQuantum dot Aluminum phthalocyanine Fluorescence resonance energy transfer Photodynamic therapy of cancers.
Photodynamic therapy (PDT) has been established as a new treatment modality for cancers during the past two decades. The principle of this modality is that a photosensitizing drug can preferably accumulate in the cancer region and produce singlet oxygen (1O2) when excited with light of appropriate wavelengths to destroy the lesion . Therefore, the efficiency of cancer inactivation is closely correlated to the light absorption of used photosensitizers (PSs). However, the light extinction coefficients of PSs are generally low, including those officially approved PSs such as Photofrin and metal phthalocyanines [2–4]. Recently, the nanotechnology with different kinds of nanoparticles has been increasingly developed, and the related applications have been expanded quickly [5–10]. The suggestion of combining nanotechnology with PDT has been proposed, and related works have been tested [11–18]. Based on the high light absorption coefficients of quantum dots (QDs), Samia et al. firstly proposed a new idea of PS-QD conjugate model, in which the QD absorbs light and then functions as a donor to transfer the energy to PS via fluorescence resonance energy transfer (FRET). They prepared QD-phthalocyanine (Pc4) conjugates in organic solution, and these conjugates achieved the high 1O2 production with the way of FRET . The other groups including our group then developed different kinds of QD-PS conjugates in aqueous solutions for their further applications in biological systems [5, 20, 21]. Since the light extinction coefficients of QDs are one order of magnitude larger than that of PSs, the conjugation of PS-QD seems a potential way to increase the PDT effect. However, the biological system is much complicated as compared to the pure solution. So far, the FRET-mediated PDT by PS-QD conjugates has not been achieved in living systems, leaving a doubt whether the PS-QD conjugates can really work in PDT. Regarding the application in biological systems such as in living cells, at least two problems should be overcome. Firstly, when conjugates pass through the plasma membranes entering the living cells, they should keep their conjugate form, which requires a stable binding between PS and QD in the conjugate. Secondly, the PS-QD conjugates are also required to maintain their physical properties in the cellular environment for carrying out the FRET. In this work, the stable conjugates of QDs with sulfonated aluminum phthalocyanines (AlPcSs) were prepared by electrostatic binding. These conjugates can load AlPcS to penetrate into cancer cells easily and then perform the FRET effectively in cellular environments. The conjugates in cells demonstrated for the first time that they can kill cancer cells via FRET-mediated PDT.
Preparation of amine-DHLA-coated CdSe/CdS/ZnS QDs
Preparation of AlPcS-QDs
The photosensitizer AlPcS (Frontier Scientific, Inc, Logan, UT, USA) could have the maximum four negative charges in its four benzene rings (Figure 1a), so that they can bind on the surfaces of QDs by an electrostatic force. The absorption and fluorescence spectra of AlPcSs in aqueous solution, measured by a spectrophotometer (Hitachi F-2500, Hitachi, Tokyo, Japan), are shown in Figure 1b. To prepare the AlPcS-QD conjugates, the mixture groups of AlPcSs and QDs with different molar ratios were made and stirred overnight in the dark at room temperature (25 °C). These mixture solutions were then centrifuged at 10,000 rpm for 30 min to separate the AlPcS-QD conjugates in the bottom of the centrifuging tubes and the unbound AlPcSs in the supernatants. The harvested AlPcS-QD conjugates were re-suspended in aqueous solution for further experiments. The FRET efficiencies of these obtained complexes were measured respectively to find out an optimal molar ratio for AlPcS-QD conjugates with the best FRET effect. From the obtained results, an optimal molar ratio of AlPcSs to QDs was found to be about 100:1.
The human nasopharyngeal carcinoma cells (KB cells) were obtained from the cell bank of Shanghai Science Academy, Shanghai, China. The cells were seeded into culture dishes containing DMEM medium with 10% calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 100 μg/ml neomycin, and incubated in a fully humidified incubator at 37 °C with 5% of CO2. When the cells reached 80% confluence with normal morphology, AlPcS-QDs were added and incubated in an incubator for 50 min. After incubation, these cells were washed three times with phosphate buffered saline (PBS) to remove unassociated AlPcS-QDs, and then, these cell samples were ready for further experiments.
Imaging measurements of AlPcS-QDs in cells
The fluorescence images of free QDs, free AlPcSs and AlPcS-QD conjugates in KB cells were measured in a laser scanning confocal microscope (LSCM) (FV300, IX71, Olympus Microscopy, Tokyo, Japan) excited by a 405-nm laser. In the measurements, the QD's PL was measured in detection channel 1 of the LSCM with a band-pass filter of 585 to 640 nm, and the fluorescence signal of AlPcSs was recorded in detection channel 2 with a 670-nm long-pass filter. Differential interference contrast (DIC) images were obtained simultaneously in a transmission channel to exhibit the cell morphology. A water immersion objective (×60) and a matched pinhole of LSCM were used in experiments.
QD's PL lifetime measurements by TCSPC
We found that two exponential components are good enough to fit the measured decay curves here. With the numerous measurements, the average PL lifetimes of QDs in different cases were determined.
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium reduction assay was used to measure PDT damaging of AlPcS-QDs on cells. The KB cells with the concentration of 3 × 104 cells/ml were equally seeded in each well of a 96-well flat-bottom tissue culture plate and allowed to attach to the plate overnight. Then, 5 μM AlPcS, 0.05 μM QDs and 0.05 μM AlPcS-QDs were added into different wells for 50-min incubation, respectively. After the incubation, the cells were washed three times with PBS to remove the unassociated compounds, and added with the fresh medium. These cells were then irradiated by a halogen lamp or a 532-nm laser at different times. After illuminations, the cells were incubated in an incubator for 24 h, and then, 10-μl MTT solution (5 mg/ml) was added into each well for incubation for 1.5 h. Finally, the 96-well plates were put into an iEMS analyzer (Laboratory Systems Group Pty Ltd., Kilsyth, Victoria, Australia) to measure optical densities (O.D) at 450 nm for each well. The cell viability in each well was determined by comparing their O.D value with that of untreated control cells in some wells in the same plate. All results were presented as the mean ± standard deviation (SD) from three independent experiments with four wells in each.
Reactive oxygen species (ROS), such as singlet oxygen (1O2) and free radicals of oxygen molecules, play an important role in PDT. When the conjugated AlPcSs in AlPcS-QDs were excited via FRET, they could emit fluorescence in one way and produce 1O2 (excited state of O2) by transferring the energy to the surrounding oxygen molecules in the other way. 1,3-Diphenylisobenzofuran (DPBF), a sensitive probe of ROS , was used to detect the ROS produced by a FRET way of AlPcS-QDs under irradiation at 532 nm. The DPBF can be quickly oxidized by ROS to become o-dibenzoylbenzene, leading to a degradation of DPBF with reduced fluorescence intensity. The fluorescence of DPBF was measured in a spectrometer (F-2500, Hitachi) with the excitation at 405 nm. In the experiment, the DPBF (12 μM) were mixed with AlPcSs (10 μM), QDs (0.1 μM) and AlPcS-QDs (0.1 μM) in aqueous solutions, respectively, and then, these samples were irradiated by a 532-nm laser (16 mW) for different times such as 20 s, 40 s, etc. The fluorescence intensity decrement of DPBF is usually used as the indicator of the ROS production during the photosensitization process of PSs. The degradation rate of the DPBF with the irradiation time is proportional to the ROS yield and, thus, can be used to evaluate the relative ROS yields for different compounds. Sodium azide (NaN3), a specific 1O2 scavenger, was also used in the experiment to confirm the 1O2 production in the FRET process of AlPcS-QDs .
Results and discussion
AlPcSs have an absorption band at around 675 nm (630 to 700 nm) in the visible region, but QDs have broad absorptions in the whole visible area (Figure 1b). A light source with a broad wavelength region may increase the PDT effect of AlPcS-QDs to cancer cells because the AlPcS moiety in conjugates can directly absorb the light around 675 nm to initiate the PDT, and the QD moiety can absorb the light of the other wavelengths shorter than the 675-nm band to conduct a FRET-mediated PDT, resulting in a combination effect to increase the PDT efficiency finally. A 150-W halogen lamp combined with a heat-isolation filter was used here to check the PDT effect of AlPcS-QDs further. The emitting spectrum of this light source is from 400 to 800 nm. Under the irradiation of this white light, the PDT efficiency of conjugates increased obviously (Figure 5b). With a 4-J/cm2 irradiation dose of this white light, AlPcS-QDs damaged most cancer cells. Therefore, fully utilizing the absorptions of both QDs and AlPcSs in conjugates is probably a good strategy for PDT. To further compare the PDT effect of AlPcS-QDs with that of free AlPcS, cells were incubated by free AlPcS (5 μM) with a prolonged incubation time (3 h) to allow more AlPcSs penetrating into the cells, and then, these cells were irradiated by the white light. As shown in Figure 5b, even at a high light dose, only about 30% cell death could be conducted in this case.
The tissue transparency window is in the region of 650 to 900 nm, so that the wavelengths in this region are optimal for carrying out PDT. However, QD's main absorption region is shorter than 600 nm, which does not match the tissue window wavelengths. Another advantage of QDs can just help them overcome this limitation. QDs have very huge two-photon absorption cross sections (thousands Goppert Mayer (GM)) in the near-infrared (NIR) wavelength region. Therefore, QDs in conjugates can be excited by two-photon excitation (TPE) with a femtosecond NIR laser and then initiate FRET-mediated PDT. Since the two-photon absorptions of PSs are very low (a few GM), the PS-QD conjugates with TPE open a new way for the so-called two-photon PDT. Our next work is already under processing toward this direction.
The main disadvantage of PSs used in PDT is skin phototoxicity because the affinity of PSs to cancers is not high, and a part of PSs remain in the skin. If the drug delivery efficiency to cancers is increased, the drug dose can be decreased, leading to a lower content of drugs in the skin, reducing skin phototoxicity. However, the accumulation of PSs in cancer is a passive targeting. Based on the passive targeting mechanism for cancers in vivo, the particles with the size of 3 to 100 nm will be accumulated more in tumors than smaller molecules due to the effect of enhanced permeability and retention. The sizes of current used PSs are all about 1 nm, so that the delivery efficiency to tumors is low. However, the size of the QDs we used is about 3.5 nm, and the sizes of PS-QD conjugates should be a little bit bigger than 3.5 nm. Therefore, with the PS-QD conjugates, better tumor accumulation is expected. The use of PS-QD conjugates may also benefit drug delivery and decrease skin phototoxicity.
AlPcS-QD conjugates were stable not only in solutions but also in cellular environments. The cellular AlPcS-QDs achieved FRET in living cells with an efficiency of 84%, reflecting that the AlPcS-QDs possess the ability to carry out PDT via FRET. Furthermore, the AlPcS-QDs killed KB cancer cells under the irradiation of a 532-nm laser with a typical light-dose-dependent PDT mode, demonstrating a new excitation way of FRET-mediated PDT by AlPcS-QDs. The ROS produced by a FRET way of AlPcS-QDs was further examined as the 1O2. With white light irradiation, the combination of direct and indirect (FRET) excitations for AlPcS-QDs can remarkably enhance PDT effect to cells. In addition, the AlPcS-QD conjugates can easily penetrate into cells, demonstrating that these conjugates are good carriers for AlPcS intracellular delivery because the cellular uptake rate for free AlPcSs is very low. The result also reflects that the positively charged QDs are probably suitable carriers to load negatively charged PSs for intracellular delivery. These results suggest that the new strategy of AlPcS-QD conjugates combined with the FRET could be a feasible modality for PDT, and this new model is worth investigating further to improve PDT effects in cancer treatments.
Ji-Yao Chen is a professor and doctorate degree holder in the State Key Laboratory of Surface Physics, Department of Physics, Fudan University. Sungjee Kim is a professor and doctorate degree holder in the Department of Chemistry, Pohang University of Science and Technology. Lei Li and Jin-Feng Zhao are master degree holders in the Department of Physics, Fudan University. Nayoun Won and Ho Jin are PhD candidates in the Department of Chemistry, Pohang University of Science and Technology.
This work was supported by the National Natural Science Foundation of China (11074053 and 31170802) and the National Research Foundation of Korea (NRF) Grant (NRF-2011-C00048).
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