NANO LETTERS
Quantum Dot anti-CD Conjugates: Are They Potential Photosensitizers or Potentiators of Classical Photosensitizing Agents in Photodynamic Therapy of Cancer?
2004 Vol. 4, No. 9 1567-1573
Rumiana Bakalova,* Hideki Ohba, Zhivko Zhelev, Toshimi Nagase, Rajan Jose, Mitsuru Ishikawa, and Yoshinobu Baba Single-Molecule Bioanalysis Laboratory, National Institute for AdVanced Industrial Science and Technology, 2217-14 Hayashi-cho, Takamatsu 761-0395, Japan Received March 8, 2004; Revised Manuscript Received June 17, 2004
ABSTRACT The present study examined the potential of quantum dot bioconjugates to sensitize cells to UV irradiation and to promote the photodynamic activity of the classical photosensitizers such as trifluoperazine (TFPZ) and sulfonated aluminum phthalocyanine (SALPC). Water-soluble CdSe nanocrystals were conjugated with anti-CD antibody with known specificity to leukemia cells. Quantum dot anti-CD conjugates were incubated with the leukemia cell line Jurkat to ensure specific interaction with the cell surface. This interaction was confirmed by fluorescent confocal microscopy. Furthermore, quantum dot anti-CD90-labeled leukemia cells were mixed with normal lymphocytes and subjected to UV irradiation in the presence or absence of a classical photosensitizer (TFPZ or SALPC). The cell fractions were separated by lectin-affinity column chromatography. The cell type was confirmed with fluorescent confocal microscopy and flow cytometry using appropriate antibodies; quantum dot anti-CD90 for leukemia cells, and PE-CD44 for normal lymphocytes. The viability of the separated cell fractions was determined using flow cytometry and the methyl tetrazolium test. The results demonstrated that quantum dot anti-CD conjugates sensitized leukemia cells to UV irradiation and promoted the effect of the classical photosensitizer SALPC. The results are discussed in the context of free radical generation during combined application of quantum dot bioconjugates and UV irradiation, as well as in the context of UV-mediated liberation of free Cd ions and their harmful effect on cell viability.
The work of Derfus and colleagues,1 provokes the hypothesis that while the cytotoxicity of quantum dots, mediated by UVirradiation, is harmful for normal cell viability, it may be useful in killing cancer cells. Quantum dots are considered to be energy donors,2,3 and the possibility for energy transfer between quantum dot particles and cell molecules (as triplet oxygen, reducing equivalents, pigments, etc.) has a potential to induce generation of reactive oxygen species and/or free radicals and to provoke apoptosis of the cells. The dual nature of UV-mediated cytotoxicity of quantum dots and their energy donor capacity could open a new area of quantum dot application in biology and medicine, as novel photosensitizers or at least as potentiators of the conventional photosensitizing drugs in photodynamic therapy of cancer. In photodynamic therapy, light, oxygen, and photosensitizing agents are combined to produce a selective therapeutic effect in the target tissue (usually localized tumor).4-6 The * Corresponding author: Rumiana Bakalova, PhD, Single-Molecule Bioanalysis Laboratory, National Institute for Advanced Industrial Science and Technology, 2217-14 Hayashi-cho, Takamatsu 761-0395, Japan. Tel./ Fax: +81-87-815 25 23; E-mail:
[email protected]. 10.1021/nl049627w CCC: $27.50 Published on Web 07/30/2004
© 2004 American Chemical Society
selectivity is based on the selective retention and concentration of photosensitizer in the cancer cells and on the direction of laser or UV-light on the sensitized cancer tissue. The photophysical processes involved in photodynamic therapy are illustrated in Figure 1, where S0 is the ground electronic singlet-state of the photosensitizer, S1 is the shortlived first excited singlet-state of the photosensitizer, and T1 is the first excited triplet-state, which is sufficiently longlived. There are two types of photodynamic reactions.7 The first type is electron or hydrogen transfer between the T1 photosensitizer and other molecules. These processes produce reactive intermediates that are harmful to cells, such as superoxide, hydrogen peroxide, and hydroxyl and hydroperoxyl radicals. The second type is an electron spin exchange between the T1 photosensitizer and triplet oxygen (3O2), resulting in the production of cytotoxic singlet oxygen (1O2). Singlet oxygen is accepted as the main mediator of photocytotoxicity in photodynamic therapy, causing biomembrane oxidation and degradation.8 During these processes the photosensitizer returns to its ground singlet-state
Figure 2. Scheme of conjugation of CdSe nanocrystals with antiCD90 antibody.
Figure 1. Photophysical processes involved in photodynamic therapy (according to Jablonski diagram).
S0 and can generate an abnormal concentration of reactive intermediates. It is important to note that the photosensitizer is degraded by light. The process, known as photobleaching, may be caused by reaction of the first or the second type. The photosensitizing capacity of a certain compound is determined by several parameters: (i) the singlet oxygen yield (Φ∆), which is the probability that the photosensitizer, after absorption of a quantum of light, converts to the T1 state and then transfers its excess energy to the triplet-oxygen, causing the formation of a singlet-oxygen; (ii) the triplet state yield (Φt), which is the probability that a photosensitizer, after absorption of a quantum of light, converts to the T1 state; (iii) the triplet state energy (∆Et) which is the difference between the energies of the S0 and T1 sates of the photosensitizer; (iv) the rate constant (kth) of the T1 state quenched by triplet oxygen; and (v) the ability of the photosensitizer to generate singlet-oxygen per irradiation power and photosensitizer concentration (R ) 1.925 × 10-5λΦ∆, where λ is irradiation wavelength and is the molar extinction coefficient of the photosensitizer at the wavelength used, expressed in M-1 cm-1).8 Do quantum dots possess a potential to be photosensitizers? The present study was designed to verify, at least partially, the ability of CdSe quantum dots to sensitize cells to UV-irradiation. Water soluble CdSe core nanocrystals were conjugated with anti-CD90 antibody with known specificity for leukemia cells. The scheme of conjugation is shown in Figure 2. The cells were derived from acute lymphoblastic leukemia (ALL). Quantum dot anti-CD90 conjugates (containing approximately 0.1 mg nanocrystals/mL) were incubated with ALLderived cells (Jurkat, 1 × 106 cells/mL) in PBS for 30 min at room temperature to guarantee a specific interaction of quantum dot anti-CD90 on the surface of leukemia cells. This interaction was confirmed by fluorescent confocal microscopy (Figure 3A). The cells were sedimented by centrifugation (1000 × g/10 min), resuspended in PBS (to 1 × 106 cells/mL), and mixed with a suspension of normal lymphocytes (derived from clinically healthy blood donors) in the same buffer (1:1, v:v). The cell mixture was further 1568
subjected to UV irradiation for 1 h (10 min irradiation, 10 min break). Leukemia cells and normal lymphocytes was separated by lectin-affinity column chromatography.9,10 CNBr-activated Sepharose 6 MB conjugated with soybean agglutinin was used as a nonmobile phase. Normal lymphocytes were easily eluted by PBS, while leukemia cells were retained on the column (Figure 3-B1) and were removed from the gel by N-acetyl-D-galactosamine (Figure 3-B2). The cell types in each eluted fraction were evaluated by fluorescent confocal microscopy using appropriate antibodies: quantum dot anti-CD90 for leukemia cells (green fluorescence) and PE anti-CD44, specific for normal lymphocytes (commercially available, red fluorescence, Figure 3-C1).9,10 The viability of leukemia cells and normal lymphocytes was determined by flow cytometry and the CellTiter Glo cell viability test (Figure 3-C2). In a parallel experiment, an aliquot of the cell suspension containing quantum dot anti-CD90-labeled leukemia cells and normal lymphocytes was incubated with 1 µM trifluoperazine (TFPZ) in RPMI-1640 medium in a humidified atmosphere (5% CO2, 36.9 °C) before being subjected to UV irradiation. TFPZ is a well-known photosensitizer of the phenothiazine family. At a concentration of 1 µM and in the absence of UV irradiation, TFPZ does not exhibit any effect on the viability of leukemia cells and normal lymphocytes. After 10 min incubation of the cell suspension with TFPZ, the cells were subjected to UV irradiation, lectinaffinity chromatography, and other procedures as described above (Figure 3). A mixture of leukemia cells and normal lymphocytes untreated with quantum dot anti-CD90 and/or TFPZ was used as control. The results in Table 1 demonstrate the potential of quantum dot anti-CD conjugates to sensitize leukemia cells to UV irradiation and/or to potentiate the effect of conventional photosensitizer TFPZ. The concentration of quantum dot anti-CD, TFPZ, or a combination of both compounds had no significant effect on the viability of nonirradiated cells. UV irradiation of the cell mixture in the absence of TFPZ and/or quantum dot anti-CD90 provoked a slight but nonsignificant decrease in cell viability. Treatment of leukemia cells with quantum dot anti-CD90 and subsequent UV irradiation of the cell mixture (leukemia plus normal cells) provoked a statistically significant decrease Nano Lett., Vol. 4, No. 9, 2004
Figure 3. Experimental protocol. (A) Interaction of quantum dot anti-CD90 conjugates with leukemia cells (A1) and normal lymphocytes (A2), detected by fluorescent confocal microscopy. Quadrant (a); green fluorescence, quadrant (b); transmission. (B) Fractionation of leukemia cells from normal lymphocytes using lectin-affinity column chromatography. B1: Retention of quantum dot anti-CD90-labeled leukemia cells on the lectin-conjugated gel beads, detected by fluorescent confocal microscopy. B2: Typical chromatogram of the separated cell fractions. (C) Verification of the type of the separated cell fractions, using fluorescent confocal microscopy (C1) and flow cytometry (C2). C1: quadrant (a), green fluorescence (of quantum dot CD90); quadrant (b), red fluorescence (of PE-CD44); quadrant (c), transmission; quadrant (d), green/red fluorescence and transmission. C2: Typical histograms from the flow cytometric analysis.
of the viability of leukemia cells, without any significant effect on the viability of normal lymphocytes. Nano Lett., Vol. 4, No. 9, 2004
Treatment of a mixture of leukemia cells and normal lymphocytes with 1 µM TFPZ and subsequent UV irradiation 1569
Table 1. Viability of Leukemia Cells and Normal Lymphocytes before and after Treatment with Quantum Dot anti-CD90 (QD-CD90), Trifluoperazine (TFPZ), and/or UV Irradiation Without UV Irradiation viable cells (%) flow cytometry
CellTiter-Glo test
cell treatment
normal cells
leukemia cells
normal cells
leukemia cells
control cells (non-treated) plus QD-CD90 plus TFPZ (1 µM) plus QD-CD90 + TFPZ (1 µM) plus anti-CD90 + TFPZ (1 µM)
92 ( 4 90 ( 7 93 ( 3 92 ( 6 91 ( 5
84 ( 11 86 ( 13 81 ( 9 81 ( 11 83 ( 8
100 102.8 ( 11.2 98.4 ( 9.3 101.7 ( 8.5 99.7 ( 6.0
100 99.3 ( 5.7 96.8 ( 6.2 94.5 ( 9.3 95.7 ( 8.8
Plus UV Irradiation-Flow Cytometric Analysis of Cell Viabilitya non-treated cells (Gr.1)
+UV-irradiation (Gr.2)
+QD-CD90 +UV-irradiation (Gr.3)
+TFPZ +UV-irradiation (Gr.4)
viable cells (%)
viable cells (%)
P-value
viable cells (%)
normal
92 ( 4
86 ( 7
NS vs Gr.1
88 ( 6
NS vs Gr.1, Gr.2
91 ( 4
NS vs Gr.1, Gr.2, Gr.3
89 ( 4
leukemia (Jurkat)
84 ( 11
72 ( 12
NS vs Gr.1
64 ( 9
p
