Bioconjugate Chem. 2007, 18, 494−499
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Water-Soluble Polymer Conjugates of ZnPP for Photodynamic Tumor Therapy M. Regehly,† K. Greish,‡,§ F. Rancan,†,| H. Maeda,*,‡,§ F. Bo¨hm,| and B. Ro¨der*,† Institut fu¨r Physik, Photobiophysik, Humboldt Universita¨t zu Berlin, Newtonstr. 15, D-12489 Berlin, Germany, BioDynamics Research Laboratory, Cooperative Research Center, Kumamoto University 2081-7 Tabaru, Mashiki-machi, Kumamoto, 861-2202, Japan, Sojo University, Faculty of Pharmaceutical Sciences, Ikeda 4-22-1, Kumamoto 860-0082, Japan, and Universita¨tsklinikum Berlin, Charite, Hautklinik, Photobiologisches Labor, Ziegelstr. 5-9, D-10117 Berlin, Germany. Received June 7, 2006; Revised Manuscript Received November 12, 2006
Zn-protoporphyrin (ZnPP) is a promising candidate for cancer therapy. It is known to inhibit heme-oxygenase-1 (HO-1), resulting in suppressed biliverdin/bilirubin production accompanying lowered antioxidative capacity. As a consequence, a significant suppression of tumor growth in vivo was reported. Recent findings also showed that ZnPP efficiently generated reactive singlet oxygen under illumination of visible light. In the present report, we describe the photosensitizing capabilities of water-soluble polymer conjugates of ZnPP as novel compounds for photodynamic therapy against solid tumors. The polymer conjugation made ZnPP water-soluble, thus possible for injection for its aqueous solution. The cellular uptake and photobiological activity of ZnPP derivatives have been tested using a human T-cell leukemia cell line in vitro and demonstrated most potent phototoxic effects of SMA-ZnPP followed by PEG-ZnPP under aerobic conditions.
INTRODUCTION Metal protoporphyrins (MePP), e.g., Zn protoporphyrin (ZnPP), exhibit many different interesting properties in terms of cancer treatment. While it is well-known that ZnPP is a strong heme oxygenase-1 (HO-1) inhibitor, it also has photosensitizing properties. It is also known that HO-1 is highly up-regulated in many cancer cells (1, 2). HO-1, also known as the heat shock protein 32 (Hsp 32), is induced by various insults such as oxystress, electromagnetic irradiation (UV and X-ray), and nitric oxide, and it is also up-regulated in the inflammatory tissues; thus, it is an inducible enzyme. HO-1 catalyzes the degradation of heme in the presence of oxygen to liberate biliverdin, iron, and CO. Biliverdin is then converted to bilirubin. The hypothesis is that inhibition of HO-1 would result in the inhibition of bilirubin production, which means that suppression and depletion of HO-1 would make cells more vulnerable to oxystress or radiation, since bilirubin is a very potent antioxidative agent. Thus, such tumor cells treated with an inhibitor of HO-1 (e.g., Zn protoporphyrin) would undergo apoptosis readily (3). On the other hand, ZnPP derivatives are known as efficient photosensitizers (4). ZnPP absorbs light in the UV-vis region and in turn changes to the first excited singlet state. In doing so, the molecules undergo intersystem crossing that leads to the population of the long-lived first excited triplet state. From this state, the energy is transferred efficiently to molecular oxygen, and thus reactive singlet molecular oxygen [1O2] (5) is generated, that would induce directly or indirectly cell death. ZnPP has a very low solubility in water at neutral pH, and hence its practical use would be limited as a pharmaceutical agent. For that reason, a water-soluble form of ZnPP was prepared by conjugating it with a poly(ethylene glycol) (PEG) chain (6). As a result, the compound became of a macromolecular nature, i.e., apparent molecular size in solution * Corresponding authors. E-mail:
[email protected] (B.R.),
[email protected] (H.M.). † Humboldt Universita ¨ t zu Berlin. ‡ Kumamoto University. § Sojo University. | Universita ¨ tsklinikum Berlin.
>70Da. In our earlier studies, it was shown that macromolecules tend to accumulate in solid tumors much more extensively, e.g., a 10-30-fold higher amount than low molecular weight drugs due to unique differences in pathophysiology and architecture of tumor blood vessels, as well as overproduction of various vascular mediators at or near the tumor site that further facilitate vascular permeability. This phenomenon was coined the EPR effect (7, 8). Now, the EPR (enhanced permeability and retention) effect is considered to be the key issue to achieve tumor-selective targeting of macromolecular drugs. On the other hand, using our more recent micellar technology with poly(styrene-maleic acid) copolymer (SMA), it becomes possible to encapsulate varieties of drugs at very high loading levels, as much as 50% (w/w) in the SMA micelle, which releases free drug in a time-dependent manner, and the micellar form of drugs also exhibits this EPR effect (9). The present study is focused on the comparative investigation of SMA-ZnPP micelles and PEG-ZnPP according to their photodynamic activity in vitro. Until now, an antitumor effect for PEG-ZnPP has been reported only in ref 10. In this earlier study, a hypothetical mechanism of antitumor activity of ZnPP was attributed to the inhibition of HO-1 (that suppresses bilirubin generation), and thus enhancement of vulnerability against oxystress (that derived via the host defense system such as macrophages) was envisaged. The aim of the present study was to investigate the ability of both compounds (PEG-ZnPP and SMA-ZnPP) for intracellular generation of singlet oxygen, that causes photodynamically induced cell death. We assume that the polymer conjugates would benefit from the EPR effect driven tumor accumulation. In this context, antitumor effect is exerted both by HO-1 inhibition and singlet oxygen generated photodynamic activity. Consequently, the antitumor effect will be exhibited in a highly tumor targeted manner. Thus, it would result in a superior therapeutic effect compared to free ZnPP.
MATERIALS AND METHODS Preparation of Polymeric Zn-Protoporphyrin. PEG-ZnPP conjugate was prepared by conjugation of succinimidyl PEG
10.1021/bc060158u CCC: $37.00 © 2007 American Chemical Society Published on Web 02/06/2007
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Water-Soluble ZnPP Conjugates
(NOF, Tokyo) to bis(ethylenediamino) protoporphyrin and the chelation of zinc into the porphyrin ring afterward. The detailed method is described in ref 7. Poly(styrene-co-maleic acid) (SMA) micelles containing ZnPP were prepared similarly to the SMA-pirarubicin micelles as reported previously (10, 11). Absorption Spectra. The absorption spectra were recorded using a commercial spectrophotometer Shimadzu UV-2501 PC at room temperature. The spectrum of free ZnPP was measured after dissolving it in ethanol. Spectra of PEG-ZnPP and SMAZnPP were taken after dissolving in H2O. Time-Resolved Singlet Oxygen Luminescence Measurement. Measurements were carried out using a nanosecond Nd: YAG laser (BM Industries, Evry Cedex, France) with an attached optical parametric oscillator (BMI). The singlet oxygen emission was detected using a liquid nitrogen cooled Ge-diode (North Coast, Inc., Santa Rosa, CA). For wavelength selection, a combination of a silicon filter and an interference filter for 1270 nm was placed in front of the diode (12). PEG-ZnPP and SMA-ZnPP were dissolved in D2O. The optical density was adjusted to 0.2 at the excitation wavelength of 550 nm. Rose Bengal served as a reference standard for the determination of singlet oxygen quantum yield (Q∆ ) 0.75) (13). Laser Flash Photolysis. The same equipment as described before was used to excite the samples for triplet-triplet absorption measurements. A continuous wave (cw) test beam was generated using an XBO lamp with the light passed through a monochromator adjusted to 480 nm. The beam traversed through the PEG- and SMA-ZnPP solutions perpendicular to the excitation beam and was detected by an avalanche Siphotodiode with an interference filter for 480 nm. Oxygen was removed by bubbling the samples with N2 gas for approximately 40 min at room temperature (14). Cell Culture and Incubation Conditions. A T-cell line of human T-lymphocytes (Jurkat cells: clone E 6-1 ATCC, human acute T-cell leukemia) were cultured in 5 mL of RPMI 1640 medium containing Glutamax-I, supplemented with 10% fetal calf serum (FCS), 100 µg/mL streptomycin, and 100 I.E./ mL penicillin in 50 mL flasks. Cells were cultured at 37 °C in 100% humidity and 5% CO2 and subcultured in new medium every 3 days. PEG-ZnPP and SMA-ZnPP were dissolved in PBS. Water-insoluble free ZnPP was dissolved in 0.1 M NaOH. Jurkat cells (2 × 105 cells/mL) were incubated in medium with four different Zn-protoporphyrin concentrations at (0.5, 1.0, 2.5, and 5 µM). All reported concentrations refer to free Znprotoporphyrin equivalents. Cells without the studied compounds were irradiated with UVA (R/UVA) or kept in the dark (R/D) and used as a reference standard. Intracellular Uptake. The amount of sensitizer uptaken by Jurkat cells was determined by measuring the fluorescence intensity of cell extracts. Cells were harvested 24 h after incubation, washed twice, and counted using a hemocytometer (Neubauer improved). The cell pellets were stored at -20 °C. Cells were then thawed, and the sensitizer was extracted after centrifugation using ethanol. The fluorescence intensity of cell extracts was measured with a fluorescence microplate reader Gemini EM (Molecular Devices, Germany), using λexc ) 420 nm and λem > 650 nm. Concentration of the sensitizers in the cell extracts was quantified by fluorescence intensity relative to standard curves after disintegration with ethanol in the case of SMA-ZnPP. UVA Irradiation of Cell Suspensions. Jurkat cells were washed, resuspended in PBS, and placed in a 24-well plate and covered with a quartz glass. A fluorescence lamp (type TLK 40 W, Philips) was used with 99.58% emission in the UVA region (315 and 400 nm) and 0.42% emission in the spectral range below 315 nm. The culture plate was placed under the lamp at 4 cm distance, and cells were irradiated for 5 min. At
Figure 1. Normalized absorption spectra of PEG-ZnPP (solid gray line) and SMA-ZnPP (solid black line) in H2O. The absorption of SMA-ZnPP in Jurkat cells (thin black line) exhibits a strong background due to light scattering by the cells. For comparison, the spectrum of ZnPP in ethanol is shown (dashed line).
these conditions, the irradiation received by the cells in the UVA range was 10.68 W/m2, and the received UVA dose was 0.3 J/cm2. Cell Survival after UVA Irradiation. The phototoxicity induced by PEG-ZnPP and SMA-ZnPP on Jurkat cells was determined by the XTT test (Roche Diagnostics GmbH, Mannheim, Germany). The assay is based on the conversion of the yellow tetrazolium salt XTT into an orange formazan dye by dehydrogenase in metabolically active cells, which was quantified by absorbance at 492 nm. The reference wavelength was at 650 nm, and the percentage of cell survival was determined as follows: (ODsample/ODdark reference) × 100 (bars; ( standard deviation, n ) 3). Caspase 3/7 Activity. Apoptosis was assessed using a fluorimetric assay kit (Apo-ONE Homogeneous Caspase-3/7 Assay, Promega GmbH, Germany), based on caspase 3/7 substrate (Z-DEVD-R110) as a profluorescent substrate. The cleavage and removal of the DEVD peptide by caspase 3 and 7 gives free rhodamine 110 of which the fluorescence intensity is proportional to caspase 3/7 activity. The fluorescence intensity was measured with a fluorescence microplate reader Gemini EM (Molecular Devices, Germany), using λexc ) 485 nm and λem > 530 nm. Cells incubated with 1.5 µM staurosporine (St) at 37 °C were used as a positive control of apoptotic cell death. Caspase 3/7 activity was detected 3-4 h after irradiation. The caspase 3/7 activity of the samples is expressed as a percentage of the positive control values 4 h after stimulation (bars; ( standard deviation, n ) 3).
RESULTS In the present study, the photophysical parameters relevant for PDT of two different water-soluble polymeric forms of Zn protoporphyrin (ZnPP), poly(ethylene glycol)-conjugated ZnPP (PEG-ZnPP), and styrene-maleic acid copolymer micelles containing ZnPP (SMA-ZnPP) were compared, and their photodynamic activity was investigated in vitro in comparison with free ZnPP. Absorption Spectra. The absorption spectrum of free ZnPP resembles that of metal-containing tetrapyrroles consisting of the Soret band and the two Q-band transitions with lower transition dipole moments and therefore lower absorbance (see Figure 1). The Soret band of PEG-ZnPP centered at 414 nm exhibits a hypsochromic shift by about 4 nm compared to that of ZnPP. For SMA-ZnPP, the blue shift is much stronger, about 27 nm. The Q-bands of PEG-ZnPP and SMA-ZnPP are redshifted by about 3 nm with respect to ZnPP. Moreover, every absorption band is broadened. In the case of SMA-ZnPP, the spectrum is a result of aggregation of many ZnPP molecules, which are tightly enclosed, in the hydrophobic inner core of the micelles. The appearance of spectral shifts may be due to
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Regehly et al. Table 1. First Excited Triplet-State Lifetimes (τT) of the Photosensitizers Obtained by Laser Flash Photolysis and Measured Singlet Oxygen Quantum Yield in H2O
sample
τT [µs] under normal air conditions
τT [µs] after 40 min N2 flushing
singlet oxygen quantum yield Φ∆
PEG-ZnPP in H2O PEG-ZnPP in JCSa SMA-ZnPP in H2O SMA-ZnPP in JCSa
2.5 ((0.2) µs 27 ((3) µs 1.4 ((0.2) µs 2.4 ((0.2) µs
406 ((40) µs 325 ((30) µs 484 ((40) µs 92 ((11) µs, 1.2 ((0.1) ms
0.17
a
Figure 2. Time-resolved singlet oxygen emission in D2O photosensitized by PEG-ZnPP and SMA-ZnPP. (Reference: Rose bengal) Inset: Magnification of the two-exponential kinetics of the singlet oxygen emission photosensitized by SMA-ZnPP.
interactions between individual dye (ZnPP) and styrene ring molecules. The absorption spectrum of SMA-ZnPP in Jurkat cells suspended in PBS buffered solution is an overlay of the free ZnPP absorption and a scattering background caused by the cells. Although the Q-band of ZnPP in cells treated with SMA-ZnPP cannot be clearly identified due to the low concentration of the drug in the cells, the peak of the Soret band is now located at the same wavelength as for free ZnPP seen in ethanol. From this observation, it can be concluded that the SMA micelles release free ZnPP molecules from their inner cores after being internalized by the Jurkat cells. For PEG-ZnPP macromolecules, the situation is quite different. The red shift of the Q-band is less pronounced and is regarded as a result of the covalent attachment of the PEG chain. Attachment of PEG chain to ZnPP does not hinder relatively free rotational and vibronic motion, which corresponds to the observed broadening of the absorption spectrum, a good contrast to SMA-ZnPP. Singlet Oxygen Measurements. It was found that PEGZnPP in D2O generates singlet molecular oxygen [1O2] with a quantum yield of 17%; the lifetime was determined to be 61 µs. The last value matches the well-known lifetime of 1O2 in D2O of 60 µs (15). Therefore, the PEG chain does not wrap the ZnPP molecule closely enough to cause efficient quenching of singlet molecular oxygen being generated. In contrast to PEG-ZnPP, the singlet oxygen generation in the case of SMAZnPP is very low, and the emission decay shows a twoexponential behavior with lifetimes of 8 µs and 50 µs (see Figure 2). The longer component of the emission is attributed to singlet oxygen, which diffused out of the micelle into the D2O environment. The shorter lifetime corresponds to singlet oxygen, which is quenched by the densely packed photosensitizer molecules within the micelle and/or the micelle-building polymers. The quantum yield of singlet oxygen generation for SMA-ZnPP was determined to be 5%. Laser Flash Photolysis. Because of the very short lifetime of singlet oxygen in viable cells, it is very hard to follow its generation via luminescence detection directly. For this reason, we used the method of laser flash photolysis for indirect determination of singlet oxygen formation via triplet lifetime measurement. This is possible because the triplet lifetimes can be related to the singlet oxygen generation. After activation of the first excited singlet state S1 via light absorption, a part of the molecules undergo transition to the first excited triplet state T1 with a quantum yield ΦT. Some of the molecules in the T1 state are deactivated via phosphorescence (kphos) or nonradiative (knonrad) transition. The fraction deactivated via interaction with molecular oxygen is referred to as fT. The energy transfer to molecular oxygen resulting in the generation of singlet molecular oxygen is described by the yield S∆. For tetrapyrroles under normal air conditions, this value is nearly unity. Therefore, the
0.05
Jurkat cell suspensions.
singlet oxygen quantum yield (Φ∆) can be calculated using the relation
Φ∆ ) ΦT‚fT‚S∆ ) ΦT‚
(
kO2[O2]
)
kphos + knonrad + kO2[O2]
‚S∆
The triplet lifetime in the presence of oxygen τO2 is given by τO2 ) (kphos + knonrad + kO2[O2])-1. Bubbling the solution with N2 removes oxygen and leads to an increase of the triplet lifetime. In this case, the lifetime is calculated by τN2 ) (kphos + knonrad)-1. The value of the singlet oxygen quantum yield can be obtained finally by
(
Φ ∆ ) Φ T‚
)
τ N2 - τ O2 τN2
‚S∆
That means that for the calculation of Φ∆ the value of ΦT must be known. For ZnPP in ethanol, a high ΦT value of 0.9 was reported (16). In the case of SMA-ZnPP in H2O, the aforementioned aggregate formation of ZnPP molecules located in the micellar core causes a strong decrease of the triplet quantum yield ΦT. In cells, the free ZnPP molecules aresat least partlys released from the micelle, and because of the unknown influence of the cellular environment on the aggregation of these molecules, ΦT is also not known. Therefore, very efficient quenching processes make it difficult to obtain the quantum yield of singlet oxygen generation in cells. On the other hand, because of the fact that S∆ of tetrapyrroles is about 1, the triplet lifetimes measured in air-liberated and deoxygenated solutions and cell suspensions provide information on the efficiency of such dyes to generate singlet molecular oxygen. For this reason, laser flash experiments were used to compare PEG-ZnPP and SMA-ZnPP according to their ability to generate singlet oxygen. In the presence of oxygen, the triplet lifetime of PEG-ZnPP in H2O was measured to τT ) 2.5 µs and the lifetime of SMAZnPP was τT ) 1.4 µs (Table 1). In an environment with low pO2, the triplet lifetime of the compounds dissolved in H2O increased to 406 µs (PEG-ZnPP) and 484 µs (SMA-ZnPP) as seen in Table 1. In comparison to these findings, the triplet-state lifetime of the two compounds accumulated in Jurkat cells under normal air conditions (aerobic) and under nitrogen (N2) bubbling of the cell suspensions were investigated. Under normal aerobic conditions, triplet lifetimes of 27 µs (PEG-ZnPP) and 2.4 µs (SMA-ZnPP) were measured (Figure 3). In the case of PEGZnPP, the larger value indicates an accumulation in cellular compartments with lower oxygen concentration or a lowered diffusion rate of reactants in regions with higher viscosity (17), although the latter possibility is more likely in this case. For PEG-ZnPP in cells after 40 min N2 flushing (removing of O2), a triplet lifetime of 325 µs was obtained (Figure 4) and a bleaching of the triplet state occurred. This can be concluded from the fact that the induced absorbance does not approach
Water-Soluble ZnPP Conjugates
Figure 3. Laser flash photolysis of SMA-ZnPP and PEG-ZnPP in Jurkat cells under normal air conditions. For SMA-ZnPP, the triplet lifetime was obtained to 2.4 µs, whereas for PEG-ZnPP, the fit yielded a value of 27 µs.
Figure 4. Laser flash photolysis of PEG-ZnPP and SMA-ZnPP in Jurkat cells after 40 min N2 flushing. The removal of oxygen leads to an increase of the triplet lifetime. The triplet lifetime of PEG-ZnPP was determined to be 325 µs. For SMA-ZnPP, a two-exponential decay was observed consisting of a short component with 92 µs and a long one with approximately 1.2 ms.
the value before excitation. This can be treated as an indication for a chemical change of the photosensitizer, whereas an additional long-lived species could not be identified. The triplet lifetime of PEG-ZnPP is in the range of the value measured in aqueous solution. On the basis of these findings, we suggest that PEG-ZnPP is preserved primarily in its monomeric watersoluble form in cells. It generates singlet oxygen under illumination in cells but with a lower quantum yield than in aqueous solution. The interpretation is consistent with the results obtained from in vitro experiments described below. For SMA-ZnPP after 40 min N2 bubbling, a two-exponential triplet decay was observed. The short component with 127 µs is significantly lower than the lifetime in H2O with oxygen removed. Such a strong reduction of the triplet lifetime may result from close association of the released ZnPP molecules. This type of situation occurs in hydrophobic environments like the lipid bilayer of cell membranes. Similar findings were reported in refs 17 and 18. This effect is due to weak interaction at high local concentrations of the photosensitizer and has to be distinguished from the aggregation of ZnPP observed in the core of SMA micelles. The latter case leads to a strong fluorescence suppression which was observed for SMAanthracycline micelles (doxorubicin and pirarubicin) as reported previously (10, 11). The longer component of the triplet decay with 1.2 ms is attributed to a bleaching process probably resulting in the formation of long-lived radicals. For SMAZnPP in cells, either singlet oxygen mediated phototoxicity (type II) or a free radical destruction mechanism (type I) under low oxygen concentration may occur. This result may reflect its biological effect. Photodynamic Activity and Cell Killing. First, the intracellular uptake of the two polymeric compounds by Jurkat cells was studied in comparison with free ZnPP at different incubation concentrations (Figure 5). The intracellular uptake of the compounds increased in parallel with the concentration of the
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Figure 5. Intracellular uptake of sensitizers by Jurkat cells. Cells were incubated with the investigated compounds at different concentrations (expressed in ZnPP equivalents). The amount of sensitizer uptaken by Jurkat cells was determined by measuring the fluorescence intensity of cell extracts as described in Materials and Methods.
Figure 6. Phototoxicity against Jurkat cells of ZnPP, PEG-ZnPP, and SMA-ZnPP. Cell survival 24 h after irradiation (radiation, +) was measured using the XTT test. Two references (R, streaked bar) were used: cells without test compounds and kept in the dark (radiation, -) and those with radiation at the same dose of UVA.
compounds in the incubation suspension. SMA-ZnPP reached higher intracellular concentrations than PEG-ZnPP and free ZnPP (in terms of ZnPP equivalents). At an incubation concentration of 5 µM, the intracellular concentration of SMAZnPP was approximately 5.4 times higher than that of PEGZnPP and more than 3.2 times higher than that of free ZnPP. This data can be explained by the fact that the molecular concentration of ZnPP in SMA micelles is much higher (loading of >40% by weight), whereas its content in PEG-ZnPP is about 5%. Both will be internalized by endocytosis. To investigate the photocytotoxicity of PEG-ZnPP and SMA-ZnPP in comparison with that of free ZnPP, Jurkat cells were incubated for 24 h with different concentrations of the compounds and irradiated at dose of 0.3 J/cm2 of UVA. Cellular viabilities were estimated by XTT assay 24 h after irradiation. Less than 5% of the reference cells lost their viability after UVA exposure (Figures 6 and 7). No cytotoxic activity by ZnPP without UVA exposure alone may indicate that HO-1 inhibition does not show a significant effect at this dose range (Figure 6), although more oxystressed systems may prove this possibility in vivo. Free ZnPP does not demonstrate a phototoxic effect under illumination. As shown in Figures 6 and 7, significantly enhanced phototoxicity was observed for SMA-ZnPP at incubation concentrations higher than 1 µM. The phototoxicity increased in a concentration-dependent manner with LD50 of about 2.5 µM. PEG-ZnPP exerted significant phototoxic activity only at concentration of 5 µM under UVA irradiation (Figure 6). Caspase 3 and 7 activities were measured 4 h after irradiation to answer the question regarding which cell death mechanism is induced by the uptake of ZnPP (Figure 7). Caspase 3 and 7 activity was detected when treated with both ZnPP compounds,
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Figure 7. Photoinduction of caspase 3/7 activity on Jurkat cells. Two references (R) were used: cells without test compounds in the dark (radiation, -) and those with radiation at the same dose of UVA (radiation, +). Cells incubated for 4 h with 1.5 µM staurosporine (St) were used as a positive control for caspase activity.
even if only a low percentage of activity (about 30% of the positive control) could be measured and only at concentrations of 5 µM for PEG-ZnPP and 2.5 µM for SMA-ZnPP. At an incubation concentration of 5 µM, only a low level of caspase activity was measured for SMA-ZnPP, whereas almost no living cells were detected with the XTT test. Thus, the caspases might have already released out of the cells. In concordance with this notion, all these cells showed Trypan blue positive staining (data not shown), indicating disruption of the integrity of cell membrane. These observations showed that cells had also undergone a necrotic cell death pathway to a considerable extent. Namely, cell viability was approximately 60% after irradiation of the cells incubated with 5 µM PEG-ZnPP, and the measurements of caspase 3 and 7 activities showed that approximately 30% of dead cells were caused by an apoptotic mechanism. These results show that SMA-ZnPP is able to initiate cell death via necrosis at higher concentrations, while PEG-ZnPP is mainly inducing the apoptotic cell death pathway.
DISCUSSION Our experimental results obtained from in vitro studies on Jurkat cell suspensions show that both SMA-ZnPP and PEGZnPP exhibit photodynamic activity. SMA-ZnPP causes about five times higher phototoxicity compared to PEG-ZnPP due to the higher uptake of ZnPP by tumor cells. This fact clearly demonstrates the advantage of SMA-ZnPP over PEG-ZnPP for photodynamic therapy. In this context, the polymeric form of ZnPP is better than free ZnPP in addition to its increased water solubility. The SMA micelle acts as a macromolecular encapsulating container for ZnPP with densely packed ZnPP molecules; it thereby seems to prevent the efficient formation of singlet oxygen to a considerable extent before delivery to tumor cells. Such dense packing of ZnPP would lead to a strong decrease of the triplet quantum yield of the photosensitizer molecules outside of cells. After incorporation into the cells via endocytosis, the SMA micelles release free ZnPP molecules in lysosomal compartments or tend to accumulate in lipophilic regions in the cells at high concentrations. The destruction of the cell membranes under UVA illumination was envisaged, as they became Trypan blue positive, indicative of a necrotic cell death pathway. The phototoxicity of SMA-ZnPP is based primarily on the generation of singlet oxygen, but probably longlived radicals are formed additionally, especially in the case of lowered oxygen concentration, such as the lipid peroxiradical (ROO·) that can also cause DNA breakage and other damage (19). Free ZnPP showed some uptake in the cells (Figure 5) though no cytotoxicity (Figure 6); this may be an arguable point that
Regehly et al.
quantification of ZnPP by fluorometry after ethanol extraction would dismiss the difference of the localization of ZnPP, i.e., one bound on a cell membrane (lipid bilayer) versus endocytotic vesicle (upon endocytosis of SMA-ZnPP). Thus, free ZnPPtreated cells may not contain real intracellular ZnPP. The comparative study of PEG-ZnPP and SMA-ZnPP shows the possibility of development of more efficient drug delivery systems for photodynamic therapy using macromolecular drug carrier systems such as SMA micelles, which exhibit tumor-selective delivery via the EPR effect, which is far better than free ZnPP or low molecular weight drugs. Moreover, by using these “intelligent” SMA micelles as a carrier system proposed in this paper, it will be possible to make a more efficient delivery system without toxicity in general. The photodynamic activity will be ideal for superficial tumors where light can penetrate, whereas on the other parts of the body, it may work by HO-1 inhibition (Hsp-32), which is considered to be one of the survival proteins of tumor cells. In addition to the comments of polymeric ZnPP in view of singlet oxygen generation, recent reports show that Zn or ZnPP can affect the signal transduction system involving downregulation of the oncogene on chronic myelogenous leukemic cells (20) or Zn transporter-mediated cytokine modulation (e.g., IL-6, etc.) (21). These could be the other alternative mechanisms to the EPR effect/HO-1 inhibition/increased ROS sensitivity in understanding the multiple effects after SMA-ZnPP uptake.
ACKNOWLEDGMENT The technical assistance of Gisela Wo¨hlecke and the support of Anneliese Powitz, and Arun Iyer for preparing SMA micelles are greatly appreciated. H.M. thanks the Ministry of Education, Science, Culture and Sports for the Cancer Research Grant for 2005-2010. F.R. and F.B. thank the DFG for financial support (BO 1353/2-2). K.G. thanks JSPS for supporting his research in Japan.
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