2.1.

Chemicals

Pc 4 was synthesized by methods previously reported.³⁵ Cholesterol (CHOL), L- $\alpha$ -dimyristoylphosphatidylcholine (DMPC), and L- $\alpha$ -dimyristoylphosphatidylethanolamine (DMPE) were obtained from Avanti Polar Lipids (Alabaster, Alabama). We obtained 10- $N$ -nonyl-acridine orange (NAO) from Invitrogen (Carlsbad, California). CL, 9,10-anthraquinone-2-sulfonate sodium salt $\left({\mathrm{AQS}}^{-}\right)$ , sodium palmitate, phosphate-buffered saline (PBS), fatty-acid-free bovine serum albumin (BSA), and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were purchased from Sigma-Aldrich (St. Louis, Missouri). Tetramethylrhodamine methylester (TMRM) was from Molecular Probes, Inc. (Eugene, Oregon). RPMI 1640, fetal bovine serum, and penicillin/streptomycin were from Hyclone (South Logan, Utah). All other compounds were the highest grade commercially available. Water was treated in a Millipore Milli-Q system (Billerica, Massachusetts).

2.2.

Estimation of the $\mathrm{p}{K}_{\mathrm{a}}$ of Pc 4

The $\mathrm{p}{K}_{\mathrm{a}}$ of Pc 4 was calculated using V8.0 Batch $\mathrm{p}{K}_{\mathrm{a}}$ Prediction Software (Advanced Development, Toronto, Ontario, Canada), with the assumption that the $\mathrm{O}\mathrm{Si}{\left(\mathrm{C}{\mathrm{H}}_3\right)}_2{\left(\mathrm{C}{\mathrm{H}}_2\right)}_3\mathrm{N}{\left(\mathrm{C}{\mathrm{H}}_3\right)}_2$ group of Pc 4 is its most basic group and that $\mathrm{C}{\mathrm{H}}_3\mathrm{O}\mathrm{Si}{\left(\mathrm{C}{\mathrm{H}}_3\right)}_2{\left(\mathrm{C}{\mathrm{H}}_2\right)}_3\mathrm{N}{\left(\mathrm{C}{\mathrm{H}}_3\right)}_2$ is a good model for this group.

2.3.

Liposome Preparation and Pc 4 Incorporation

Large unilamellar vesicles were prepared with a Mini-Extrudor from Avanti Polar Lipids, Inc. Typically, an aliquot of lipid solution in absolute ethanol was evaporated to form a thin film of lipid. All excess ethanol was removed during evaporation. PBS $(\mathrm{pH}=7.4)$ , composed of $0.1\mathrm{M}$ $\mathrm{Na}{\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_4$ , $0.1\mathrm{M}$ ${\mathrm{K}}_2\mathrm{H}\mathrm{P}{\mathrm{O}}_4$ , and $0.15\mathrm{M}$ NaCl, was added to hydrate the film. Aliquots of the mixture were extruded through a membrane of pore diameter $\cong 100\mathrm{nm}$ (Whatman^®, Clifton, New Jersey) at $15°\mathrm{C}$ above the lipid-transition temperature³⁶ $\left(T_c\right)$ of DMPE $(T_c=49°\mathrm{C})$ . To avoid the loss of Pc 4 through its absorption to the extrusion membrane, Pc 4 was incorporated into liposomes after extrusion from a stock solution of Pc 4 in tetrahydrofuran (THF)/ethanol (EtOH) (1:1), and the liposome-Pc 4 complex was incubated at $15°\mathrm{C}$ above the $T_c$ of DMPE for $30\min$ . The resultant liposomes were determined to be $125\text{to}200\mathrm{nm}$ in diameter by dynamic light scattering on a Brookhaven Instruments Corporation 90 Plus Particle Sizing Analyzer (Holtsville, New York).

2.4.

Stock Solutions of Pc 4

Stock solutions were prepared in 1:1 THF/EtOH for the liposome studies and in dimethylformamide (DMF) for the cellular studies. The solutions were stored at $4°\mathrm{C}$ and protected from ambient light.

2.5.

Spectroscopic Studies

Absorption spectra were recorded on a Varian Cary 50 Bio UV-Vis Spectrophotometer (Varian, Palo Alto, California). Fluorescence spectra were recorded on a Varian Cary Eclipse Fluorescence Spectrophotometer. Pc 4 was excited at $610\mathrm{nm}$ (Q band), and the emission spectra were recorded between 620 and $800\mathrm{nm}$ . Measurements on liposomes were performed above the lipid transition temperature using a Quartz SUPRASIL (QS) controlled-temperature cell with a $10\text{-}\mathrm{mm}$ path length (Hellma USA, Plainview, New Jersey).

2.6.

Binding Experiments

Measurements were made at a fixed concentration of Pc 4 $\left(6\mu \mathrm{M}\right)$ and increasing liposome concentrations from $0\mathrm{mM}$ to the concentration at which maximum association was attained. In water, Pc 4 is insoluble, highly aggregated, and nearly nonfluorescent. On the addition of liposomes, only Pc 4 monomers bind and become fluorescent. Association constants for Pc 4 were calculated as

2.7.

Fluorescence-Quenching Experiments

Quenching of Pc 4 fluorescence was studied in liposomes with or without CL, as described elsewhere.³⁷ Briefly, increasing concentrations of ${\mathrm{AQS}}^{-}$ $\left(0\text{to}13\mathrm{mM}\right)$ were used to quench Pc 4 fluorescence in liposomes without or with 20% CL. Experiments were carried out at $\left[\mathrm{Pc}4\right]=6\mu \mathrm{M}$ , $\left[\text{lipid}\right]=2.1\mathrm{mM}$ . The lipid concentration used was that corresponding to the maximum incorporation of Pc 4 into liposomes as monomer (in the plateau region of association plots). Pc 4 was excited at $610\mathrm{nm}$ and the fluorescence emission was recorded in the range of $620\text{to}800\mathrm{nm}$ . These determinations were performed at $15°\mathrm{C}$ . The bimolecular quenching rate constant $K_{\mathrm{q}}$ was calculated from

2.8.

Cell Culture

MCF-7c3 cells were grown in RPMI (Roswell Park Memorial Intitute) 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in an atmosphere of 5% $\mathrm{C}{\mathrm{O}}_2∕95\mathrm{\%}$ air at $37°\mathrm{C}$ in a humidified incubator. Cells were used for experiments when they were in exponential growth.

2.9.

Confocal Microscopy

MCF-7c3 cells were plated in $35\text{-}\mathrm{mm}$ glass-bottomed tissue-culture dishes (MatTek Corp., Ashland, Massachusetts) at $2\times {10}^5$ cells per dish. Prior to addition of Pc 4, the cultures were treated for $1\mathrm{h}$ to block mitochondrial function, either by heating at $65°\mathrm{C}$ or by the addition of $10\mu \mathrm{M}$ CCCP, a mitochondrial uncoupler, which abolishes the mitochondrial membrane potential. After treatment, the medium was removed and the cultures were incubated with $200\mathrm{nM}$ Pc 4 in complete medium for $1\mathrm{h}$ to allow cell uptake of the photosensitizer. This time had been previously shown to allow maximum uptake. Then, the complete medium was removed and replaced with serum-free RPMI $\left(1\mathrm{mL}\right)$ . Imaging was carried out with a $63\times$ numerical aperture (NA) 1.4 oil immersion planapochromat objective on a Zeiss LSM 510 confocal microscope (Thornwood, New York) in the Case Comprehensive Cancer Center Confocal Microscopy Core Facility. A $\mathrm{He}∕\mathrm{Ne}$ laser supplied the $633\text{-}\mathrm{nm}$ excitation light to excite Pc 4, and a $650\text{-}\mathrm{nm}$ long-pass filter was used to collect emission. Cells without pretreatment but with Pc 4 were included as controls.

2.J.

Monitoring Mitochondrial Membrane Potential

Cells were grown as above for $48\mathrm{h}$ , then loaded with $100\mathrm{nM}$ TMRM for $30\min$ at $37°\mathrm{C}$ . The medium was removed, and the cells were kept in $10\mathrm{nM}$ TMRM in calcium buffer ( $130\mathrm{mM}$ NaCl, $5\mathrm{mM}$ KCl, $1.5\mathrm{mM}$ $\mathrm{Ca}{\mathrm{Cl}}_2$ , $1\mathrm{mM}$ $\mathrm{Mg}{\mathrm{Cl}}_2$ , $25\mathrm{mM}$ Hepes, and 0.1% BSA). Images of TMRM fluorescence were collected using $561\text{-}\mathrm{nm}$ excitation light from a solid state diode laser, and a $580\text{to}650\text{-}\mathrm{nm}$ bandpass filter and carried out with a $100\times$ NA 1.4 oil immersion planapochromat objective. After recording the first image, $10\mu \mathrm{M}$ CCCP was added to the cells in calcium buffer containing TMRM $\left(10\mathrm{nM}\right)$ . Control cells received $10\mathrm{nM}$ TMRM in calcium buffer, but without CCCP. Imaging was performed at $5\text{-}\mathrm{s}$ intervals over a period of $1\min$ or until depolarization was observed. For evaluation of the effect of heat, we used flow cytometry and showed that 100% of the cells were dead after $1\mathrm{h}$ at $65°\mathrm{C}$ .

2.K.

Flow Cytometry

Cells were plated in $60\text{-}\mathrm{mm}$ tissue culture dishes at $6\times {10}^5$ cells per dish and allowed to grow for $24\mathrm{h}$ . Albumin-bound fatty acids were prepared by stirring fatty acid sodium salts at $37°\mathrm{C}$ in a molar ratio of 2:1 fatty acid to BSA. (Ref. 30). The solution was filtered through a $0.22\text{-}\mu \mathrm{m}$ filter. Cells were serum-starved in RPMI containing 0.5% fatty acid-free BSA for $12\mathrm{h}$ , then incubated with BSA conjugated to palmitate $\left(0.1\mathrm{mM}\right)$ for up to $8\mathrm{h}$ . The final concentration of BSA was always adjusted to 0.5%. After incubation with BSA-fatty acid, the medium was removed and the cells were incubated with Pc 4 $\left(200\mathrm{nM}\right)$ or NAO $\left(200\mathrm{nM}\right)$ in RPMI for 60 or $30\min$ , respectively, times previously shown to allow maximum uptake, and then harvested by trypsinization. Flow cytometric analysis was performed in the Case Comprehensive Cancer Center Flow Cytometry Core Facility, using a BD^™ LSR II Flow Cytometer (San Jose, California). NAO was excited by a laser $(488\pm 5\mathrm{nm})$ , and fluorescence emission was collected with a $525\pm 5\text{-}\mathrm{nm}$ bandpass filter. Pc 4 was excited by a UV laser $\left(355\mathrm{nm}\right)$ , and fluorescence emission was collected with a $650\text{-}\mathrm{nm}$ long-pass filter. Autofluorescence of control cells was subtracted. Cells not exposed to BSA-palmitate were used as controls.

2.L.

Measurements of CL Level

Lipid extraction and separation by TLC were used to confirm the decrease of CL content of the variously treated MCF-7c3 cells. Cells were treated as described for flow cytometry. After trypsinization and counting of the cells, identical numbers of control and palmitate-treated cells were centrifuged and resuspended into $1.5\mathrm{mL}$ of milliQ water saturated with NaCl. Phospholipids were extracted by vortexing with $3\mathrm{mL}$ of chloroform:methanol [2:1, v/v]. After centrifugation, the recovered organic phase was dried with $\mathrm{Mg}\mathrm{S}{\mathrm{O}}_4$ , filtered using cotton, and evaporated under nitrogen. Each dried extract was dissolved in chloroform $\left(25\mu \mathrm{L}\right)$ and applied to a silica gel TLC plate. The mobile phase consisted of methanol/chloroform/acetic acid/water³⁹ $(3∕0.52∕0.36∕0.12)$ . TLC was visualized by iodine vapors to detect total lipid.³⁴ Commercial CL served as a standard. Densitometric analysis of the bands was accomplished with the Image Quantum TL v 2005 software (Amersham Biosciences Corp., Sunnyvale, California).

2.M.

Pc 4-Mediated Photo-oxidation of CL in Liposomes

Liposomes $\left(0.2\mathrm{mM}\right)$ were prepared as already described but containing 80% DMPC and 20% CL and loaded with $6\mu \mathrm{M}$ Pc 4. Aliquots of $1\mathrm{mL}$ were transferred into $35\text{-}\mathrm{mm}$ tissue culture dishes and exposed to $100\mathrm{mW}∕{\mathrm{cm}}^2$ red light produced by a light-emitting diode array (EFOS, Mississauga, Ontario, Canada, ${\lambda }_{\max }$ $670\text{to}675\mathrm{nm}$ ) at room temperature for $40\min$ . Control samples were kept in the dark. Lipids were extracted from the liposome solution with $2\mathrm{mL}$ chloroform:methanol [2:1]. CL was separated by TLC as described above. The CL spot was scraped into a $0.6\text{-}\mathrm{mL}$ Eppendorf tube and extracted with $100\mu \mathrm{L}$ chloroform:methanol [1:1]. The chloroform:methanol solution of CL was analyzed by Thermo Finnigan LCQ Advantage spectrometer (Waltham, Massachusetts) using negative-ion electrospray ionization-mass spectrometry (ESI-MS) with direct infusion.

3.1.

Spectroscopic Studies

Figure 1 shows the changes in fluorescence spectrum of Pc 4 on titration with lipid. The addition of liposomes results in an increase in the fluorescence intensity at a fixed Pc 4 concentration until a maximum fluorescence intensity is reached, at which point Pc 4 is maximally monomerized, and a $1\text{to}2\text{-}\mathrm{nm}$ blue shift in the peak wavelength has occurred, denoting incorporation of Pc 4 into the liposomes. The blue shift is typical of the change in shape of spectra for other phthalocyanines upon addition of lipid.³⁷ Similar changes were found for all lipid compositions studied (Table 1 ). The identical shape of the emission spectra in PBS and liposomes and the nonsignificant wavelength shift indicates that the monomer fluorescent specie is the same in both conditions.

Fig. 1

Increase in Pc 4 fluorescence intensity with increasing liposome concentration. In this example, the liposomes contained 45% DMPC, 45% DMPE, 10% CL and $\left[\mathrm{Pc}4\right]=6\mu \mathrm{M}$ . (a) Fluorescence spectra of Pc 4 in PBS with no lipid or increasing lipid concentration $\left[\mathrm{L}\right]=0.102$ ; 0.412; 0.630; 0.889; 1.120 and $1.5510\mathrm{mM}$ , and (b) titration curve for no lipid up to $\left[\mathrm{L}\right]=2.5\mathrm{mM}$ .

Table 1

UV absorbance and fluorescence data for Pc 4 in liposomes of different compositions.

3.2.

Determination of Binding Constants

The major polar lipid components found in mitochondria from liver, heart, and kidney are CL, phosphatidyl choline, and phosphatidyl ethanolamine; these are present^{23, 40} in a molar ratio of 1:4:4. We chose to work with liposomes of similar lipid composition to mitochondria, using DMPC and DMPE in a ratio of 1:1 and adding CL (or not), but keeping the total lipid concentration constant, to determine whether Pc 4 displayed any preferential binding to CL-containing liposomes.

On the basis of fluorescence spectra, it is possible to compare the degree of incorporation of Pc 4 as monomer into liposomes with different phospholipid compositions. The association constants were calculated by hand according to Eq. 1; they were also calculated by plotting $(F-F_0)∕(F_{\infty }-F_0)$ versus [L] and fitting to Eq. 1 using Origin software (OriginLab Corporation, Northampton, Massachusetts). Only minor differences in $K_{\mathrm{a}}$ values were obtained using the two calculation methods. The averages of $K_{\mathrm{a}}$ values for Pc 4 in 0, 10, and 20% CL were $2.1\times {10}^3$ $(n=2)$ , $1.7\times {10}^3$ $(n=1)$ , and $2.4\times {10}^3$ $(n=2)$ ${\mathrm{M}}^{-1}$ , respectively (Table 2 ). Therefore, no trend in binding affinity versus CL content was observed, which suggests that Pc 4 does not show preferential binding to CL in the liposomal system. Since our earlier studies using confocal microscopy showed that Pc 4 did not localize to the plasma membrane,^{10, 11} liposomes containing DMPC, DMPE, and cholesterol were also studied. Cholesterol is a major lipid of the plasma membrane of human cells. With liposomes containing 30 or 45% cholesterol, the average of $K_{\mathrm{a}}$ values for Pc 4 were $1.3\times {10}^3$ $(n=2)$ and $2.1\times {10}^3$ $(n=2)$ ${\mathrm{M}}^{-1}$ , respectively. The similarity of these constants to the prior results (Table 2) suggests that Pc 4 has similar affinities to different lipids in our system. Furthermore, the mean and standard deviation of all of the results is $1.9\pm 0.7{\mathrm{M}}^{-1}$ $(n=9)$ . Since all but one of the individual values fall within the standard deviation, it appears that there is no significant influence of lipid composition on the affinity of Pc 4 for liposomes. Thus, another factor besides CL is likely to be responsible for the preferential localization of Pc 4 in the mitochondrial and ER membranes.

Table 2

Binding constants for Pc 4 in liposomes of different compositions.

3.3.

Fluorescence-Quenching Experiments

Since there is a blue shift of $1–2\mathrm{nm}$ in the emission of Pc 4 when CL is introduced to the liposomes (Fig. 2 and Table 1), fluorescence quenching was studied to explore the possibility of a dissimilar distribution of Pc 4 in liposomes of different composition. The quenching of the fluorescence of Pc 4 was measured in DMPC liposomes with and without CL using ${\mathrm{AQS}}^{-}$ as quencher, which doesn’t penetrate the lipid bilayer. The fluorescence quenching data, as analyzed by the Stern-Volmer equation [Eq. 2], gave quenching constants $K_{\mathrm{q}}$ ( $\text{mean}\pm \text{standard}$ deviation) of $(4.4\pm 0.9)\times {10}^9{\mathrm{M}}^{-1}$ $(n=3)$ and $(4.3\pm 0.7)\times {10}^9{\mathrm{M}}^{-1}$ $(n=3)$ in liposomes with and without CL, respectively. Since the $k_{\mathrm{q}}$ values are essentially identical, these data suggest that the presence of CL in the lipid bilayer does not affect the distribution of Pc $4\text{molecules}$ in the liposome. The $k_{\mathrm{q}}$ values are in agreement with the values found³⁷ for other phthalocyanines in liposomes ( $4.6\times {10}^9$ , $9.6\times {10}^9$ , and $7.8\times {10}^9{\mathrm{M}}^{-1}$ for three different zinc phthalocyanines).

Fig. 2

Fluorescence spectra of $\left[\mathrm{Pc}4\right]=6\mu \mathrm{M}$ in (dashed line) liposomes at 0% CL and $\left[\mathrm{L}\right]=2.23\mathrm{mM}$ and (solid line) liposomes at 20% CL and $\left[\mathrm{L}\right]=1.33\mathrm{mM}$ .

3.4.

Role of Mitochondrial Membrane Potential in the Preferential Localization of Pc 4 in Mitochondria

Aside from the presence of CL, another factor that could account for the preferential localization of Pc 4 in mitochondria is its cationic character. Hydrophobic compounds with delocalized positive charges can be driven across the inner membrane lipid bilayer by the mitochondrial electrochemical potential established by the proton-pumping mechanism of respiratory complexes.⁴¹ Using V8.0 Batch $\mathrm{p}{K}_{\mathrm{a}}$ Prediction Software, the calculated $\mathrm{p}{K}_{\mathrm{a}}$ of Pc 4 was approximately 9.6. Although the positive charge is localized, rather than delocalized as in mitochondrion-targeting dyes such as rhodamine-123 and JC-1, we considered the possibility that the preferential uptake of Pc 4 required a strong mitochondrial membrane potential.⁴¹ Two methods were used to block mitochondrial function and/or uncouple oxidative phosphorylation: heating cells and introducing an uncoupler, CCCP. As shown in Fig. 3 the distribution of Pc 4 $\left(200\mathrm{nM}\right)$ in individual cells treated with heat or CCCP was similar to that in the control cells, with Pc 4 bound to intracellular membranes and excluded from the nucleus; the distributions were similar to those previously published.^{10, 11, 12, 13, 14} Although these images have not been quantified, individual cells appear to have similar levels of Pc 4 fluorescence irrespective of the loss of some cells from the CCCP-treated population. The same pattern was found for a lower concentration $\left(50\mathrm{nM}\right)$ of Pc 4 (data not shown).

Fig. 3

Influence of mitochondrial membrane potential on the binding of Pc 4 to mitochondria. (A) Confocal images of intracellular distribution/accumulation of Pc 4. MCF-7c3 cells were plated at $2\times {10}^5$ per $35\text{-}\mathrm{mm}$ dish, allowed to attach overnight $\left(16\text{to}18\mathrm{h}\right)$ , and then treated for $1\mathrm{h}$ in one of three ways: (a) and (b) untreated cells, (c) and (d) cells heated at $65°\mathrm{C}$ , and (e) and (f) cells incubated with CCCP $\left(10\mu \mathrm{M}\right)$ . After that, the medium was removed and the cultures were incubated $1\mathrm{h}$ with Pc 4 $\left(200\mathrm{nM}\right)$ in a $37°\mathrm{C}$ incubator. All cells were washed twice in PBS and overlaid with phenol red-free Hank’s balanced salt solution prior to imaging. (B) Mitochondrial membrane potential visualized by confocal microscopy. Cells were grown as already described, then loaded with $100\mathrm{nM}$ TMRM for $30\min$ at $37°\mathrm{C}$ . The medium was removed, and the cells were kept in $10\mathrm{nM}$ TMRM in calcium buffer. After recording the first image, $10\mu \mathrm{M}$ CCCP was added to the cells in calcium buffer containing TMRM $\left(10\mathrm{nM}\right)$ . Control cells received $10\mathrm{nM}$ TMRM in calcium buffer but without CCCP. Imaging was performed at $5\text{-}\mathrm{s}$ intervals over a period of $1\min$ or until depolarization was observed. Upper panel from left to right: (a) cells before adding CCCP, $10\mu \mathrm{M}$ ; (b) and (c) $48\mathrm{s}$ and $1\min$ after adding CCCP, respectively; (d) $1\min$ after adding CCCP in a different field. Lower panel from left to right: control cells (e) 0, (f) 1, and (g) $2\min$ , after the start of imaging; and (h) $8\min$ after the start of imaging but in a different field.

We performed experiments to monitor mitochondrial membrane potential using TMRM. Polarized mitochondria take up the cationic dye TMRM, which is released after depolarization. Using confocal microscopy, we evaluated the mitochondrial level of TMRM, as a measure of mitochondrial membrane potential. Figure 3 shows the loss of the mitochondrial membrane potential after adding CCCP $\left(10\mu \mathrm{M}\right)$ , the same concentration used in the experiments to evaluate Pc 4 localization [Fig. 3]. Even $200\mathrm{nM}$ CCCP was able to completely depolarize the mitochondrial membranes [Fig. 4 ]. Control cells loaded with vehicle alone did not release TMRM. For evaluation of the effect of heat, we used flow cytometry and showed that 100% of the cells were dead after $1\mathrm{h}$ at $65°\mathrm{C}$ [Fig. 4]. From these experiments, we concluded that the mitochondrial membrane potential appears not to be indispensable for Pc 4 uptake into mitochondria.

Fig. 4

(A) Mitochondrial membrane potential visualized by confocal microscopy. Cells were grown as described in methods for $48\mathrm{h}$ , then loaded with $100\mathrm{nM}$ TMRM for $30\min$ at $37°\mathrm{C}$ . The medium was removed, and the cells were kept in $10\mathrm{nM}$ TMRM in calcium buffer. After recording the first image, $200\mathrm{nM}$ CCCP was added to the cells in calcium buffer containing TMRM $\left(10\mathrm{nM}\right)$ . Imaging was performed at $5\text{-}\mathrm{s}$ intervals until depolarization was observed: (a) cells before adding CCCP; (b) and (c) $48\mathrm{s}$ and $1\min$ , respectively, after adding CCCP; and (d) $1\min$ after adding CCCP in a different field. (B) Flow cytometric evaluation of the effect of heat on cells. Graphs of side scatter (SSC) versus forward scatter (FSC) of (a) control cells kept at $37°\mathrm{C}$ and (b) cells after $1\mathrm{h}$ at $65°\mathrm{C}$ . Dead cells have lower FSC and higher SSC than living cells.

3.5.

Role of CL Level in the Preferential Localization of Pc 4 in Mitochondria

CL is a critical phospholipid of the mitochondrial inner membrane. We therefore asked whether reducing the CL content would affect the ability of Pc 4 to bind to the cells. Two laboratories have reported reduced mitochondrial levels of CL on exposure of rat neonatal cardiomyocytes or human breast cancer MDA-MB-231 cells to palmitate.^{30, 42} Because both studies found that the loss of CL on addition of this saturated fatty acid is followed by apoptosis, the MTT assay was used to identify a concentration where the CL level was reduced but the cells remained viable. With concentrations of palmitate $⩽0.5\mathrm{mM}$ , no toxicity was observed in MCF-7c3 cells for incubation times up to $6\mathrm{h}$ , but toxicity was found at longer times (data not shown). Using this condition, we extracted phospholipids from control and palmitate-treated cells, separated the phospholipids by TLC, and quantified the amount of CL using ImageQuant software based on the iodine staining.³⁴ As shown in the TLC plate of Fig. 5 , CL was well separated from the other phospholipids. Duplicate samples and duplicate controls were studied in each of three experiments. Using the phosphatidyl choline spot of each lane to normalize the amount of CL, we find that under the chosen conditions, palmitate-treated cells had $38\pm 16\mathrm{\%}$ $(n=6)$ of the CL content of untreated cells.

Fig. 5

Separation of cellular lipids by TLC. MCF-7c3 cells were treated as described in Sec. 2. Phospholipids were extracted with chloroform/methanol (2:1, v/v) from cells suspended in saturated saline solution. After that, the organic phase was recovered and dried under nitrogen. Each dried extract was dissolved in $25\mu \mathrm{L}$ chloroform and applied to a silica TLC plate. The mobile phase consisted of methanol/chloroform/acetic acid/water $(3∕0.52∕0.36∕0.12)$ . TLC was developed by iodine vapors to detect total lipid. Commercial CL was analyzed similarly as a standard. Lanes are 1, control cells; 2 and 3, palmitate-treated cells; 4, control cells plus addition of CL standard; and 5, CL standard. CL, cardiolipin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine.

Cells treated with palmitate for $6\mathrm{h}$ to reduce their CL content and control cells were then evaluated for the uptake of Pc 4 and NAO by flow cytometry. When the control cells were exposed to a range of NAO concentrations from $20\text{to}300\mathrm{nM}$ , a linear increase in fluorescence was observed in the cells, without evidence of saturation (data not shown). For study of uptake of NAO into palmitate-treated cells, $200\mathrm{nM}$ NAO was used, a concentration that was within the linear range of the measurements. For $200\mathrm{nM}$ NAO, uptake into palmitate-treated cells was not significantly different (i.e., $93\pm 23\mathrm{\%}$ ; $n=6$ ) from that into control cells. The data agree with previous findings^{43, 44} that NAO cannot be used to quantify CL. For Pc 4 $\left(200\mathrm{nM}\right)$ , the mean channel fluorescence in palmitate-treated cells was found to be $94.9\pm 5.4\mathrm{\%}$ $(n=6)$ of the values for nontreated cells, indicating that there was no significant difference in the uptake of Pc 4 into cells as a function of palmitate treatment.

Although no specific binding between Pc 4 and CL was found, oxidation of CL was demonstrated to occur in the liposome system using ESI-MS and ESI-MS/MS. For these experiments, higher doses of Pc 4 and light were used than are typically applied in our cellular studies. Note, however, that the actual photosensitizer concentration at the critical sites is not known either in cells or within the liposomes, so doses were chosen to enable identification of products of CL oxidation. The mass spectrum of CL isolated from the Pc 4-containing liposomes after photoirradiation showed two major oxidized species with mass increments of $+32$ and $+64$ from the unmodified tetralinoleoyl CL (Fig. 6 ). The MS/MS spectra suggest that the $+32$ and $+64$ are mono- and dihydroperoxidized CL species, respectively. These could be produced directly by PDT-generated singlet oxygen, or they might result from radical chain reactions initiated and propagated within the CL pool.^{28, 29}

Fig. 6

ESI-MS spectra of CL extracted from liposomes. Liposomes with Pc 4 were prepared as described in Sec. 2. Control liposomes were kept in the dark in the presence of Pc 4, while experimental liposomes were irradiated with $100\mathrm{mW}∕{\mathrm{cm}}^2$ red light at room temperature for $40\min$ . The peak at $m∕z$ 1447.8 is ${\left[\mathrm{M}\text{-}{\mathrm{H}}^{+}\right]}^{-}$ of tetralinoleoyl CL.

Disclosures

Two of the authors, MEK and NLO, are inventors on patents concerning Pc 4-PDT and are associated with Fluence Therapeutics, Inc., a company developing photodynamic therapy with the photosensitizer Pc 4.