The porphyrazines (pzs), a class of porphyrin analogues, are being investigated for their potential use as tumor imaging/therapeutic agents. We here examine six peripherally-functionalized M[pz(AnB4-n)] pzs with n=4, 3, or 2 (in a trans conformation) and M = H2 or Zn, where A is an [S((CH2)2O)4Me]2 unit and B is a fused beta,beta'-diisopropyloxybenzo group. Cell viability/proliferation assays and fluorescence microscopy were carried out in both tumor and normal cells. Dark toxicity studies disclosed that four of the compounds exhibited toxicity in both normal and tumor cells; one was nontoxic in both normal and tumor cells, and one was selectively toxic to normal cells. Additionally, three of the pzs showed enhanced photo-induced toxicity with these effects in some cases being observed at treatment concentrations of up to ten-fold lower than that needed for a response in Photofrin. All six compounds were preferentially absorbed by tumor cells, suggesting that they have potential as in vitro diagnostic agents and as aids in the isolation and purification of aberrant cells from pathological specimens. In particular, two promising diagnostic candidates have been identified as part of this work.
The porphyrazines (pzs), a class of porphyrin analogues, are being investigated for their potential use as tumor imaging/therapeutic agents. We here examine six peripherally-functionalized M[pz(AnB4-n)] pzs with n=4, 3, or 2 (in a trans conformation) and M = H2 or Zn, where A is an [S((CH2)2O)4Me]2 unit and B is a fused beta,beta'-diisopropyloxybenzo group. Cell viability/proliferation assays and fluorescence microscopy were carried out in both tumor and normal cells. Dark toxicity studies disclosed that four of the compounds exhibited toxicity in both normal and tumor cells; one was nontoxic in both normal and tumor cells, and one was selectively toxic to normal cells. Additionally, three of the pzs showed enhanced photo-induced toxicity with these effects in some cases being observed at treatment concentrations of up to ten-fold lower than that needed for a response in Photofrin. All six compounds were preferentially absorbed by tumor cells, suggesting that they have potential as in vitro diagnostic agents and as aids in the isolation and purification of aberrant cells from pathological specimens. In particular, two promising diagnostic candidates have been identified as part of this work.
Porphyrin antitumor agents have been widely studied in recent years, especially in the area of photodynamic therapy (PDT), where the development of the clinically used porphyrin, haematoporphyrin
derivative (HpD, or Photofrin), has garnered significant interest [1, 2]. However, limitations in the design, synthesis, and biological properties of Photofrin have led to the development of many second-generation PDT agents, including a number of porphyrin derivatives [3-9]. One such class of compounds being investigated as an alternative is the porphyrazines (pzs), porphyrin analogues in which the meso (CH) groups are replaced by nitrogen atoms linking the pyrrole rings (Scheme 1, Section 3), thereby resulting in compounds with chemical and physical properties distinct from those of the
porphyrins [10]. The presence of the meso-nitrogen
atoms results in the pzs possessing intense long-wavelength absorbance and
emission bands, photophysical properties that are intrinsically superior to
those of the porphyrins. Furthermore, the porphyrazines are prepared by the
templated cyclization of maleonitrile derivatives, while porphyrins are
synthesized by the condensation of aldehyde and pyrrole derivatives. This
difference allows pzs to be readily prepared with S, N, or O heteroatoms
attached to the macrocycle core, while porphyrins cannot. These heteroatom
substituents enable the photophysical properties of the pzs to be tuned such
that they exhibit optimal near-IR absorbance and emission in the 700–900 nm
window, and allow for tuning of their singlet oxygen quantum yields from
essentially off to on—characteristics which make the pzs attractive
candidates for biomedical applications [11-14].
Scheme 1
Synthetic route to porphyrazines 5–10.
Because of the novelty of these compounds, initial biocompatibility studies involving the pzs have focused upon determining how specific pz structures selectively alter biological functions [15, 16], leading to the establishment of “structure-function rules,” which will ultimately be used to predict future biologically active pzs. To this end, we recently
reported the biological activity of a suite of three anionic H2[pz(A
n
B
)] pzs, in which , 3, or 2 (trans), A was an [S-R]2 unit with , and B was a fused -dialkoxybenzo
group. These three anionic compounds showed a systematic increase in toxicity
as (and in turn, the number of R groups) was increased, and one pz () was found to be selectively toxic to tumor cells. Herein we similarly describe the biocompatibility of a group of M[pz(An B)]
pzs, where M = H2 or Zn, , 3, or 2 (trans), A is an [S-R]2 unit with R = [(CH2)2O]4Me, and B is again a fused -dialkoxybenzo group (5–10, Scheme 1).1As with the aforementioned anionic porphyrazines, the pzs examined here, with thiotriethoxy(ethoxy)methyl groups appended to their periphery, allow us to study the biological effect of a
specific functional group. By varying , we can simultaneously vary the
core pz structure and the number of appended R groups, resulting in each
compound possessing slightly different chemical and physical properties,
thereby impacting the biological effects of each.This study also marks the first biocompatibility experiments involving metallated pzs. As previously reported, the singlet oxygen quantum yields (Φ
Δ) of the free-base (H2) pzs increase systematically as is decreased; the same trend is true
for metallated pzs, but the presence of Zn in the pz core increases the value
of Φ
Δ approximately five-fold over that of the analogous H2pzs [12]. Herein we report the concentration/time-dependent cytotoxicity and phototoxicity measurements, and present confocal fluorescence microscopic visualization, for pzs 5–10 in A549 tumor and WI-38 VA13 normal cells.
2. MATERIALS AND METHODS
2.1. Chemical reagents and instrumentation
All chemicals were purchased from Aldrich Chemical Company “Wis, USA” and used
as received. Baxter silica gel (60 Å; 230–400 mesh) was used for column
chromatography. A Hewlett-Packard HP8452A diode-array spectrophotometer was
used to record electronic absorption spectra, and electronic emission spectra
were recorded using a Photon Technology International QM2 fluorescence
spectrometer. 1H and 13CNMR spectra were obtained using
a Varian Inova 500 MHz spectrometer. Electrospray ionization mass spectra
(ESI-MS) were recorded using a Finnegan LCQ Advantage mass spectrometer, and
matrix-assisted laser desorption ionization-time of flight mass spectra
(MALDI-TOF-MS) were recorded using a PE Voyager DE-Pro MALDI-TOF mass
spectrometer; α-cyano-4-hydroxycinnamic acid was used as the matrix for
MALDI-TOF experiments.
2.2. Porphyrazine synthesis
Disodium 1,2-dicyano-1,2-ethenedithiolate (Na2MNT) [17] and
1-imino-4,7-bis(1-methylethoxy)-1H-isoindolin-3-amine (diiminoisoindoline, 1) [11] were prepared as previously reported.
During the purification of each pz, distinct bands were observed and pooled
together during column chromatography; collected fractions were monitored by
UV-visible spectroscopy. Each pz was prepared as a 5 mM working stock solution
in dimethyl sulfoxide (DMSO) for use in the tissue culture experiments.
Sodium hydroxide (12 g, 0.3 mol) dissolved in water (55 mL) and tetra(ethylene glycol) monomethyl ether (2) (41.7 g, 0.2 mol) in THF (55 mL) were placed in a flask, and the mixture was
stirred and cooled in an ice bath. To the mixture was added dropwise -toluenesulfonyl choride (41.9 g, 0.22 mol) in THF (65 mL) over 30 minutes with continuous stirring and cooling of the mixture below
5°C.
The solution was stirred an additional 2 hours at ∼0–5°C, at
which time the mixture was poured into ice water (200 mL) and extracted twice
with CH2Cl2 (200 mL). The combined organic
extract was washed twice with water and once with a saturated NaCl solution
(brine), and then dried over Na2SO4. After evaporation of the solvent,
the resulting residue was purified by chromatography on silica gel (4% MeOH in CH2Cl2 eluant) to yield the tosylate 3 (63 g, 87%) as a colorless viscous oil: 1HNMR (500 MHz, CDCl3) δ 2.43
(s, 3H, ArCH3), 3.35 (s, 3H, OCH3), 3.52 (t, 2H, CH2), (m, 10H, CH2), 3.67 (t, 2H, CH2), 4.14 (t, 2H,
CH2), 7.33 (d, 2H, ArH), 7.78 (d, 2H, ArH); 13CNMR (125 MHz, CDCl3) 21.8, 59.1, 68.8, 69.4, 70.6, 70.8, 71.2, 71.6, 71.9, 72.2, 128.1, 130.0, 133.1, 145.0; ESI-MS (m/z) calculated for C16H27O7S [M + H]+ 363.44, found 363.
Mg turnings (0.1 g, 4 mmol) and I2 (0.01 g, mol) in
-PrOH (50 mL) were heated at reflux for 24 hours under N2 to prepare Mg(OPr)2. MNT[S(C2H4O)4Me]2 (4) (5.23 g, 0.01 mol) was added, and the reaction was heated at reflux for 7 hours. The yellow reaction mixture gradually turned
green. After 2 hours, the reaction mixture was a deep blue color. Heating and
stirring were continued for a total 7 hours after which the solvent was removed
under reduced pressure. The resulting residue was dissolved in CH2Cl2 (20 mL). TFA (2 mL) was slowly added, and the solution was stirred for 1 hour. After dilution in CH2Cl2 (100 mL), the mixture was washed 4 times with a large amount of water to remove any residual TFA, dried over Na2SO4, and rotary evaporated. The resulting residue was purified via column chromatography (4% MeOH in CH2Cl2 eluant) to produce 5 (628 mg, 12% yield) as a dark blue solid: UV-vis (CH2Cl2) (log ɛ) 360 (4.66), 502 (2.23), 640 (3.10), 710 (3.99) nm; 1HNMR (500 MHz, CDCl3) δ-1.13 (br s, 2H,
NH), 3.32 (s, 24H, –OCH3), 3.40 (t, 16H, CH2), 3.55 ∼ 3.59 (m, 64H, CH2), 3.65 (t, 16H, CH2), 3.99 (t, 16H, CH2), 4.28 (t, 16H, CH2); 13CNMR (125 MHz, CDCl3) 34.76, 59.23, 70.68, 70.72, 70.74, 70.77, 71.11, 72.09, 140.56; MALDI-TOF-MS (m/z) calculated for C88H155N8O32S8 [M + H]+ 2093.72, found 2093.66; calculated for C88H154N8NaO32S8 [M + Na]+ 2115.71, found 2116.48.
2.6. {2-{2-[2-(2-Methoxyethoxy)ethoxy]ethoxy}ethylthio}porphyrazines 6 and 7
Mg turnings (0.3 g, 12 mmol) and I2 (0.03 g, mol) in
-PrOH (150 mL) were heated at
reflux for 24 h under N2 to prepare Mg(OPr)2. MNT[S(C2H4O)4Me]2 (4) (5.23 g, 0.01 mol) and diiminoisoindoline (1) (5.23 g, 0.02 mol) were added, and the reaction was heated at reflux for 7 hours, during which time the solution went from orange to blue-black.
The solvent was removed under reduced pressure, and the residue was dissolved
in CH2Cl2 (30 mL). TFA (3 mL) was slowly added, and the
solution was stirred for 1 hour. After dilution in CH2Cl2 (200 mL), the mixture was washed 4 times with a large amount of water to remove any residual TFA, dried over Na2SO4, and rotary evaporated. The resulting residue was purified via column
chromatography (4% MeOH in CH2Cl2 eluant)
to yield 6 (260 mg, 4.3%) as a dark blue solid and 7 (477 mg, 6.2%) as a dark green solid, as well as trace amounts of 5.
The three metallated pzs were each prepared analogously to zinc pzs previously
described [11]. A solution of zinc chloride (20.4 mg, 0.15 mmol) in methanol (10 mL) was added to a solution of metal-free porphyrazine (0.05 mmol) in CH2Cl2 (30 mL). The reaction mixture was heated to reflux for 30 minutes or until zinc insertion was completed, as monitored by spectrophotometry. The reaction mixture was diluted with CH2Cl2 (60 mL), washed with water to remove excess zinc chloride, dried with Na2SO4,
and evaporated to dryness under vacuum. The resulting residue was purified by chromatography on silica gel (4% MeOH in CH2Cl2 eluant) to yield the
desired Zn porphyrazine in quantitative yield.
All media and supplements were purchased from Invitrogen “Calif, USA” except where noted. Phosphate-buffered saline (PBS) solution was prepared by adding Na2HPO4
7 H2O (54.0 g), KH2PO4 (5.0 g), KCl (5.0 g), and NaCl (200.0 g) to 25 L of distilled water. A 2.0 g/L solution of
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in PBS was
prepared and sterile filtered (0.2 μm pore size) prior to use in the
cytotoxicity assays. Haematoporphyrin derivative (HpD, or Photofrin; QLT
Phototherapeutics, Inc. “Vancouver, BC, Canada” ) was used as a positive control in the phototoxicity assays; concentrations of HpD were calculated using an assigned molecular weight of 600.The following cell lines were utilized in this study: A549 (humanlung adenocarcinoma), WI-38 VA13 (SV40 transfected fibroblast-like human embryonic cell line), five human head and
neck squamous cell carcinomas (SCC016, SCC040, SCC056, SCC114, SCC116), and
three humanbreast adenocarcinomas (BT-20, T-47D, and MCF-7). All cell lines
were obtained from American Type Culture Collection “Va, USA.” The A549 and T-47D cell lines were maintained in RPMI 1640 media supplemented with 10% fetal calf serum
heat inactivated at 56°C for 30 minutes, 2 mM L-Glutamine, 100 μg/mL Streptomycin, 100 U/mL Penicillin, and 2.5 mcg/mL Amphotericin B solution. All other cell lines were maintained in minimumessential medium (MEM) with Earle’s salts supplemented with 10% fetal calf serum heat inactivated at 56°C for 30 minutes, 2 mM L-Glutamine, 100 μg/mL Streptomycin, 100 U/mL Penicillin, and 2.5 mcg/mL Amphotericin B solution, as well as 100 μM MEM nonessential amino acids and 1 mM Sodium Pyruvate (CellGro, Inc. “Va, USA”) in distilled water. Cells were grown at 37°C in a humidified atmosphere containing 5% CO2.
2.14. Cytotoxicity assays
Cells were seeded (100 μL) into 96-well microtiter plates and grown until they were
approximately 70% confluent. The plates were then treated in the dark (to avoid
photosensitized killing) with the appropriate concentration of pz or a volume
of DMSO equivalent to the volume of compound added at these concentrations. Pz
and DMSO solutions were added as 100 μL aliquots to the original media in
the well. The subsequent 200 μL of media was thoroughly mixed,
after which time 100 μL was removed, resulting in a final
volume of 100 μL in each well; final pz/DMSO concentrations ranged from 50–1.56 μM. The media were removed at designated time points (24, 48,
72, 96, or 120 hours), and 100 μL of MTT/PBS solution was added to each well.
The plates were then incubated for an additional 5 hours at 5% CO2 and
37°C. During this time, the tetrazolium ring of the MTT molecules is cleaved by
the mitochondrial dehydrogenases of viable cells, resulting in the formation of
purple formazan crystals. Following incubation, the supernatant was decanted,
and 100 μL of DMSO was added to each well to dissolve any formazan crystals.
The absorbance was then read at 540 nm for each well. Each data point
represents the average of at least four microtiter wells for each plate, and at
least three independent trials were carried out for each experiment. Individual
trials were normalized and averaged such that the final reported values
represent a minimum of 12 independent values for each reported condition for
each cell line.
2.15. Phototoxicity assays
Two identical 96-well plates were seeded with A549 and WI-38 VA13 cells grown to
∼70% confluency as described above. The cells were then treated with the
desired concentration of pz or HpD and incubated for additional 4 hours.
Aluminum foil was then wrapped around the sides and top of the first plate,
leaving the bottom of the plate uncovered in order to enable light penetration.
The second plate was completely wrapped in aluminum foil to inhibit light from
reaching the cells; this plate served as the dark control. The two plates were
then placed on top of a standard X-ray illuminator (consisting of four 15 W
bulbs, ∼3600 total lumens) and exposed to 10 minutes of light. Following the
light treatment, the plates were placed back into the incubator for 24 hours, after
which time MTTcytotoxicity assays were performed.
2.16. Imaging assays
A549 and WI-38 VA13 cells were plated onto sterilized
glass coverslips in mm dishes and grown at 37°C in a humidified atmosphere
containing 5% CO2. Upon reaching 60–80% confluency, the cells were
treated with the desired pz at 25 μM and incubated in the dark under the
same conditions for an additional 4 hours. Negative controls were prepared by
treating cells with an amount of DMSO equivalent to that of the 25 μM pz sample.Confocal microscopic images of the red pz emission were obtained at
room temperature with a Zeiss 510 LSM confocal microscope. Following the 4-hour
incubation period of the cells with the pz, the supernatant was decanted and
the cells were washed twice with PBS. The washed coverslips were then inverted
onto microscope slides, and the cells were imaged live in PBS. Cells were
excited with an argon-ion laser line at 488 nm, and fluorescence was detected
with a long-pass 505 nm filter. All images were taken using the same detector
gain and amplitude settings.
3. RESULTS AND DISCUSSION
3.1. Pz synthesis and properties
The six compounds examined in this study are of the form M[pz(An
B
)], where M = H2 or Zn, A is [S((CH2)2O)4CH3]2, and B is a fused 4,7-bis(isopropyloxy)benzo group, with (5, 8), (6, 9), and the trans form of (7, 10).
Scheme 1 shows the reaction scheme utilized to prepare the pzs.In order to attach the desired tetra(ethylene glycol) monomethyl ether, (CH2CH2O)4Me, functional group onto the periphery of a pz, it was first necessary to prepare 2-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}ethyl-4-methylbenzenesulfonate
(3) via tosylation of the commercially available tetra(ethylene glycol) monomethyl ether (2). MNT[S(C2H4O)4Me]2 (4) was then prepared by treating Na2MNT with two
equivalents of 3 in acetone at reflux for 12 hours.Magnesium-templated Linstead macrocyclization [18] of MNT[S(C2H4O)4Me]2 (4), followed by demetallation in
trifluoroacetic acid and purification via column chromatography, led to the
formation of the H2[pz(A
4)], 5, in
12% yield. The H2[pz(A
3
B)] (6,
4% yield) and trans-H2[pz(A
2
B
2)] (7, 6% yield) pzs were prepared via a mixed cyclization of
MNT[S(C2H4O)4Me]2 (4) and 1,3-diiminoisoindoline (1), reacted in a 2 : 1 stoichiometric ratio, and followed by demetallation with trifluoroacetic acid. Column chromatography was then used to separate and
purify the two compounds. It is noted that the cis-H2 [pz(A)] compound was not produced during this reaction, indicating that under these
reaction conditions, the diiminoisoindoline (1) reacts
preferentially with its cocyclization partner, 4, resulting in only the trans form of the M[pz(A)]. The yield of the trans-macrocycle
was further improved by the use of 0.38 equivalents of Mg per mole of combined
cyclization partners, and by heating the reaction mixture for 7 hours at reflux
in -propanol. The corresponding zinc analogues—8, 9,
and 10—were prepared by
reacting each of the free-base pzs in a solution of zinc chloride in CHCl3/MeOH
at reflux for 30 minutes. All three compounds were obtained in quantitative
yield.Each compound is freely soluble in DMSO. By utilizing different combinations of M (H2 or Zn) and (4, 3, or 2, in a trans conformation), we can predictably vary the core
macrocycle structure, resulting in different optical properties for each of the
six pzs. Previous work has shown that the optical properties of the pzs are
dependent upon M and , but independent of R [10,11,19,20]. Figure 1 shows
typical spectra obtained in CH2Cl2 for the six pzs presented
here. All six compounds exhibit an intense B (Soret) band at ∼350 nm with high extinctions (∼50,000 M−1cm−1),
but each has a different Q-band region, depending upon M and . For the free-base pzs, split Q-bands are observed for both and (trans), with H2[pz(A
3
B)]
exhibiting a maximum absorption of ∼700 nm (ɛ
∼45,000 M−1cm−1)
and H2[pz(A
2
B
2)] having
well-defined peaks at ∼654 nm and 798 nm (ɛ
∼50,000 M−1cm−1 for both),
respectively; in contrast, the symmetrical H2[pz(A
4)]
() exhibits only a single Q-band with a maximum absorption at ∼712 nm (ɛ
∼35,000 M−1cm−1). As expected, similar
Q-band patterns are observed in the three Zn[pz(A
n
B
)]pzs, but the peaks are shifted ∼30 nm towards the blue. Zn[pz(A
4)] has a single Q-band absorption at 672 nm (ɛ
∼47,000 M−1cm−1); both Zn[pz(A
3
B)] and Zn[pz(A
2
B
2)] have split Q-bands, with the former exhibiting a maximum absorption at 676 nm (ɛ
∼50,000 M−1cm−1) and the latter having peaks at 666 nm (ɛ
∼54,000 M−1cm−1) and 761 nm (ɛ
∼45,000 M−1cm−1), respectively. All
six compounds also exhibit dual fluorescence, as shown in Figure 1: for the
free-base pzs with , 3, and 2, short wavelength (uv) fluorescence is observed at = 410, 428, and 440 nm, respectively; and long wavelength (nir) fluorescence occurs at = 737, 800, and 827 nm,
respectively; for the Zn pzs with , 3, and 2, uv emission is
observed at = 435, 444, and 440 nm, respectively; and nir emission occurs at = 713, 754, and 795 nm, respectively [10]. Both emissions can be
generated with excitation wavelengths to the blue of ∼400 nm, while only the
nir luminescence is produced upon excitation to the red of ∼450 nm. Unlike
previously reported pzs in which broadening and aggregation were often observed
[15, 16], the uv-visible spectra of 5–10 in DMSO looked nearly identical to the spectra obtained in CH2Cl2.
Figure 1
Absorbance (solid lines) and emission (dashed lines)
spectra of H2[pz(A
n
B
)] and Zn[pz(A
n
B
)] pzs in CH2Cl2.
Earlier work with the pzs has also found that the singlet oxygen quantum yields (Φ
Δ) for a series of M[pz(An
B
)], where A = [S-R]2 and B is a fused dialkoxybenzo group,
are dramatically affected by both M and , but are independent of the identity
of the R group [12]. For a given M, the quantum yield is found to increase as
the value of is decreased. Therefore, the
H2[pz(A
2
B
2)], 7, has the highest quantum yield of the three free-base pzs (Φ
Δ
∼ 0.130), with 6 (Φ
Δ
∼ 0.026) and 5 (Φ
Δ
∼ 0.0074) have markedly lower values. Introducing zinc into the core of
the pz results in significantly higher yields than those found in the analogous
free-base compounds. Thus the Zn[pz(A
2
B
2)], 10, possesses the highest singlet oxygen quantum yield (Φ
Δ
∼
0.370) of the six compounds presented in this study, while the quantum yields
for 9 (Φ
Δ
∼ 0.110) and 8 (Φ
Δ
∼ 0.037) are accordingly lower.
3.2. Quantitative in vitro effect of porphyrazines
MTT assays were used to test the
proliferation/viability of A549 and WI-38 VA13 cells grown in the
presence of compounds 5–10 over a 72-hour time period.
Cells were initially treated with a pz concentration of 50 μM, and MTT assays were performed at 24, 48, and 72 hours (Figure 2).
Figure 2
MTT time course plot of A549 (□) and WI-38 VA13 (Δ) cells exposed to 50 μM pz. Dotted lines = control (cells + DMSO), solid lines = cells + pz.
In order to confirm that any toxicity
observed was due to the pzs and not the DMSO solvent, control cells were
exposed to a volume of DMSO equivalent to the volume of pzs at 50 μM; cells exposed to DMSO exhibited normal growth behavior.
Unlike previously reported pzs which exhibited little cellular toxicity after a
24-hour exposure [15, 16], four of the six pzs tested here exhibited marked
toxicity to both normal and tumor cells within the first 24 hours upon 50 μM exposure. (We note here that the 50 μM treatment dose was chosen because it corresponds to the
approximate amount of HpD typically needed to observe a clinical response in
patients undergoing PDT treatments.) Cells grown in the presence of H2[pz(A
2
B
2)], 7, and the M[pz(A
3
B)] pzs, 6 (M = H2) and 9 (M = Zn), showed moderate-to-large decreases in viability after 24 hours, while cells treated with Zn[pz(A
2
B
2)], 10,
were almost completely killed within the first 24 hours of exposure. An additional
24 hours of exposure to pzs 6, 7, and 9 resulted in both normal and tumor cells being nearly completely killed.The proliferation/viability of cells exposed to the M[(A
4)] pzs, 5 (M = H2) and 8 (M = Zn), contrasted sharply to that of
the M[pz(A
3
B)] and M[pz(A
2
B
2)] pzs. Compound 8 showed little-to-no toxicity in either cell line
over the entire time course of 72 hours, while 5 was found to be
selectively toxic to normal cells. Nearly 80% of the WI-38 VA13 normal cells
were killed within 24 hours upon exposure to 5, while no
appreciable toxicity was seen for tumor cells exposed to 5 for 72
hours. The nontoxic behavior found with cells exposed to 5 (tumor
only) and 8 (both tumor and normal) prompted the study of longer
treatment times for these two compounds. Additional MTT assays were therefore
carried out at 96 and 120 hours for both 5 and 8.
However, as shown in Figure 2, these longer exposure times did not result in
any additional observed toxicity.The
selective killing of the normal cells upon exposure to 5 prompted us to
test this particular compound against other tumor cell lines to determine if
the resistance of the A549 cells was consistent across different tumor cell
lines and types. Table 1 shows the percent viability of human breast
adenocarcinomas (BT-20, T-47D, and MCF-7) and human head and neck squamous cell
carcinomas (SCC016, SCC040, SCC056, SCC114, and SCC116) exposed to 5 at 50 μM for 72 hours, relative to DMSO-treated control cells. In
general, very little toxicity was observed for any of the eight additional
tumor cell lines, as growth rates of the pz-treated cells were similar to the
growth rates of the DMSO-treated controls.
Table 1
Percent cell viability of various cell lines
exposed to 50 μM 5 for 72 hours, relative to DMSO-treated cells.
Cell Line
Cell Line Description
Percent Cell Viability (%)
SCC016
Human tongue squamous cellcarcinoma
91.2±19.2%
SCC040
Human tongue squamous cellcarcinoma
95.3±23.8%
SCC056
Human tongue squamous cellcarcinoma
94.8±20.5%
SCC114
Human floor of mouth squamous cellcarcinoma
78.2±17.0%
SCC116
Human alveolar ridge squamous cellcarcinoma
102.0±10.2%
BT-20
Human breast adenocarcinoma
118.4±4.5%
MCF-7
Human breast adenocarcinoma
77.7±19.6%
T-47D
Human breast adenocarcinoma
106.4±8.4%
A549
Human pulmonary adenocarcinoma
92.7±23.6%
WI-38 VA13
Human embryonic fibroblast
20.9±23.3%
3.3. Dose-dependent effects of porphyrazines
As described above, exposure to the M[pz(A
3
B)]
and M[pz(A
2
B
2)] pzs at concentrations
of 50 μM resulted in significant toxicity in both tumor and normal
cells (Figure 2). To determine the toxicity limits of each pz, MTT assays were
carried out in both A549 tumor and WI-38 VA13 normal cells at varying
concentrations (1.56–50 μM) for 72 hours. Figure 3 shows the dose-dependent results as percent cell viability, calculated relative to the
viability of the control DMSO-treated cells. (Note that treated cells growing
at an equivalent rate to the DMSO control cells would have a 0% change in cell
viability; treated cells which grew slower and/or were less viable than the
control cells would have a negative percent change in cell viability.)
Figure 3
Percent cell viability, relative to DMSO control, of A549 (black bars) and WI-38 VA13 (gray bars) cells after a 72-hour exposure to pzs at varying concentrations. (Note that a 0% change in the percent cell viability correlates to cells which grew at the same rate as the control cells; negative values correlate to cells which grew at rates below those of the control cells.)
With the exception of the M[pz(A
4)]
pzs, the thiotriethoxy(ethoxy)methyl-appended pzs were generally found to
exhibit significant toxicity even at lower concentrations. Both of the M[pz(A
2
B
2)]
pzs, 7 and 10, caused nearly 100% cell killing at
treatment concentrations of 12.5 μM and above. Significant toxicity was
still observed for both tumor and normal cell lines exposed to 6.25 μM 10; however, A549 tumor cells exposed to 7 at 6.25 μM showed only very little toxicity (∼10% killed), while
normal cells exposed to the same concentration resulted in nearly 80% of the
cells dying. Slight toxicity was still observed in both normal and tumor cells
exposed to the lowest concentration of 10 tested (1.56 μM), while normal and tumor cells exposed to 7 at a treatment concentration of 1.56 μM showed essentially normal growth
upon a 72-hour exposure.While 7 and 10 showed a somewhat similar dose-dependent proliferation/viability behavior, the
two M[pz(A
2
B
2)] pzs, 6 and 9, differed depending upon the identity of M. The H2 [pz(A
3
B)], 6, was found to exhibit a growth behavior very similar to that
observed for its H2[pz(A
2
B
2)] analogue, 7. Significant toxicity was found in both tumor and normal cells exposed to 6 at concentrations of 12.5 μM and above for 72 hours; and similar
to 7, the normal cells showed selective toxicity at treatment
concentrations of 6.25 μM and below. However, unlike cells
exposed to 7, normal cells treated with 6 at 1.56 μM still exhibited a moderate amount of toxicity (∼30% cell
death). The Zn[pz(A
3
B)] analogue, 9, showed
significantly less toxicity at the higher treatment concentrations (25 and 12.5 μM) in both normal and tumor cells than that observed in cells
treated with the free-base 6 at the same concentrations. However,
slight toxicity (∼10–20% cell killing) was still observed for both tumor and
normal cells exposed to low concentrations of 9, whereas A549tumor cells exposed to 6 at treatment concentrations of 6.25 μM and below exhibited normal growth behavior.Based upon the 50 μM time-course plots in Figure 2, it was expected that the dose-dependence results of the M[pz(A
4)] pzs (Figure 3) would generally show little toxicity. To this end, the free-base 5 exhibited selective toxicity towards WI-38 VA13 cells at concentrations of 25 μM and higher upon a 72-hour exposure; at treatment concentrations of 12.5 μM and below, any toxicity observed in either the tumor or normal cells fell within the experimental error limits for
normal growth (i.e., within the error range for a 0% change in cell viability).
Both normal and tumor cells exposed to the Zn[pz(A
4)] pz, 8,
also exhibited normal growth behavior at all concentrations studied.
3.4. Photosensitizing effects of the porphyrazines
In addition to the dark toxicity studies described above
(Figures 2 and 3), light-dependent proliferation/viability studies were
employed to determine if the compounds possessed additional photosensitizing
properties. Due to the high levels of toxicity
observed in the MTT dose-dependence studies (Figure 3), a shorter incubation time
and a lower treatment concentration were employed for these compounds than used
in the biological studies of previously reported pzs [15, 16]. Cell lines were
treated with 25 μM
of 5–10 and 25 or 50 μM HpD as a reference for 4 hours and
were then exposed to white light for a period of 10 minutes. The microtiter
plate serving as the dark control was completely wrapped in aluminum foil so
that the cells would avoid any light exposure, but would still be exposed to
any potential heat being generated by the bulbs of the X-ray illuminator during
the 10-minute treatment period. The cells were incubated overnight following
the light treatment, and MTT assays were performed after 24 hours. Figure 4 shows data comparing no light treatment
versus cells exposed to 10 minutes of white light.
Figure 4
MTT results for A549 (a) and WI-38 VA13 (b) cells exposed to pz (25 μM) or HpD (25 or 50 μM) for 4 hours, followed by white light exposure for 0 (black bars) or 10 (gray bars) minutes. Control cells were treated with an amount of DMSO equivalent to 25 μM pz.
Both
untreated (data not shown) and DMSO-treated cells were used as controls;
similar results were obtained for both; therefore, Figure 4 shows only the
DMSO-treated cells.As expected, growth of the DMSO-treated cells was not adversely
affected by the additional light exposure. However, unlike previously studied
pzs [15, 16], three of the compounds reported here—7, 9,
and 10—showed a distinct
photosensitizing effect in the A549 tumor cells. It could not be determined if
a similar effect occurred in the WI-38 VA13, due to the inherent toxicity of 7, 9, and 10 in the normal cells; absorption readings
at the 0-minute light-exposure time point were extremely low for these three
compounds in the WI-38 VA13 cell line (Figure 4). It is noted that, of the six
pzs tested in this study, the three exhibiting the phototoxic effect in A549
cells correspond to the three compounds having the highest singlet oxygen
quantum yield (Φ
Δ). Accordingly, the compounds with relatively low Φ
Δ values—5, 6,
and 8—did not show any
appreciable phototoxic effect in either cell line, as viability levels remained
steady with and without light treatment. Interestingly, no appreciable light
effect was observed for either tumor or normal cells exposed to 25 μM HpD (a dose
that is two-fold below the typical clinical treatment dose); however, treatment
of cells with HpD at the usual clinical dose, 50 μM, results in substantial photo-induced
cell death in both A549 and WI-38 VA13 cells, as shown in Figure 4.Additional dose-dependent phototoxicity studies were carried
out for 7, 9, and 10 in A549 cells to
determine if a light-dependent response could be observed for the pzs below the
25 μM treatment
dose employed in Figure 4. Cells were treated with the pzs at concentrations of
25–1.56 μM
for 4 hours and then exposed to either 0 or 10 minutes of white light. MTT
assays were carried out after an overnight incubation (Figure 5). As expected
from the results of Figure 4, Photofrin did not show any light-induced toxicity
at treatment concentrations of 25 μM or below (data not shown). However,
significant phototoxicity was still observed in A549 cells treated with 7 at concentrations below 25 μM upon a 10-minute white light exposure;
a treatment dose of 12.5 μM resulted in additional 60% of the
cells being killed when exposed to light, while cells treated with 6.25 μM 7 resulted in approximately 40% cell death, relative to the dark toxicity (i.e.,
0 minute white light exposure). Even at the lowest treatment concentration of 7 studied, 1.56 μM, a moderate amount (∼15–20% cell death) of toxicity was still observed. In contrast, both pzs 9 and 10 exhibited far less
sensitivity than 7. Moderate phototoxicity (∼30% cell death) was
observed in A549 cells exposed to 9 at 12.5 μM, but little
effect was detected at doses of 6.25 μM and below. Cells treated with 10 did not show any additional phototoxicity at doses
below 25 μM.
Figure 5
MTT results for A549 cells exposed to varying concentrations of pz or DMSO for 4 hours, followed by white light exposure for 0 (black bars) or 10 (gray bars) minutes. Line graphs are overlaid for clarity.
3.5. Cellular uptake of porphyrazines
Experiments were carried out to
assess the cellular uptake and general localization behavior of each of the six
pzs. Figure 6 presents the confocal microscopic images of A549 and WI-38 VA13
cells exposed to each compound for 4 hours at 25 μM. Due to the rapid onset of toxicity
caused by these compounds, as discussed above, the cells were treated at a
lower concentration and for a shorter period of time than the conditions used
in previous cellular uptake studies of
the pzs [15].
Figure 6
Cellular uptake of pzs using false-white confocal fluorescence microscopy images of A549 (top) and WI-38 VA13 (bottom) cells treated with 25 μM agent: (a) DMSO control,
(b) 5, (c) 6, (d) 7, (e) 8, (f) 9, (g)
10. Scale bar = 10 μm.
Moderate autofluorescence was
observed for the control (DMSO-treated) cells in both tumor and normal samples.
All six of the pzs showed enhanced intracellular luminescence in A549 cells
upon treatment with compound; each of the six compounds displayed similar
fluorescence intensity and exhibited uniform staining of the cytoplasm with no
uptake by the nucleus. In contrast, WI-38 VA13 cells showed little-to-no
measurable increase in fluorescence upon treatment of the cells with the pzs.
Significant vacuole formation was also observed in both tumor and normal cells
exposed to 6, 9, and 10.
4. Conclusions
Recent work has sought to study the
structure-function relationships of the porphyrazines (pzs) in biological
systems, in an effort to develop these compounds for use as therapeutic and
diagnostic agents [15, 16]. Herein we continue our structure-function studies
of the porphyrazines by examining a series of six new M[pz(An
B
)] porphyrazines, where M = H2 or Zn, A is [S((CH2)2O)4CH3]2, B is a fused 4,7-bis(isopropyloxy)benzo
group, and , 3, or 4 (Scheme 1). The thiotetra(ethylene glycol)
monomethyl ether functional group is hydrophilic in nature, while the pz core
is hydrophobic; thus for a given M, as the value of is increased in the series, the resulting compounds progressively become more hydrophilic in
character. Furthermore, by introducing zinc into the core of the pzs (8–10), the solubility of the compound and the singlet oxygen quantum yield are both
enhanced, relative to their free-base analogues. The zinc pzs reported here are
the first metallated pzs to be tested for their biological behavior. Concentration/time-dependent
MTT proliferation/viability assays were carried out, both in the presence and
absence of white light, in order to measure the dark and photoinduced toxicity
of each pz in normal (WI-38 VA13) and tumor (A549) cell lines, and confocal
microscopy was used to determine the cellular uptake and localization of each
compound.The dark
toxicity studies of the six pzs revealed a dose-dependent response in which
cellular toxicity increased in both normal and tumor cell lines as was
decreased (Figures 2 and 3). With the exception of 8, all of the
pzs were found to be toxic to normal cells at higher concentrations (25 μM and above); and for the M[pz(A
3
B)]
and M[pz(A
2
B
2)] pzs, significant toxicity
was also observed in the tumor cells at higher concentrations. In general, for
a given concentration, the observed toxicity was found to be greater in the
normal cells than in the tumor cells. Furthermore, in the case of the M[pz(A
3
B)] and M[pz(A
2
B
2)] pzs, for a given , toxicity was generally found to be higher for the free-base compounds than for the metallated pzs.A particularly interesting result from the dark toxicity studies is the finding that 5 is selectively toxic to normal cells. As shown in Figure 2 and Table 1, 5 was found to be nontoxic across a number of different tumor cell lines and tumor types (both adenocarcinomas and squamous cell carcinomas), suggesting
that this compound might possess multiple uses as a diagnostic agent. First, when attempting to establish a cell line derived from a humantumor (for purposes of preparing a cell line that will grow continuously in the
laboratory), it is necessary to selectively remove and purify the tumor cells
from the tumor sample removed from the patient, which contains a mixture of normal
and cancerous tissue. This procedure is often very difficult and
time-consuming; if 5 were added during this process, one could
theoretically remove the normal cells more quickly, leading to faster
purification of the desired tumor cell line. Second, the results of many
patient biopsies are unclear as to whether a particular tissue sample is
malignant or benign; in such unclear cases, one could treat the biopsy sample
with 5 to verify the malignancy of the tissue.The photo-induced toxicity studies (Figure 4) found further toxicity in A549 cells exposed to the pzs with higher singlet oxygen quantum yields—7, 9, and 10. These three compounds are the first members of the M[pz(An
B
)] subclass to exhibit photosensitivity [15, 16]. The result for 7 is particularly interesting, considering that previously studied H2[pz(A
2
B
2)] pzs all contained A groups that resulted in aggregation of the
compound in aqueous media, thereby suppressing singlet oxygen generation. The
result observed here suggests that we have achieved the proper
hydrophobic/hydrophilic balance necessary for sufficient solubility in cellular
environments, and this enhanced solubility is likely a key contributing factor
in the observed photosensitivity of 7 at relatively low doses
(Figure 5). Furthermore, the observation that 6 (H
2A3B) did not exhibit a light-dependent toxicity, while 9
(ZnA3B) did show a response, suggests that the inherent singlet oxygen quantum yield of the free-base A3B structure may not be high enough to be applicable
in PDT applications. Likewise, no photo-induced toxicity was observed for the
M[pz(A
4)] pzs, 5 and 8,
regardless of the identity of M. Therefore, the development of future pzs for
PDT applications will focus mainly on members of the A2B2 (both free-base and metallated) and metallated A3B subfamilies.
However, while members of the M[pz(A
4)] and H2[pz(A
3
B)]
pz subfamilies may not provide any additional PDT activity, it is still
important to study pzs belonging to these subfamilies in an effort to discover
new selectively toxic antitumor agents.Confocal
fluorescence microscopy images revealed that all six of the compounds prepared
in this study were preferentially incorporated into the A549 tumor cells and
localized in the cytoplasm. The results of the cellular uptake studies further
suggest that 8 is a particularly promising imaging agent since it
was found to be nontoxic upon exposure to both tumor and normal cells and was
selectively taken up by tumor cells. As mentioned above, vacuole formation was
observed in both the tumor and normal cells upon exposure to 6, 9,
and 10, suggesting that these compounds may be inducing terminal
differentiation, a specialized form of apoptosis [21, 22]. Future work will
more closely examine the mechanism by which the pzs in this report (5–7, 9, 10) cause cell death. By determining the role of
the pzs in cell destruction, we may be able to slightly alter the structure of
the compounds in order to minimize toxicity (while maintaining the proper pz
solubility and selective tumor uptake), thus leading to many other potential
imaging agents from this class of compounds.The results of
this study are the first steps in the search for useful tumor specific imaging
agents and compounds for use in purifying cell lines from clinical samples. If
the issue of nonselective toxicity can be overcome, the path to developing
porphyrazines as useful antitumor drugs (both traditional and PDT-activated)
will be opened up.
Authors: Neal D Hammer; Sangwan Lee; Benjamin J Vesper; Kim M Elseth; Brian M Hoffman; Anthony G M Barrett; James A Radosevich Journal: J Med Chem Date: 2005-12-29 Impact factor: 7.446
Authors: Efstathia G Sakellariou; Antonio Garrido Montalban; Hubert G Meunier; Garry Rumbles; David Phillips; Richard B Ostler; Klaus Suhling; Anthony G M Barrett; Brian M Hoffman Journal: Inorg Chem Date: 2002-04-22 Impact factor: 5.165
Authors: Benjamin J Vesper; Sangwan Lee; Neal D Hammer; Kim M Elseth; Anthony G M Barrett; Brian M Hoffman; James A Radosevich Journal: J Photochem Photobiol B Date: 2006-01-04 Impact factor: 6.252
Authors: Sangwan Lee; Robert Stackow; Colleen Trevithick-Sutton; Christopher S Foote; Anthony G M Barrett; Brian M Hoffman Journal: Photochem Photobiol Date: 2003-01 Impact factor: 3.421
Authors: Evan R Trivedi; Allison S Harney; Mary B Olive; Izabela Podgorski; Kamiar Moin; Bonnie F Sloane; Anthony G M Barrett; Thomas J Meade; Brian M Hoffman Journal: Proc Natl Acad Sci U S A Date: 2009-12-23 Impact factor: 11.205
Authors: Evan R Trivedi; Zhidong Ma; Emily A Waters; Keith W Macrenaris; Rohit Subramanian; Anthony G M Barrett; Thomas J Meade; Brian M Hoffman Journal: Contrast Media Mol Imaging Date: 2014 Jul-Aug Impact factor: 3.161
Scheme 1
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6