Carbon-1 versus Carbon-3 Linkage of d -Galactose to Porphyrins: Synthesis, Uptake, and Photodynamic Efficiency

Article abstract of DOI:10.1021/acs.bioconjchem.7b00636

The use of glycosylated compounds is actively pursued as a therapeutic strategy for cancer due to the overexpression of various types of sugar receptors and transporters on most cancer cells. Conjugation of saccharides to photosensitizers such as porphyri

Full text of DOI:10.1021/acs.bioconjchem.7b00636

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Article
Carbon-1 versus carbon-3 linkage of D-galactose to
porphyrins: Synthesis, uptake, and photodynamic efficiency
Patrícia Manuela Ribeiro Pereira, Waqar Rizvi, Naga Venkata Satya Dinesh Kumar Bhupathiraju,
Naxhije Berisha, Rosa Fernandes, Joao P. C. Tome, and Charles Michael Michael Drain
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00636 • Publication Date (Web): 09 Jan 2018
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Carbon-1 versus carbon-3 linkage of D-galactose to porphyrins:
Synthesis, uptake, and photodynamic efficiency
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§ #
§
§
Patrícia M. R. Pereira†‡§#, Waqar Rizvi , N. V. S. Dinesh K. Bhupathiraju , Naxhije Berisha , Rosa
⊥
‡+
†
§*
Τ
∥
Fernandes , João P.C. Tomé , Charles Michael Drain
†QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
‡Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3000-548 Coim-
bra, Portugal
+Centre for Neuroscience and Cell Biology - Institute for Biomedical Imaging and Life Sciences (CNC.IBILI), Research
Consortium, University of Coimbra, 3004-504 Coimbra, Portugal
§Department of Chemistry and Biochemistry, Hunter College of the City University of New York, New York, NY 10065,
USA
⊥Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, New York, NY 10016, USA
∥CNC.IBILI, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal
ΤCQE, Departamento de Engenharia Química, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portu-
gal
ABSTRACT: The use of glycosylated compounds is actively pursued as a therapeutic strategy for cancer due to the overexpression
of various types of sugar receptors and transporters on most cancer cells. Conjugation of saccharides to photosensitizers such as
porphyrins provides a promising strategy to improve the selectivity and cell uptake of the photosensitizers, enhancing the overall
photosensitizing efficacy. Most porphyrin-carbohydrate conjugates are linked via the carbon-1 position of the carbohydrate because
this is the most synthetically accessible approach. Previous studies suggest that carbon-1 galactose derivatives diminish binding since
the hydroxyl group in the carbon-1 position of the sugar is a hydrogen bond acceptor in the galectin-1 sugar binding site. We therefore
synthesized two isomeric porphyrin-galactose conjugates using click chemistry: one linked via the carbon-1 of the galactose, and one
linked via carbon-3. Free base and zinc analogs of both conjugates were synthesized. We assessed the uptake and photodynamic
therapeutic (PDT) activity of the two conjugates in both monolayer and spheroidal cell cultures of four different cell lines. For both
the monolayer and spheroid models, we observe that the uptake of both conjugates is proportional to the protein levels of galectin-1
and the uptake is suppressed after pre-incubation with an excess of thiogalactose, as measured by fluorescence spectroscopy. Com-
pared to the carbon-1 conjugate, the uptake of the carbon-3 conjugate was greater in cell lines containing high expression levels of
galectin-1. After photodynamic activation, MTT and lactate dehydrogenase assays demonstrated that the conjugates induce photo-
toxicity in both monolayers and spheroids of cancer cells.
family, has a key role in different events associated with cancer
biology including tumor transformation. Galectin-1 has been
INTRODUCTION
Galectins are animal lectins with high affinity for β-galac-
exploited as a target for anticancer drugs, since its expression is
tose-containing oligosaccharides.1 Galectin-1, a member of this
higher in tumors compared to normal tissues.2
Drugs and other molecules can be taken up by cells via active
or passive transport mechanisms, or combination of both.3 Pas-
sive mechanisms involve simple diffusion of the molecule
through the cell membrane and depends on the molecular hy-
drophobicity,4 e.g. the octanol/water partition coefficient, while
active uptake requires the target molecules to be recognized by
specific receptors and other intermolecular interactions. Por-
phyrinoids and other photosensitizers (PS) can be targeted to-
ward cell receptors by appending appropriate targeting moie-
ties. For example, chromophores appended with carbohydrates
are directed to specific cell surface carbohydrate receptors or
carbohydrate-binding proteins overexpressed in cancer cells.
After light activation, glyco-PS can sensitize the formation of
Figure 1. Tetrathioglycosylated porphryins.
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improve affinity for galactose-binding proteins, galactosylated
reactive oxygen species and induce cytotoxicity.5-16 Simple
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click-type reactions were reported to append polyglycerol den-
dendritic motifs appended to porphyrinoids were developed to
enable multivalent interactions.26, 27 Monovalent galactose de-
rivatives attached at carbon-3 yielded compounds with greater
affinity to galectin-1 than the corresponding carbon-1 deriva-
tives.24 Therefore, we hypothesized that attachment of a porphy-
rin through the carbon-3 of galactose will result in improved
uptake to cancer cells overexpressing galectin-1 protein.28 This
report is the first direct comparison of the uptake and PDT effi-
ciency of carbon-1 versus carbon-3 linked porphyrin-galactose
conjugates.
drimers, glucose, polyethylene glycols, polyamines and poly-
17-20
saccharides to porphyrinoids.5,
Since most glycosylated
porphyrinoids have O- linkages where hydrolysis diminishes in
vivo effectiveness, non-hydrolysable linkages can be more ef-
fective.5
PS such as porphyrins are commonly used in photodynamic
therapy (PDT).6-9, 11, 16, 21 PDT is a rapidly growing modality to
treat age-related macular degeneration, cancer, and various skin
disorders using visible light.4, 21, 22 Previously, our group ap-
pended PEGs, polyamines, lysines, alkanes, fluorous alkanes,
sugars, and nucleotides with S-, O- and N- linkages to porphy-
rins.5, 23 The PDT activity of many of these derivatives was stud-
ied with a variety of cell lines, and the glycosylated compounds
affect both necrosis and apoptosis depending on PS concentra-
tion and light flux.23 We reported the synthesis of nonhydrolyz-
able tetrathioglucose and tetrathiogalactose porphyrins21 con-
nected through carbon-1 of the sugar (Figure 1). The glucose
derivative proved to be selective towards cancer cells overex-
pressing glucose transporters and an effective PDT agent in
vitro using cell lines such as MDA-MB-231 human breast can-
cer, whereas the galactose derivative was much less effective.21
In addition to upregulation of glucose transporters expression,
such as Glut-1, many cancer cells exhibit overexpression of ga-
lectin-1. Galactose derivatives linked at the carbon-1 position
bind to galectins 200 fold less than corresponding carbon-3 de-
rivatives.24
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We attached galactose units through carbon-3 or carbon-1 to
a Zn(II) porphyrin by click chemistry to yield (C1-Gal)4-ZnPor
and (C3-Gal)4-ZnPor, respectively. Removing the metal ion
yields the (C3-Gal)4-Por and (C1-Gal)4-Por derivatives
(Scheme 1). Porphyrins conjugated with galactose through the
carbon-3 of the sugar have not been previously explored in
PDT. The main focus of the present research was to study the
photophysical properties, in vitro cell uptake, and photody-
namic efficiency of the carbon-1 versus the carbon-3 conju-
gates. In vitro cell studies were performed using HCT-116 co-
lon cancer cells, MCF-7 breast cancer cells, UM-UC-3 bladder
cancer cells and HeLa cervical cancer cells, which exhibit dif-
ferent levels of the galectin-1 protein (HeLa > UM-UC-3 >
HCT-116>MCF-7 cells). In addition to the monolayer cancer
cell culture studies, we performed studies using 0.05 mm3 three-
dimensional spheroids because they more closely mimic the tu-
mor microenvironment and diffusion processes that take place
in solid tumors; this 3D-spheroid culture model enables more
accurate predictions of in vivo of photosensitizer efficacy.29-31
Recently, we have observed that monolayer cultures and tumor
spheroids differ in glucose metabolism, endogenous reactive
Galactosylated photosensitizers have demonstrated an excel-
lent potential in PDT due to their ability to target galactose-
binding proteins such as galectin-1.25 Photosensitizers linked to
galactose via N-, S-, C- and O- bonds at carbon-1 (anomeric) or
at carbon-6 of the sugar to the porphyrinoids are reported.6 To
Scheme 1. Synthesis of porphyrin-galactose conjugates.
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Table 1. Photophysical properties of C-1 and C-3 porphyrin derivatives.
2
3
4
5
DLS (PDI) mean diame-
ter & poly dispersity in-
dexd (nm)
Emissionb λmax (nm)
c
Compound
Solvent
UV-visiblea λmax (nm)
ΦF
424 nm excitation
6
7
8
9
(C1-Gal)4-ZnPor (3) DMSO
431, 568, 608
609, 656
604, 651
655, 718
0.035
0.03
-
Ethanol
425, 564, 604
74(0.39)
PBS buffer
410, 425, 446, 571, 613
0.002
16, 38 (0.39)
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(C3-Gal)4-ZnPor (6) DMSO
430, 568, 608
425, 564, 604
610, 650
602, 651
0.03
-
Ethanol
0.025
0.003
79, 164 (0.23)
24 (0.33)
PBS buffer
DMSO
424, 440, 453, 454, 573, 617 655, 723
(C1-Gal)4-Por (4)
(C3-Gal)4-Por (7)
424, 523, 559, 588, 614
419, 523, 562, 599, 651
610, 654, 715
605, 654
0.05
0.05
0.01
-
Ethanol
80 (0.30)
24 (0.34)
PBS buffer
402, 429, 530, 556, 603, 655 658, 734
DMSO
424, 524, 560, 588, 613
419, 522, 563, 602, 657
608, 654, 714
604, 654
0.06
0.06
0.008
-
Ethanol
67 (0.23)
28 (0.33)
PBS buffer
410, 427, 530, 567, 602, 659 657, 718
ab Spectra in supporting information, cΦF are ± 10%, dDLS histograms in supporting information.
oxygen species (ROS), and galectin-1 and GLUT-1 protein lev-
els.28 Here, we report that even though the cell uptake for car-
bon-3 conjugates is significantly greater than the carbon-1 de-
rivatives and proportional to the protein levels of galectin-1, the
PDT activities are about the same.
and porphyrin aromatic peaks were found between 110-158
ppm. HRMS is consistent with the reported structures.
After deprotection of the sugar hydroxyl groups on porphy-
rins 2 and 5, no peaks between 1.8-2.2 ppm in the 1H NMR and
no carbonyl peaks in the 13C NMR were observed, indicating
that the acetate groups were effectively removed. The free base
porphyrins are characterized by the UV-visible spectra, the ob-
servation of the pyrrole N-H at -2.7 ppm, and HRMS.
RESULTS AND DISCUSSION
Synthesis and Characterization of the conjugates
The synthesis and characterization of porphyrin 1 is typical
for tetraaryl porphyrins, and proceeds in a ca. 33% overall yield.
The zinc complex was made to avoid copper metalation of the
macrocycle during the next step wherein copper salts were used
for the click reaction, because copper (II) porphyrins are diffi-
cult to demetalate and have complex photophysics.32 Append-
ing the commercially available or synthesized azido sugars to
compound 1 were carried out using standard click chemistry
conditions in 85-90% yields of compounds 2 and 5 without
transmetalation of the macrocycle. Deprotection by removal of
the acetate groups on the sugars was efficiently accomplished
with sodium methoxide in methanol. Herein, a large excess of
the base was avoided to prevent hydrolysis of the sugars, and
Sephadex columns were used for purification. The zinc was re-
moved using 1:1 methanol:TFA in about 6 hr without hydroly-
sis of the sugars.
The 1H NMR characterization of porphyrins 2 and 3 showed
characteristic peaks for β pyrrolic porphyrin at ~ 8.5 ppm, 2,6-
and 3,5- phenyl protons at ~ 8.1 and 7.2 ppm, galactopyranose
peaks around 4.0-5.6 ppm, and the acetate peaks were found
between 1.8-2.2 ppm. Due to the anomeric galactopyranoside
(1,2,4,6-tetra-O-acetyl-3-azido-3-deoxy-D-galactopyranose),
porphyrin 5 has a distinct multiplicity compared to porphyrin 2.
13C NMR confirmed the formation of the products where the
carbonyl carbon peaks of acetates were found around 170 ppm
Photophysics
The photophysical properties of chromophores such as por-
phyrins with polar substituents depend on the solvent. The am-
phipathic nature of sugar substituted porphyrin allows it to par-
tition into lipophilic, hydrophilic, or amphipathic cellular struc-
tures.5 Therefore, we have studied the photophysical properties
in DMSO, ethanol and DPBS (pH = 7.4). The electronic spectra
of the free base and metalated derivatives are significantly dif-
ferent. Zn analogues show red-shifted Soret bands and the typ-
ical two Q-bands between the 500-700 nm, while freebase ana-
logues show the expected four Q bands. The absorbance and
fluorescence emission spectra for the four compounds are cor-
respondingly different and have a strong solvent dependence
(Table 1).
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Compared to the fluorescence in DMSO, the fluorescence
compounds reported herein, the high local concentration of un-
bound dye around the cells increases passive diffusion, so up-
take also depends on the hydrophobicity of the molecules.37
2
emission of both the free bases and the zinc complexes are ca.
80-90% quenched in PBS. There are negligible Stokes shifts for
all compounds in DMSO and ethanol solvents, indicating min-
imal specific solvent-solute interactions with the macrocycle ef-
fecting vibrational processes. Excitation spectra of all com-
pounds indicate the presence of only one species (data not
shown). The 0.05 and 0.06 fluorescence quantum yields for
(C1-Gal)4-Por (4) and (C3-Gal)4-Por (7) is similar to other
free base glycosylated porphyrins, and almost twice the zinc
complexes.5, 33 Because of the orthogonal phenyl groups, the
1,2,3-triazole and hydrophilic sugar molecules has minimal ef-
fect on photophysical properties, but their presence modulates
the amphipathicity to promote solvent dependence. The split
Soret bands in the UV-visible spectra in PBS at ca. 0.1 µM of
all four conjugates (Supporting Information) indicate some de-
gree of aggregation even at this low concentration. Thus, some
of the quenching observed in PBS is due to the internal shading
and the branching deactivation of aggregates.
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4
5
6
7
8
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Table 2. Partition coefficient (log P n-octanol/PBS) of the
galactose-conjugates.
Compound
logP±S.D.
-1.36±0.05
-1.50±0.12
-0.99±0.01
-1.10±0.10
(C1Gal)4-Por (4)
(C3Gal)4-Por (7)
(C1Gal)4-ZnPor (3)
(C3Gal)4-ZnPor (6)
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Octanol-PBS partition coefficient values
Octanol-PBS partition coefficient values were determined for
(C1-Gal)4-ZnPor, (C1-Gal)4-Por, (C3-Gal)4-ZnPor and (C3-
Gal)4-Por using the standard shake flask method.5 Data show
that the values of log P are somewhat greater for (C3-Gal)4-Por
compared to (C1-Gal)4-Por (Table 2), indicating that (C1-
Gal)4-Por has somewhat less water solubility compared with
the corresponding (C3-Gal)4-Por. As observed for many other
tetraarylporphyrins, the partition coefficients were greater for
the free base porphyrins compared with Zn-porphyrin indicat-
ing the free bases are ca. 33% more soluble in PBS.4 These dif-
ferences in PS intrinsic lipophilicity can result in different sub-
cellular localization and mechanisms of cell death after photo-
activation.
All deprotected compounds show normal UV-visible spectra
in DMSO as expected for typical solvated porphyrins (Support-
ing Information). In ethanol, the peaks are somewhat broadened
indicating some level of aggregation. In PBS buffer, (C1-Gal)4-
ZnPor (3), (C1-Gal)4-Por (4) and (C3-Gal)4-Por (7) are
broadened and have shoulders to the blue and red of the main
absorption in the UV-visible spectra indicating both J and H ag-
gregates. The exception is (C3-Gal)4-ZnPor (6), which the
electronic spectra indicates only J aggregates in PBS. To inves-
tigate the aggregation, the DLS studies were done in PBS buffer
and ethanol. All compounds in PBS buffer yield 16-30 nm di-
ameter aggregates. Larger 44-80 nm aggregates are observed in
ethanol (Supporting Information). Aggregates less than ca. 50
nm are known to be taken up by cancer cells.6
In vitro studies with monolayer and spheroids
Despite the fact that the uptake of carbon-3 conjugates is
greater relative to the carbon-1 conjugates in the four cell lines,
compounds 6 and 7 are not significantly better PDT agents in
both monolayer and spheroid cell cultures. Since the free base
(C1-Gal)4-Por and (C3-Gal)4-Por and the zinc complexes (C1-
Upon irradiation with 550 ± 10 nm light, DMF solutions of
1
galactose-conjugated porphyrins catalyze the formation of O2
1
as indicated by the O2 scavenger 1,3-diphenylisobenzofuran
(DPBF) to a similar extent as the reference tetraphenyl-porphy-
rin (TPP) (Supporting Information).34 Consistent with similar
porphyrinic compounds in this solvent the Zn(II) complexes
(C1-Gal)4-ZnPor and (C3-Gal)4ZnPor have about the same
1O2 yields as the free base compounds (C1-Gal)4-Por and (C3-
Gal)4-Por measured by this method even though the heavy
atom effect increases intersystem crossing to the triplet state.35
Importantly, there is no significant difference between the C1
and the C3 isomers. Though there are different values in the
literature, both TPP and ZnTPP have 1O2 quantum yield (ϕΔ) of
ca. 0.52 ± 0.12 in DMF.34-36 Thus, both the free base and the
Zn(II) C1- and C3 porphyrins have about the same ϕΔ as the
TPP reference in DMF.
1
Gal)4-ZnPor and (C3-Gal)4-ZnPor have similar O2 quantum
yields (vide supra),38, 39 these observations suggest that there are
important differences in uptake and intracellular transport be-
tween the carbon-1 and carbon-3 conjugates.
Uptake/binding
It is generally accepted that there is a correlation between the
expression level of receptors on the cell membrane and the up-
take and/or binding of drugs appended to the ligands for the re-
ceptor. However, nonspecific partition into the membrane is
also possible with hydrophobic or amphipathic molecules. Tar-
geted porphyrin conjugates increase the local concentration of
the dye around cancer cells, and when the binding constant is
not large, there is an equilibrium between the free and bound
conjugate. For dyes weakly bound to receptors, such as the
Figure 2. Intracellular uptake of galactose-porphyrins by mono-
layer cultures of HCT-116, MCF-7, UM-UC-3 or HeLa cancer
cells. The concentration of porphyrins was determined by fluo-
rescence spectroscopy after incubation of cancer cells with 9 μM
of galactose-porphyrins for 4 h and the results were normalized
to protein quantity. Data are the means ± S.D. of at least three
independent experiments performed in triplicate. **(p<0.001)
significantly different from the corresponding C3-Gal4-Por.
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Previous studies reported this lack of association between up-
take and phototoxicity of galactose-conjugated PS.26 Factors
such as different amounts of intracellular ROS and/or induction
of a protective cellular mechanism after PDT can explain the
reasons by which PSs with different uptake exhibit similar pho-
totoxicity after PDT.26 Additionally, differences in the intracel-
lular localization of photosensitizers galactose-conjugated sug-
ars can result in different cell death processes.40
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Carbon-1 and carbon-3 galactose-porphyrins accumu-
late in cancer cells growing as monolayer and spheroid
cultures and are non-toxic in the dark
Figure 3. Intracellular uptake of galactose-porphyrins by spheroid
cultures of HCT-116, MCF-7, UM-UC-3 or HeLa cancer cells.
The concentration of porphyrins was determined by fluorescence
spectroscopy after incubation of cancer cells with 9 μM of galac-
tose-porphyrins for 4 h and the results were normalized to protein
quantity. Data are the means ± S.D. of at least three independent
experiments performed in triplicate. *(p<0.05), **(p<0.001) sig-
nificantly different from the corresponding C3-Gal4-Por.
To evaluate the photodynamic potential of the newly synthe-
sized carbon-1 and carbon-3 galactose-porphyrins, we first
evaluated their accumulation in monolayer and spheroid cul-
tures by fluorescence spectroscopy and microscopy (Figures
S32-S36) and dark cytotoxicity (Figures S37-S44). Monolayer
cultures of cancer cells were incubated with increasing concen-
trations (2.25, 4.5 and 9 µM) of (C1-Gal)4-ZnPor, (C1-Gal)4-
Por, (C3-Gal)4-ZnPor and (C3-Gal)4-Por in PBS containing a
maximum of 0.5% v/v dimethyl sulfoxide, DMSO, for up to 4
h. Cancer cell-derived spheroids were incubated with the high-
est concentration (9 μM) of each photosensitizer.
No toxicity was observed in untreated cells (up to 4 h) in the
presence of 0.5% (v/v) DMSO in the incubation medium.28
Moreover, the galactose-porphyrins showed no significant dark
cytotoxicity in monolayer and spheroid cultures at the 9 µM
maximum concentration tested (Figures S37-S44).
The uptake by HCT-116, UM-UC-3 and HeLa cancer cells
growing as monolayers or spheroids was greater for carbon-3
porphyrins than for carbon-1porphyrins (Figures 2,3). The up-
take is proportional to the expression of the receptors, and com-
petition experiments demonstrated that a pre-incubation with
thiogalactose decreased the uptake of both the carbon-1 and car-
bon-3 galactose-porphyrins in UM-UC-3 cell cultures (Support-
ing Information).41 Data showed that the reduction in uptake
was higher for carbon-3 when compared with carbon-1 galac-
tose-porphyrins. The uptake of galactose-porphyrins was
greater in HCT-116, HeLa or UM-UC-3 cancer cells growing
as monolayers than with monolayers of MCF-7 breast cancer
cells. MCF-7 breast cancer cells have lower levels of galectin-
1 protein compared to HCT-116, UMUC-3 and HeLa cancer
cells.28 When spheroids were used as cell culture model, a de-
crease in the uptake of galactose-porphyrins was observed in
HeLa cancer cells.
Figure 4. Cytotoxicity at 24 h after PDT with galactose-porphy-
rins in cancer cells growing in monolayers, determined using the
MTT assay. Cells were incubated with 9 µM PS for 4 h and PDT
was performed during 30 min at 0.44 mW/cm2. Data are means ±
S.D. of at least three independent experiments performed in trip-
licate. ***(p<0.0001) significantly different from control cells.
Galactose-porphyrins induce cytotoxicity in monolayer
and spheroid cultures after photodynamic activation
After performing uptake assays, we investigated the effect of
galactose-porphyrins mediated PDT efficacy in monolayer and
spheroid cultures. The cells were treated 48 h after plating, with
9 µM of galactosylated porphyrins for 4 h, and then exposed to
light (420-700 nm) delivered at 0.44 mW/cm2 during 30 min
(0.792 J/cm2). The 48 h time point following cell seeding was
chosen based on the time needed for spheroids of the different
cell lines to form and achieve reproducible and similar com-
pactness (based on spheroid volume and shape) in each well.28
In all monolayer cultures, the light activation of galactose-por-
phyrins induced reduction of cell viability.
Figure 5. Cytotoxicity at 24 h after PDT with galactose-porphy-
rins in cancer cells growing in spheroids, determined using the
LDH assay. Spheroids were incubated with 9 µM PS for 4 h and
PDT was performed during 30 min at 0.44 mW/cm2. Data are
means ± S.D. of at least three independent experiments performed
in triplicate. *(p<0.05), ***(p<0.0001) significantly different from
control cells.
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Bioconjugate Chemistry
As shown in Figure 4, the phototoxicity of carbon-3 porphy-
PerkinElmer UV/Vis spectrophotometer. Steady-state fluores-
cence (emission) spectra were measured with a Fluorolog τ-3,
Jobin-SPEX Instruments S.A., Inc.
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2
3
4
5
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7
8
rins in cancer cells growing as monolayers was similar to car-
bon-1 porphyrins. As expected, the phototoxicity was greater in
monolayer cultures of HCT-116, UM-UC-3 and HELA com-
pared to MCF-7 cells because of the reduced galectin-1 expres-
sion level in the latter.28 When PDT assays used spheroid cul-
tures, the release of LDH into the culture medium was increased
compared with control cells (Figure 5). After PDT, LDH re-
leased by HCT-166, UM-UC-3 and HeLa spheroids was greater
than that by MCF-7 spheroid cultures. However, the amount of
LDH leakage into the extracellular medium was lower for all
cancer spheroid cultures when compared to monolayer counter-
cultures. In spheroid cultures of HCT-166, UM-UC-3 and
HELA the PDT efficacy is slightly greater for the carbon-3 de-
rivative than for the carbon-1 derivatives, and again there is lit-
tle effect on spheroids of MCF-7.
5,10,15,20-tetrakis(4-propargyloxyphenyl)porphyrin
was
synthesized from commercially available 4-propargyloxy ben-
zaldehyde and pyrrole.43 Zn complexation was carried out using
Zn(OAc)2 in CH2Cl2 at room temperature overnight resulting in
98% overall yield of the metalloporphyrin 1. 1,2,4,6-tetra-O-
acetyl-3-azido-3-deoxy-D-galactopyranose was prepared using
the published procedure,42 and characterization was in agree-
ment with the published spectra. The 1-azido-1-deoxy-β-D-ga-
lactopyranoside tetraacetate is commercially available. Either
1,2,4,6-tetra-O-acetyl-3-azido-3-deoxy-β-D-galactopyranose
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or
2,3,4,6-tetra-O-acetyl-1-azido-1-deoxy-β-D-galactopyra-
nose (2.6 equivalents) were then refluxed overnight with one
equivalent of porphyrin 1 using one equivalent Cu(0), 0.2
equivalents of CuSO4.5H2O, and 0.5 equivalents of Na-ascor-
bate dissolved in a mixture of THF/water resulting in porphy-
rins 2 and 5 in good yields after silica column purification using
CH2Cl2/EtOAc (80:20) as eluent.
CONCLUSION
Carbon-1 and carbon-3 galactose-porphyrins demonstrate
high accumulation and phototoxicity in cell lines (monolayers
or spheroids) containing high expression of galectin-1 protein.
The octanol/PBS partition coefficients indicate that the carbon-
1 compounds are somewhat more lipophilic than the carbon-3
derivatives and indicates facilitated diffusion as a mode of up-
take. Carbon-1 and carbon-3 galactose-porphyrins demon-
strated low uptake in MCF-7 cancer cells, which contain low
levels of galectin-1 protein. In cell lines containing high levels
of galectin-1 (HCT-116, HeLa and UM-UC-3), carbon-3 com-
pounds demonstrated greater uptake compared with carbon-1
galactose-porphyrins. The greater uptake of carbon-3 compared
with carbon-1 galactose-porphyrin can be explained by its
higher water solubility, as well as be the availability of the hy-
droxyl group at carbon-1 to bind the carbohydrate-recognition
domain of galectin-1 protein.24 The similar photodynamic effi-
ciency of carbon-1 and carbon-3 galactose-porphyrins in cell
lines containing different levels of galectin-1 protein can be a
result of differences in intracellular localization, as well as dif-
ferences in cellular oxygenation, cell death mechanism, and cel-
lular antioxidant responses upon light activation.
5,10,15,20-tetrakis(4-propargyloxyphenyl)porphyrinato
Zn(II), compound 1
To 100 mg (0.12 mmol) of 5,10,15,20-tetrakis(4-propar-
gyloxyphenyl)porphyrin in 20 mL CH2Cl2 was added 300 mg
Zn(OAc)2. The reaction mixture was stirred at room tempera-
ture overnight. The obtained mixture was evaporated using ro-
tary evaporator under reduced pressure followed by a silica
chromatography with CH2Cl2/EtOAc (95:5) as eluent resulting
in compound 1 in 98% yield (105 mg, 0.117 mmol).1H NMR
(500 MHz, CDCl3) δ 8.97 (s, 8H), 8.13 (d, J=8.35 Hz, 8H), 8.35
(d, J1=8.35 Hz, 8H), 4.98 (s, 8H), 2.69 (s, 4H),); 13C NMR (125
MHz, CDCl3) δ 156.4, 134.5, 134.4, 118.5, 112.0, 77.6, 74.8,
55.1.
5,10,15,20-tetrakis[N-(2',3',4',6'-O-acetyl-1'-β-D-galac-
topyranosyl)-(4''-methylenoxytriazole)-phenyl]porphy-
rinato Zn(II), compound 2
Compound 1 (52.5 mg, 0.058 mmol), 1-azido-1-deoxy-β-D-
galactopyranoside tetraacetate (56.24 mg, 0.15 mmol,) and (3.6
mg, 0.058 mmol) Cu(0) were dissolved in a mixture of
THF/water (6 mL, 3:1), under inert gas atmosphere. To the
above mixture, a solution of CuSO4.5H2O (2.9 mg, 0.0116
mmol) and Na-ascorbate (5.75 mg, 0.029 mmol) in THF/water
(6 mL, 3:1) was added and the reaction mixture was refluxed
overnight. Then the reaction mixture was cooled to room tem-
perature and extracted using ethyl acetate and brine. The or-
ganic layer was washed twice with brine and the combined or-
ganic layers were dried over Na2SO4 and the solvent was evap-
orated under reduced pressure. The crude product was then pu-
rified by silica chromatography. First using CH2Cl2 as eluent to
remove any unreacted starting materials then followed by 80:20
(CH2Cl2:EtOAc) to obtain the desired product in 90% yield
(125 mg, 0.052 mmol). 1H NMR (500 MHz, CDCl3) δ 8.95 (s,
8H), 8.13 (d, J=7.8 Hz, 8H), 7.77 (s, 4H), 7.24 (d, J= 7.8 Hz,
8H), 5.59 (t, J=9.2 Hz, 4H), 5.53 (t, J=2.9 Hz, 4H), 5.42-5.45
(m, 4H), 5.21 (dd, J1= 2.95, J2= 2.95 Hz, 4H), 4.67 (s, 8H), 4.16-
4.20 (m, 8H), 4.09-4.13 (m, 4H), 2.20 (s, 12H), 2.05 (s, 12H),
2.00 (s, 12H), 1.80 (s, 12H). 13C NMR (125 MHz, CDCl3)
δ170.3, 170.1, 169.9, 169.3, 157.7, 150.4, 144.5, 136.4, 135.7,
131.7, 120.9, 120.3, 112.9, 88.2, 86.1, 72.8, 70.7, 68.1, 66.9,
61.2, 61.2, 20.6, 20.6, 20.6, 20.9. HRMS (ESI) m/z calcd. for
C112H112N16O40Zn ([M+2H]2+), 1194.3352, found 1194.3325.
EXPERIMENTAL PROCEDURES
Commercially available reagents such as trifluoroaceticacid
(TFA), sodium methoxide, and solvents such as methanol,
DMSO, and CH2Cl2, were purchased from Sigma-Aldrich,
Fisher Scientific or Acros Organics and used without further
purification. 1,2,4,6-tetra-O-acetyl-3-azido-3-deoxy-D-galac-
topyranose was prepared using the published procedure.42
5,10,15,20-tetrakis(4-propargyloxyphenyl)porphyrin was syn-
thesized using a known procedure.43 Analytical thin-layer chro-
matography (TLC) was performed on polyester-backed TLC
plates 254 (precoated, 200 μm, Sorbent Technologies), and sil-
ica gel 60 (70−230 mesh, Merck) was used for column chroma-
1
tography. H and 13C NMR spectra were obtained using a
Brüker Avance III 400 MHz and Brüker Avance DRX
500MHz; chemical shifts are expressed in ppm relative to
CDCl3 (7.26 ppm, 1H; 77.0 ppm,13C), (CD3)2CO (2.05 ppm, 1H;
1
29.84 and 206.26 ppm, 13C), CD3OD (3.31 and 4.78 ppm, H;
49.2 ppm, 13C). Mass analyses were conducted at the CUNY
Mass Spectrometry Facility at Hunter College on an Agilent
iFunnel 6550 Q-ToF LC/MS System (for HRMS-ESI). The
electrospray ionization was run in methanol, with 0.1% (v/v)
formic acid. UV-visible spectra were recorded on a Lambda 35
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Bioconjugate Chemistry
Page 8 of 12
5,10,15,20-tetrakis[N-(1'-β-D-galactosyl)-(4''-meth-
ylenoxytriazole)-phenyl]porphyrinato Zn(II),
compound 3
5,10,15,20-tetrakis[N-(3'-β-D-galactosyl)-(4''-meth-
ylenoxytriazole)-phenyl]porphyrinato Zn(II), com-
pound 6
1
2
3
4
5
6
7
8
Compound 2 (62.5 mg, 0.026 mmol) was dissolved in meth-
anol/CH2Cl2 (3:1, 10 mL). To this mixture was added sodium
methoxide (0.5 M solution in methanol, 3 mL). The reaction
mixture was stirred at room temperature for 4 h and then the
solvent was evaporated using rotary evaporator. The resulting
crude product was passed through Sephadex LH-20 using
MeOH as eluent to yield (C1-Gal)4-ZnPor (3) in 84% yield
(37.39 mg, 0.021 mmol). 1H NMR (400 MHz, CD3OD) δ 8.85
(s, 8H), 8.54 (s, 4H), 8.13 (d, J= 7.92 Hz, 8H), 7.51 (d, J= 8Hz,
8H), 5.62 (d, J= 9.04 Hz, 4H), 5.51 (s, 8H), 4.20 (t, J= 9.12 Hz,
Compound 5 (62.5 mg, 0.026 mmol) was dissolved in meth-
anol/CH2Cl2 (3:1, 10 mL). To this mixture was added sodium
methoxide (0.5 M solution in methanol, 3 mL). The reaction
mixture was stirred at room temperature for 4 h and then the
solvent was evaporated using rotary evaporator. The resulting
crude product was passed through Sephadex LH-20 using
MeOH as eluant to give (C3-Gal)4-ZnPor (6)in 79% yield
(35.16 mg, 0.020 mmol). 1H NMR (500 MHz, CD3OD) δ 8.81
(bs, 8H), 8.02 (bs, 8H), 7.65 (bs, 4H), 7.28 (bs, 8H), 5.45-5.47
(m, 4H), 5.31-5.33 (m, 8H), 4.90-5.12 (m, 24H); 13C NMR (125
MHz, CD3OD) δ 159.8, 151.7, 141.6, 137.3, 136.6, 132.5,
131.3, 121.5, 113.8, 63.4. HRMS (ESI) m/z calcd. for
C80H80N16O24Zn ([M+H]+), 1715.4905, found 1715.4864;
calcd. for C80H80N16O24Zn ([M+Na]+), 1736.4745, found
1736.4662.
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4H), 3.81 (bs, 4H), 3.81-3.82 (m, 4H), 3.63-3.68 (m, 12H); 13
C
NMR (100 MHz, CD3OD) δ 158.3, 150.11, 143.5, 136.0, 135.7,
131.9, 124.1, 120.3, 113.4, 88.8, 79.0, 74.4, 70.0, 69.1, 62.1,
61.1. HRMS (ESI) m/z calcd. for C80H80N16O24Zn ([M+H]+),
1715.4905, found 1715.4893.
5,10,15,20-tetrakis[N-(1'-β-D-galactosyl)-(4''-meth-
ylenoxytriazole)-phenyl]porphyrin, compound 4
5,10,15,20-tetrakis[N-(3'-β-D-galactosyl)-(4''-meth-
ylenoxytriazole)-phenyl]porphyrin, compound 7
(C1-Gal)4-ZnPor (3) (37.39 mg, 0.021 mmol) was dissolved
in methanol/TFA (1:1, 10 mL). The reaction mixture was stirred
at room temperature overnight and then the solvent was evapo-
rated using rotary evaporator. The resulting crude product was
washed with CH2Cl2 and acetone several times to yield (C1-
Gal)4-Por (4) in 90% yield (31.18 mg, 0.018 mmol) as green
(C3-Gal)4-ZnPor (6) (35.16 mg, 0.020 mmol) was dissolved
in methanol/TFA (1:1, 10 mL). The reaction mixture was stirred
at room temperature overnight and then the solvent was evapo-
rated using rotary evaporator. The resulting crude product was
washed with CH2Cl2 and acetone several times resulting in (C3-
Gal)4-Por (7) in 87% yield (28.71 mg, 0.017 mmol) as a green
solid. 1H NMR (500 MHz, DMSO-d6) δ 8.86 (s, 8H), 8.14 (s,
12H), 7.47 (bs, 8H), 5.54 (s, 8H), 3.76-4.79 (m, 28H), -2.82 (bs,
2H); 13C NMR (500 MHz, DMSO-d6) δ 158.5, 158.2, 158.1,
158.0, 157.7, 135.4, 133.8, 120.3, 119.6, 117.9, 115.6, 113.2,
76.5, 72.9; HRMS (ESI) m/z calcd. for C80H82N16O24 ([M+H]+),
1652.5791, found 1652.5735; calcd. for C80H82N16O24
([M+Na]+), 1674.5610, found 1674.5545.
1
solid. H NMR (500 MHz, CDCl3) δ 8.87 (bs, 8H), 8.60 (s, 4H),
8.18 (bs, 8H), 7.53 (bs. 8H), 5.72-5.76 (m, 8H), 5.49-5.62 (m,
8H), 4.47-4.59 (m, 4 H), 4.13-4.28 (m, 8H), 3.80-3.87 (m, 8H);
13C NMR (125 MHz, CDCl3) δ 158.1, 156.5, 156.2, 142.7,
124.0, 123.9, 113.1, 88.2, 87.6, 78.5, 74.4, 73.7, 73.0, 69.3,
68.9, 68.6, 68.5, 68.0, 61.3, 60.5, 54.9. HRMS (ESI) m/z calcd.
for C80H82N16O24 ([M+H]+), 1652.5791, found 1652.5771.
5,10,15,20-tetrakis[N-(1',2',4',6'-O-acetyl-3'-β-D-galac-
topyranosyl)-(4''-methylenoxytriazole)-phenyl]porphy-
rinato Zn(II), compound 5
Photophysics
UV-visible and fluorescence measurements were performed
on diluted solutions, typically about 0.1 μM, of compounds in
ethanol, dimethylsulfoxide (DMSO) and Dulbecco's phosphate
buffered saline (DPBS without CaCl2 and MgCl2 pH = 7.4 with
2% v/v DMSO). The UV-visible spectra were obtained from
350 nm to 750 nm using 1 cm quartz cuvettes. For steady-state
fluorescence spectroscopy, samples were excited at 425 nm for
all solvents at concentrations where absorbancies are less than
0.1. For emission spectra, both the excitation and detection
monochromators had a band-pass of 1 nm. The corrected emis-
sion (for instrument response) and absorption spectra were used
to calculate the quantum yield. Fluorescence quantum yields
were determined for (C3-Gal)4-Por (7), (C3-Gal)4-ZnPor (6),
(C1-Gal)4-Por (4) and (C1-Gal)4-ZnPor (3), solutions relative to
a tetraphenylporphyrin standard in toluene, which has a fluores-
cence quantum yield of 0.11 in this and the other solvents.5, 44
All experiments were carried out on the same day, using identi-
cal concentrations to minimize any experimental errors. (Sup-
porting Information).
Compound 1 (52.5 mg, 0.058 mmol), 1,2,4,6-tetra-O-acetyl-
3-azido-3-deoxy-D-galactopyranose (56.24 mg, 0.15 mmol)
and Cu(0) (3.6 mg, 0.058 mmol) were dissolved in a mixture of
THF/water (6 mL, 3:1), under inert gas atmosphere. To the
above mixture, a solution of CuSO4.5H2O (2.9 mg, 0.0116
mmol) and Na-ascorbate (5.75 mg, 0.029 mmol) in THF/water
(6 mL, 3:1) was added and the reaction mixture was refluxed
overnight. Then the reaction mixture was cooled to room tem-
perature and extracted using ethyl acetate and brine. The or-
ganic layer was washed twice with brine and the combined or-
ganic layers were dried over anhydrous Na2SO4 and the solvent
was evaporated under reduced pressure. The crude product was
then purified by silica chromatography. First using CH2Cl2 as
eluent to remove any unreacted starting materials then followed
by CH2Cl2/EtOAc (80:20) to obtain the desired product in 84%
yield (116 mg, 0.048 mmol).
1H NMR (500 MHz, CDCl3) δ 8.93 (bs, 8H), 8.11 (bs, 8H),
Dynamic light scattering (DLS) was used to determine aggre-
gate size in the different solvents using a Malvern nano
Zetasizer, modeled as a sphere and reported as the diameter.
Samples were sonicated for about a minute and filtered with hy-
drophilic 200nm filters before measurements.
7.42-7.49 (m, 4H), 7.12-7.27 (m, 8H), 5.09-5.53 (m, 12H),
4.65-4.72 (m, 8H), 3.86-4.34 (m, 12H), 1.67-2.14 (m, 48H); 13
C
NMR (100 MHz, CDCl3) δ 170.6, 169.5, 169.4, 168.6, 162.8,
143.7, 142.8,132.0, 132.0, 130.5, 126.0, 123.3, 115.1, 115.0,
90.1, 72.0, 71.5, 68.6, 66.0, 63.0, 62.0, 61.9, 60.4, 57.9, 20.98,
20.8, 20.4, 20.2, 20.2. HRMS (ESI) m/z calcd. for
C112H112N16O40Zn ([M+2H]+2), 1216.3161, found 1216.4143.
The partition coefficient (P) values were measured in an n-
octanol/PBS (1:1, v/v) mixture at a concentration of 10 μM. Af-
ter shaking the solution at room temperature, the two different
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Bioconjugate Chemistry
Speed Wavelength Source, Molecular Devices) equipped with
phases were obtained by centrifugation and the concentration of
1
2
3
4
5
6
7
8
the PS into each phase was determined by UV-Vis using the
molar absorptivity values.
Andor iXon EMCCD camera. For DAPI detection, the speci-
men was excited at 405 nm and light emitted was collected with
a 460/50 nm band pass filter. Porphyrins fluorescence was ob-
tained using 640 nm excitation and a 685/40 nm emission band
pass filter.
For the singlet oxygen experiments, DMF solutions contain-
ing 50 μM of DPBF and 5 μM of porphyrins were irradiated at
room temperature and under gentle magnetic stirring. DPBF de-
pletion was monitored by measuring the decrease in absorbance
(415 nm) at 2, 4, 6, 8 and 10 min after light irradiation. Irradia-
tion with 0.49 mW/cm2 light (measured at the front of the cu-
vette) from a xenon arc lamp in Horiba SPEX fluorometer with
the monochonometer set at 550 nm and the band pass set at 10
nm to minimize absorption by DPBF.
PDT assays
After monolayers or spheroid cultures were incubated with 9
μM of porphyrins for 4 h, the cultures were gently rinsed with
PBS, and then PDT assays were performed. The cells were ex-
posed to light (420-700 nm) emitted by OLED Lumiblade Brite
FL300-wm Level 4 (OLED Works, Rochester, NY, USA) de-
livered at 0.44 mW/cm2 during 30 min (0.792 J/cm2). The con-
trols were performed by keeping monolayers or spheroids in the
dark for the same durations and under the same conditions as
the irradiated cells. Triplicate wells were established under each
experimental condition and each experiment was repeated at
least three times. Cytotoxicity was determined in monolayer or
spheroid cultures 24 h after treatment using the 3-[4,5-dime-
thylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT,
Sigma) and lactate dehydrogenase (LDH) assays, respec-
tively.28
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Cell lines and culture
UM-UC-3 bladder cancer cell line, HeLa cervical cancer cell
line, HCT-116 colon cancer cell line and MCF-7 breast cancer
cell line were obtained from the American Type Culture Col-
lection (ATCC®, Manassas, VA, USA).All batches of culture
media were supplemented with 10% (v/v) of fetal bovine serum
(Life Technologies, Carlsbad, CA, USA), 100 U/mL penicillin,
100 µg/mL streptomycin and 0.25 µg/mL amphotericin B
(Sigma). HCT-116 colon cancer cells, MCF-7 and MDA-MB-
231 breast cancer cells were cultured in Dulbecco´s Modified
Eagle´s Medium (DMEM, Sigma). UM-UC-3 bladder cancer
cells were cultured in Eagle´s Minimum Essential Medium
(EMEM; Corning, NY, USA) with 1.5 g/L sodium bicarbonate,
non-essential amino acids, L-glutamine and sodium pyruvate.
HeLa cervical cancer cells were cultured in DMEM (Corning)
with 4.5 g/L glucose, and L-glutamine without sodium py-
ruvate. All cells were maintained at 37 ºC in a 5% CO2 humid-
ified atmosphere. Agarose-coated 96-well plates were used for
spheroid cultures as previously reported in theliterature.28
Briefly, 5000 HCT-116 cells per well, 15000 cells MCF-7 per
well, 20000 UM-UC-3 cells per well and 20000 HeLa cells per
well were plated on agarose-coated microplates and left for 48
h undisturbed to obtain viable spheroids with volume ≈ 0.05
mm.3
MTT and lactate dehydrogenase assays
The MTT assay was used to determine metabolic activity of
cells growing in monolayers by measuring their ability to re-
duce the yellow-colored MTT to a colored formazan using a
microplate reader (PowerWave HT Microplate Spectrophotom-
eter). The data were expressed in percentage of control (i.e. op-
tical density of formazan from control cells). The CytoTox 96®
Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI,
USA) was used to determine cytotoxicity in spheroid cultures.
The LDH activity was normalized to protein concentration and
the data were normalized to the maximal LDH release.
Statistical analysis
Statistical analysis was performed using GraphPad Prism.
Student’s t-test was used to compare the treatment effects with
that of control. P-value was considered at the 5% level of sig-
nificance of the data. All graphs and statistics were prepared
using the GraphPad Prism 5.0 software.
Cellular uptake
After incubation of monolayers or spheroids of cancer cells
with the photosensitizer in the dark, the cells were washed with
PBS buffer (pH 7.60, 10 mM NaH2PO4, 70 mM Na2HPO4 and
145 mM NaCl) and scrapped in 1% (m/v) sodium dodecyl sul-
fate (SDS; Sigma) in PBS buffer at pH 7.0. The concentration
of the porphyrins was determined by spectrofluorimetry using
a microplate reader (Gemini EM Microplate Spectrofluorome-
ter) with the excitation and emission filters set at 410 nm and
702 nm, respectively. The results were normalized for protein
concentration using the bicinchoninic acid reagent (Pierce,
Rockford, IL, USA). For the competition experiments, mono-
layers of UM-UC-3 cells were pre-incubated with the thiogalac-
tose sugar (100 μM or 1.00 mM) for 1h. Next, those cells were
incubated for 3h with Pors at a concentration of 9 μM.
ASSOCIATED CONTENT
Supporting Information
NMR spectra, UV-visible absorbance spectra, fluorescence emis-
sion spectra, mass spectra, DLS, uptake, dark toxicity.
AUTHOR INFORMATION
Corresponding Author
cdrain@hunter.cuny.edu
Present Addresses
For evaluation of cellular uptake by fluorescence micros-
copy, cells were grown on coated glass coverslips with poly-L-
lysine (Sigma). The cells were incubated with 9 μM porphy-
rins for 4 h, at 37°C. After incubation, cells were washed twice
with PBS and fixed with 4% paraformaldehyde (PFA; Merck,
Darmstadt, Germany) for 10 min at room temperature. The sam-
ples were then rinsed in PBS, and mounted in Vecta SHIELD
mounting medium containing 4´,6-diamidino-2-phenylindole
(DAPI; Vector Laboratories, CA, Burlingame) for visualization
under a confocal microscope (Nikon Eclipse Ti With Ultra High
†Patricia M.R. Pereira present address: Department of Radiology,
Memorial Sloan Kettering, New York, NY 10065, USA.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manu-
script. #These authors contributed equally.
Funding Sources
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Bioconjugate Chemistry
Page 10 of 12
A. (2007) In vitro phototoxicity of glycoconjugated porphyrins and
This work was supported by the National Science Foundation
CHE-1610755, Hunter College science infrastructure is supported
by the NSF, the National Institute on Minority Health and Health
Disparities 8G12 MD007599, and the City University of New
York. The authors also acknowledge Faculdade de Ciências e
Tecnologia, Universidade Nova de Lisboa for the doctoral research
SFRH/BD/85941/2012 (to PMRP). Thanks are due to FCT/MEC
for the financial support to QOPNA (FCT UID/QUI/00062/2013),
chlorins in colorectal adenocarcinoma (HT29) and retinoblastoma
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IBILI
(FCT
UID/NEU/04539/2013) and
CQE
(FCT
UID/QUI/0100/2013) research units, through national funds and
where applicable co-financed by the FEDER, within the PT2020
Partnership Agreement.
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ACKNOWLEDGMENT
“We thank Professor Eileen Jaffer, Department of Molecular Ther-
apeutics at Fox Chase Cancer Center of Temple University Health
Systems, for reading the manuscript and insightful comments.”
ABBREVIATIONS
GLUT1, glucose transporter 1; PDT, Photodynamic Therapy; PS,
Photosensitizer; ROS, Reactive Oxygen Species, DMSO
dimtheylsulfoxide.
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