Photochemical internalization of tamoxifens transported by a "trojan-horse" nanoconjugate into breast-cancer cell lines

Article abstract of DOI:10.1002/anie.201500183

Photochemical internalization (PCI) has shown great promise as a therapeutic alternative for targeted drug delivery by light-harnessed activation. However, it has only been applicable to therapeutic macromolecules or medium-sized molecules. Herein we desc

Full text of DOI:10.1002/anie.201500183

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Chemie
DOI: 10.1002/anie.201500183
Targeted Drug Delivery
Photochemical Internalization of Tamoxifens Transported by
a “Trojan-Horse” Nanoconjugate into Breast-Cancer Cell Lines**
Theodossis A. Theodossiou,* A. Ricardo GonÅalves, Konstantina Yannakopoulou,
Ellen Skarpen, and Kristian Berg
Abstract: Photochemical internalization (PCI) has shown
great promise as a therapeutic alternative for targeted drug
delivery by light-harnessed activation. However, it has only
been applicable to therapeutic macromolecules or medium-
sized molecules. Herein we describe the use of an amphiphilic,
water-soluble porphyrin–b-cyclodextrin conjugate (mTHPP-
bCD) as a “Trojan horse” to facilitate the endocytosis of CD-
guest tamoxifens into breast-cancer cells. Upon irradiation, the
porphyrin core of mTHPP-bCD expedited endosomal mem-
brane rupture and tamoxifen release into the cytosol, as
documented by confocal microscopy. The sustained complex-
ation of mTHPP-bCD with tamoxifen was corroborated by 2D
NMR spectroscopy and FRET studies. Following the applica-
tion of PCI protocols with 4-hydroxytamoxifen (4-OHT),
estrogen-receptor b-positive (Erb+, but not ERbÀ) cell groups
exhibited extensive cytotoxicity and/or growth suspension even
at 72 h after irradiation.
activation enables spatiotemporal specificity and control over
the intracellular drug release. The principle of PCI lies in drug
endocytosis. Cells are treated with an amphiphilic PS with
high affinity for the plasma membrane (e.g. Amphinex), so
that the PS subsequently remains in the membranes of the
endosomal vesicles following endocytosis. The cells are
simultaneously treated with drugs or molecules that cannot
passively enter cells owing to their unfavorable properties
(e.g. hydrophilicity, large size, or negative charge). The drugs
are endocytosed and thus confined in endosomes and
lysosomes with photosensitizers anchored on their mem-
branes. Upon selective irradiation with light of the appro-
1
priate wavelength, the formed ROS, and especially O2, may
cause lipid peroxidation[4] to the endosomal membranes. This
transformation potentiates membrane rupture with concom-
itant release of the endosomal load, thus leading to the
cytoplasmic release of a drug at high concentrations specif-
ically in irradiated tissues.
P
hotodynamic therapy of cancer (PDT)[1] is a treatment
Until now, PCI has been developed and documented for
the delivery of therapeutic biomacromolecules and medium-
sized molecules, which are unable to enter cells in any other
manner than endocytosis. However, the majority of the most
efficient, widely used, and approved cancer therapeutics are
small molecules, which can freely enter both target and
nontarget cells by passive diffusion. The use of PCI technol-
ogy for the specific delivery of such molecules to cancer
lesions in doses much higher than those used in chemotherapy
would minimize side effects to normal tissues. There are
numerous other techniques based on small-molecule caging
with photocleavable cages or containers;[5] however, PCI is
unique in its use of the endocytic vesicles as photolabile
confinement compartments for the drugs to be delivered.
The synthesis of nanosized molecular carriers that are
capable of transporting and releasing chemotoxic agents into
a biological target, preferably in a controlled manner, is an
important objective of contemporary drug-delivery research.
The ideal carrier would combine different functionalities,
sufficient water solubility, and the capacity to be internalized
by cells in vitro and in vivo. Cyclodextrins (CDs), water-
soluble macrocyclic oligosaccharides and approved drug
excipients, are commonly used to facilitate drug dissolution
in aqueous solutions though inclusion-complex formation as
the major mechanism. The covalent attachment of a CD
moiety to a PS has been envisaged as a strategy to combine
the drug-encapsulation and solubilization capacity of the CD
moiety with the photosensitizing/fluorescence-imaging prop-
erties of the PS moiety, especially in the case of nonionic
porphyrins, which are notorious for their lack of aqueous
solubility. 5,10,15,20-Tetrakis(m-hydroxyphenyl)-21,23H-por-
modality which utilizes a photosensitive drug (photosensi-
tizer, PS), light of the appropriate wavelength, and molecular
oxygen as the terminal acceptor of energy or electrons to form
singlet oxygen or other deleterious reactive oxygen species
(ROS). These species cause local irreversible photodamage to
biological substrates within the region of irradiation. A
promising evolution of PDT came through the concept of
photochemical internalization (PCI).[2] PCI is actually a light-
controlled drug- and gene-delivery modality[3] in which light
[*] Dr. T. A. Theodossiou, Dr. E. Skarpen, Prof. K. Berg
Department of Radiation Biology (T.A.T., K.B.) and Department of
Biochemistry (E.S.), Institute for Cancer Research
The Norwegian Radium Hospital, Oslo University Hospital
Montebello, 0379 Oslo (Norway)
E-mail: Theodossis.Theodossiou@rr-research.no
A. R. GonÅalves, Dr. K. Yannakopoulou
Institute of Nanoscience and Nanotechnology
NCSR “Demokritos”, Patriarchou Gregoriou and Neapoleos
Ag. Paraskevi, Attiki, 15310 (Greece)
[**] K.B. and T.A.T. gratefully acknowledge the European Community for
financially supporting this research through the Marie Curie Intra-
European Fellowship HYPERTAM (327075). K.Y. and A.R.G. thank
the Marie Curie Program No. 237962 CYCLON (FP7-PEOPLE-ITN-
2008) for financial support to MC fellow A.R.G. and funding of the
research. Moreover, A.R.G. is indebted to Assoc. Prof. Helena
Cabral-Marques, Faculty of Pharmacy, University of Lisbon, for
helpful discussions and support. Finally, we thank Seahorse
Biosciences for the kind loan of a Seahorse XFe96 Analyzer, which
made the metabolic studies possible.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201500183.
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phyrin (mTHPP, Scheme 1a) is a nonionic, water-insoluble
PS, the porphyrin analogue of the clinically used chlorin
temoporfin (marketed as Foscan). We recently reported[6] the
efficient synthesis and molecular characterization of
solubilizer. pMbCD has been shown to form very strong
inclusion complexes with porphyrins in aqueous environ-
ments.[12] In good agreement with the previously reported
studies, strong diastereoisomeric 1:1 inclusion complexes
between mTHPP-bCD and pMbCD were formed with K11
=
3.8(Æ 1.6) ꢀ 106 mÀ1 (R2 = 0.9954), thus resulting in a solubility
of approximately 2 mm. NMR spectra unequivocally showed
that the bCD cavity of mTHPP-bCD remained available for
further complex formation, as the pMbCD unit was found to
be stationed exclusively on the mTHPP moiety (Figure 1; see
Figure 1. Solution structure of mTHPP-bCD/pMbCD/NDMTAM as
a 1:1 mixture of diastereoisomeric complexes, as derived from NMR
spectroscopic data. bCD: light gray; pMbCD: dark gray.
Scheme 1. Chemical structures of a) mTHPP and b) mTHPP-bCD.
also the Supporting Information, including Figures S1–S5).
Moreover, the mTHPP-bCD/pMbCD nanosystem in 4%
dimethyl sulfoxide (DMSO) either in phosphate-buffered
saline solution (PBS) or in D2O was shown to encapsulate
NDMTAM via either one of its unsubstituted phenyl groups,
and the drug solubility increased by a factor of four, thus
demonstrating the capability of the conjugate, even in
complex with pMbCD, to solubilize the drug by inclusion
(see Figure S6).
Fçrster resonance energy transfer (FRET) studies were
performed between mTHPP-bCD and NDMTAM covalently
attached to FITC (NDMTAM-FITC). The FRET wavelength
was set to 488 nm, which is optimal for FITC but not for the
porphyrin moiety (minimal absorbance). The strong FRET
observed from NDMTAM-FITC to mTHPP-bCD (Figure 2)
indicated that the NDMTAM-FITC moiety remained encap-
sulated, in good agreement with 2D NOESY spectra (see
Figure S6), and further showed that the complexation is not
abolished in serum-containing media.
Representative confocal images of MCF7 estrogen-recep-
tor b-positive (Erb+) and MDA-MB-231 (ErbÀ) breast
human carcinoma cells loaded with mTHPP-bCD/
NDMTAM-FITC, pre- and postirradiation, are shown in
Figure 3. The quenching of FITC fluorescence before irradi-
ation can be attributed either to FRET from the FITC to the
mTHPP moiety owing to their close proximity (as in solution,
Figure 2) or to p–p interactions and the formation of
aggregates that favor energy dissipation through nonirradia-
tive processes. FITC fluorescence is also known to be
attenuated at the pH value of lysosomes. The red mTHPP-
bCD fluorescence is punctate (Figure 3, middle column), in
a mTHPP-bCD conjugate (Scheme 1b), which displayed
improved emission properties in aqueous media as compared
to those of mTHPP alone and aqueous solubility in the
biologically relevant micromolar range. Moreover, mTHPP-
bCD proved to be a photoresponsive multifunctional nano-
carrier with the combined capacity to release ROS and
transport a nitric oxide releasing guest.[6]
In the current study, we investigated the PCI potential of
the mTHPP-bCD conjugate with tamoxifens as guest mole-
cules. Tamoxifen (TAM) is a nonsteroidal selective estrogen-
receptor modulator (SERM) applicable to the treatment of
estrogen-responsive cancers, such as breast cancer.[7]
N-Desmethyltamoxifen (NDMTAM) and trans-4-hydroxy-
tamoxifen (4-OHT), the major metabolites of tamoxifen,
exhibit estrogen-receptor affinities comparable to that of 17b-
estradiol.[8] TAM binds to cell estrogen receptors (ERs)
stimulating cytostasis[7b] or cell-apoptosis propagation.[9]
TAM-induced cell death has, however, been observed both
in estrogen-receptor-positive and estrogen-receptor-negative
cell lines,[10] thus suggesting a nongenomic component of
TAM action. In a previous study,[11] we demonstrated that
fluorescein isothiocyanate (FITC)-conjugated NDMTAM
localized in cell mitochondria and the endoplasmic reticulum.
To document the formation of inclusion complexes
between mTHPP-bCD and NDMTAM by NMR spectrosco-
py, a gigantic leap in the aqueous solubility of mTHPP-bCD
was required, from micromolar to millimolar concentrations.
This solubility improvement was accomplished by the use of
heptakis(2,3,6-O-methyl)-b-cyclodextrin (pMbCD, in which
the “p” stands for “per”) as an effective yet noninterfering
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are liberated into the cell cytosol. In both cell lines, it was
NDMTAM-FITC fluorescence that increased after irradia-
tion (Figure 3). This result is attributed to dislocation of the
NDMTAM-FITC molecules from the mTHPP-bCD cavities.
The dislodged NDMTAM-FITC molecules are redistributed
perinuclearly, most likely to mitochondria and/or the endo-
plasmic reticulum, which have been previously shown to be
the subcellular accumulation loci for NDMTAM-FITC.[11] On
the other hand, mTHPP-bCD fluorescence did not exhibit
any radical relocalization following irradiation. The PCI-
induced translocation of drugs and the photosensitizer itself
into the cytosol has been found to take some time and may
take as long as 1 h.[13] The singlet oxygen formed has a very
short lifetime (submicrosecond level) but induces lipid-
peroxidation chain reactions that may last for approximately
20–40 min.[14] Thus, the present results are consistent with the
photochemically induced release of tamoxifen from mTHPP-
bCD within the endocytic vesicles.
Unbound tamoxifen can freely pass cellular membranes,
and this process is rapid (Figure 3). Initially, the photo-
cytotoxicity light-dose response curves (PDT effect) were
determined for cells treated with mTHPP-bCD (10 mm)
overnight (see Figure S7). From these curves, the LD50
values were extracted for both cell lines (4 JcmÀ2 for MCF7
cells, 2.7 JcmÀ2 for MDA-MB-231 cells). The PCI effect of the
complex mTHPP-bCD(10 mm)–4-OHT(7.5 mm) was subse-
quently assessed in the two cell lines. We used four treatment
groups: incubation with the vehicle (medium controls), 4-
OHT (7.5 mm), mTHPP-bCD (10 mm; PDT group), and
mTHPP-bCD(10 mm)–4-OHT(7.5 mm) (PCI group) for 16 h.
All cell groups (except dark controls) were irradiated at their
LD50 light-dose values. We subjected the cells to standard
MTT assays at 24, 48, and 72 h postirradiation to determine
their viability. The toxicity data for the control experiments in
the dark for all the above groups show (see Figure S8) that all
compounds have limited chemical toxicity: In MCF7 cells the
maximal toxicity in the dark was seen for incubation with the
complex mTHPP-bCD(10 mm)–4-OHT(7.5 mm) (ꢀ 40%),
whereas for MDA-MB-231 the equivalent value was about
20%. Accordingly, the corresponding chemical toxicities for
incubation with mTHPP-bCD were found to be about 20 and
10%, respectively.
Figure 2. FRET spectra between mTHPP-bCD and NDMTAM-FITC in
RPMI 1640 medium containing 10% fetal calf serum. NDMTAM-FITC
(7.5 mm) fluorescence occurs at lex =488 nm (black dashed line).
mTHPP-bCD fluorescence is shown as a gray line with a dash–dot
pattern (registered following excitation at 410 nm of a 10 mm mTHPP-
bCD solution). The same solution upon excitation at 488 nm yielded
the spectrum depicted with a solid gray line. Fluorescence of the
mTHPP-bCD (10 mm)/NDMTAM-FITC (7.5 mm) complex is shown as
a solid black trace (lex =488 nm, FRET). Inset: Fluorescence of 1 mm
NDMTAM-FITC (lex =488 nm).
The phototoxicities for the four treatment groups are
shown in Figure 4. The two cell lines behaved quite differ-
ently. In the MCF7 PDT cell group, profound phototoxicity
was observed at 24 h postirradiation (ca. 70%). This photo-
toxicity, however, was gradually abrogated at 48 and 72 h,
presumably owing to the proliferation of surviving cells. In the
PCI group, cell death was much more profound (ca. 80%) and
remained unchanged at both the 48 and 72 h assay time
points. This result implies a profound PCI effect in Erb+ cells,
which are genomically affected by the SERM 4-OHT. In this
sense, PCI presumably suppressed the abrogation of the PDT
effect by the release in the cell cytosol of substantial amounts
of 4-OHT, which engaged the available subcellular SERM
targets. The fact that 4-OHT alone conferred no substantial
toxicity to MCF7 cells in the absence or presence of light
shows that the regime of administration (7.5 mm, 16 h) was
sublethal to the cells and the sustained toxicity conferred to
Figure 3. Confocal microscopy images of MCF7 and MDA-MB-231
cells treated with mTHPP-bCD/NDMTAM-FITC overnight (16 h). MCF7
cells were irradiated with a 2 JcmÀ2 dose and MDA-MB-231 cells with
a 1.4 JcmÀ2 dose of red light, and images were acquired approximately
4 min later (see Figure S9). The micrographs of cells prior to and after
light exposure are not from the same area of the dish.
agreement with subcellular accumulation in endocytic vesi-
cles, as also shown by our colocalization studies with dextran-
FITC (see Figure S12). Upon irradiation, monomeric photo-
sensitizer, relocated quite possibly in close proximity to the
endosomal membranes, is activated and rupture the endo-
somal membranes according to the PCI principle.[2] Upon
endosomal-membrane compromise, the endocytosed species
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Figure 5. Changes induced by mTHPP-bCD PDT and mTHPP-bCD–
4-OHT PCI in the respiration of MCF7 cells. Cell respiration was
monitored by measurement of the oxygen-consumption rate (OCR)
with a Seahorse XFe96 Analyzer. The equilibrated basal respiration
values of interest are highlighted in white, and the response to
mitochondrial stress induced by various electron-transport-chain (ETC)
inhibitors is shown for all cell groups.
We also performed metabolic studies with a Seahorse
XFe96 Analyzer. The profound effects of mTHPP-bCD–
4-OHT PCI on MCF7 respiratory capacity are shown in
Figure 5. The basal respiration, as reflected by the oxygen-
consumption rate (OCR), underwent a very profound
decrease following PCI treatment. The effect was even
greater than that in the PDT (mTHPP-bCD-vehicle-treated)
group. In contrast, the glycolytic rate remained comparable
between the treatment groups (see Figure S10). The above
metabolic changes were observed 1 h after irradiation of the
PDT and PCI groups; no significant respiratory (OCR)
changes between the control, tamoxifen (4-OHT), PDT
(mTHPP-bCD + light), and PCI (mTHPP-bCD–4-OHT+
light) were observed in the case of MDA-MB-231 cells,
which exhibited an approximately fivefold lower basal
respiration than that of MCF7 cells (see Figure S11A). The
MCF7 respiratory changes are attributed to the large 4-OHT
payload released intracellularly by PCI, whereas in the case of
incubation with free 4-OHT, the internalized amount was
limited by equilibrium dynamics. The corresponding meta-
bolic studies for MDA-MB-231 cells (see Figure S11) showed
no significant discrepancies between treatment groups and
thus confirmed the differential effect of the controlled release
of the 4-OHT payload within MCF7 cells. In all metabolic
studies, the inhibitors helped establish that the respiration was
not uncoupled from ATP production and that the respiration
was not completely inhibited.
Figure 4. Photocytotoxicity to A) MCF7 and B) MDA-MB-231 cells
following overnight (16 h) incubation with medium only (CTRL),
4-OHT (7.5 mm), mTHPP-bCD (10 mm), and mTHPP-bCD(10 mm)–
4-OHT(7.5 mm). All groups were irradiated at the LD50 light doses (see
the Supporting Information). The MTT assays were performed at 24,
48, and 72 h postirradiation.
the PCI group is solely attributable to the large amounts of 4-
OHTreleased intracellularly upon cell irradiation. In the case
of MDA-MB-231 cells, the nongenomic photochemically
induced chemotoxicity was less profound, but it was also
gradually abrogated (lower at 72 h). Further clonogenic
survival studies (see Figure S13) confirmed that the PCI
effects shown in Figure 4 are not cytostatic, but clearly
cytotoxic, since only about 5% of the PCI-treated MCF7 cells
retained the ability to form clones. In the case of MDA-MB-
231 cells, PCI did not add significantly to the PDT cytotox-
icity.
The main advantage of using the mTHPP-bCD conjugate
as a host for 4-OHT is the fact that PCI up to now has only
been applicable to a large variety of macromolecules
(> 1000 Da) that do not readily transverse the plasma
membrane, including immunotoxins based on type I ribo-
some-inactivating protein toxins, poly- and oligonucleotide
therapeutics, and chemotherapeutic agents, such as bleomycin
(1400 Da).[15] PCI has until now not been applicable to small
molecules, such as 4-OHT, which instead of being endocy-
tosed, freely penetrate the cell membranes owing to their
physicochemical properties (e.g. small size, hydrophobicity,
positive charge).
In the present study, the mTHPP-bCD conjugate func-
tioned like a “Trojan horse” to keep the complexed tamoxifen
moieties “hidden” so that they would be endocytosed
together with the carrier macromolecule conjugate and
remain confined until the complex was released from the
endocytic vesicles upon irradiation. PCI has the unique
advantage of controlled drug delivery and release upon
activation with light. Also, drugs can be released specifically
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Received: January 8, 2015
Published online: && &&, &&&&
Keywords: antitumor agents · cyclodextrins · drug delivery ·
.
photochemical internalization · porphyrinoids
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Communications
Targeted Drug Delivery
Smuggled within enemy walls: A por-
phyrin–b-cyclodextrin (mTHPP–bCD)
conjugate was used to “smuggle” small
tamoxifen (TAM) molecules into cells by
endocytosis on the command of light
(see picture; FITC=fluorescein isothio-
cyanate). Upon irradiation with red light,
the porphyrin ruptured the endosomal
membranes, and the large tamoxifen
payload that was released caused sub-
stantial and permanent cytotoxicity to
tamoxifen-sensitive MCF7 cells.
T. A. Theodossiou,* A. R. GonÅalves,
K. Yannakopoulou, E. Skarpen,
K. Berg
&&& — &&&
Photochemical Internalization of
Tamoxifens Transported by a “Trojan-
Horse” Nanoconjugate into Breast-
Cancer Cell Lines
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