Thiophene-based compounds as fluorescent tags to study mesenchymal stem cell uptake and release of taxanes

Article abstract of DOI:10.1021/bc5000498

Human mesenchymal stem cells (hMSC) are multipotent cells that display the unique ability to home and engraft in tumor stroma. This remarkable tumor tropic property has generated a great deal of interest in many clinical settings. Recently, we showed that hMSC represent an excellent base for cell-mediated anticancer therapy since they are able to internalize paclitaxel (PTX) and to release it in an amount sufficient to inhibit tumor cell proliferation. In order to shed light on the dynamics of drug uptake and release, in the present paper we describe the synthesis of two novel thiophene-based fluorophore-paclitaxel conjugates, namely PTX-F32 and PTX-F35, as tools for in vitro drug tracking. We aimed to study the ability of these novel derivatives to be efficiently internalized by hMSC and, in a properly engineered coculture assay, to be released in the medium and taken up by tumor cells. In order to ensure better stability of the conjugates toward enzymatic hydrolysis, the selected oligothiophenes were connected to the taxol core at the C7 position through a carbamate linkage between PTX and the diamino linker. Antiproliferative experiments on both tumor cells and stromal cells clearly indicate that, in good correlation with the parent compound, cells are sensitive to nanomolar concentrations of the fluorescent conjugates. Moreover, in the coculture assay we were able to monitor, by fluorescence microscopy, PTX-F32 trafficking from hMSC toward glioblastoma U87 tumor cells. Our work paves the way for novel possibilities to perform extensive and high quality fluorescence-based analysis in order to better understand the cellular mechanisms involved in drug trafficking, such as microvescicle/exosome mediated release, in hMSC vehicle cells.

Full text of DOI:10.1021/bc5000498

Communication
pubs.acs.org/bc
Thiophene-Based Compounds as Fluorescent Tags to Study
Mesenchymal Stem Cell Uptake and Release of Taxanes
S. Duchi,†,§ P. Dambruoso,‡ E. Martella,†,§ G. Sotgiu,‡ A. Guerrini,‡ E. Lucarelli,† A. Pessina,∥ V. Cocce,∥
̀
A. Bonomi,∥ and G. Varchi*,‡
†Osteoarticular Regeneration Laboratory, Rizzoli Orthopaedic Institute, 40136 Bologna, Italy
‡Italian National Research Council, Institute of Organic Chemistry and Photoreactivity, 40129 Bologna, Italy
§Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, 40136 Bologna, Italy
∥Department of Biomedical, Surgical, Dental Sciences, University of Milan, Milan, Italy
S
* Supporting Information
ABSTRACT: Human mesenchymal stem cells (hMSC) are
multipotent cells that display the unique ability to home and
engraft in tumor stroma. This remarkable tumor tropic
property has generated a great deal of interest in many clinical
settings. Recently, we showed that hMSC represent an
excellent base for cell-mediated anticancer therapy since they
are able to internalize paclitaxel (PTX) and to release it in an
amount sufficient to inhibit tumor cell proliferation. In order
to shed light on the dynamics of drug uptake and release, in
the present paper we describe the synthesis of two novel thiophene-based fluorophore−paclitaxel conjugates, namely PTX-F32
and PTX-F35, as tools for in vitro drug tracking. We aimed to study the ability of these novel derivatives to be efficiently
internalized by hMSC and, in a properly engineered coculture assay, to be released in the medium and taken up by tumor cells. In
order to ensure better stability of the conjugates toward enzymatic hydrolysis, the selected oligothiophenes were connected to
the taxol core at the C7 position through a carbamate linkage between PTX and the diamino linker. Antiproliferative experiments
on both tumor cells and stromal cells clearly indicate that, in good correlation with the parent compound, cells are sensitive to
nanomolar concentrations of the fluorescent conjugates. Moreover, in the coculture assay we were able to monitor, by
fluorescence microscopy, PTX-F32 trafficking from hMSC toward glioblastoma U87 tumor cells. Our work paves the way for
novel possibilities to perform extensive and high quality fluorescence-based analysis in order to better understand the cellular
mechanisms involved in drug trafficking, such as microvescicle/exosome mediated release, in hMSC vehicle cells.
fluorescence imaging. Probe brightness and photostability are
crucial features to this aim. Moreover, drug conjugation with
the fluorochrome should be easily achievable and the steric
hindrance generated by the fluorophore should not completely
withdraw and/or modify drug activity.
Two of the three reactive hydroxyl groups can be suitably
used for paclitaxel derivatization, e.g., C2′ and C7 (Figure 1).
The reactivity of these positions follows the sequence C2′ >
C7. According to the literature on PTX derivatives,9,10 the free
2′-hydroxyl group (or an easily hydrolyzable moiety at that
position) is required for effective microtubule binding.11 On the
other hand, literature reports describe the general maintenance
of cytotoxic activity upon C7 esterification.12
uman mesenchymal stem cells (hMSC) are multipotent
Hcells that can self-renew and at the same time
differentiate into multiple lineages. During the past several
years, hMSC have generated a great deal of interest in many
clinical settings, including regenerative medicine, immune
modulation, and tissue engineering.1 Moreover, hMSC have
the unique ability to migrate along chemokine gradients to sites
of inflammation/injury and to tumor’s stroma, making them
useful tools for the targeted delivery of therapeutics to cancer
cells.2−4 To this end, hMSC have been engineered to express
proteins that induce tumor cell death,4,5 or more recently to
carry prodrugs,6 nanoparticle loaded drugs,2 photosensitizers,7
and conventional anticancer drugs8 directly into the tumor.
With regard to the latter application, we recently reported on
the ability of hMSC to internalize paclitaxel (PTX) and to
release it in an amount sufficient to inhibit tumor cell
proliferation.8 The experiment on tumor cells was conducted
by using the 24 h-conditioned medium obtained from hMSC
loaded with the drug.
Based on these considerations, we selected the commercially
available Flutax-213 (Figure 1) to attempt the study of PTX
trafficking from hMSC toward tumor cells through fluorescence
imaging. Flutax-2 is modified at the C7 position with the dye
Received: February 3, 2014
Revised: March 12, 2014
Published: March 14, 2014
Fluorescent tagged drugs are useful tools to visualize drug
trafficking dynamics (cell uptake and release) through
© 2014 American Chemical Society
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Figure 1. Paclitaxel (PTX), Flutax-2, PTX-F32, and PTX-F35 structures.
Scheme 1. PTX-F32 and PTX-F35 Synthesis
Oregon Green 488 through an ester bond (Figure 1).
Unfortunately, in our hands, this compound did not provide
the desired results in terms of both fluorescence stability and
intracellular localization.8 In fact, in our work we showed a
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Figure 2. PTX-F32 and PTX-F35 spectral analysis performed with confocal laser microscopy and NIS-Elements AR software on loaded hMSC.
Figure 3. Representative pictures of U87-RFP tumor cells treated with 4 μM PTX-F32 (blue) and 4 μM PTX-F35 (green) and observed from 6 h
after drug loading until 6 days after. Scale bar is 50 μm.
main Golgi staining by Flutax-2 instead of a microtubule
network distribution. This behavior could be due to the low
enzymatic stability of the ester linkage connecting Oregon
Green with PTX, which in turn could compromise its proper
visualization in our hMSC-tumor cell migration experiments.
Thus, we envisioned the need to design and synthesize novel
fluorescent PTX derivatives. To this end we selected
oligothiophenes as the fluorescent probes, which are chemically
stable molecules characterized by a common fluorescent
skeleton that can be suitably functionalized for several different
applications including organic electronics14,15 and biomedical
applications.16 They display intense absorption bands whose
wavelength can be tailored to fall anywhere from the UV to the
deep-red spectrum by carefully choosing the number of
aromatic rings and by introducing suitable substituents at the
α positions (Figure 1).17 Thiophene-based fluorophores (TbF)
have already been used for the efficient labeling of proteins,
DNA, and cells,18 while to our knowledge, no reports describe
the conjugation of anticancer drugs with TbF. Recently, we
reported the synthesis of TbF bearing electron donors and
electron-acceptor moieties able to interact through the π-
conjugated core (Figure 1).17 These compounds are charac-
terized by an efficient emission in the blue-orange interval and
can be conveniently used to label biologically active molecules.
In this view, we first protected the C2′-hydroxyl of the taxol
side chain with triethylsilyl chloride (TES-Cl) and, after
activation of the C7-hydroxyl group with carbonyl diimidazole,
the C2′-OTES-C7-imidazolyl taxol derivative (3) was trans-
formed in the corresponding C2′-OTES-C7-(4-aminobutyl
carbamoyl)-taxol derivative (4) using 1,4-diaminobutane. 2′-
OH TES deprotection followed by fluorophore amidation, e.g.,
F32 and F35,17 afforded the desired fluorescent taxoids, PTX-
F32 and PTX-F35 (Scheme 1), in acceptable overall yields (see
ESI for experimental details). The normalized absorption and
emission spectra of PTX-F32 and PTX-F35 have been
recorded in CH2Cl2 and are depicted in Figure S3. Confocal
microscopy spectral analysis detected comparable emission
spectra profile in hMSC loaded for 24 h with the two
compounds (Figure 2). A carbamate linkage between PTX and
the diamino linker was intentionally selected because of its
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Figure 4. Co-culture assay between hMSC and U87-RFP tumor cells. Time-lapse epifluorescence imaging of hMSC primed for 24 h with PTX-F32
(panel A) and with PTX-F35 (panel B) co-cultured with U87-RFP cells. hMSC have been plated on the left inserts; U87-RFP on the right ones.
Scale bar is 100 μm.
higher stability toward enzymatic hydrolysis as compared, for
example, to the corresponding ester.19 It is worth mentioning
that optical properties of TbFs were not substantially altered
upon conjugation with PTX,17 and the observed hypsochromic-
shift is in agreement with literature data.20 In particular, large
Stokes shifts are observed for the two novel derivatives, i.e., 108
and 142 nm for PTX-F32 and PTX-F35, respectively, while
they showed good photostability under constant illumination
with a fluorescent microscope excitation source (data not
shown).
fibroblasts (hSDFs), widely recognized as carriers for cancer
therapy,21 hMSC freshly isolated from two donors (hMSC1,
hMSC2), human T-cell leukemia MOLT-4, and the glioblas-
toma U87-RFP cell line (Table S1). The generally lower
activity of PTX-F35 and PTX-F32 compared to PTX can be
ascribed to a slightly modified engagement of the taxanes to
microtubules. However, our synthesized fluorescent conjugates
showed that modification at the C7 position reduces but does
not abrogate their antiproliferative activity. In fact, in good
correlation with native PTX, both tumor cells and stromal cells
proved to be sensitive to nanomolar concentrations of the
fluorescent conjugates.
We then evaluated the proliferation inhibition rate of our
compounds on four different cell lines, e.g., human skin-derived
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Figure 5. Co-culture assay between hMSC and U87-RFP tumor cells. Time-lapse epifluorescence imaging of hMSC primed for 24 h with vehicle
DMSO 0.2% (top panels) and with 2 μM PTX (bottom panels) co-cultured with U87-RFP cells. hMSC have been plated on the left inserts; U87-
RFP on the right one. Scale bar is 100 μm.
Fluorescence imaging was then used to investigate the
compounds’ behavior in tumor cells. To this end, we treated
U87-RFP glioblastoma cells with 4 μM PTX-F32 (blue in
Figure 3), with 4 μM PTX-F35 (green in Figure 3) and PTX 4
μM (Figure 3) as the control. Images were acquired starting
from 6 h after the compound initial loading over time up to 6
days in cell culture. In spite of the results obtained with the
MTT assay (Table S1), we used a higher PTX-F concentration
in order to better visualize their intra- and subcellular
localization with conventional fluorescence microscopy.11 We
observed an increase in fluorescence level within the incubation
time from 6 h (29.2 7.1 and 20.2 6.5 mean fluorescence
intensity PTX-F32 and PTX-F35, respectively) up to 24 h
(39.2 13.3 and 34.5 9.6 mean fluorescence intensity PTX-
F32 and PTX-F35, respectively). At this time point tumor cells
were efficiently stained while their morphology appeared
strongly affected (see magnifications in Figure 3). After several
days, cells still showed a good intracellular fluorescence signal
(see 6 days in Figure 3), while the morphology was highly
compromised. These results demonstrate that our compounds
retain an acceptable cytotoxic activity toward tumor cells, while
their fluorescence is stable enough to perform long-time
experiments. Fluorescence imaging was then used to investigate
the compounds’ trafficking between hMSC and U87-RFP
tumor cells. For all the experiments described in the following
section, hMSC are intended to be hMSC1, which resulted in
lower sensitivity to PTX and PTX-F treatment (Table S1). To
this aim, we first evaluated whether the novel PTX-F conjugates
were efficiently internalized by hMSC. Accordingly, hMSC
were exposed to PTX-F32 and PTX-F35 (4 μM) for 24 h and
immunostained for confocal microscopy imaging. β-Tubulin
staining demonstrated that PTX-F32 distributes along the
microtubule networks (white arrows in Figure S4) and in
vesicle-like structures (white asterisks in Figure S4), and not
only in Golgi apparatus as previously observed with Flutax-2.8
Moreover, the internalization of the fluorescently labeled PTX
derivatives did not strongly affect β-tubulin organization even if
a rounded-shaped morphology was observed for treated cells
(Figure S5).
Finally, we optimized an in vitro assay to determine whether
our fluorescent taxanes are valuable tools for visualizing their
migration through cells. This model mimics the physiologic
hMSC-tumor cells interaction in a 2D system. We therefore
primed hMSC with 4 μM PTX-F32 and PTX-F35 for 24 h, and
meanwhile, we co-plated U87-RFP cells, keeping them initially
separated by inserts. After the 24 h of incubation, hMSC were
washed several times to eliminate potential unloaded drugs, and
inserts were removed, making hMSC free to migrate toward
tumor cells (Figure 4). Time-lapse imaging was performed over
24 h. Non-conjugated PTX was used as the control (Figure 5),
confirming it to be efficiently released from hMSC, since co-
cultured tumor cells appear to be strongly affected (Figure
5).8,21 As shown in Figure 4A, the time-lapse imaging of PTX-
F32 loaded hMSC revealed that the fluorescent taxane was
released and uploaded by tumor cells within 6 h. After 24 h
observation, U87-RFP cells become round-shaped and detach
from plate differently from DMSO control and similarly to PTX
loaded hMSC (Figure 5). It is worth noting that after 24 h both
hMSC and U87-RFP are still equally stained (see magnifica-
tions of Figure 4A), but PTX-F32 fluorescence results were
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much weaker compared to those at time 0. These data confirm
that PTX-F32 has been released from hMSC in the medium
and taken up by tumor cells. The same experiment performed
with PTX-F35 loaded hMSC revealed that the compound is
not completely released by the host cells within 24 h (Figure
4B). Right after incubation, hMSC start to suffer and detach,
being unable to efficiently transfer the drug to U87-RFP cells
that in turn are motile, proliferative, and only slightly
fluorescent (Figure 4B). In order to verify that this behavior
was not caused by the higher potency of PTX-F35 compared to
PTX-F32 (Table S1), we decided to repeat the experiment
using a lower concentration of PTX-F35 (e.g., 2 μM instead of
4 μM). However, even under these conditions PTX-F35 was
only barely able to cross over the medium to reach U87-RFP
cells, which after 24 h were faintly stained displaying only
weakly compromised morphology (Figure S6).
In summary, we described the synthesis of novel thiophene
based fluorophore−paclitaxel conjugates as promising tools for
in vitro drug tracking. We demonstrated that such compounds
could be effectively internalized in several cell lines. Further, in
the case of PTX-F32, we were able to monitor, by fluorescence
microscopy, the fluorescent taxane trafficking from hMSC
toward tumor cells. The different behavior of PTX-F35 loaded
hMSC co-cultured with U87 cells could be ascribed either to a
different mechanism of action or, more likely, to the specific
nature of the thiophene fluorophore that might induce
aggregate formation through π-stacking interactions. However,
the high tunability of these fluorophores in terms of chemical
and photochemical properties, paves the way to future
investigations in the field. In view of the high expectations of
hMSC use as drug carriers, our work suggests a novel possibility
to perform extensive and high quality fluorescence-based
analysis to better understand the cellular mechanisms involved
in drug trafficking, such as microvescicle/exosome mediated
release.22
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures, spectroscopic data, and copies of
1H/13C NMR of new compounds; additional figures and tables
on biological experiments. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +39 0516398283; Fax +39 0516398349; E-mail: greta.
varchi@isof.cnr.it.
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Author Contributions
S. Duchi, P. Dambruoso, and E. Martella equally contributed to
this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Research was supported by Progetto FIRB-Accordi di
programma 2010 COD. RBAP10447, Italian Ministry of Health
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ABBREVIATIONS
PTX, Paclitaxel; hMSC, human Mesenchymal Stem Cell;
Thiophene-based fluorophore, TbF
■
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