In situ activation of a doxorubicin prodrug using imaging-capable nanoparticles

Article abstract of DOI:10.1039/c6cc01024e

A general strategy for image-guided prodrug activation using fluorescently-labeled magnetic iron oxide nanoparticles is described. It is based on a recently developed concept in bio-orthogonal inverse-electron demand Diels-Alder chemistry, which is termed

Full text of DOI:10.1039/c6cc01024e

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In situ activation of a doxorubicin prodrug using imaging-capable
nanoparticles
a
a
a*
a*
Irfan Khan , Paul F. Agris , Mehmet V. Yigit and Maksim Royzen
Received 00th January 20xx,
Accepted 00th January 20xx
A general strategy for image-guided prodrug activation using fluorescently labeled magnetic iron oxide nanoparticles is
described. It is based on a recently developed concept in bio-orthogonal inverse-electron demand Diels Alder chemistry
termed ‘click-to-release’. To illustrate a potential new biomedical application of the chemistry, the nanoparticles were
modified with tetrazine, as well as near infrared fluorescent (NIRF) cy5.5 dye, while doxorubicin was converted into a
prodrug. The nanoparticles taken up by the MDA-MB-231 breast cancer cells efficiently converted the prodrug of
doxorubicin into the biologically active chemotherapeutic doxorubicin form.
DOI: 10.1039/x0xx00000x
www.rsc.org/
9
surface of nanomaterials. We illustrate how this new
Introduction
development in IEDDA chemistry can be used for image-guided
prodrug activation inside of cancer cells. This strategy has the
The inverse electron demand Diels-Alder (IEDDA) reaction
between trans-cyclooctene (TCO) and tetrazine has been
explored for a number of biomedical applications capitalizing
on fast reaction kinetics, as well as bio-orthogonality of the
potential for converting systemically administered prodrugs
into image-guided targeted chemotherapeutics.
The conventional chemotherapy relies on the systemic
injection of active drugs into the blood circulation en route to
the cancerous tissues, where they will bind the intended target
and exhibits their therapeutic functions. An ideal drug would
concentrate only at its intended target, but the vast majority
of the existing small molecule drugs distribute throughout the
1
chemistry to functional groups found in biological milieu. The
majority of the described constructs involve TCO-conjugated
antibodies that deliver the bio-orthogonal reagent to antigen-
2
specific cells or tissues. Meanwhile, tetrazine is attached to
therapeutic or imaging modalities, thereby facilitating targeted
drug delivery, radio-imaging, or pathogen detection.
entire body. Because of
a
lack of specificity of
chemotherapeutic agents for the targeted pathological site,
large doses are typically required to achieve effective local
Recently IEDDA chemistry has also been explored on the
surface of nanomaterials, such as magnetic nanoparticles
1
0
concentrations. These high doses chemotherapeutic drugs
often result in non-specific toxicity and other adverse side
effects.
(
MNPs). These applications are particularly intriguing, as bio-
orthogonality of TCO and tetrazine are complemented by the
non-toxic dextran-coated MNPs that were clinically used for
3
lymph node imaging. Enhanced permeability and retention
Meanwhile, systemically administered non-toxic MNPs
accumulate primarily in liver, spleen and lungs but also home
(EPR) effect and receptor-mediated endocytosis facilitate MNP
1
1
in cancerous tissues due to EPR effect. Uptake of tetrazine
and NIRF-labeled MNPs by the cancer cells can be monitored
in vitro by microscopy or in vivo using T2-weighted MRI. Once
localization of MNPs inside the cancer cells is confirmed, they
can act as molecular triggers capable of activating systemically
delivery of therapeutic payloads to cancerous cells and tumor
4
tissues. In addition these MNPs are T2-weighted MRI contrast
5
agents, capable of non-invasive imaging. In recent reports,
tetrazine-conjugated MNPs have been utilized for the
6
detection of Gram-positive bacteria, profiling of glioblastoma
7
or erythrocyte-derived or microvesicles. Similar constructs
administered
chemotherapeutic
prodrugs
containing
releasable TCO. Thus, the development of image-guided,
activatable prodrug delivery approaches will minimize the
side-effects related to drug’s penetration of healthy cells and
will allow precise control over prodrug activation in the target
cancer cells.
were also reported to be diagnostic tools for the detection of
8
lung cancer.
The current work describes a new biomedical application
for tetrazine and NIRF-modified MNPs using recently reported
‘
click-to-release’ chemistry, which is yet to be explored on the
The procedure described herein consists of two steps and
involves two reagents: MNPs modified with tetrazine and NIRF
cy5.5 dye, (MNP-cy5.5-tet) and a prodrug of doxorubicin,
formed by modification of the latter with releasable TCO
(
DOX-TCO). Cytotoxicity of doxorubicin is significantly
attenuated by the chemical modification, Figure S1 and S2
.
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MNP-cy5.5-tet acts as
a molecular trigger capable of coupling, the nanoparticles were purified by size exclusion
converting DOX-TCO into doxorubicin (DOX) inside of live cells. chromatography. The MNPs with cy5.5 label show strong
The image-guided activation of DOX-TCO can be selectively fluorescence emission at 710 nm which was absent with the
achieved inside of cells upon systemic administration of MNP- unmodified MNP, Figure 3b. Ratiometric functionalization of
cy5.5-tet
.
MNP accumulation can be monitored by MNPs with variable amounts of Tet-NHS and cy5.5-NHS is
fluorescence microscopy. Once, adequate accumulation is shown in Figure 3c. Difference in the visual appearance (Figure
confirmed, DOX-TCO is systemically administered at the 3c) and spectrophotometric properties of the five different
concentration that correlates to its IC20 value. The prodrug’s sets of MNP suggests that the amount of each functional
therapeutic activity multiplexes when it reaches the cells moiety on the surface can be controlled by the ratiometric
containing MNP-cy5.5-tet that triggers the prodrug’s approach (Figure 3d). Modulation of the two functional groups
conversion into doxorubicin which is considerably more allows to achieve the construct with optimal fluorescence for
cytotoxic than the DOX-TCO
.
validating MNP cellular uptake and capability to trigger DOX-
The underlying mechanism of prodrug activation is TCO activation for optimal therapeutic outcome. In the
illustrated in Figure 1. The multivalency of MNPs allows subsequent studies, such construct was deemed to be MNPs
loading of up to about 60 tetrazines per nanoparticle. After the synthesized using 1:9 ratio of cy5.5-NHS to Tet-NHS.
bio-orthogonal IEDDA reaction (‘click’ step) the cycloaddition
product spontaneously tautomerizes, thereby converting DOX-
TCO into DOX inside the target cells (‘release’ step). Herein, we
describe the synthesis of MNP-cy5.5-tet and DOX-TCO and
illustrate how image-guided prodrug activation can be
achieved in MDA-MB-231 human breast cancer cells.
DOX
DOX
NH
N
N
N
N
O
NH
O
O
H
Click
-N
Release
O
N
N
NH
N
+
+
DOX
N
N
2
-CO2
N
N
H
N
N
N
N
DOX-TCO
Tet
Tet
Tet
Tet
Figure 1. Illustration of how click-to-release chemistry is utilized on the surface
of MNPs for activation of the DOX-TCO prodrug.
Results and Discussion
Figure 3. (a) Illustration of MNP functionalization with cy5.5-NHS and tetrazine-
Synthesis of DOX-TCO and MNP-cy5.5-tet
NHS to form MNP-cy5.5-tet. (b) The unmodified nanoparticles lack any
fluorescence, but MNP-cy5.5-tet is fluorescent (exct: 675 nm, emm; 710 nm).
The cy5.5 and tetrazine content on MNP surface can be controlled
ratiometrically and monitored (c) visually and (d) spectroscopically.
The prodrug of doxorubicin, DOX-TCO, was synthesized as
shown in Figure 2. The amine group of DOX was modified with
9
TCO, using releasable carbamate linker. The details of the
Prodrug activation in vitro and in live cells
synthesis are described in the supporting information. This
structural modification substantially lowered potency of the
Prodrug activation was first tested in vitro. Aqueous
solutions of MNP-cy5.5-tet (2 µM) were treated with 0.2 µM
drug. The cytotoxicity studies, shown in Figures S1 and S2
,
DOX-TCO for variable amounts of time. The solutions were
separated from nanoparticles using amicon tubes. The filtrates
were concentrated and analyzed by HPLC. The HPLC spectra of
five different supernatant solutions, collected after different
incubation times, are shown in Figure 4. The prodrug
activation begins almost immediately after addition of DOX-
TCO to MNP-cy5.5-tet. Complete conversion of DOX-TCO to
DOX has been observed after 1 h of incubation.
revealed that that DOX-TCO is 5-times less cytotoxic than DOX
to MDA-MB-231 cells. From the MTT assays, the IC50 values of
DOX and DOX-TCO were determined to be 97 and 480 nM,
respectively.
Figure 2. Synthesis of cleavable DOX-TCO prodrug from DOX
.
Meanwhile, the nanoparticles capable of activating DOX-
TCO were synthesized, as described in Figure 3a. The
polydextran coated iron oxide nanoparticles, averaging 25 nm
in diameter with about 180 amine termini on the surface were
used. The amine groups were concomitantly coupled to Tet-
NHS and cy5.5-NHS in PBS buffer (pH 7.4). After the 24 h
Figure 4. Activation of DOX-TCO prodrug using MNP-cy5.5-tet. The HPLC data
showing the disappearance of the DOX-TCO peak and appearance of the DOX
peak over time.
2
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The prodrug is efficiently taken up by the MDA-MB-231 does not spontaneously get hydrolyzed into DOX in MDA-MB-
cells (Figure 5a). The image-guided in situ prodrug activation 231 cell lysate for as long as four days of incubation, Figure S3
.
was subsequently tested using the same cell line. The extent of intracellular activation of DOX-TCO by MNP-
Approximately, 1000 cells were grown in 25 mm MatTek glass cy5.5-tet was assayed by measuring Caspase-3 activity
bottom dishes and treated with MNP-cy5.5-tet (2
h. The nanoparticle uptake was confirmed by fluorescence of Caspase-3 activity in cells treated with either MNP-cy5.5-
microscopy measurements, shown in Figure 5b. Subsequently, tet DOX-TCO, or the combination of the nanoparticles and the
the cells were treated with DOX-TCO (0.2 M) for 48 h. This prodrug. Cells treated with MNP-cy5.5-tet alone showed
µM) for 24 indicative of apoptosis. Figure 7a illustrates the relative levels
,
µ
concentration corresponds to the IC80 value of the prodrug. minimal Caspase-3 activity, thus proving that the nanoparticles
Meanwhile, complete conversion of the prodrug to DOX would are not cytotoxic. Meanwhile, the combination therapy of
correspond to the IC20 value of the latter. Thus, the activated MNP-cy5.5-tet and the prodrug showed activity analogous to
prodrug is expected to be considerably more cytotoxic. the positive controls, supplied in the kit. The prodrug alone
Fluorescence microscopy confirmed that MNP-cy5.5-tet, as showed significantly lower Caspase-3 activity.
well as DOX-TCO were taken up by cells (Figure 5c). After the
Cell viability assay (MTT) was carried out in parallel to
prodrug treatment, the cells were grown for another 24 h in confirm the findings of the Caspase-3 activity assay. As shown
DMEM to evaluate the cytotoxicity of the activated prodrug.
in Figure 7b, cells treated with MNP-cy5.5-tet, followed by
DOX-TCO have similar levels of viability as the cells treated
with DOX
. Meanwhile, cells treated with either the
nanoparticles or the prodrug alone have significantly higher
levels of viability.
Figure 7. a) Caspase and b) cell viability assay (MTT) results demonstrate that
the apoptosis of cells incubated with MNP-cy5.5-tet and DOX-TCO is significantly
higher than the cells treated with MNP-cy5.5-tet and DOX-TCO separately.
Figure 5. Confocal microscopy images of MDA-MB-231 breast cancer cells
incubated with (a) DOX-TCO only, (b) MNP-cy5.5-tet only, and (c) MNP-cy5.5-
tet, followed by DOX-TCO. The cy5.5 and DOX-TCO channels show nanoparticle
and pro-drug uptake, respectively.
Conclusions
Additionally, flow cytometry (FACS) data indicated that
In summary, a novel biomedical application of the bio-
orthogonal ‘click-to-release chemistry has been described. To
our knowledge this is the first study describing multiplexing
the therapeutic pontential of a chemotherapeutic prodrug
using nanoparticles functionalized with tetrazines. The concept
of image-guided in situ prodrug activation has been illustrated
using TCO-modified doxorubicin. Meanwhile, tetrazine-
functionalized MNPs served as image-guided triggers, capable
of activating the prodrug inside of live breast cancer cells. As
described above, this strategy has the potential for converting
systemically administered prodrugs into image-guided
targeted chemotherapeutics. Because of its modular nature,
this methodology should be translatable to other
chemotherapeutic agents modified with the releasable-TCO, as
well as other nano-platforms.
almost all of the cells (99.9%) internalized MNP-cy5.5-tet
(
Figure 6b) and DOX-TCO
component separately. The negative control, shown in Figure
suggests that the cells without any treatment do not display
(Figure 6c) when treated with each
6
a
any inherent cy5.5 or doxorubicin fluorescent signals. Finally,
the FACS analysis of the cells treated with both MNP-cy5.5-tet
and DOX-TCO exhibited fluorescent signals in both emission
channels (Figure 6d).
Figure 6. Flow cytometry histograms of MDA-MB-231 breast cancer cells
incubated with (b) MNP-cy5.5-tet only, (c) DOX-TCO only, and (d) MNP-cy5.5-
tet, followed by DOX-TCO
.
Prodrug activation and ensuing release of DOX is expected
to trigger apoptosis upon reaching cytotoxic concentrations
inside the cells. Control experiments confirmed that DOX-TCO
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M. Hay, D. W. Thomas, J. L. Craighead, C. Economides, J.
Rosenthal, Nat. Biotech. 2014, 32, 40.
Footnote
M. Royzen and M. V. Yigit are corresponding authors. M. 11 (a) Z. Medarova, W. Pham, C. Farrar, V. Petkova, A. Moore,
Nat. Med, 2007, 13, 372; (d) M. V. Yigit, S. K. Ghosh, M.
Royzen supervised the bioconjugation studies. M. V. Yigit
Kumar, V. Petkova, A. Kavishwar, A. Moore, Z. Medarova,
Oncogene, 2013, 32, 1530.
supervised nanoparticle synthesis and characterization studies.
Together they have designed the project, supervised in vivo
studies, analyzed the data and wrote the paper. I. Khan
performed all the experiments and helped in writing the
paper.
Acknowledgements
The authors would like to thank BD Biosciences for their
financial support of this work. This work was also supported by
start-up funds of M. Royzen and M. V. Yigit provided by the
State University of New York at Albany.
Notes and references
‡ The supporting information provides the details of DOX-TCO
and MNP-cy5.5-tet syntheses. It also provides the details of the
MTT and Caspase-3 activity assays.
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