Non-invasive, real-time reporting drug release in vitro and in vivo

Article abstract of DOI:10.1039/c4cc09920f

We developed a real-time drug-reporting conjugate (CPT-SS-CyN) composed of a near-infrared (NIR) fluorescent cyanine-amine dye (CyN), a disulfide linker, and a model therapeutic drug (camptothecin, CPT). Treatment with dithiothreitol (DTT) induces cleavage of the disulfide bond, followed by two simultaneous intramolecular cyclization reactions with identical kinetics, one to cleave the urethane linkage to release the NIR dye and the other to cleave the carbonate linkage to release CPT. The released CyN has an emission wavelength (760 nm) that is significantly different from CPT-SS-CyN (820 nm), enabling easy detection and monitoring of drug release. A linear relationship between the NIR fluorescence intensity at 760 nm and the amount of CPT released was observed, substantiating the use of this drug-reporting conjugate to enable precise, real-time, and non-invasive quantitative monitoring of drug release in live cells and semi-quantitative monitoring in live animals. This journal is

Full text of DOI:10.1039/c4cc09920f

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Non-invasive, real-time reporting drug release
in vitro and in vivo†
Cite this:DOI: 10.1039/c4cc09920f
a
a
b
bc
a
a
Yanfeng Zhang, Qian Yin, Jonathan Yen, Joanne Li, Hanze Ying, Hua Wang,
a
c
bcde
ab
Received 11th December 2014,
Accepted 17th March 2015
Yuyan Hua, Eric J. Chaney, Stephen A. Boppart
and Jianjun Cheng*
DOI: 10.1039/c4cc09920f
www.rsc.org/chemcomm
We developed a real-time drug-reporting conjugate (CPT-SS-CyN) pro-drug and conjugated nanomedicine are in their inactive
composed of a near-infrared (NIR) fluorescent cyanine-amine dye form, it is also essential to ensure the conjugated, inactive drug
(
CyN), a disulfide linker, and a model therapeutic drug (camptothecin, can actually be released in vivo and become therapeutically
9
–15
CPT). Treatment with dithiothreitol (DTT) induces cleavage of the active.
However, conventional methods to assess the drug
disulfide bond, followed by two simultaneous intramolecular cycliza- release and track the dynamics of active drug concentrations
2
,16–21
tion reactions with identical kinetics, one to cleave the urethane are very complicated.
Harvested tissues generally need to
linkage to release the NIR dye and the other to cleave the carbonate be homogenized and cells need to be lysed in order to release
22–29
linkage to release CPT. The released CyN has an emission wavelength the agents from certain intracellular compartments.
During
(760 nm) that is significantly different from CPT-SS-CyN (820 nm), these processes, especially in the process of cell lysis, the intact
enabling easy detection and monitoring of drug release. A linear drug carriers (e.g., micelles or liposomes) will also be disrupted,
relationship between the NIR fluorescence intensity at 760 nm and posing great difficulty in differentiating the released active agents
the amount of CPT released was observed, substantiating the use of from those inactive agents remaining in the carriers at the point
this drug-reporting conjugate to enable precise, real-time, and non- of tissue harvesting. As such, development of minimally invasive
invasive quantitative monitoring of drug release in live cells and semi- or non-invasive methods to confirm the drug release and deter-
quantitative monitoring in live animals.
mine the active drug concentration kinetics in localized tissues is
of great benefit.
Near-infrared (NIR) fluorescence imaging has been widely used
30
Accurate assessment and monitoring of the concentration of
functional agents in biological tissues is important in biology for monitoring biological processes in living objects because of its
1
–4
and medicine. In anticancer drug development and clinical deep tissue penetration capability of NIR signal and the effective
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1–37
cancer treatment specifically, the therapeutic benefit of drugs is elimination of tissue auto fluorescence interference.
a function of the concentration-time profile in tumor tissues.
Compared
5
–8
to other molecular imaging techniques such as magnetic resonance
Therefore, it is crucial to know the change of drug concen- imaging (MRI), positron emission tomography (PET), and computed
tration in the plasma and local tissues over a certain time- tomography (CT), NIR fluorescence imaging has great potential to
1
course. As the vast majority of therapeutic agents used in track some biological processes noninvasively, in real time, and
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8–42
with sequential, longitudinal monitoring capability.
Using
a
NIR fluorescence imaging as a readout tool, which has been
used in drug delivery applications, would be of particular interest
Department of Materials Science and Engineering, University of Illinois at
Urbana–Champaign, 1304 West Green Street, Urbana, IL 61801, USA.
E-mail: jianjunc@illinois.edu; Fax: +1 217-333-2736; Tel: +1 217-244-3924
Department of Bioengineering, University of Illinois at Urbana–Champaign,
Urbana, IL 61801, USA
43–46
for tracking the kinetics of drug release in vivo.
Here, we
b
c
report the design and use of CPT-SS-CyN to enable precise, real-
time, and non-invasive monitoring of drug release quantita-
tively in live cells and semi-quantitatively in vivo. CPT-SS-CyN
has a built-in disulfide bond, which can be cleaved through
reduction in the cells. The cleavage of the disulfide bond controls
the two subsequent elimination reactions that are responsible for
the concurrent release of the drug and reporting dye molecule.
CPT-SS-CyN is composed of a near-infrared fluorescent
cyanine-amine dye (CyN), a reductive-responsive disulfide lin-
ker, and a model therapeutic drug (camptothecin, CPT). The
Biophotonics Imaging Laboratory, Beckman Institute for Advanced Science and
Technology, University of Illinois at Urbana–Champaign, Urbana, IL 61801, USA
Department of Electrical and Computer Engineering,
d
e
University of Illinois at UrbanaÀChampaign, Urbana, Illinois 61801, USA
Department of Internal Medicine, University of Illinois at UrbanaÀChampaign,
Urbana, Illinois 61801, USA
†
Electronic supplementary information (ESI) available: Experimental details,
including the synthesis and characterization of prodrugs, degradation of prodrugs,
and analysis of fluorescence arising process in vitro and in vivo. See DOI: 10.1039/
c4cc09920f
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Fig. 1 (a) Fluorescence intensity changes of CPT-SS-CyN (10 mM) upon
DTT treatment (1 mM) for up to 120 min. The excitation wavelength was
6
50 nm. (b) The correlation of fluorescence increase at 760 nm (release of
CyN from CPT-SS-CyN, 10 mM) with the release of CPT from CPT-SS-CyN
in presence of DTT (1 mM).
of the released CPT was found to have a linear correlation with the
2
normalized increase in fluorescence intensity at 760 nm (R =
0
.9989). In the absence of DTT, however, neither CPT release nor
Scheme 1 (a) Schematic illustration of real-time tracking of active drug
release in vivo. (b) Proposed mechanism for concurrent NIR/CPT release
from CPT-SS-CyN triggered by DTT/GSH.
enhanced fluorescence intensity at 760 nm was observed. In
comparison, CPT-CC-CyN without a disulfide linker (10 mM)
showed no release of CPT or CyN in the presence of 100-fold
DTT concentration (1 mM) (Fig. S9b†). We thus consider that the
change in fluorescence emission at 760 nm can act as a direct
on-off signal to determine CPT release.
To demonstrate our hypothesis that the active drug release
and kinetics could be quantitatively assessed by monitoring the
NIR fluorescence signal changes in live cells, Hoechst pre-
stained HeLa cells in serum free DMEM medium was incubated
with CPT-SS-CyN (0.5 mM), and NIR fluorescence imaging was
collected in situ with an emission filter of 680–750 nm on a GE
In-Cell Analyzer at different time points (Fig. 2a). Significant
disulfide linker between the drug and the NIR dye is responsive to
intracellular reducing agents such as glutathione (GSH), cysteine
47–50
(Cys) and thioredoxin (Trx).
Treatment with these reducing
reagents induces cleavage of the disulfide bond, followed by two
subsequent intramolecular cyclization reactions, one to cleave the
urethane linkage to release the NIR dye and the other to cleave
the carbonate linkage to release CPT with 1,3-oxathiolan-2-one as
the same byproduct of the two cyclization reactions. The released
NIR dye has a maximum emission wavelength (760 nm) that is
significantly different from CPT-SS-CyN (820 nm), and the release
of the NIR dye can thus be used to monitor the release of CPT
in vitro and in vivo (Scheme 1).
Synthesis details of CPT-SS-CyN can be found in the ESI†
(
Scheme S1–S5 and Fig. S1–S5). The chemical structure of
1
13
CPT-SS-CyN was confirmed by H NMR, C NMR and ESI-MS
analyses (Fig. S4 and S5†). Reversed-phase high-performance
liquid chromatography (RP-HPLC) analysis showed over 95%
purity of the obtained CPT-SS-CyN (Fig. S6†). Following the
same method, we synthesized CPT-CC-CyN as the non-cleavable
control (Scheme S1, ESI†). We first used RP-HPLC and ESI-MS
to confirm the trigger-induced degradation of CPT-SS-CyN
upon the addition of dithiothreitol (DTT). As shown in the
RP-HPLC spectrum, the single peak of CPT-SS-CyN at 40.6 min,
corresponding to the major peak of 1220.5 m/z in the ESI-MS
spectrum, decreased significantly upon treatment with DTT
Fig. 2 (a) Fluorescence microscopy images of HeLa cells incubated with
CPT-SS-CyN using the GE In-Cell Analyzer. The cells were incubated with
serum free DMEM medium containing CPT-SS-CyN (0.5 mM), and then the
(
Fig. S6c and S6d†). Released CPT (16.2 min) and CyN (44.2 min) images were obtained at each time point (10 min, 2, 3, and 4 h) at 37 1C.
The nucleus was stained with Hoechst. The cell images were obtained using
the DAPI channel (excitation at 320–370 nm, emission at 430–580 nm)
and the Cy5 channel (excitation at 640–660 nm, emission at 680–750 nm).
were detected (Fig. S6†), demonstrating the successful disulfide
cleavage of CPT-SS-CyN followed by the two concurrent elimina-
tion reactions as shown in Scheme 1.
(b) Relationship between average fluorescence intensity per cell (background
We then used a combined RP-HPLC and fluorimetric analysis
fluorescence signal has been subtracted) and the amount of CPT released
to further confirm that the release of CPT occurred concurrently per HeLa cell. (c) Amount of CPT released from CPT-SS-CyN with/without
BSO/DEM inhibitors and CPT-CC-CyN with incubation time in HeLa cells.
with the observed fluorescence changes (Fig. S7 and S8†). At
different time points, amount of CPT released was measured by
RP-HPLC and fluorescence intensity of degraded products was
measured at 760 nm on a fluorescence spectrometer. As shown
The cells were incubated with serum free Opti-MEM medium containing
CPT-SS-CyN (0.5 mM), and then the images were obtained every 10 min at
3
7 1C. The nucleus were stained with Hoechst. The cells were imaged using
the DAPI channel (excitation at 320–370 nm, emission at 430–580 nm) and
in Fig. 1, when CPT-SS-CyN was treated with DTT, the percentage the Cy5 channel (excitation at 640–660 nm, emission at 680–750 nm).
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increase in fluorescence signal was observed in HeLa cells after 2 h
incubation, indicating the uptake of CPT-SS-CyN and release of
CyN and CPT in cells. The gradual increase in the NIR fluorescence
intensity in the range of 680–750 nm was observed over time. In
comparison, no fluorescence signal in the range of 680–750 nm
was observed in HeLa cells after treatment with CPT-CC-CyN
(0.5 mM) for 5 h (Fig. 2c, and Fig. S9c†). To demonstrate that the
fluorescent enhancement in HeLa cells was due to reductive
degradation of CPT-SS-CyN, L-buthionine sulfoximine (BSO,
100 mM) and diethylmaleate (DEM, 300 mM) were used as GSH
inhibitors to investigate whether inhibition of GSH levels in HeLa
cells would reduce or slow down the degradation of the reporting
51
conjugate. As shown in Fig. 2c, fluorescence signal in the range
of 680–750 nm greatly decreased in BSO/DEM treated HeLa cells
after 5 h incubation with CPT-SS-CyN (0.5 mM).
To evaluate whether CPT-SS-CyN enables quantitative assess-
ment of released CPT via following the fluorescence changes in
live cells, we measured the concentrations of the released CPT in
lysed HeLa cells via liquid chromatography-multiple-reaction
monitoring-mass spectrometry (LC-MRM/MS) and the fluores-
cence intensity change at 680–750 nm per cell via GE In-Cell
Analyzer (Fig. S10–S14†), and then sought inter-correlation of
these two parameters. As shown in Fig. 2b, a linear relationship
2
(
R = 0.98) between the average fluorescence intensity per cell and
the amount of released CPT per cell was observed, demonstrating
that the NIR fluorescence intensity change can be utilized as a
diagnostic tool for quantitative CPT release studies. This calcula-
Fig. 3 Real-time reporting of CPT release from CPT-SS-CyN in female
athymic nude mice bearing human A375 melanoma. (a) Fluorescence
tion was further validated by correlating the average fluorescence spectra (CyN channel) of tumor tissues and whole body NIR fluorescence
images of athymic nude mice bearing human A375 melanoma following a
single intratumoral injection of CPT-SS-CyN (5 nmol per tumor, the scale
intensity with the amount of released CyN in the living cells.
Fig. S12† showed the average fluorescence intensity enhance-
bar is 1.0 cm). Images taken at 10 min, 2 h, and 5 h by a Maestro in vivo
ment at Cy5 channel (excitation at 640–660 nm, emission at
whole-animal fluorescence imaging system (excitation at 615–665 nm,
6
(
80–750 nm) analyzed by GE In-Cell Analyzer correlates linearly
emission at 680–950 nm) were shown. (b) Average fluorescence intensity
R = 0.98) with the amount of CyN released per cell, as determined profile at 740 nm of tumor tissues after injection of CPT-SS-CyN.
2
by fluorescence spectrometer. Fig. S13† showed a linear relation-
ship between the average amount of CyN per cell analyzed by
fluorescence method and average amount of CPT per cell analyzed During the entire imaging acquisition period (5 h), strong and
by LC-MRM/MS. The real-time tracking of active CPT release in steady fluorescence signal enhancement was observed at 10 min
the living cells was further investigated. As shown in Fig. 2c, the post injection, and the fluorescence spectra of tumor tissues
À17
amount of actively released CPT increased from 0.094 Â 10
showed a noticeable shift from B820 nm (CPT-SS-CyN emission)
À17
mole per cell at 10 min post incubation to 5.223 Â 10
mole per to B740 nm (CyN emission). The increase in the fluorescent
cell at 5 h post incubation. However, negligible enhancement of the intensity of CyN at 740 nm over time in tumor tissues was showed
average fluorescence intensity per cell was observed when HeLa in Fig. 3b. The fluorescence intensity at 5 h post injection
cells were incubated with CPT-CC-CyN, indicating minimal release increased by 5 times compared to that at 10 min post-injection.
of CyN and CPT. These studies and experimental results demon- Based on the obtained linear relationship of CPT release and CyN
strated the potential of CPT-SS-CyN prodrug as a diagnostic tool for fluorescence intensity enhancement from the in vitro CPT-SS-CyN
real-time tracking of CPT release quantitatively in live cells.
(Fig. S13†), the amount and kinetics of active CPT release thus
We next assessed the drug-reporting capability of CPT-SS- can be monitored semi-quantitatively in vivo. We evaluated the
CyN in vivo. Athymic nude mice bearing human A375 melanoma cytotoxicity of CPT, CyN, CPT-SS-CyN, and CPT-CC-CyN using
were intratumorally injected with CPT-SS-CyN (5 nmol of CyN, in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
25 mL DMSO/PBS (1 :10 v/v)) and in vivo whole-body fluorescence assay (Fig. S15†). Both the NIR dye (CyN) and the control prodrug
imaging was taken on an in vivo imaging system (Maestro, CRi, (CPT-CC-CyN) showed negligible cytotoxicity against HeLa cells.
Inc., excitation filter: 615–665 nm, emission filter: 680–950 nm). While CPT-SS-CyN showed comparable cytotoxicity to free CPT due
Image collection started simultaneously as the intratumoral injec- to the intracellular GSH induced CPT release.
tion of CPT-SS-CyN and continued for 5 h. Fig. 3a showed the
In summary, we developed a real-time drug-reporting system
fluorescence spectra of tumor tissues and the whole-body images (CPT-SS-CyN) composed of a disulfide bond as a cleavable linker,
of mice at 10 min, 2 h, and 5 h post injection of CPT-SS-CyN. a cyanine-amide moiety as a near-infrared (NIR) fluorescence
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