Direct fluorescence monitoring of the delivery and cellular uptake of a cancer-targeted RGD peptide-appended naphthalimide theragnostic prodrug

Article abstract of DOI:10.1021/ja303998y

Presented here is a multicomponent synthetic strategy that allows for the direct, fluorescence-based monitoring of the targeted cellular uptake and release of a conjugated therapeutic agent. Specifically, we report here the design, synthesis, spectroscopic characterization, and preliminary in vitro biological evaluation of a RGD peptide-appended naphthalimide pro-CPT (compound 1). Compound 1 is a multifunctional molecule composed of a disulfide bond as a cleavable linker, a naphthalimide moiety as a fluorescent reporter, an RGD cyclic peptide as a cancer-targeting unit, and camptothecin (CPT) as a model active agent. Upon reaction with free thiols in aqueous media at pH 7.4, disulfide cleavage occurs. This leads to release of the free CPT active agent, as well as the production of a red-shifted fluorescence emission (λ_max = 535 nm). Confocal microscopic experiments reveal that 1 is preferentially taken up by U87 cells over C6 cells. On the basis of competition experiments involving okadaic acid, an inhibitor of endocytosis, it is concluded that uptake takes place via RGD-dependent endocytosis mechanisms. In U87 cells, the active CPT payload is released within the endoplasmic reticulum, as inferred from fluorescence-based colocalization studies using a known endoplasmic reticulum-selective dye. The present drug delivery system (DDS) could represent a new approach to so-called theragnostic agent development, wherein both a therapeutic effect and drug uptake-related imaging information are produced and can be readily monitored at the subcellular level. In due course, the strategy embodied in conjugate 1 could allow for more precise monitoring of dosage levels, as well as an improved understanding of cellular uptake and release mechanisms.

Full text of DOI:10.1021/ja303998y

Article
pubs.acs.org/JACS
Direct Fluorescence Monitoring of the Delivery and Cellular Uptake
of a Cancer-Targeted RGD Peptide-Appended Naphthalimide
Theragnostic Prodrug
Min Hee Lee,† Jin Young Kim,‡ Ji Hye Han,‡ Sankarprasad Bhuniya,† Jonathan L. Sessler,§,⊥
Chulhun Kang,*,‡ and Jong Seung Kim*,†
†Department of Chemistry, Korea University, Seoul, 136-701, Korea
‡The School of East-West Medical Science, Kyung Hee University, Yongin, 446-701, Korea
§Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712-0165, United States
⊥Department of Chemistry, Yonsei University, 262 Seonsanno Sinchon-dong, Seodaemun-gu, Seoul 120-749, Korea
S
* Supporting Information
ABSTRACT: Presented here is a multicomponent synthetic strategy that allows for
the direct, fluorescence-based monitoring of the targeted cellular uptake and release of
a conjugated therapeutic agent. Specifically, we report here the design, synthesis,
spectroscopic characterization, and preliminary in vitro biological evaluation of a RGD
peptide-appended naphthalimide pro-CPT (compound 1). Compound 1 is a
multifunctional molecule composed of a disulfide bond as a cleavable linker, a
naphthalimide moiety as a fluorescent reporter, an RGD cyclic peptide as a cancer-
targeting unit, and camptothecin (CPT) as a model active agent. Upon reaction with
free thiols in aqueous media at pH 7.4, disulfide cleavage occurs. This leads to release
of the free CPT active agent, as well as the production of a red-shifted fluorescence
emission (λmax = 535 nm). Confocal microscopic experiments reveal that 1 is
preferentially taken up by U87 cells over C6 cells. On the basis of competition
experiments involving okadaic acid, an inhibitor of endocytosis, it is concluded that
uptake takes place via RGD-dependent endocytosis mechanisms. In U87 cells, the active CPT payload is released within the
endoplasmic reticulum, as inferred from fluorescence-based colocalization studies using a known endoplasmic reticulum-selective
dye. The present drug delivery system (DDS) could represent a new approach to so-called theragnostic agent development,
wherein both a therapeutic effect and drug uptake-related imaging information are produced and can be readily monitored at the
subcellular level. In due course, the strategy embodied in conjugate 1 could allow for more precise monitoring of dosage levels, as
well as an improved understanding of cellular uptake and release mechanisms.
drug is taken up and released is operational, being inferred from
increased activity or improved cell adhesion, rather than
measured directly.7−10 This has made it difficult to establish
precisely when, where, and how the pharmaceutically active
payload is delivered to a cell. It would thus be desirable to
develop an RGD-based drug delivery system that contains both
an active drug (for efficacy) and a fluorophore (for ease of
monitoring uptake and delivery). In principle, this would
permit drug delivery and release to be monitored directly. It
would also allow the power of theragnostics (combined therapy
and diagnostics) to be extended to the subcellular level by
enhancing efficacy while facilitating imaging. Here, we report a
system that meets these design criteria. Specifically, we detail
the design, synthesis, characterization, and optical properties of
an RGD peptide-appended naphthalimide pro-camptothecin
delivery agent (conjugate 1), as well as the results of a
INTRODUCTION
■
In recent years, targeted drug delivery systems have been
extensively investigated in an effort to improve chemotherapy
treatment regimens.1 The use of ligands selective for cell-type2,3
and conjugation strategies that allow for the selective targeting
of a drug have emerged as attractive approaches in this overall
context.4 Cyclic peptides containing an RGD (Arg-Gly-Asp)
sequence are particularly effective targeting agents. These
sequences are recognized and internalized by αvβ3 integrin,5 a
well-known tumor-associated receptor. The receptor is highly
expressed on activated endothelial cells in several tumors,
playing predominant roles in tumor-induced angiogenesis and
growth.6 In fact, cyclic RGD peptides conjugated with
anticancer drugs, such as doxorubicin,7 camptothecin,8 and
paclitaxel,9 have been shown to have improved therapeutic
activity in vitro and in vivo relative to the corresponding free
drugs.7−10 Typically, the targeted drug is linked to an RGD
carrier with a cleavable linker allowing conversion to the active
drug form inside the cells.11 In general, the conclusion that the
Received: April 26, 2012
Published: May 29, 2012
© 2012 American Chemical Society
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preliminary in vitro biological evaluation using the U87 and C6
cell lines. As detailed below, this multicomponent system shows
off-on fluorescence changes that coincide with drug release
inside the cells.
RESULTS AND DISCUSSION
■
Conjugate 1 is composed of camptothecin (CPT), an
antitumor inhibitor of topoisomerase I,12 a disulfide linker
cleavable by thiols that are relatively abundant in tumor cells,
such as glutathione (GSH) or thioredoxin (Trx),13 and a
naphthalimide moiety that gives rise to a strong, red-shifted
fluorescence signal upon disulfide cleavage.14 This elaborated
system was thus expected to act as an effective drug delivery
agent capable of allowing concurrently both selective cell
targeting and fluorescent-based monitoring of drug release.
Compound 1 was prepared by the synthetic route outlined in
Scheme 1. The naphthalimide derivatives 3−5 were prepared
Scheme 1. Synthetic Route to 1
Figure 1. (a) Absorption and (b) fluorescence spectra of 2 (10.0 μM)
recorded in the presence and absence of GSH (5.0 mM). (c)
Fluorescence changes of 2 (10.0 μM) seen upon treatment with
increasing concentrations of GSH (0−60 equiv). Inset: Change in
fluorescence intensity at 535 nm as a function of GSH concentration.
(d) The fluorescence response of 2 (10.0 μM) with and without GSH
(1.0 mM) as a function of pH. All data were acquired 2 h after GSH
addition at 37 °C in PBS buffer (pH 7.4) containing 16% (v/v) of
DMSO. Excitation was effected at 430 nm.
0.99619) with GSH concentration (0−5 equiv) (see also the
inset to Figure 1c).
To evaluate the possibility of interference from other
biologically relevant analytes, the reactions of 2 with various
thiols, nonthiol amino acids, and metal ions were investigated
under the same conditions. Upon the addition of cysteine
(Cys) or homocysteine (Hcy) to 2, similar changes in the
absorption and emission features are seen as in the case of GSH
(cf., Supporting Information Figures S1 and S2, respectively).
On the other hand, no appreciable spectroscopic changes were
seen upon exposure to thiol-free amino acids or biologically
relevant metal ions.
The pH dependence of the GSH-induced increase in the
fluorescence intensity at 535 nm was also investigated. As
shown in Figure 1d, in the absence of GSH, compound 2 is
stable between pH 2 and 9, whereas in the presence of GSH a
large fluorescence enhancement is seen over a pH range of 5−
11. Taken in concert, these findings provide support for the
notion that the disulfide-linked control system 2 will undergo
thiol-induced cleavage in biological milieus but not degrade in
the presence of various potential interferants, including metal
cations.
Reverse-phase HPLC and ESI-MS analyses were used to
confirm the anticipated CPT release as the result of disulfide
bond cleavage following exposure to GSH. Under conditions of
our analysis, aqueous solutions of 2 give rise to a single peak at
14.3 min in HPLC chromatogram and a major peak at 917.3
m/z corresponding to [2 + Na]+ in the ESI-MS spectrum
(Figure S3, Supporting Information). As can be seen from an
inspection of Figure S3a and S3b, in the presence of GSH (0.1
mM), the peak intensity at 14.3 min, corresponding to 2 (0.1
mM), decreases and new strong fragments peaks appear that
elute at 10.1 and 11.3 min, respectively. These new fragment
peaks are well matched with those from CPT and 4,
by adapting previously published procedures.14,15 CPT was
reacted with triphosgene followed by treatment with 5 to afford
2 in moderate yield. Hydrolysis of 2 with TFA/DCM, followed
by reaction with c(RGDyK), gave the RGD-containing
conjugate 1. The chemical structure of 1 was confirmed by
1H NMR, 13C NMR, MALDI-TOF, and ESI-MS analyses (cf.,
Supporting Information).
Precursor 2 stands as a convenient RGD-free analogue of 1.
It was used to confirm that cleavage could be effected by
treatment with thiols. This was done by treating aqueous
solutions of 2 with 0−60 equiv of glutathione (GSH) and
monitoring the changes in the absorption and fluorescence
spectra. As shown in Figure 1a and 1b, the broad absorption
and emission bands at 370 and 473 nm, characteristic of 2,
undergo a red-shift to 430 and 535 nm, respectively, upon
treatment with 5.0 mM GSH. The new emission band at 535
nm produced as the result of this treatment corresponds to the
4-aminonaphthalimide derivative 4 (Figure 1c).14 Importantly,
the fluorescence enhancement at 535 nm increases linearly (R =
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respectively (Figure S4, Supporting Information). In addition,
ESI-MS spectral analysis revealed the presence of fragments
following exposure of 2 to GSH that are likewise consistent
with CPT ([M − H] = 347.1 m/z) and 4 ([M − H] = 339.2
m/z) (cf., Figure S3c).
Together, these results lead us to suggest that the disulfide
bond present in 2 is cleaved by GSH. On the basis of the
chemistry involved, this scission is expected to be followed by
intramolecular cyclization and cleavage of the neighboring
carbamate bond. This, in turn, will serve to release CPT and
produce the fluorescent product 4, as shown in Figure 2. The
Figure 3. (a) Fluorescence response of 1 (0.1 mM) with and without
GSH (0.1 mM). Excitation was effected at 430 nm. (b) CPT released
from 1 (0.1 mM) as a function of time in the presence and absence of
GSH (0.1 mM). CPT in RP-HPLC chromatograms was detected by
UV absorption using 370 nm as the interrogation wavelength. All data
were measured at 37 °C in PBS buffer (pH 7.4) containing 16% (v/v)
DMSO.
When treated with 1, a strong increase in the fluorescence
intensity was seen in the case of the U87 cells, whereas in the
case of the C6 cells only a weak fluorescence signal was seen,
even after 60 min incubation (Figure 4). In contrast, analogue
Figure 2. Proposed reaction mechanism of 1 and 2 with GSH under
physiological conditions.
fluorescence intensity is thus expected to be directly propor-
tional to the amount of CPT released from 2 (and 1) as the
result of GSH-induced disulfide bond cleavage. Because thiol
species, including GSH, are more abundant in tumor cells than
in normal cells this release strategy is appealing as an easy-to-
monitor drug delivery system (DDS) that, in due course, could
prove applicable to cancer theragnostics.16
To confirm that the pro-CPT RGD-containing conjugate 1
undergoes GSH-induced disulfide cleavage, the reaction of 1
with GSH was tested using conditions identical to those used in
the study of 2. In the case of 1, the reaction products proved to
be the expected free CPT (the payload), as well as the
naphthalimide derivative 7, as inferred from RP-HPLC and
ESI-MS analyses (Figure S6, Supporting Information; see also
Figure 2 and Scheme S1, Supporting Information). Absorption
and fluorescence spectral changes were found to be similar to
those of 2 (Figure S5, Supporting Information; see also Figure
1).
Figure 4. Confocal microscopy images of U87 and C6 cells treated
with 1. The cells were incubated with PBS containing 1 (5 μM), and
then the images were obtained at each time point (0, 30, and 60 min).
The bottom panels show an overlay of the image with a nonconfocal
phase contrast image. Cell images were obtained using excitation at
458 nm and a long-path (>505 nm) emission filter.
Further confirmation that upon disulfide bond cleavage CPT
release occurs concurrent with the observed fluorescence
changes came from a combined RP-HPLC and fluorimetric
time-dependent analysis of conjugate 1. As can be seen from an
inspection of Figure 3, upon treating conjugate 1 with GSH, the
amount of CPT released was found to correlate with the
observed increase in fluorescence intensity at 535 nm (arising
from 7). In contrast, in the absence of GSH, monitoring
solutions of 1 as a function of time revealed neither CPT
release nor an enhancement in the fluorescence intensity at 535
nm. We thus consider that the fluorescence changes at 535 nm
act as a direct off-on signal that is diagnostic of CPT release.
To demonstrate the role of the RGD moiety in guiding
conjugate 1 to αvβ3 integrin-rich tumor cells, compounds 1 and
2 were incubated with two different cell lines, namely U87 and
C6. These two cell lines were chosen because the expression
level of αvβ3 integrin in U87 cells is much higher than in C6.17
2, lacking an RGD-guiding group, displays a strong fluorescence
response with essentially identical behavior being seen for both
cell lines (Figure S7, Supporting Information). This marked
difference in emissive behavior ion the case of these two cell
lines is consistent with the appealing suggestion, namely that
the selective uptake of 1 seen in the case of the U87 cells can be
traced to integrin receptor-mediated endocytosis.
To provide further evidence for the proposed endocytic
process, the cellular uptake of 1 was studied in the presence of
an endocytosis inhibitor, okadaic acid.18 The results obtained
from these studies are shown in Figure 5. We found that the
fluorescence intensity of 1 in U87 cells decreases as the
concentration of okadaic acid is increased (0−30 nM). In
contrast, a strong fluorescence signal was observed for 2 even in
the presence of okadaic acid. Considered in conjunction with
the results shown in Figures 4 and S7, these findings provide
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rather than the ER (panel 2-A). Because the ER membrane is
contiguous with the inner nuclear membrane (INM), it may be
presumed that molecules destined for the INM diffuse through
the ER membrane.19 We, therefore, suggest that thiol-induced
disulfide cleavage occurs in the ER in the case of 1; this
cleavage serves to release the CPT molecule, which presumably
diffuses into the cell nucleus where it functions as an inhibitor
of topoisomerase I.
The efficacy of conjugates 1 and 2, and thus the inferred level
of CPT delivered, was evaluated using standard cell viability
protocols. The results are summarized in Figure 7. When the
Figure 5. Confocal microscopy images of U87 cells treated with 1 and
2. The cells were preincubated with media containing okadaic acid (0,
15, and 30 nM) for 30 min at 37 °C. The media were replaced with
PBS containing 1 and 2 (5 μM, respectively) prior to measurement.
Cell images were obtained using excitation effected at 458 nm and a
long-path (>505 nm) emission filter.
support for the design expectation that cellular uptake into U87
cells occurs via αvβ3 integrin-mediated endocytosis. The net
result is cell-dependent CPT drug release and off-on
fluorescence changes that are ascribed to thiol-induced disulfide
bond cleavage.
To identify the cellular location of 1 after αvβ3 integrin-
mediated endocytosis, colocalization experiments using endo-
plasmic reticulum (ER), lysosome, and mitochondria-selective
staining reagents were performed. As can be seen from an
inspection of Figure 6, the fluorescence ascribable to 1
colocalizes well with ERtracker (panel 1-A), but does not
match what is observed in the case of Mito- or with Lysotracker
(panels 1-B and 1-C). In contrast, the use of 2 gives rise to
fluorescence enhancement in the mitochondria (panel 2-C),
Figure 7. Cell viability of 1 and 2 at different concentrations. The U87
cells were seeded at 1 × 104 cells/well on a 96-well plate. When 80%
confluency was reached, the cells were treated with different
concentrations of 1 and 2 dissolved in DMEM media for 48 h.
After this incubation time, an MTT assay was performed. The data are
presented as mean
SD (n = 5). The statistical signification was
marked as * for p < 0.001, compared with the normal cells according
to Student's t test.
U87 cells were treated with 1 (1.0−50.0 μM), a dose-
dependent decrease in cell viability was clearly observed (49−
29% of cell viability in 1.0 × 104 cells/well). The
pharmaceutical activity of 1 is evident at 1.0 μM with a cell
viability level of 49% being reached within 48 h (Figure 7a).
Conjugate 2 was tested in an analogous manner but gave rise to
a lower degree of cytotoxicity (e.g., 76% cell viability at 1.0 μM
after 48 h; Figure 7b). These results are fully consistent with
the design expectations and provide experimental support for
the suggestion that a major pathway of intracellular delivery of
1 involves integrin receptor-mediated endocytosis. Compound
2 does not benefit from such a localization mechanism and is
less active. The superior pharmaceutical activity found for 1
relative to 2 is thus explained by the fact that the CPT released
in the ER is more potent than when it is released in the
mitochondrion.
Figure 6. Colocalization studies of 1 and 2 carried out using ER
Tracker Red (0.01 μM) (A) or Lyso Tracker Red DND-99 (0.1 μM)
(B), Mito Tracker Red FM (0.1 μM) (C) in U87 cells. Panels show
overlay of the merged images with the corresponding nonconfocal
phase contrast images. The cells were separately pretreated with the
trackers in question, and then 1 or 2 (10 μM, respectively) was added.
Fluorescence images of 1 and 2 are labeled with green signals while
those of ER-, Mito-, or Lysotracker are labeled with red signals. In the
merged image, the yellow regions highlight the colocalized areas of the
corresponding dyes, whereas the green colored areas (black arrows)
indicate the locations of 1 or 2 alone. Images of the cells were obtained
using excitation wavelengths of 458 and 543 nm, and a band-path
(505−530 nm, green signal) and a long-path (>585 nm, red signal)
emission filters, respectively.
The suggested drug delivery mechanism of 1 is shown in
Scheme 2. Conjugate 1 selectively penetrates into tumor cells
through, presumably, integrin receptor-mediated endocytosis.
In the ER, disulfide bond cleavage occurs upon exposure to
thiols. This releases the CPT and induces a concomitant
fluorescence off-on signal response within the ER. This precise
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Synthesis of 7. To a mixture of 6 (2.0 mg, 0.7 μmol) and DIPEA
(2.5 μL, 1.4 μmol) in DMF (0.5 mL) was added TSTU (1.6 mg, 5.5
μmol), and the reaction mixture was stirred at room temperature. After
2 h of stirring, c(RGDyK) (4.7 mg, 5.5 μmol) in DMF was added. The
reaction mixture was stirred under nitrogen overnight. The volatile
components were removed, and the residue was purified using HPLC
(VP-ODS, 4.6 × 150 mm for the stationary phase; buffer A, H2O;
buffer B, CH3CN for the mobile phases). Compound 7 was eluted at
5−90% of buffer B for 5−20 min and was lyophilized, affording
yellowish fluffy powder (2.0 mg, 33%). The HPLC chromatogram of 7
is shown Figure S19, Supporting Information (>98% purity). HPLC
retention time: 9.1 min, ESI-MS m/z (M+) calcd 885.9, found 886.4
(M + H+).
Scheme 2. The Integrin Receptor Is Highly Expressed on the
Surface of Tumor Cells
a
Synthesis of 5. To a mixture of 4 (1.0 g, 2.9 mmol) and
triphosgene (2.6 g, 8.7 mmol) in 20 mL of dry toluene was added
DIPEA (1.5 mL, 8.7 mmol) dropwise. The resulting solution was
heated at reflx for 3 h. After cooling to room temperature, the reaction
mixture was flushed with nitrogen gas. After removal of unreacted
phosgene gas (CAUTION: TOXIC) and neutralization in an NaOH
bath, a solution of 2,2′-dithiodiethanol (2.3 g, 14.5 mmol) in distilled
THF/DCM (v/v, 1:1) was added to the mixture. The reaction mixture
was stirred overnight. The solvent was evaporated, at which point
CH2Cl2 (100 mL) and water (100 mL) were added, and the organic
layer was collected. The CH2Cl2 layer was dried over anhydrous
MgSO4. After removal of the solvent, the crude product was purified
over silica gel using ethyl acetate/hexane (v/v, 1:1) as the eluent to
yield 5 as a yellowish oil (91.5 mg, 60%). ESI-MS m/z (M+) calcd
520.1, found 519.1 (M − H+). 1H NMR (CDCl3, 400 MHz): δ 8.54−
8.48 (m, 2 H); 8.25 (t, 2 H, J = 9.6 Hz); 7.99 (br s, 1 H); 7.69 (t, 1 H,
J = 8.3 Hz); 4.54 (t, 2 H, J = 5.9 Hz); 4.41 (t, 2 H, J = 7.4 Hz); 3.96
(br t, 2 H, J = 5.2 Hz); 3.06 (t, 2 H, J = 6.3 Hz); 2.95 (t, 2 H, J = 6.0
Hz); 2.68 (t, 2 H, J = 7.4 Hz); 2.56 (br s, 1 H); 1.42 (s, 9 H). 13C
NMR (CDCl3, 100 MHz): 170.9, 164.1, 163.6, 153.2, 139.3, 132.6,
131.5, 128.9, 126.7, 123.1, 117.7, 117.1, 81.1, 64.0, 60.7, 41.7, 37.6,
36.4, 34.0, 28.2 ppm
a
The RGD-containing conjugate 1 is thought to be selectively
internalized in the cells by receptor-mediated endocytosis. Upon
target-specific internalization, this RGD conjugate (1) provides a new
fluorescence emission at 535 nm (off-on signal) and releases CPT due
to cleavage of the disulfide bond by GSH, a species that is
overexpressed in tumor cells (vide supra).
localization leads us to propose that conjugates such as 1 could
be used to produce both an imaging and therapeutic effect in
various targeted cells. The initial demonstration of this
principle, as reported here, thus helps to define what could
be a new chapter in theragnostic drug development, namely
one where the key elements of sensing and therapy are
effectively “miniaturized” such that they function at the cellular
level.
Synthesis of 2. To a mixture of CPT (50 mg, 0.1 mmol) and
DMAP (123 mg, 0.7 mmol) in 20 mL of dry chloroform was added
triphosgene (0.2 g, 0.5 mmol) dropwise. The resulting solution was
heated for 3 h. After being cooled to room temperature, the reaction
mixture was flushed with nitrogen gas. After removal of unreacted
phosgene gas (CAUTION: TOXIC) and neutralization in an NaOH
bath, compound 5 (70 mg, 0.1 mmol), DMAP (35 mg, 0.2 mmol), and
DIPEA (25 μL, 0.1 mmol) were added to the mixture. The reaction
mixture was stirred overnight. The solvent was evaporated, at which
point CH2Cl2 (100 mL) and water (100 mL) were added, and the
organic layer was collected. The CH2Cl2 layer was dried over
anhydrous MgSO4. After removal of the solvent, the crude product
was purified over silica gel using ethyl acetate/hexane (v/v, 4:1) as the
eluent to yield 2 as a yellowish solid (60 mg, 47%). ESI-MS m/z (M+)
CONCLUSIONS
■
We have described the synthesis, characterization, spectro-
scopic properties, and biological applications of 1, an RGD
peptide-appended naphthalimide pro-CPT agent. Reaction of 1
with GSH induces disulfide bond cleavage followed by
intramolecular cyclization and cleavage of the neighboring
carbamate bond. This serves to release the CPT and leads to
fluorescence enhancement. From in vitro cellular experiments,
we conclude that conjugate 1, bearing a cyclic RGD peptide
subunit, is selectively internalized into the tumor U87 cells via
αvβ3 integrin-mediated uptake as evidenced by competitive
okadaic acid treatment. This uptake permits an off-on
observation of the fluorescence changes (signal monitored at
535 nm) upon reaction with GSH (a species overexpressed in
cancer cells). In the case of 1, disulfide bond cleavage occurs in
the ER to release the CPT, which in turn diffuses into the
nucleus of the U87 cell. This would be an ideal theragnostic
delivery system that provides specific tumor targeting and
which permits the concentration of free drug to be monitored
by fluorescence signaling changes.
1
calcd 894.2, found 923.2 (M − H+). H NMR (CDCl3, 400 MHz): δ
8.57−8.49 (m, 2 H); 8.38 (s, 1 H); 8.30 (d, 1 H, J = 8.4 Hz) 8.22−
8.16 (m, 2 H); 8.05 (s, 1 H); 7.95 (d, 1 H, J = 8.2 Hz); 7.86−7.82 (m,
1 H); 7.71−7.67 (m, 2 H); 7.32 (s, 1 H); 5.37−5.03 (m, 4 H); 4.58−
4.30 (m, 6 H); 3.07−2.95 (m, 4 H); 2.70 (t, 2 H, J = 7.4 Hz); 2.27−
2.02 (m, 2 H); 1.43 (s, 9 H); 0.98 (t, 3 H, J = 7.4 Hz). 13C NMR
(CDCl3, 100 MHz): 170.7, 167.4, 164.0, 163.5, 157.1, 153.9, 152.9,
152.1, 148.9, 146.5, 145.6, 139.4, 132.4, 131.3, 130.9, 129.6, 128.9,
128.4, 128.3, 126.8, 126.5, 123.1, 123.0, 120.0, 117.6, 116.5, 95.9, 80.8,
78.4, 66.9, 66.5, 63.0, 50.0, 37.6, 36.5, 36.3, 33.9, 31.8, 28.1, 7.7 ppm
Synthesis of 1. A solution of TFA/DCM (v/v, 1:1) was added to
compound 2 (9 mg, 0.1 mmol). After 30 min of stirring, the volatiles
were removed under reduced pressure. The crude product was
confirmed by MS analysis (Figure S14, Supporting Information) and
then used directly for the next reaction. The crude product was
dissolved in DMF (0.5 mL), and DIPEA (3.5 μL, 0.2 mmol) was
added. TSTU (3.0 mg, 0.1 mmol) was then added, and the mixture
was stirred at room temperature. After 2 h of stirring, c(RGDyK) (8.5
mg, 0.1 mmol) in DMF was added. The reaction mixture was stirred
under nitrogen overnight. Solvent was removed, and the residue was
purified using HPLC (C18, 3.5 μm, 4.6 × 150 mm for the stationary
EXPERIMENTAL SECTION
■
β-Ala-OtBu15 and naphthalimide derivatives 3, 4, and 614 were
prepared by procedures reported earlier. Peptide c(RGDyK) was
purchased from FutureChem Co., Ltd.
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phase; buffer A, H2O containing 0.1% TFA; buffer B, CH3CN
containing 0.1% TFA were used as the mobile phases). Target 1 was
eluted at 5−70% of buffer B for 5−30 min and was lyophilized, which
afforded a yellowish fluffy powder (8.0 mg, 28%). The HPLC
chromatogram of 1 is shown Figure S16, Supporting Information
(>97% purity). HPLC retention time: 29.0 min, MALDI-TOF MS m/
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the CRI project (20120000243) of
the National Research Foundation of Korea. J.L.S. thanks the
WCU (World Class University) program (R32-2010-000-
10217-0) administered through the National Research
Foundation of Korea funded by the Ministry of Education,
Science and Technology (MEST).
+
z (M+) calcd 1440.5, found 1440.1 (M ).
Synthetic Materials and Methods. All reactions were carried out
under nitrogen atmosphere. Silica gel 60 (Merck, 0.063−0.2 mm) was
used for column chromatography. Analytical thin layer chromatog-
raphy was performed using Merck 60 F254 silica gel (precoated sheets,
0.25 mm thick). 1H and 13C NMR spectra were recorded in CDCl3 or
CD3OD (Cambridge Isotope Laboratories, Cambridge, MA) on
Varian 300 and 400 MHz spectrometers. All chemical shifts are
reported in ppm value using the peak of residual proton signals of
TMS as an internal reference. Reverse-phase HPLC experiments were
conducted using a Agilent HPLC (Agilent 1100 series) with a Zorbax
C18 (3.5 μm, 4.6 × 150 mm), Shim-pack VP-ODS (4.6 × 150 mm)
column for analytical, Waters HPLC (Waters 600) with XBridge C18
(5 μm, 19 × 150 mm) column for preparative separation. The flow
rates for analytical and preparative HPLC were 1.0 mL/min and 6.0
mL/min, respectively. For the mobile phase, buffer A (water with 0.1%
v/v TFA) and buffer B (acetonitrile with 0.1% v/v TFA) were used to
provide the solvent gradient. ESI mass spectrometric analyses were
carried out using an LC/MS-2020 Series (Shimadzu) instrument.
MALDI-TOF mass spectral analyses were carried out at the National
Center for Interuniversity Research Facilities in Seoul National
University.
Spectroscopic Materials and Methods. Stock solutions of
biologically relevant analytes [thiols, Val, Tyr, Thr, Tau, Ser, Pro, Phe,
Met, Lys, Leu, Ile, His, Gly, Gluc, Glu, Gln, Asp, Asn, Arg, Ala, Trp,
Zn(II), Na(I), Mg(II), K(I), Fe(III), Fe(II), Cu(II), and Ca(II)] were
prepared in triple distilled water. Stock solutions of 1 and 2 were also
prepared in triple distilled water. All spectroscopic measurements were
performed under physiological conditions (PBS buffer containing 16%
(v/v) of DMSO, pH 7.4, 37 °C). Absorption spectra were recorded on
an S-3100 (Scinco) spectrophotometer, and fluorescence spectra were
recorded using an RF-5301 PC spectrofluorometer (Shimadzu)
equipped with a xenon lamp. Samples for absorption and emission
measurements were contained in quartz cuvettes (3 mL volume).
Excitation was provided at 430 nm with excitation and emission slit
widths of 3 and 1.5 nm, respectively.
Preparation of Cell Cultures. The C6 rat glioma and U87 human
glioma cells were maintained and subcultured every other day in
DMEM supplemented with 10% fetal bovine serum and 1%
penicillin−streptomycin at 37 °C in 5% CO2 and 95% ir environment.
The cells were seeded on 24-well plates and stabilized for overnight.
Compounds 1 and 2 were applied to the cells to monitor their uptake
and drug release as discussed in the main text above. In some
experiments, the cells were incubated with media containing okadaic
acid, Mito-, Lyso-, or ERtracker prior to treatment with 1 or 2. Then
the cells were briefly washed with 1 mL of PBS and were then treated
with 1 or 2 in PBS. After incubation, residual quantities of 1 or 2 that
were not taken up in the cells were removed by washing the cells three
times with PBS before the cells were placed in 1 mL of a PBS solution.
Fluorescence images were taken using a confocal laser scanning
microscope (Zeiss LSM 510, Zeiss, Oberko, Germany).
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ASSOCIATED CONTENT
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* Supporting Information
Additional spectra (UV/vis absorption, fluorescence, NMR,
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AUTHOR INFORMATION
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Corresponding Author
kangch@khu.ac.kr; jongskim@korea.ac.kr
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