Biotin-guided anticancer drug delivery with acidity-triggered drug release

Article abstract of DOI:10.1039/c5cc03003j

A novel biotin-guided anticancer drug delivery system, prodrug 9, consisting of biotin, nitrobenzene, and doxorubicin, with acid-triggered drug releasing capability was synthesized. Its cellular uptake and anticancer activity are selective to the HepG2 cell line over the WI-38 cell line, as revealed by fluorescence confocal microscopic experiments and MTT assay.

Full text of DOI:10.1039/c5cc03003j

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DOI: 10.1039/C5CC03003J
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Biotin-guided anticancer drug delivery with acidity-
triggered drug release
Cite this: DOI: 10.1039/x0xx00000x
Soyeon Park,a Eunjin Kim,b Won Young Kim,a Chulhun Kang,*,b and Jong Seung
Kim*,a
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
induced electron transfer (PET) to induce fluorescence quenching.10
A novel biotin-guided anticancer drug delivery system,
prodrug 9, consisting of biotin, nitrobenzene, and
doxorubicin, with acid-triggered drug releasing capability
was synthesized. Its cellular uptake and anticancer activity
are selective to the HepG2 cell line over the WI-38 cell line, as
revealed by fluorescence confocal microscopic experiments
and MTT assay.
The PET effect is then diminished by cleavage of the bond between
the fluorophore and the prodrugꢀbearing quencher in acidic
conditions enhancing the fluorescence of the drug (Dox in this case).
In this context, we herein present a novel DDS with minimized
complexity in size, but still capable of targeting tumor sites and
releasing anticancer drugs under the tumor tissue’s acidic conditions.
The DDS we have designed is composed of three parts: a
fluorescence quencher moiety, a tumorꢀtargeting group, and an acidꢀ
triggered releasable anticancer drug, as shown in Scheme 1.
Nitrobenzene is adopted as the quencher. Biotin, which has often
been used as a cancerꢀtargeting moiety, selectively guides the DDS
to tumor cells and markedly enhances the water solubility of the
prodrug.11 Dox, linked to the nitrobenzene–biotin scaffold by an
acidꢀcleavable hydrazone bond, is employed as the pHꢀresponsive
part. The combination of these parts is expected to provide selective
tumor targeting and Dox release with dramatic fluorescence
enhancement upon activation by the acidic environment of the tumor
cells (Scheme 1), allowing for the realꢀtime assessment of its drug
release efficiency by imaging the changes in the fluorescence
intensity in situ.
Drug delivery systems (DDS) that are capable of selective targeting
and realꢀtime monitoring of drug release have drawn much attention
owing to their capability of reporting the drug delivery location and
delivered amount in situ,1 allowing determination of the drug
delivery kinetics. Moreover, since these drug delivery systems can
be modified by specific external or internal stimuli to selectively
release their drugs at malignant cancer cells, the cancer cells can be
selectively killed without affecting normal cells, minimizing
undesirable side effects.
Diverse drug release mechanisms for DDS have been reported
that utilize the different environments of tumor cells compared to
normal cells, e.g., lower pH,2 hypoxia,3 overexpressed enzymes,4 or
higher levels of thiols.5 Among them, the lower pH environment
draws particular interest as an anticancer drugꢀreleasing mechanism
because both primary and metastasized tumors have a lower pH than
nonꢀmalignant tissues.6 Thereby, a DDS with acidꢀtriggered drug
release capability can selectively deliver drugs to tumor tissues.
Furthermore, this system may have an additional advantage of
minimizing nonꢀselective delivery since the blood stream has a
relatively high pH and the DDS would not release drug molecules
prior to reaching the target tissues.
Monitoring drug release from DDSs has often been performed
through Forster resonance energy transfer (FRET),7 aggregationꢀ
induced emission (AIE),8 or a quenching system,9 consisting of a
fluorophore, sometimes with a quencher. These ways usually have
Scheme 1 Illustration of functioning mechanism of acidꢀactivated
complicated chemical structures, often resulting in increased tumorꢀtargeted prodrug
hydrophobicity accompanied by decreased bioavailability, as well as
9 at the tumor site.
poor control of drug release. Therefore, introducing a minimally
sized quencher moiety on the fluorescent drug molecule, e.g.,
doxorubicin (Dox) would be an ideal way to obtain efficient
monitoring capabilities in a DDS. Nitrobenzene is a wellꢀknown
small fluorescent quencher that is able to lower the LUMO level of
aromatic molecules, making it an excellent acceptor in photoꢀ
Prodrug 9 was prepared following the synthetic route shown in
Scheme 2. First, the biotin derivative 2 was synthesized in two steps,
and nitrobenzene moiety 6 was prepared in four steps from 2ꢀ
hydroxyꢀ4ꢀnitrobenzoic acid. Compounds 2 and 6 were then coupled
by a click reaction in DMF to produce compound 7. Finally, prodrug
9 was synthesized by attaching Dox to the biotinꢀconjugated
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nitrobenzene moiety 7 in two steps. The structures of all the
1
synthesized compounds were confirmed by H, 13C NMR, and ESIꢀ
mass spectrometry (see Fig. S1–S21).
O
O
NH
O
O
OH
HN
NH
N3
NaN3, H2O
60oC
S
Cl
N3
O
HN
HO
HO
EDCI, DMAP, DMF, rt
S
2
1
O
O
Br
DMAP, DCC
THF, reflux
O
OH
OH
+
K2CO3, ACN, rt
O2N
OH
O2N
OH
3
O
O
O
H
O
N
O
TFA, DCM
NH2NHBoc
HATU, DIPEA, DMF
N
O
OH
O
H
O
O2
N
O2N
O
O2N
4
5
6
O
H
N
O
S
N
H
2, Ascorbic acid sodium salt
NH
CuSO4.5H2O, DMF, Ar, rt
O
O
O2
N
O
N
O
H
N
O
S
N
N
Fig. 1 (a) Fluorescence emission spectra of free doxorubicin and
7
O
O
prodrug
9 (5 ꢁM); λex = 501 nm. (b) Fluorescence changes of prodrug 9
NH2
(5 ꢁM) at various times (0–400 min) at pH 7.4 and (c) at pH 5.0 (0–400
min, overnight); λex = 501 nm. (d) Fluorescence intensity changes using
N
H
TFA, DCM
Doxorubicin
TFA, MeOH
NH
O
O2
N
N
N
O
H
prodrug
9 (5 ꢁM) at 558 nm at various times (0–400 min) at pH 7.4, pH
O
N
N
6.5, and pH 5.0. All spectra were recorded in PBS buffer at 37°C. FL:
8
fluorescence.
NH2
O
HO
8 (M + K+), respectively (graph = cationic measurement). This
provided further strong evidence that prodrug 9 releases strongly
fluorescent Dox through cleavage of the hydrazone bond of prodrug
9 in acidic conditions.
O
OH
OH
O
O
OH
O
O
OH
N
O
O
S
N
H
NH
Next, we investigated the antitumor efficacy of prodrug 9 towards
cancer cells. To evaluate the cytotoxicity of prodrug 9, MTT assay
was carried out in two different cell lines, biotin receptorꢀpositive
HepG2 cells and negative WIꢀ38 cells as shown in Fig. 2. Upon
treatment of free Dox alone, both cell lines were highly susceptible
to the drug treatment (Fig. 2(a)). In the case of prodrug 9 treatment,
the cytotoxicity was reduced against both cell lines compared to the
cytotoxicity of Dox alone, but the HepG2 cells were more
susceptible to the prodrug than the WIꢀ38 cells (Fig. 2(b)). This
result demonstrates that prodrug 9 can exhibit antitumor activity in
biotin receptorꢀpositive cells, the HepG2 cell line in this study,
obviously due to selective uptake by the biotin receptor.
O
O2N
N
N
O
H
N
N
9
Scheme 2 Synthetic route of the acidꢀactivated tumorꢀtargeted prodrug
9.
To examine whether the hydrazone bond between the biotin–
nitrobenzene conjugate and Dox of prodrug 9 is cleavable in acidic
environments, the fluorescence properties of prodrug 9 were
measured in different pH conditions. In a neutral solution (pH 7.4),
compound 9 exhibited much weaker emission peak at 558 nm than
did free Dox at the same concentration as shown in Fig. 1. The
decreased fluorescence intensity of the Dox unit in 9 is presumably
quenched by the nitrobenzene moiety through PET mechanism. As
predicted, prodrug 9 shows no change in its fluorescence spectrum
for at least 7 h under normal physiological pH conditions (pH 7.4),
whereas the fluorescence intensity gradually increased in low pH
conditions (pH 5.0) in a timeꢀdependent manner (Fig. 1b and c). The
fluorescence intensity at 558 nm clearly depends on the acidity of
the solution (Fig. 1d). These results can envisage that the hydrazone
bond of prodrug 9 could remain stable during blood circulation, then
be cleaved and release Dox upon arrival at tumor cells which in
general are in an acidic environment. Such a tendency demonstrates
that the cleavage of the hydrazone bond will release the active drug,
Dox, intracellularly and simultaneously enable us to monitor the
distribution of the drug through the fluorescence enhancement of
Dox.
Fig. 2 Cytotoxicity of (a) free doxorubicin and (b) prodrug
9 in HepG2
cells and WIꢀ38 cells after 48ꢀh incubation.
To get insight into the selective uptake of prodrug 9 by biotin
receptorꢀpositive cells over biotin receptorꢀnegative cells, confocal
microscopy was carried out for both Dox and prodrug 9 using
HepG2 and WIꢀ38 cells. As shown in Fig. 3, after 1 h incubation
with free Dox, the fluorescence of Dox was observed in both HepG2
cells and WIꢀ38 cells, with little bit more intense fluorescence in the
WIꢀ38 cells. Conversely, in the presence of prodrug 9, the
fluorescence of Dox released from prodrug 9 was much stronger in
the HepG2 cells than that in WIꢀ38 cells. These confocal
microscopic images provide evidence of prodrug 9 being selectively
In order to confirm the release of Dox from 9 in acidic conditions,
compound 9-containing solution incubated at pH 5.0 for 3 days was
analyzed by mass spectrometry, and the data are shown in Fig. S22.
We observed molecular ion peaks of m/z 566.35 and m/z 587.25,
which correspond to the molecular ion peaks of Dox (M + Na+) and
2 | J. Name., 2012, 00, 1-3
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DOI: 1C0.O10M39M/CU5CNCI0C3A00T3IOJ N
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show that prodrug 9 preferentially targets biotinꢀpositive cancer cells,
where the prodrug is accumulated and is activated by acidic
conditions to deliver the drug (Dox) to the cancer cells with
fluorescence enhancement. The delivery of the Dox may result in
apoptosis and eventually inhibition of tumor growth in tumor
models. Such theranostic strategies based on acidityꢀtriggered drug
release with markedly fluorescence changes could be simple and
efficient ways for the early diagnosis and precise treatment of
cancer.
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Fig. 3 Confocal microscopy images of HepG2 and WIꢀ38 cells treated
with free Dox and prodrug
excitation wavelength of 488 nm. Dox: doxorubicin.
9 at 5 μM for 1 h. Image was acquired at an
<
Graphical Abstract>
In conclusion, a novel biotinꢀguided anticancer drug delivery
system, prodrug 9, consisting of biotin, nitrobenzene, and Dox, was
synthesized. The drug release was mediated by acidꢀtriggered
hydrazone bond cleavage and accompanied by the enhanced
fluorescence intensity of Dox which was initially quenched by a
nitrobenzene moiety. In the cytotoxicity experiment, biotin receptorꢀ
positive HepG2 cells were found to be more sensitive to prodrug 9
than biotin receptorꢀnegative WIꢀ38 cells. Hence, prodrug 9 can be a
promising candidate as a selective tumorꢀtargeted drug delivery
system with applications in anticancer therapy and imaging as a
theranostic tool.
This work was funded by CRI project (No. 2009ꢀ0081566, J.S.K.)
and the Basic Science Research Program through the National
Research Foundation of Korea (2014R1A2A1A11052325, C.K.).
Notes and references
a Department of Chemistry, Korea University, Seoul 136-701, Korea. E-mail:
jongskim@korea.ac.kr; Fax: +82-2-3290-3121
b
School of East-West Medical Science, Kyung Hee University, Yongin
446-701, Korea. E-mail: kangch@khu.ac.kr.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
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