A triple-targeting delivery system carrying two anticancer agents

Article abstract of DOI:10.1039/d1ob01089a

To improve tumor selectivity, a triple-targeting delivery system (Oct-FK(PBA-Az)-Dox) carrying two anticancer agents (apoptozole (Az) and doxorubicin (Dox)) was designed and synthesized. The results showed that both anticancer agents in Oct-FK(PBA-Az)-Dox are liberated in the presence of both H₂O₂and cathepsin B, which are normally present at high levels in tumors.

Full text of DOI:10.1039/d1ob01089a

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A triple-targeting delivery system carrying two
anticancer agents†
Cite this: DOI: 10.1039/d1ob01089a
Hui Li, Chang-Hee Lee and Injae Shin
*
Received 5th June 2021,
Accepted 1st July 2021
DOI: 10.1039/d1ob01089a
rsc.li/obc
To improve tumor selectivity, a triple-targeting delivery system utilized as a ligand for targeting SSTRs in delivery systems.
(Oct-FK(PBA-Az)-Dox) carrying two anticancer agents (apoptozole This synthetic peptide recognizes SSTR2 relatively selectively
(Az) and doxorubicin (Dox)) was designed and synthesized. The over other SSTRs and its conjugates with anticancer or
results showed that both anticancer agents in Oct-FK(PBA-Az)-Dox imaging agents enter cells via SSTR-mediated endocytosis.6
are liberated in the presence of both H2O2 and cathepsin B, which Thus, SSTRs were chosen as the 1st target of the delivery
are normally present at high levels in tumors.
system developed in this study (Fig. 1).
Several previous studies have shown that H2O2 is present at
high levels in most types of tumors.7 On this basis, H2O2 was
chosen as the 2nd target of the new targeting delivery system.
Furthermore, the cysteine protease cathepsin B, present in
lysosomes, is upregulated in various tumors, and is involved in
tumor invasion and metastasis.8 This protease is also often
upregulated in premalignant lesions and is involved in local
invasive stages of tumors.8 Accordingly, the lysosomal cathep-
sin B was selected as the 3rd target of our new delivery system.
In previous studies, we showed that apoptozole (Az), an
inhibitor of Hsp70, induces lysosomal membrane permeabili-
zation and thereby enhances lysosome-mediated apoptotic
cancer cell death (Fig. 2a).9 In addition, doxorubicin (Dox, an
inhibitor of topoisomerase II) displays potent anticancer
activity against various cancer cells.10 Furthermore, we also
showed that the combined treatment of Az and Dox promotes
enhanced apoptosis of cancer cells.9a As a result, Az and Dox
Over the past several decades, a variety of potential chemother-
apeutic agents have been developed to treat tumors.1 A major
goal of these efforts is the discovery of safe and efficacious
anticancer drugs, which have enhanced selectivities against
cancer cells over normal cells and, thus, display minimal side
effects.2 In an effort to improve the tumor selectivity of anti-
cancer agents, drug delivery systems that target cell-surface
receptors and/or enzymes overexpressed in cancer cells have
been developed.3 This approach contributes to improving the
selectivity of cancer chemotherapeutic agents. However, the
tumor selectivities of drug delivery systems developed to date
are still not high and their cross-reactivity with normal cells
remains problematic. To overcome the limitations of current
drug delivery systems and further improve tumor selectivity,
we designed and synthesized the new triple-targeting delivery
system Oct-FK(PBA-Az)-Dox, which contains two anticancer
agents. The results of this investigation showed that both anti-
cancer agents in this substance are released in the presence of
both H2O2 and cathepsin B, each of which is produced at high
levels in cancer cells.
Somatostatin receptors (SSTRs) are upregulated in many
types of tumors.4 Particularly, SSTR2 is most frequently over-
expressed on the surfaces of various cancer cells and, thus, is a
good target for agents that selectively deliver drugs to tumors.
Octreotide (Oct), a synthetic cyclic octapeptide which pharma-
cologically mimics the natural ligand somatostatin,5 has been
Department of Chemistry, Yonsei University, Seoul 03722, Republic of Korea.
E-mail: injae@yonsei.ac.kr
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1ob01089a
Fig. 1 Schematic representation of the cancer cell-specific triple-tar-
geting delivery system carrying two anticancer agents.
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Fig. 2 (A) Structure of the triple-targeting delivery system (Oct-FK(PBA-Az)-Dox). (B) Sequential cleavage of Oct-FK(PBA-Az)-Dox by H2O2 and
cathepsin B to liberate Az and Dox.
were utilized as anticancer agents in the new triple-targeting which reacted with p-nitrophenyl chloroformate to afford a
delivery system.
mixed carbonate 6. To prepare the Az-conjugated compound
Utilizing the strategy and components described above, we 7′, 6 was reacted with Az and the crude mixture was subjected
designed the triple-targeting delivery system Oct-FK(PBA-Az)- to flash column chromatography. It was found that the phenyl-
Dox (Fig. 2a). The system consists of (1) Oct acting as an SSTR boronate ester moiety in 7′ is partially hydrolyzed to form
ligand, (2) a H2O2-responsive phenylboronic acid (PBA) moiety, phenylboronic acid (PBA) during purification by flash column
(3) a dipeptide Phe-Lys (FK) serving as a cathepsin B substrate, chromatography. Thus, both TBS and pinacol ester groups in
and (4) two anticancer agents. Because they are covalently 7′ were removed under weakly acidic conditions to afford 7
linked to the delivery system, both Dox and Az in Oct-FK that was readily purified by flash column chromatography.
(PBA-Az)-Dox are expected to have almost no antitumor
activity. It is anticipated that following its binding through Oct
The Dox-conjugated intermediate 10 was synthesized from
which was prepared by using known procedure
8
a
to upregulated SSTRs, Oct-FK(PBA-Az)-Dox will be internalized (Scheme 2).6a Specifically, the benzyl alcohol moiety in 8
into lysosomes of cancer cells via SSTR-mediated endocytosis. reacted with p-nitrophenyl chloroformate to form a mixed car-
The PBA moiety of Oct-FK(PBA-Az)-Dox will then be cleaved by bonate, which upon treatment with doxorubicin produced 9.
reaction with H2O2 present at high levels in cancer cells, con- The 4-methoxytrityl (MTT) protecting group in 9 was carefully
comitantly releasing the anticancer agent Az and producing removed under weakly acidic conditions to yield intermediate
Oct-FK-Dox (Fig. 2b). Subsequently, the cathepsin B induced 10. It should be noted that the glycosidic linkage in Dox is
cleavage of the C-terminus of the dipeptide in Oct-FK-Dox will cleaved when more strongly acidic conditions are employed.
lead to the release of Dox. Free Az and Dox will kill cancer cells
effectively and selectively.
Coupling of 7 to 10 was then accomplished by initially acti-
vating the hydroxyl group in 7 with p-nitrophenyl chlorofor-
To assess this proposal, the triple-targeting delivery system, mate and subsequent reaction of the resulting carbonate with
Oct-FK(PBA-Az)-Dox, was prepared by using the route shown in 10 to produce 11 (Scheme 3). Finally, the alkynylated com-
Schemes 1–3. In the sequence, the two hydroxyl groups in the pound 11 was subjected to click chemistry with azide-
benzaldehyde derivative 1 were first protected with TBS to appended Oct (Oct-N3), which was synthesized by using con-
generate 2 (Scheme 1). The phenolic TBS group in 2 was selec- ventional
Fmoc/tBu
solid-phase
peptide
synthesis
tively removed by reaction with NaH to form a mono-TBS pro- (Scheme S1†),6a to form Oct-FK(PBA-Az)-Dox. The reaction
tected product 3.11 The reaction of 3 with 4-bromomethyl- mixture containing Oct-FK(PBA-Az)-Dox was purified by pre-
phenylboronic acid pinacol ester under basic conditions gener- parative HPLC and analysed by MS. Also, all intermediates in
ated boronate 4. Reduction of 4 with NaBH4 produced 5, the synthetic pathway were characterized by NMR and MS.
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Scheme 1 Synthesis of compound 7.
Scheme 2 Synthesis of compound 10.
We then explored the release of Dox and Az from Oct-FK FK-OH, were liberated from Oct-FK(PBA-Az)-Dox by sequential
(PBA-Az)-Dox by reversed-phase HPLC analysis of mixtures of treatment with H2O2 and cathepsin B. In contrast, free Dox
Oct-FK(PBA-Az)-Dox treated with H2O2 and/or cathepsin B was not generated when Oct-FK(PBA-Az)-Dox was sequentially
(Fig. 3). The findings showed that the incubation of Oct-FK incubated with H2O2 and cathepsin B in the presence of
(PBA-Az)-Dox with H2O2 leads to the production of Az and CA-074-Me, a selective inhibitor of cathepsin B.12 The find-
Oct-FK-Dox (Fig. 2B). However, neither Az nor Dox was ings clearly indicate that both Dox and Az are released from
released when a mixture of Oct-FK(PBA-Az)-Dox was treated the new delivery system only in the presence of H2O2 and
with cathepsin B, indicating that this substance possessing a cathepsin B.
PBA-capped lysine is not hydrolytically cleaved by the
In conclusion, we designed and prepared a novel delivery
enzyme. Importantly, both Dox and Az, along with Oct- system containing a triple-targeting site composed of a tumor-
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Scheme 3 Synthesis of Oct-FK(PBA-Az)-Dox.
selective SSTR ligand, a peptide substrate for cathepsin B and
a H2O2-responsive PBA moiety, along with two anticancer
agents (Az and Dox). We demonstrated that both of the linked
anticancer agents are released from Oct-FK(PBA-Az)-Dox only
in the presence of H2O2 and cathepsin B. Cell and in vivo
studies are now underway to assess the usefulness of a novel
delivery system for the treatment of cancer.
Conflicts of interest
The authors declare no competing interests.
Acknowledgements
This study was supported financially by the National Research
Foundation of Korea (grant no. 2020R1A2C3003462).
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