Folate Receptor Targeting and Cathepsin B-Sensitive Drug Delivery System for Selective Cancer Cell Death and Imaging

Article abstract of DOI:10.1021/acsmedchemlett.0c00031

In this work, a folate receptor (FR)-mediated dual-targeting drug delivery system was synthesized to improve the tumor-killing efficiency and inhibit the side effects of anticancer drugs. We designed and synthesized an FR-mediated fluorescence probe (FA-Rho) and FR-mediated cathepsin B-sensitive drug delivery system (FA-GFLG-SN38). FA-GFLG-SN38 is composed of the FR ligand (folic acid, FA), the tetrapeptide substrate for cathepsin B (GFLG), and an anticancer drug (SN38). The rhodamine B (Rho)-labeled probe FA-Rho is suitable for specific fluorescence imaging of SK-Hep-1 cells overexpressing FR and inactive in FR-negative A549 and 16-HBE cells. FA-GFLG-SN38 exhibited strong cytotoxicity against FR-overexpressing SK-Hep-1, HeLa, and Siha cells, with IC50 values of 2-3 μM, but had no effect on FR-negative A549 and 16-HBE cells. The experimental results show that the FA-CFLG-SN38 drug delivery system proposed by us can effectively inhibit tumor proliferation in vitro, and it can be adopted for the diagnostics of tumor tissues and provide a basis for effective tumor therapy.

Full text of DOI:10.1021/acsmedchemlett.0c00031

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Letter
Folate Receptor Targeting and Cathepsin B Sensitive Drug
Delivery System for Selective Cancer Cells Death and Imaging
Xiangmei Jin, Jun Zhang, Xiaoyan Jin, Lan Liu, and Xizhe Tian
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00031 • Publication Date (Web): 18 May 2020
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Folate Receptor Targeting and Cathepsin B Sensitive Drug Delivery
System for Selective Cancer Cells Death and Imaging
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Xiangmei Jin†, Jun Zhang‡, Xiaoyan Jin†, Lan Liu‡*, and Xizhe Tian†*
†Department of Chemistry, Yanbian University, Yanji 133000, Jilin, China
‡Department of Pathology, Affiliated Hospital of Yanbian University, Yanji 133000, Jilin, China
Supporting Information Placeholder
ABSTRACT: Folate receptor (FR)-mediated dual-targeting drug delivery system are synthesized to improve tumor-killing
efficiency and inhibit the side-effects of anticancer drugs. In this paper, we designed and synthesized FR-mediated fluorescence
probe (FA-Rho) and FR-mediated cathepsin B sensitive drug delivery system (FA-GFLG-SN38). FA-GFLG-SN38 is composed of
ligand (folic acid, FA) of folate receptor (FR), tetrapeptide substrate (-GFLG-) for cathepsin B and an anticancer drug (SN38). FA-
Rho is suitable for specific fluorescence imaging of SK-Hep-1 cells to overexpress FR, and inactive in FR negative A549 and 16-
HBE cells. FA-GFLG-SN38 exhibited strong cytotoxic against FR-overexpressed SK-Hep-1, HeLa and Siha cells with IC50 of 2-3
μM, with no effects on FR negative A549 and 16-HBE cells. The experimental results show that FA-CFLG-SN38 drug delivery
system proposed by us could effectively inhibit tumor proliferation in vitro, it can be adopted for the diagnostics of tumor tissues
and provide a basis for effective tumor therapy.
KEYWORDS: Drug delivery system; Folate receptor; Cathepsin B; Rhodamine B; SN38
Tumor tissues are distinguished from normal by atypical
growth, metabolic disorders, aberrant gene expression, and
abnormal expression and activity of related enzymes in itself
and surroundings.1 These characteristics lead to abnormal
proliferation and metastasis, as well as weaken the antitumor
efficacy of conventional treatments. 2 Ehrlich was the first to
put forward the option of targeted drug therapy for improving
diagnosis and treatment of malignant tumors.3 An ideal drug
delivery system distributes antitumor drugs specifically to
target cells, tissues or organs to achieve selective drug
delivery, controlled release, and toxicity reduction.4,5 Tumor
receptor-mediated targeted therapies include those involving
folate receptor (FR),6 epidermal growth factor receptor,7 sialic
acid glycoprotein receptor,8 glycyrrhetinic acid receptor,9 and
transferrin receptor systems.10 In contrast to lightly expressed
in normal tissues, FR is over two orders of magnitude higher
expressed on the surface of tumor cells such as ovarian cancer
and colon cancer.11 FA selected as the raw material for the
synthesis for tumor-targeting drug delivery systems, due to its
characteristics of relatively low cost, non-toxicity, low
immunity, stability and easy modification. Xu et al. prepared a
glutathione-responsive prodrug based on FA and camptothecin
(CPT) via disulfide bonds. The resulting FA-CPT prodrug
showed higher specificity and cytotoxicity in FR-positive KB
tumor cells rather than FR-negative A549 tumor cells.12
Therefore, FR-mediated active drug delivery system could
improve cancer-targeting ability and reduce the side-effects of
anticancer drugs.13
7-Ethyl-10-hydroxycamptothecin (SN38) is an active
metabolite of irinotecan,14 and also the substrate of p-
glycoproteins, multidrug resistance-associated proteins 2 and
breast cancer resistance protein efflux pumps.15 SN38 exerts
strong inhibitory activity on DNA topoisomerase I with 100-
1000 times greater than irinotecan in cytotoxicity, 16 and it is
used as an anticancer drug against malignant tumors in vitro17,
such as colorectal, lung, lymphoma, gastric, cervical, and
ovarian cancers.18,19 Its clinical application is limited, however,
by poor water solubility and instability.20 The water solubility
of SN38 could be improved through several modifications,
including liposome formulation21, antibody drug conjugates22,
and PEG functionalization23. Currently, several delivery
systems for irinotecan and its active metabolite SN38 have
been developed, and the delivery system of liposome
preparation SN38 (LE-SN38) has been studied in clinical
trials.24 LE-SN38 has been evaluated for mCRC following
oxaliplatin progression in phase ii studies, and the results
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showed that LE-SN38 had acceptable toxicity, but its
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1) FA plays a targeting role in drug-specific guidance to FR-
positive cancer cells;11 2) CTSB, which is overexpressed in
cancer cells, is used to release SN38 under conditions of weak
acidity to improve selectivity and reduce the side-effects of
antitumor drugs;30 3) a relatively long oligoethyleneglycol
tether increases the solubility of drugs, allowing longer
circulation owing to reduction in renal clearance, enzymatic
degradation and immune cell recognition of drugs.23 We
anticipated that SN38 is inactive when linked to the FR-
mediated delivery system. The FA-GFLG-SN38 drug delivery
system binds to FR on the surface of cancer cells, and it is
internalized into cells via FR-mediated endocytosis. Then, the
drug released via a 1,6-elimination process and become active
within the cells when the C-terminus of the tetrapeptide
chain(-GFLG-) in the system cleaved by cancer cells
overexpressing CTSB, thus resuming therapeutic activity. In
addition, the FA-Rho fluorogenic probe without a CTSB-
responsive tetrapeptide was prepared. Although non-cleavable
conjugate internalized into cells via FR-mediated, and
rhodamine B (Rho) would be accumulated in lysosomes due to
the lack of a tetrapeptidic substrate of CTSB. In the research
described below, we synthesized and explored the
effectiveness of the dual target delivery system. The results
suggest that the new dual-target delivery system can indeed be
used to selective killing or image cancer cells.
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therapeutic activity can not reach the prescribed criteria.
Therefore, the development of an effective SN38 drug
delivery system has important clinical significance.
Existing types of tumor microenvironment stimulation
include responses to pH, reduction, oxidation, and enzymes.25-
27
Tumor tissues generate abnormal expression or activity of
specific enzymes, such as matrix metalloproteinases,28
cytosolic phospholipase A2,29 and cathepsin B (CTSB).30
CTSB as a potential marker for tumor screening and treatment
target, has attracted extensive research attention in recent year.
The protease specifically hydrolyzes peptides, such as Leu-
Leu, Arg-Arg, Ala-Leu, Phe-Arg, Phe-Lys, Gly-Phe-Leu-Gly,
and Ala-Leu-Ala-Leu et al,31, 32 Gly-Phe-Leu-Gly (-GFLG-) is
the most commonly used CTSB-responsive substrate among
them. Leveraging significant differences in the concentrations
and activities of enzymes between tumor and normal tissues,
effective enzyme-responsive antitumor drug delivery systems
based on the high selectivity of enzymes can be developed.33
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In order to improve the imaging method and tumor
selectivity of treatment, we designed FR-mediated dual-
targeting delivery system that could target cancer cell more
accurately. FA-N3 was coupled to form the ligand component
of FR. The antitumor drug SN38, tetrapeptide chain -GFLG-,
and oligoethyleneglycol tether were used to form the drug
component. The ligand and drug underwent click reactions to
generate the FA-GFLG-SN38 dual-targeted delivery system.
Compared with SN38, our system has a number of advantages:
A schematic representation of FA-GFLG-SN38 synthesis is
shown in Scheme 1A. Alkyne-linked peptides (alkyne-Gly-Ph
e-Leu-Gly) possessing a C-terminal hydrazide were assembled
Scheme 1. Synthesis of (A) FA-GFLG-SN38 and (B) FA-Rho.
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fluorescence signal was not enhanced in the absence of CTSB,
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confirming obvious quenching of FA-GFLG-SN38.
Fluorescence increased with time in CTSB solutions, which
was caused by enzymatic cleavage of the tetrapeptide GFLG
in FA-GFLG-SN38 and subsequent release of SN38 (λex = 365
nm, λem = 540 nm). In contrast, no increase in fluorescence
was observed upon treatment of FA-GFLG-SN38 with CTSB
in the presence of the inhibitor CA-074 Me. These findings
indicate that the SN38 is inactive when linked to the delivery
system, and upon its CTSB-triggered release from the FA-
GFLG-SN38, it restores its fluorescence and therapeutic
activity.
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Figure 1. Release of the SN38 drug from FA-GFLG-SN38
promoted by CTSB. (A) The images of different solutions (a:
SN38; b: FA-GFLG-SN38; c: FA-GFLG-SN38 with CTSB; d:
FA-GFLG-SN38 with CTSB and CA-074 Me) under visible light
and UV irradiation. (B) Release of SN38 from FA-GFLG-SN38
monitored using a fluorometer (λex = 365 nm, λem = 540 nm). (C)
Release of SN38 from FA-GFLG-SN38 in the presence of CTSB
monitored using RP-HPLC (detection at 254 nm) and Mass (a =
FA-GFLG-SN38, b = FA-linker-Gly-Phe-OH ([M + Na]: m/z =
1114.7), c = SN38).
Scheme 2. Cleavage of FA-GFLG-SN38 by CTSB.
on a solid support in the presence of 1-hydroxybenzotriazole
To further verify the CTSB-triggered release of SN38 from
FA-GFLG-SN38, mixtures obtained from treatment of FA-
GFLG-SN38 in the presence of CTSB were characterized and
monitored using RP-HPLC and mass spectrometry. The results
showed that SN38 (retention time = 23.6 min, [M + H]: m/z =
393.1) was efficiently released from FA-GFLG-SN38
(retention time = 26.4 min, [M + Na]: m/z = 1764.3) and
produced along with FA-linker-Gly-Phe-OH (retention time =
21.4 min, [M + Na]: m/z = 1114.7) in the presence of CTSB
(Figure 1C). Mixtures were further subjected to liquid
chromatography-mass spectrometry, which revealed the
presence of cyclic dipeptides (Leu-Gly: retention time = 34.0
min, [M + H]: m/z = 171.2). The tetrapeptidic substrate in FA-
GFLG-SN38 was enzymatic hydrolyzed by CTSB into Gly-
Phe and Leu-Gly, and the drug released via a 1,6-elimination
process when the C-terminus of the dipeptide (Leu-Gly) in the
delivery system was cleaved (Scheme 2). The above
observations demonstrate that FA-GFLG-SN38 is cleaved by
CTSB to release FA-linker-Gly-Phe-OH, cyclic dipeptide
(Leu-Gly), PABOH, and SN38.
(HOBt),
diisopropylethylamine
(DIEA)
and
N,N′-
diisopropylcarbodiimide (DIC). Assembled alkyne-linked
peptides were released from the solid support by treatment
with 10% trifluoroacetic acid (TFA) in CH2Cl2. The alkyne-
linked peptides and 4-aminobenzyl alcohol (PABOH) formed
an amide group. Bromination of the hydroxyl group of alkyne-
GFLG-PABOH, followed by nucleophilic substitution and
binding of SN38 yielded alkyne-GFLG-PAB-SN38. FAγ-
COOH had less steric hindrance compared to α-COOH and
could be selectively activated by controlling the molar ratio of
FA and N,N′-dicyclohexylcarbodiimide (DCC). FA-N3 was
obtained via formation of an amide bond with NH2-linker-N3
at a FA:DCC ratio of 1:1.34 The resulting mixture was purified
using semi-preparative reversed-phase high performance
liquid chromatography (RP-HPLC) and lyophilized. The
retention time of FA-N3 on analytical RP-HPLC was 19.2 min
and comparison of peak areas disclosed 74.58% FAγ-
coupling. The alkyne-GFLG-SN38 was reacted with FA-N3
under click conditions (CuSO4 and sodium ascorbate), purified
with semi-preparative HPLC and lyophilized. The retention
time of FA-GFLG-SN38 was 25.44 min on analytical RP-
HPLC.
To determine the effect of FR specificity on the cellular
internalization rate of the delivery system, we compared
fluorescence of SK-Hep-1, A549 and 16-HBE cells
immediately after incubation with FA-Rho. Rhodamine B
exhibits fluorescence in both its free and conjugate forms.35 It
was expected that FA-Rho accumulation in cells would occur
through FR-mediated endocytosis, followed by increased
fluorescence emission in cells caused by rhodamine B localize.
The synthetic pathway of FA-Rho is presented in Scheme
1B. Alkyne-Rho and reacted with FA-N3 under click
conditions (CuSO4 and sodium ascorbate). The resulting
mixture was purified with semi-preparative RP-HPLC and
lyophilized. The retention time of FA-Rho was 26.5 min on
PR-HPLC.
36
The results of confocal microscopy analysis of the treated
In order to verify whether the FA-GFLG-SN38 is
responsive to CTSB, we applied fluorescence spectroscopy to
detect the fluorescence changes of FA-GFLG-SN38 in the
presence of CTSB. As shown in Figure 1A, free SN38 showed
yellow native fluorescence under UV irradiation. FA-GFLG-
SN38 displayed no fluorescence due to quenching of SN38
fluorescence through binding with other conjugates. Treatment
of FA-GFLG-SN38 with CTSB at 37°C was characterized
using fluorescence spectroscopy (Figure 1B). The
SK-Hep-1 cancer cells show that a red fluorescence signal is
detected mainly in the lysosomes (Figure 2). After pre-
incubation with CA-074 Me (20 μM) and subsequent
treatment with 5 μM FA-Rho, strong red fluorescence signals
were observed in SK-Hep-1 cancer cells. However, when SK-
Hep-1 cells were incubated with FA for 1 h and then treated
with FA-Rho, fluorescence signal associated with rhodamine
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Hoechst 33342 (1 μg/mL) were used to stain the nucleus. The
incubated cells were imaged with confocal microscopy.
B were attenuated. The results showed that rhodamine B was
enriched in cells due to endocytosis of FR. In addition, faint
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probe the expectations, we compared the fluorescence of SK-
Hep-1, A549, and 16-HBE cells immediately after incubation
with 5 μM FA-GFLG-SN38. As shown in Figure 3, strong
green fluorescence was detected in nuclei of treated SK-Hep-1
cancer cells, indicating that released SN38 does indeed spread
to and localize in the nucleus. However, before treatment with
5 μM FA-GFLG-SN38, the fluorescence intensity of the SK-
Hep-1 cancer cells pre-incubated with 3 mM FA was
significantly reduced. On the other hand, SK-Hep-1 cancer
cells preincubated with CA-074 Me (20 μM) and subsequent
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treatment with
5
μM FA-GFLG-SN38 exhibit faint
fluorescence signals. The above results indicated that although
FA-GFLG-SN38 was degraded in SK-Hep-1 cancer cells, in
the presence of CA-074 Me, the coupling was not cleaved by
intracellular cathepsin B and released free SN38. A549 cells
were incubated with FA-GFLG-SN38, faint fluorescence
signals was observed in the lysosomes, phenomena which are
similar to those promoted by incubation mixtures of cancer
cells with FA-Rho in the presence of FA. In addition, no
fluorescence was detectable in FA-GFLG-SN38 treated 16-
HBE normal cells owing to lack of FR. Taken together, the
results show that both FR and CTSB activity are required for
the release of free SN38 from FA-GFLG-SN38 in SK-Hep-1
cells where it accumulates in the nucleus.
Figure 2. Fluorescence imaging of cells treated with FA-Rho.
SK-Hep-1 cancer cells were incubated with (a) 5 μM FA-Rho for
24 h, (b) 3 mM FA for 1 h and then 5 μM FA-Rho for 24 h, (c) 20
μM CA-074 Me for 24 h and then 5 μM FA-Rho for 24 h. (d)
A549 cancer cells were incubated with 5 μM FA-Rho for 24 h. (e)
16-HBE normal cells were incubated with 5 μM FA-Rho for 24 h.
Hoechst 33342 (1 μg/mL) were used to stain the nucleus. The
incubated cells were imaged with confocal microscopy.
fluorescence was detectable in FA-Rho treated A549 cancer
cells or 16-HBE normal cells owing to low expression levels
of FR. The above observations clearly suggest that the FA-
Rho exerts selective fluorescence imaging on cancer cells
through FR-mediated endocytosis.
The anti-tumorigenic activity of FA-GFLG-SN38 was
evaluated by measuring the viability of cancer (HeLa, Siha,
A549, and SK-Hep-1) and normal (16-HBE) cells using the
MTT assay (MTT
=
3-(4,5-dimethylthiazol-2-yl)-2,5-
The usefulness of FA-GFLG-SN38 for fluorescence
detection of cancer and normal cells was investigated. Unlike
rhodamine B, the fluorescence of SN38 depends on existing in
its free or conjugate form, when the conjugated system was
modified, the SN38-conjugate will be in its “Turn-OFF”
state,37 through enzymatically-cleavable group would
transform the system into a “Turn-ON” state. We infer that if
free SN38 is released from the FA-GFLG-SN38 binding under
the action of lysosomal CTSB, it should accumulate in the
nucleus of its action due to its fast diffusion kinetics.38 To
diphenylte-trazolium bromide). It is well known that HeLa
cells express FR-positive, while A549 cells are FR-negative.39
The results indicated that FA-GFLG-SN38 had a significant
anti-tumorigenic activity against SK-Hep-1, HeLa, and Siha
cells with an IC50 values of 2–3 μM (Figure 4A). FA-GFLG-
SN38 reduced the cell viability of A549 cells by 51% under
concentration of 20 μM, while that of 16-HBE normal cells at
the same concentration decreased by 24%. The low toxicity of
FA-GFLG-SN38 to A549 and 16-HBE cells is due to the
deficiency of FR, which is consistent with the described
phenomenon of fluorescence imaging. These results indicate
preferential activity of FA-GFLG-SN38 against FR-positive
cells. Next, we examined the anti-proliferative activity of free
SN38 on cancer and normal cells. Free SN38 exhibited strong
cytotoxic against cancer and normal cells with an IC50 values
of 0.3-1 μM (Figure 4B). These results suggest that FA-
GFLG-SN38 selectively targets cancer cells containing FR
and CTSB, and FA-GFLG-SN38 could be used as an effective
system for targeted therapy of tumor cells. results show that
both FR and CTSB activity are required for the release of free
SN38 from FA-GFLG-SN38 in SK-Hep-1 cells where it
accumulates in the nucleus.
Figure 3. Fluorescence imaging of cells treated with FA-
GFLG-SN38. SK-Hep-1 cancer cells were incubated with (a) 5
μM FA-GFLG-SN38 for 24 h, (b) 3 mM FA for 1 h and then 5
μM FA-GFLG-SN38 for 24 h, (c) 20 μM CA-074 Me for 24 h and
then 5 μM FA-GFLG-SN38 for 24 h. (d) A549 cancer cells were
incubated with 5 μM FA-GFLG-SN38 for 24 h. (e) 16-HBE
normal cells were incubated with 5 μM FA-GFLG-SN38 for 24 h.
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1.Chen, Z. H.; Yu, Y. P.; Tao, J.; Liu, S.; Tseng, G.; Nalesnik, M.;
Figure 4. Cancer and 16-HBE cells were incubated with
various concentrations of (A) FA-GFLG-SN38 or (B) free SN38
for 72 h. Cell viability was measured with the MTT assay.
Hamilton, R.; Bhargava, R.; Nelson, J. B.; Pennathur, A.; Monga, S.
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In summary, we have successfully developed two FR-
mediated delivery systems. FA-Rho probe is suitable for
specific fluorescence imaging of SK-Hep-1 cancer cells for
overexpressing FR, and usually inactive in FR negative cells
(A549 and 16-HBE cells). The FR-mediated cathepsin B
sensitive drug delivery system (FA-GFLG-SN38) is composed
of ligand (FA) of FR, tetrapeptide substrate (-GFLG-) for
cathepsin B and an anticancer drug (SN38). The experimental
results showed that FA-GFLG-SN38 is an effective anti-
tumorigenic agent. FA-GFLG-SN38 integrates features of
enzymatically-triggered drug release, fluorescence imaging
and targeted drug delivery into one system. Our preliminary
studies suggest that SN38 is released from FA-GFLG-SN38 in
the presence of CTSB and emits fluorescence, which can be
effectively applied for cancer cells for overexpressing FR. The
FA-GFLG-SN38 delivery system exhibited strong cytotoxic
against SK-Hep-1, HeLa and Siha cells of IC50 = 2–3 μM, with
no effects on FR negative A549 and 16-HBE cells, thus
minimizing the toxic side-effects of anticancer drugs. Based
on the collective findings, we propose that the newly
developed dual-targeted drug delivery system provides an
effective framework for future potential applications to tumor
detection and targeted therapy in vivo.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Experimental procedures, chemical characterization, biochemical
methods and additional data (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail: xizhet@ybu.edu.cn and lliu@ybu.edu.cn
ORCID
Xizhe Tian: 0000-0001-9608-1180
Notes
The authors declare that there is no conflicts of interest.
ACKNOWLEDGMENT
This study was supported financially by the National Science
foundation of China (grant no. 21462045 and 31760330).
ABBREVIATIONS
FR, folate receptor; FA, folic acid; SN38, 7-ethyl-10-
hydroxycamptothecin; CTSB, cathepsin B; GFLG, Gly-Phe-Leu-
Gly; Rho, rhodamine B; HOBt, 1-hydroxybenzotriazole; DIEA,
diisopropylethylamine; PABOH, 4-aminobenzyl alcohol; DIC,
N,N′--diisopropylcarbodiimide; TFA, trifluoroacetic acid; DCC,
N,N′-dicyclohexylcarbodiimide; RP-HPLC, reversed-phase high
performance
liquid
chromatography;
MTT,
3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide.
17. Liu, H.; Lu, H.; Liao, L.; Zhang, X.; Gong, T.; Zhang, Z. Lipid
Nanoparticles Loaded with 7-Ethyl-10-Hydroxycamptothecin-
Phospholipid Complex: in vitro and in vivo Studies. Drug Deliv. 2015,
22, 701–709.
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