Synthesis and anti-cancer evaluation of folic acid-peptide- paclitaxel conjugates for addressing drug resistance

Article abstract of DOI:10.1016/j.ejmech.2019.03.031

The drug resistance and the poor water solubility are major limitations of paclitaxel (PTX) of based chemotherapy. To conquer the two problems, targeting folate (FA) receptor PTX-lytic peptides conjugates were synthesized and evaluated. Compared with PTX, FA-P3-PTX and FA-P7-PTX displayed significantly enhanced cell toxicity in many cancer cells, particularly drug resistant cancer cells MCF-7/PTX. FA-P7-PTX possessed stronger effect on cell toxicity (IC₅₀ = 2.92 ± 0.2 μM), membrane disrupting activity and pro-apoptosis in MCF-7/PTX cells than FA-P3-PTX. Further investigation displayed that the anti-cancer mechanisms of FA-P3-PTX and FA-P7-PTX might be a mitochondrial impairment and caspase-3-dependent apoptotic cell death. Furthermore, the in vivo antitumor efficacy study confirmed that FA-P7-PTX performed more stronger potency in inhibition of tumors growth than PTX. The study demonstrated that conjugate FA-P7-PTX with superior properties for antineoplastic activity, which makes it a promising potential candidate for drug-resistant cancer therapy.

Full text of DOI:10.1016/j.ejmech.2019.03.031

Accepted Manuscript
Synthesis and anti-cancer evaluation of folic acid-peptide- paclitaxel conjugates for
addressing drug resistance
Yuxuan Dai, Xingguang Cai, Xinzhou Bi, Chunxia Liu, Na Yue, Ying Zhu, Jiaqi Zhou,
Mian Fu, Wenlong Huang, Hai Qian
PII:
S0223-5234(19)30249-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.031
EJMECH 11201
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 December 2018
Revised Date: 13 March 2019
Accepted Date: 13 March 2019
Please cite this article as: Y. Dai, X. Cai, X. Bi, C. Liu, N. Yue, Y. Zhu, J. Zhou, M. Fu, W. Huang, H.
Qian, Synthesis and anti-cancer evaluation of folic acid-peptide- paclitaxel conjugates for addressing
drug resistance, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.03.031.
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ACCEPTED MANUSCRIPT
Graphical Abstract for
Synthesis and anti-cancer evaluation of folic acid-peptide- paclitaxel conjugates
for addressing drug resistance
Yuxuan Dai a, Xingguang Cai a, Xinzhou Bi a, Chunxia Liu a, Na Yue a, Ying Zhu a, Jiaqi Zhou a,
Mian Fu a, b, Wenlong Huang a, c, Hai Qian a, c
a
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
b Department of Biochemistry and Molecular Biology, Nanjing Medical University, 140 Hanzhong
Road, Nanjing 210029, PR China.
c
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
Corresponding author: Hai Qian, Centre of Drug Discovery, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.
Tel: +86-25-83271051; Fax: +86-25-83271051.
E-mail: qianhai24@163.com (H. Qian).

ACCEPTED MANUSCRIPT
Synthesis and anti-cancer evaluation of folic acid-peptide-
paclitaxel conjugates for addressing drug resistance
Yuxuan Dai a, Xingguang Cai a, Xinzhou Bi a, Chunxia Liu a, Na Yue a, Ying Zhu a, Jiaqi Zhou a,
Mian Fu a, b, Wenlong Huang a, c, Hai Qian a, c
a
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
b Department of Biochemistry and Molecular Biology, Nanjing Medical University, 140 Hanzhong
Road, Nanjing 210029, PR China.
c
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
Abstract
The drug resistance and the poor water solubility are major limitations of
paclitaxel (PTX) of based chemotherapy. To conquer the two problems, targeting
folate (FA) receptor PTX-lytic peptides conjugates were synthesized and evaluated.
Compared with PTX, FA-P3-PTX and FA-P7-PTX displayed significantly enhanced
cell toxicity in many cancer cells, particularly drug resistant cancer cells MCF-7/PTX.
FA-P7-PTX possessed stronger effect on cell toxicity (IC50 = 2.92 ± 0.2 uM),
membrane disrupting activity and pro-apoptosis in MCF-7/PTX cells than
FA-P3-PTX. Further investigation displayed that the anti-cancer mechanisms of
FA-P3-PTX and FA-P7-PTX might be a mitochondrial impairment and
caspase-3-dependent apoptotic cell death. Furthermore, the in vivo antitumor efficacy
study confirmed that FA-P7-PTX performed more stronger potency in inhibition of
Corresponding author: Hai Qian, Centre of Drug Discovery, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.
Tel: +86-25-83271051; Fax: +86-25-83271051.
E-mail: qianhai24@163.com (H. Qian).
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tumors growth than PTX.
The study demonstrated that conjugate FA-P7-PTX with superior properties for
antineoplastic activity, which makes it a promising potential candidate for
drug-resistant cancer therapy.
Keywords
Lytic peptides, Folate receptor targeted, Membranolytic, Apoptosis, Drug
resistance, Paclitaxel, Conjugates of paclitaxel
1. Introduction
Cancer is a leading cause of morbidity and mortality globally, and is responsible
for an estimated 9.6 million deaths in 20181. Chemotherapy is an important approach
used in cancer therapy, while the development of multidrug resistance (MDR)2 is a
major cause of failure in cancer treatment3. The most common mechanism of cancer
MDR is the overexpression of energy-dependent efflux pumps such as P-glycoprotein,
a main member of adenosine triphosphate-binding cassette (ABC) transporters4-5.
Consequently, it is imperative to develop alternative new potent and less toxic
chemotherapeutic agents.
Paclitaxel (PTX)6 is a potent anticancer agent frequently used for treatment of a
wide variety of human malignancies such as breast, ovarian, lung, and other cancers6-7.
However, the application of PTX was limited due to its poor aqueous solubility8,
systematic toxicity and acquired drug resistant9. As a substrate of P-glycoprotein10,
antitumor efficacy of PTX is reduced considerably owing to MDR11-12. The initial
formulation of paclitaxel was Cremophor EL- paclitaxel (CrEL–paclitaxel)13, the
widely used formulation in the clinical setting14. But, due to its undesirable toxicity,
many formulations have already been developed to ameliorate systematic toxicity,
such as Lipusu (liposomal PTX), Abraxane (PTX nanoparticles)15, Genexol-PM16
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(polymer micelles)16-17, and NK105 (a PTX-incorporating micellar nanoparticle)18.
Compared to PTX, these formulations reduced systematic toxicity and improved
water solubility, however, they are not efficient for MDR tumor.
Peptide carrier-mediated drug delivery has been used as a powerful strategy to
increase the drug’s water solubility19, optimize to improve cellular uptake20, to
achieve cell penetration or selectivity21, and to reduce the side effects of cytotoxic
drugs22. Meanwhile, peptide mediated drug delivery system, as one of the main pillars,
is applied to overcome MDR11, 23. Many researchers have put their mind to
constructing new antitumor drug conjugates to solve the drug resistance of tumors24.
For instance, Sheng et al. designed a peptide-DOX conjugate and successfully used it
to reverse the drug resistance of breast cancer cells25. Lytic peptides, consist of basic
amino acids and about half hydrophobic amino acids, are cationic and amphipathic26,
which could arrange into amphipathic structure and display potent cell penetration
and disruption of cell membrane27. The tumor cell membranes comprise a high
proportion of net negative charges so that the cationic lytic peptides could selectively
bind with tumor cells by the electrostatic interactions28-29. Accordingly, lytic peptides
could be used as efficient tools for delivering pharmaceuticals into cells. The strategy
that conjugate PTX to lytic peptide can inhibit drug resistance by enhancing the
transport PTX into tumor cells, as well as improving water solubility to avoid the use
of Cremophor EL.
Folate (FA) receptors are attractive molecular targets for anticancer drug deliver
y
30, 31, which are characteristically overexpressed in many solid tumors including
breast32, hepatic carcinoma and others33, while its expression is low in normal
tissues31. Folate conjugation offers some functional roles, namely, selective delivery
of nonspecific drugs into cancer cells34, reducing adverse side effects on health cells
and facilitating cell internalization via receptor-mediated endocytosis35. For example,
vintafolide, a folate-conjugated vinca alkaloid desacetylvinblastine hydrazide, is
currently undergoing phase II clinical studies in patients with platinum-resistant
ovarian cancer36 and non-small cell lung cancer37.
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We have previously reported that structural optimized lytic peptides I-3 and I-7
can be used as cell-disrupting peptides and molecular carriers38. Meanwhile, PTX, a
firstline antitumor drug, its poor aqueous solubility (no more than 0.004 mg/mL)8 and
acquired drug resistant need to be addressed urgently. In this work, we choose the
16-site cysteine-substituted I-3 and I-7 (namely P3 and P7, respectively) served as
peptide backbone and we designed a novel folate targeting peptide-PTX conjugates to
achieve selective tumor delivery, enhance cellular uptake, make FA-P3/P7-PTX
conjugates water-soluble and overcome drug resistance. The conjugates were
evaluated for the antiproliferative activity in different cancer cell lines, the inhibitory
rate of tubulin polymerization, hemolytic toxicity and water solubility (more than 20
mg/mL, Fig. S5). Furthermore, we assessed the conjugates for their cellular uptake,
Membrane permeability, pro-apoptosis, alternation of mitochondrial membrane
potential, rat plasma stability and cell apoptosis pathway in PTX resistant
MCF-7/PTX cells. Finally, we researched the most optimized conjugate in vivo
antitumor efficacy compared with free PTX.
2. Results and discussion
2.1 Synthesis of FA-targeting conjugates
Scheme 1. Synthesis Route of Paclitaxel maleimide. aReagents and conditions: (a) glacial acetic
acid, reflux; (b)SOCl2, DCM, N2; (c)TEA, DCM, N2
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Scheme 2. Synthesis route of N10- TFA-Pteroic Acid. (a). (i) TFAA, (ii) 3% TFA.
Scheme 3. Solid phase synthesis of conjugates. (a) Sodium phosphate buffer (pH 7.0), 25 .
Novel peptide-drug conjugates based on PTX coupled with lytic membrane
peptide and folic acid modifiers were designed and synthesized (Table 1 and Fig.S1).
The objective was to combine a set of desirable physicochemical, biochemical, and
chemotherapeutic properties into a single integrated macromolecule structure. As
shown in Scheme 1, PTX was modified by maleimide, since maleimide undergoes
Michael additions with thiol groups at neutral pH. Cysteine containing peptides were
synthesized by using a solid phase methodology according to the previously reported
procedure39. As shown in Scheme 2 and Scheme 3, the thiol group in cysteine moiety
was used to conjugate with PTX-maleimide and N-terminal of peptide was used for
conjugation of folic acid. The purities of the four conjugates were all above 95%. The
observed multiply charged ions of the conjugates were shown in Table 2 and Fig.S6,
by ESI mass spectrometry.
Table 1 Structures of the conjugates
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Compounds
P3-PTX
R
Xaa
H
FA-P3-PTX
H
P7-PTX
FA-P7-PTX
Table 2. Characterization of the conjugatesa
Mass (Da)
Molecular
formula
C188H272N40O40S
Molecular
weight
Compounds
FA-P3-PTX
Calculated
[M+4H]4+
942.1
Observed
[M+4H]4+
942.5
3764.54
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[M+5H]5+
[M+4H]4+
753.9
[M+5H]5+
[M+4H]4+
754.7
837.2
836.0
C
169H254N33O35S
3341.15
P3-PTX
[M+5H]5+
[M+4H]4+
[M+5H]5+
[M+4H]4+
[M+5H]5+
669.0
984.2
787.5
878.1
702.7
[M+5H]5+
[M+4H]4+
[M+5H]5+
[M+4H]4+
[M+5H]5+
669.9
984.9
788.3
878.9
703.2
C188H272N52O40S
C169H254N45O3S
3933.63
3509.23
FA-P7-PTX
P7-PTX
aUPLC conditions: 10–80% acetonitrile (mobile phase A: water with 0.1% formic acid, mobile phase B:
acetonitrile with 0.1% formic acid) in 10 min at a flow rate of 0.3 mL/min with ultraviolet (UV) detection at 214
nm with the use of Waters ACQUITY UPLCBEH C18 column (1.7 × 50 mm, Waters)
2.2 Cytotoxicity and Hemolytic Activity
Table 3 Anti-tumor activity of the conjugates
IC50 (µM)
Compounds
MCF-7
MCF-7/PTX
K562
A2780
SKOV3
5.92±0.84
7.17±0.77
5.49±0.36
6.61±0.94
HUVEC
1.79±0.09#
2.15±0.18
1.39±0.12#
1.98±0.14
4.54±0.71**,
6.11±0.61**
1.95±0.20*,
2.69±0.19
48.55±2.94**,
#
#
##
5.38±0.25
5.90±0.92
FA-P3-PTX
P3-PTX
FA-P7-PTX
P7-PTX
P3
31.60±1.88**
2.92±0.2**,
5.53±0.76**
3.85±0.9*
1.42±0.08**,
2.79±0.17
45.21±1.79**,
37.22±2.36**
##
##
##
3.82±0.29**
13.65±1.30**
9.49±0.80**
5.80±0.40
12.31±0.96** 15.24±1.53**
13.56±1.72**
11.50±1.37**
2.85±0.40
14.69±1.10** 40.17±1.92**
13.58±1.28** 44.53±2.61**
8.61±0.86**
1.17±0.29
17.08±1.32**
54.8±2.83
P7
PTX
6.29 ± 0.73
9.39±0.98
#
a Notes: PTX: paclitaxel. *P < 0.05. **P < 0.01 vs PTX; P < 0.05. ##P < 0.01 (FA-P3-PTX vs P3-PTX or
FA-P7-PTX vs P7-PTX).
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Fig. 1 Hemolysis effects on RBCs of the conjugates. Various concentrations of P3-PTX,
FA-P3-PTX, P7-PTX and FA-P7-PTX were incubated with RBCs. The results shown all the
tested peptides exhibited modest hemolytic activity
The anticancer activities of the conjugates were evaluated using various cancer
cells (MCF-7, MCF-7/PTX, K562, A2780 and SKOV3). The IC50 values are listed in
Table 3, and PTX was used for comparison. All the conjugates exhibited improved
cytotoxic effects on various cancer cells. According to the results, all the conjugates
showed significantly stronger antiproliferative activity than former lytic peptides (P3
and P7), and FA-P3-PTX and FA-P7-PTX showed more excellent antiproliferative
activity than P3-PTX and P7-PTX in FA-overexpressing cancer cells MCF-7 (1.79
uM versus 2.15 uM; 1.39 uM versus 1.98 uM), MCF-7/PTX (4.54 uM versus 6.11 uM;
2.92 uM versus 5.53 uM), A2780 (1.95 uM versus 2.69 uM; 1.42 uM versus 2.79 uM),
respectively. Thus, the conjugate FA-P3-PTX and FA-P7-PTX exhibited great
antiproliferative activity on folate receptors overexpressing cancer cells, and almost
equal potency to both drug resistant and –sensitive cells. Meanwhile, the conjugates
showed weak toxicity to the normal cell lines HUVEC. To assess the safety profile of
the designed conjugates, we examined their hemolytic activity using RBCs. As
depicted in Fig. 1, all the tested peptides exhibited modest hemolytic activity.
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2.3 Concentration Dependent Cellular Uptake
Fig. 2 Concentration-dependent cellular uptake of P3FITC, P3-PTXFITC and FA-P3-PTXFITC by drug
resistant MCF-7/PTX cells. (A) Representative flow cytometry spectra of the cells treated with 1,
2, or 6 µM different drugs for 2 h, and untreated cells as control; (B) quantitative comparison of
the fluorescence intensity in the studied from endocytosed drugs after 2 h treatment with 1, 2, or 6
µM drugs.; (C) and (D) Cell viability of drug sensitive MCF-7 cells (C) and resistant MCF-7/PTX
cells (D) after treatment with PTX, P3-PTX, or FA-P3-PTX at three different concentrations (1, 2,
and 6 µM).
To obtain quantitative information on the concentration-dependent cellular
uptake, FITC labeled conjugates were synthesized (Table S1). We used flow
cytometry to track endocytosed conjugates in drug resistant breast MCF-7/PTX
cancer cells.
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The respective low fluorescent intensities suggested low accumulated
concentration of the dye or drug within cells. Simultaneously, the increase in
fluorescence intensity for labelled conjugates implied that an active cellular uptake
mechanism may be involved. In cases of the conjugates, it was evident that the
endocytosis of FA-P3-PTXFITC was higher than P3-PTXFITC at the same concentration,
visibly suggested that the conjugation FA has a significant influence on the cellular
uptake (Fig. 2A and 2B). Next, we performed cytotoxicity experiments of the
conjugates against MCF-7 and MCF-7/PTX cells, to study the relationship of cellular
uptake and cytotoxicity. For the drug resistant MCF-7/PTX cells, the cytotoxicity of
free PTX dropped dramatically, with no significant cytotoxicity observed (Fig. 2C and
2D). In contrast, conjugate FA-P3-PTX showed consistent cytotoxicity against both
the sensitive and resistant cell lines. This result indicated that acquired drug resistant
not greatly affected the antitumor activities of the drug conjugates, as is the case for
free PTX.
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2.4 Membrane Permeability
Fig. 3 (A) LDH leakage assay of drug conjugates; (B) AO/EB staining images in MCF-7/PTX
cells after treatment with 3 µM of PTX, P7, P3-PTX, FA-P3-PTX, P7-PTX and FA-P7-PTX,
respectively. 0.1% DMSO served as control.
To research if the conjugates reserved property with the disruptive membrane of
lytic peptides, we designed lactate dehydrogenase (LDH) leakage assay as previous
reported 40-41. Membrane lysis activity of the conjugates confirmed by measuring
release of the ubiquitous, cytoplasmic enzyme LDH in the culture medium.
MCF-7/PTX cells were incubated with different concentrations of drug conjugates
(FA-P3-PTX, P3-PTX, FA-P7-PTX and P7-PTX,) and the results exhibited that the
drug conjugates caused the release of the cytosolic LDH enzyme in a
concentration-dependent manner (Fig. 3A). Notably, as shown in Table 3 and Fig. 3A,
FA-P3-PTX and FA-P7-PTX possessed more excellent anti-cancer activity in
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MCF-7/PTX cells and stronger disruptive membrane activity. To further validated,
visualization of AO/EB double staining test were utilized to detect membrane
integrity in MCF-7/PTX cells. AO can stain both live and dead cells and presents
green fluorescence. EB stains only apoptotic or necrotic cells that have lost their
membrane integrity and shows red fluorescence. As shown in Fig. 3B, MCF-7/PTX
cells displayed red fluorescence all after treatment with the drug conjugates (3 uM of
drug conjugates) for 12 h.
Thus, these results demonstrated that the drug conjugates with the character of
lytic peptides could seriously disrupt the membrane and alter the penetrability to
strengthen the cytotoxicity activity of conjugates. FA-P3-PTX and FA-P7-PTX drug
conjugates were selected for further investigation. In addition, the clearly
morphological features of AO/EB double staining preliminary indicated that the drug
conjugates can induce apoptosis of MCF-7/PTX cells.
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2.5 Pro-apoptotic Assay
Fig. 4 The conjugates induced apoptosis in MCF-7/PTX cells. (A) Cells were treated with 3 uM of
conjugates for 12 h. Control samples were treated with 0.1% DMSO; (B) Cells were treated with
0.5, 1, 2, 4, and 6 uM of FA-P7-PTX. Percentage of apoptosis was measured by flow cytometry;
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(C) Quantitative data from the apoptosis measurement described in (A), *P < 0.05. **P < 0.01.
***P < 0.001 vs control, #P < 0.05 (FA-P7-PTX vs FA-P3-PTX).; (D) Quantitative data from the
apoptosis measurement described in (B).
To further confirm the nature of drug resistance cell death induced by
compounds, the effect on drug resistant MCF-7/PTX cells apoptosis analysis was
determined indirectly using Flow Cytometer with Annexin-FITC/PI staining.
Preliminary flow cytometric analysis MCF-7/PTX cell lines treated under identical
conditions with all the drug conjugates (3 uM) evidenced that FA drug conjugates
were able to induce the more pro-apoptotic, while 3 µM of FA-P3-PTX and
FA-P7-PTX induced 34.6 and 41.6 % MCF-7/PTX cell apoptosis respectively (Fig.
4A and 4C).
Thus, FA-P7-PTX possessed stronger pro-apoptotic activity compared to
FA-P3-PTX. As shown in Fig. 4B and 4D, apoptosis ratio appeared
concentration-dependent increase after MCF-7/PTX cells were treated with
FA-P7-PTX with concentration of 0.5 uM to 6 uM.
2.6 Cellular Colocalization
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Fig. 5. Conjugates induced mitochondrial dysfunction. Fluorescence microscope analysis of
cellular mitochondrial transmembrane potential level by JC-1 staining after 3 uM of PTX,
FA-P3-PTX and FA-P7-PTX treatment for 12 h. 0.1% DMSO was served as control.
Mitochondria are the essential parameter of the two main apoptotic signaling
pathway, namely the intrinsic and extrinsic pathways 42-43. To recognize if FA-P3-PTX
and FA-P7-PTX induce apoptosis by targeting mitochondria, we measured the loss of
mitochondrial transmembrane potential by a fluorescence dye JC-1, a mitochondrial
membrane-potential-sensitive dye, which act as reliable indicator of the dissipation of
mitochondrial membrane, observed as red fluorescence to green44. As shown in Fig. 5,
a significant alteration of fluorescence owing to loss of mitochondrial membrane
potential was observed following MCF-7/PTX cells treatment with FA-P3-PTX and
FA-P7-PTX compared with control (Fig. 7B) indicating that these novel drug
conjugates induced apoptosis by targeting the mitochondria.
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Collectively, the results provide evidence that drug conjugates FA-P3-PTX and
FA-P7-PX are able to induce cell death by apoptosis through a mitochondrial
dependent pathway with FA-P7-PTX being the most active of the series.
2.7 Western Blot Analysis of Caspase-3, Cytochrome-C and Folate receptor.
Fig. 6 (A) The western blot analysis of cleaved-caspase-3 for MCF-7/PTX cells; (B) The western
blot analysis of Cytochrome C. (C) The western blot analysis of folate receptor on the cell lines.
GADPH was probed as loading control.
The disrupted mitochondrial can release pro-apoptotic factors such as
cytochrome c and other apoptosis inducing factors45. Caspases also are the important
parameter of initiation and performance of apoptosis and the apoptotic signaling
pathway consist of the extrinsic and intrinsic pathways, which are dependent on the
cleavage of caspases46 . The activation of Caspase-3 is a crucial regulatory protein of
cell apoptosis. To further explain the mechanism of drug resistance cells apoptosis
induced by FA-P3-PTX and FA-P7-PTX, the activities of cleaved caspase-3 and
cytochrome C were examined. As shown in Fig. 6A and 6B, MCF-7/PTX cells
treatment with 3 µM of FA-P3-PTX and FA-P7-PTX for 2 h, exhibits dramatic
enhancement in cleaved caspase-3 and Cytochrome-C in the cytoplasm. As shown in
Fig. 6C, the results shown that MCF-7, MCF-7/PTX and A2780 had higher
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expression quantities of folate receptor than that of other cells.
This result further confirms that FA-P3-PTX and FA-P7-PTX induces
mitochondrial dysfunction and caspase-3-dependent apoptotic cell death in
MCF-7/PTX cells.
2.8 Tubulin Polymerization Assay
Fig 7. In vitro tubulin polymerization induced by 3 uM of PTX and FA-P7-PTX.
The results shown that at equimolar concentrations, polymerization induced by
FA-P7-PTX was slightly lower than PTX. This may not affect the antitumor activity
of FA-P7-PTX.
2.9 Stability of the FA-P7-PTX in Rat Plasma
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Fig.8 Time-dependent stability of P7 and FA-P7-PTX in rat plasma monitored
over 24 h.
FA-P7-PTX were incubated with plasma over 24 h and compared with P7.
Aliquots were removed at different time points (0.1,0.5, 1, 2, 3, 4, 8, 12, 24, h) for
analysis using UPLC/MS. Fig. 8 displayed the time profile of conjugates degradation.
As expected, FA-P7-PTX was more stable to proteolytic degradation compared to P7.
FA-P7-PTX possessed a half-life of ∼5.9 h at 37 °C, while P7 the half-life was 0.8 h.
These data revealed that FA-P7-PTX is stable enough to allow sufficient time for
cancer cell targeting.
2.10 In Vivo Antitumor Efficacy
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Fig. 9 (A) Tumor volume curve of H22 mice model over time when treated with FA-P7-PTX
(12 umol/kg), PTX (12 umol/kg) or 0.9% saline. Tumor volumes were measured by calipers;
(B) Tumor weight and body weight. *P < 0.05. **P < 0.01 vs PTX, Values represent the means ±
SD. (C) Photos of tumors separated from mice.
To study the anticancer activity of FA-P7-PTX in vivo, we performed
tumor-bearing mice model with H22 cells by administering once every two days
peritumoral injection of FA-P7-PTX (12 umol/kg), PTX (12 umol/kg, as the positive
control), or 0.9% saline as the negative control for 2 weeks. Compared with control
group, the tumor volumes of the FA-P7-PTX group were dramatically reduced by 69%
with no significant variation in mouse body weight (Fig. 9). Meanwhile，FA-P7-PTX
exhibited stronger inhibitory effects on tumor volume compared with PTX (69%
versus 49%). The result confirmed that FA-P7-PTX possessed higher potency in
slowing the growth of solid tumors.
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3. Conclusion
In conclusion, we have successfully developed novel folate receptors targeted
drug conjugates by incorporating lytic peptides, which serves as a linker of the FA and
PTX(Table1). FA-P3-PTX and FA-P7-PTX showed significantly higher
antiproliferative activity than PTX in MCF-7/PTX and A2780 cells when compared
with PTX which showed comparable activity against MCF-7, K562 and SKOV3 cells
(Table 3). FA-P3-PTXFITC exhibited higher cellular uptake in MCF-7/PTX cells as
shown by flowcytometry when compared with P3-PTXFITC, which demonstrated that
the increased cellular uptake dependent on FR (Fig. 2). FA-P3-PTX and FA-P7-PTX
presented stronger membrane disrupting activity in MCF-7/PTX cells with a
concentration-dependent manner. The AO/EB double staining test also ascertained
evidence of the disruption to MCF-7/PTX cell membranes (Fig. 3). Meanwhile,
FA-P3-PTX and FA-P7-PTX exhibited little hemolysis activity against the RBCs (Fig.
1). The Annexin-FITC/PI staining proved that the pro-apoptosis proportion of
FA-P7-PTX was much greater than the control and better than that of FA-P3-PTX
with a dose-dependent manner (Fig. 4). Compared to FA-P3-PTX, FA-P7-PTX
revealed much stronger effects on cell toxicity, membrane disrupting activity and
pro-apoptosis in MCF-7/PTX cells. The anti-cancer mechanisms of FA-P3-PTX and
FA-P7-PTX might be a mitochondrial impairment and caspase-3-dependent apoptotic
cell death (Fig. 5 and Fig. 6). Meanwhile, the rat plasma stability revealed that
FA-P7-PTX exhibited favorable plasma stability over P7 (Fig.8). Furthermore, the in
vivo antitumor efficacy study confirmed that FA-P7-PTX performed more stronger
potency in inhibition of tumors growth than PTX (Fig. 9).
In summary, the present work signifies that the conjugate FA-P7-PTX may be a
promising candidate for drug-resistant cancer therapy.
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4. Experimental section
4.1 Materials and Animals
All reagents were purchased as reagent grade and used without further
purification.
Nα-Protected amino acids, Fmoc-Arg(Pdf)-OH, Fmoc-Leu-OH,
Fmoc-Val-OH and Rink amide MBHA resin (loading 0.557 mmol g_1), were
purchased from GL Biochem Ltd. (Shanghai, China). Fmoc-Glu-OtBu, Fmoc-Lys
(Boc)-OH, Fmoc-Phe-OH, Fmoc-Cys(Trt)-OH and N-Hydroxybenzotriazole (HOBt),
were purchased from Nanjing Peptide Biotech Ltd. N， N-diisopropylcarbodiimide
(DIC), trifluoroacetic acid (TFA), N, N-diisopropylethyl amine (DIPEA) and
Trifluoroacetic anhydride(TFAA) were purchased from Energy chemical. Paclitaxel
was purchased from Ark Pharm. Pteroic acid was purchased from Bide Pharmatech.
Fluorescein isothiocyanate isomer (FITC) was purchased from Aladdin. All other
reagents, unless otherwise indicated, were obtained from Sigma-Aldrich Co. (Saint
Louis, MO) and used as received. LDH Activity Assay Kit was purchased from
Nanjing Jiancheng Bioengineering Institute. RIPA buffer, Membrane Potential Assay
Kit and BCA Protein Assay Kit were purchased from Beyotime Biotechnology.
Annexin V–FITC/PI Detection Kit and Cell Cycle Detection Kit were purchased from
KeyGEN BioTECH. Rabbit polyclonal antibody (CASP3 polyclonal antibody,
GAPDH polyclonal antibody, Cytochrome C) were purchased from ABclonal.
Microwave procedures were performed in the Microwave Peptide Synthesizer (CEM,
Matthews, NC, USA).
4.2 Synthesis of the peptides
The peptides were synthesized by the standard solid-phase peptide synthesis
protocol of standard Fmoc strategy on Rink amide MBHA resin, as already described
in previous research47. HBTU/HOBt/DIPEA was used for the coupling procedure of
amine acid and piperidine/DMF (20%, v/v) was used for deprotection of N-terminal
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Fmoc group. For peptides P3 and FA-P3, the Dde protection of Lys3 was removed by
hydrazine/DMF (2% , v/v) for 3 times for 10 min at 25 . The result was detected by
Kaiser test. Then Fmoc-β-Ala-OH was coupled by DIC/HOBt. Fmoc group was
removed and FITC labeling was performed with the solution of FITC (4 equiv) and
DIPEA (12 equiv) in DMF in the dark overnight. Completed Rink amide MBHA
resins were treated with Reagent
K
(TFA/EDT/water/phenol/thioanisole,
82.5:2.5:5:5:5) for 4 h. Cold Et2O was used to separate out the crude peptide, which
was purified by preparative RP-HPLC. The reaction mixture was filtered and washed
by DCM two times.
4.3 Synthesis of the PTX maleimide (5)
General procedure for preparation of compound 3 has been reported in previous
research39. Then, compound 3 (422 mg, 2 mmol) was dissolved in 20 ml of anhydrous
DCM which was then reacted with 10 equiv of SOCl2 at 0 °C for 1 h to produce the
desired intermediate compound 4. PTX (854mg, 1mmol) and 350ul of trimethylamine
(TEA) were dissolved in 10ml of anhydrous DCM, and then compound 4 was added
at 0 under Ar atmosphere. The resulting solution was stirred for 2 h at the dark. The
reaction mixture with Ar atmosphere protection was evaporated under vacuum.
Compound 5 was obtained by column chromatography (DCM: Methanol, 20:1) with a
yield of 58% (Scheme 1). 1H NMR (300 MHz, DMSO-d6) δ 9.29-9.26 (d, J=9.24 Hz,
1H), 8.00-7.96 (t, J=12.56 Hz, 3H), 7.88-7.86 (d, J=6.12 Hz, 2H), 7.77-7.64 (m, 3H),
7.58-7.45 (m, 7H), 7.18-7.20 (t, J=6.07 Hz, 1H), 6.99 (s, 2H), 6.30 (s, 1H), 5.84-5.78
(t, J=18.04 Hz, 1H), 5.57-5.51 (t, J=18.16 Hz, 1H), 5.43-5.33 (m, 2H), 4.93-4.90 (m,
2H), 4.64 (s, 1H), 4.12-4.02 (m, 3H), 3.60-3.58 (d, J=6.78 Hz, 1H), 3.28 (m, 1H),
2.89 (s, 2H), 2.73 (s, 2H), 2.31 (s, 3H), 2.25 (s, 3H), 1.77-1.71 (m, 3H), 1.60-1.40 (m,
6H), 1.30-1.19 (m, 5H), 1.03-1.01 (d, J=6.46 Hz, 6H). MS (ESI) m/z calcd for
[C57H62N2O17 + Na]+ calcd. for 1070.12; found, 1070.5.
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4.4 Synthesis of N10- TFA-Pteroic Acid
Pteroic acid (624 mg, 2 mmol) was dissolved in 4ml of trifluoroacetic anhydride
(TFFA) and stirred under nitrogen in the dark for 2 h. The mixture was concentrated
under reduced pressure by rotary evaporator. Then, 20 ml of 3% TFA was added, and
the resulting solution was neutralized with NH4OH(aq). 590 mg of pale yellow solid
(compound 7) was obtained (yield 72%, Scheme 2). H1 NMR (400 MHz, DMSO-d6)
δ 4.48 (m, 2H); 6.66-6.65 (d, J = 4.27 Hz, 2H); 7.68-7.65 (d, J = 12.04 Hz, 2H);
8.67-8.64 (m, 2H); 11.53 (s, 1H); MS (ESI) m/z calcd for [C16H12F3N6O4 + H]+ calcd.
for 409.29; found, 409.6.
4.5 Synthesis and purification of the conjugates
The
peptides
(P3:
LVKRFKKFFRKLKKCVLL-NH2,
with cysteine were synthesized
P7:
on
LVRRFRRFFRRLRRCVLL-NH2)
Rink-amide-MBHA (4-methyl-benzylhydrylamine) resin using Fmoc-based solid
phase peptide synthesis (SPPS) as previously reported47.
0.1 mmol of the cysteine-containing peptides (P3, P7, FA-P3, FA-P7, FITC- AP3
β
and FITC- AFA-P3) were dissolved in 5 mL of 0.05 mol·L−1 sodium phosphate buffer
β
(pH 7.0), then, added methanol solution of compound 5 (107 mg, 0.1 mmol) slowly.
The reaction mixture was stirred at 25 for 12 h until the reaction was complete
(Scheme 3). Purification of crude conjugate was carried out by RP-HPLC on a C18
column (Shimadzu LC-10). RP-HPLC conditions: 10–80% acetonitrile (mobile phase
A: water with 0.1% TFA, mobile phase B: acetonitrile with 0.1% TFA) in 60 min at a
flow rate of 10 mL/min with ultraviolet (UV) detection at 214 nm. All the conjugates
purities were monitored by UPLC/MS (Waters UPLC with the ACQUITY TQD;
Waters Corporation, Milford, MA, USA) with a Waters ACQUITY UPLCBEH C18
column (1.7 × 50 mm,Waters). The purities of the conjugates were above 95%.
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4.6 Cell Culture
Human ovarian cancer cell lines A2780 and SKOV3, leukemia cells K562,
human breast cancer cell lines MCF-7, paclitaxel resistant sub-line MCF-7/PTX,
human gastric mucosal epithelial cells GES-1 and mouse H22 hepatoma cells, were
obtained from KeyGEN BioTECH (Nanjing, China). K562, MCF-7, MCF-7/PTX,
H22 and GES-1 cells were cultured in RPMI 1640 supplied with 10% fetal calf serum
(FBS, Hyclone Laboratories). A2780 cells were grown in DMEM supplemented with
10% FBS. SKOV3 cells were cultured in McCoy’s 5A supplemented with 10% FBS.
All media were supplemented with 1% penicillin/streptomycin antibiotics
(GibcoBRL). All the cells were incubated at 37 °C in a humidified 5% CO2.
4.7 Cytotoxicity Assay
Cell viability was evaluated by MTT method with a minor modification38. cells
during logarithmic growth phase were seeded in 96-well plates at 6 × 103 cells per
well and incubated overnight. The conjugates were added to cells with various final
concentrations (ranging from 0.4 uM to 50 uM) and incubated in a 37 °C incubator
containing 5% CO2 for 48 h. MTT dye (10 µL of 5 mg/mL in PBS) was added to each
well 4 h. The plates were then centrifuged at 1500 rpm for 10 min and the supernatant
was removed without distracting the formazan precipitate and cells in the wells. Next,
150 µL of DMSO was added to dissolve formazan crystals and the plates agitated on a
plate shaker for 5 min. The absorbance at 490 nm was detected in a microplate reader
(Thermo, USA). The IC50 values of conjugates were calculated by GraphPad Prism
7.0 software (San Diego, CA, USA) on the basis of the dose−response curves. Assays
were examined three times.
4.8 Hemolysis Activity
Wistar rats (male, 18−22 g, Certificate number: NO.201824693) were purchased
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from the Comparative Medical Center of Yangzhou University (Jiangsu, China). All
the animals involved were treated in accordance with protocols approved by the
ethical committee of China Pharmaceutical University. All animal experimental
protocols adhered to the Guide for the Care and Use of Laboratory Animals published
by the National Institutes of Health (NIH publication 85-23, revised 1986). Hemolytic
activity was determined by a standard procedure against mouse erythrocytes. Briefly,
the erythrocytes were diluted to 5 × 108/mL, and 250 µL of erythrocytes solution were
incubated for 1 h with various concentrations of conjugates at 37 °C. Intact
erythrocytes were centrifuged at 1500 rpm for 10 min, and the absorbance of the
supernatant at 540 nm was measured by a microplate reader (Thermo, USA). 1%
Triton X-100 was set as a positive control, and PBS buffer served as a negative
control.
4.9 Cellular uptake of conjugates
To investigate if the cell penetration efficiency of the conjugates would be
affected by folate receptor, the cellular uptake of the FITCFA-P3-PTX, FITCP3-PTX,
and FITCP3 were compared. Adherent MCF-7/PTX cells were seeded overnight in
12-well plates and and then treated with labelled conjugates at various concentrations
(1, 2, and 6 µM) for different periods of time for 2 h. Then cells were collected and
washed with cold PBS for three times. The cellular fluorescence intensities of about
10000 cells treated by respective compounds were analyzed each time using a flow
cytometer.
4.10 LDH leakage assay
The LDH leakage assay was used to determine the membrane integrity by using
a commercial LDH Activity Assay Kit (Nanjing Jiancheng Bioengineering Institute)48.
The LDH assay was accomplished according to the manufacturer’s protocols. Briefly,
MCF-7/PTX cells were seeded in a 96-well plate (6 × 103 cells per well) and
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incubated for 24 h. Next, the serum-free medium containing the various
concentrations of conjugates was added and incubated for 12 h. The plates were
centrifuged (3000 × g, 10 min) and supernatants were transferred to a new 96-well
plate. PBS buffer was added as a negative control which was taken as no leakage. The
absorbance was detected by microplate reader at 450 nm. The cells treated with 1%
Triton X-100 represented 100% leakage.
4.11 AO/EB Double Staining Assay
MCF-7/PTX cells was used for AO/EB double staining. Briefly, MCF-7/PTX
cells were grown in a 24-well plate at 5 × 104 cells per well, cultured for 24 h. Then
the cells were treated with various concentration of conjugates for 12 h, respectively.
Next, the cells were stained with 20 µg/ml of AO/EB solution in the dark for 10 min.
Subsequently, excess AO/EB dye mixture was washed with cold PBS. Fluorescence
images were obtained with fluorescence microscopy (Nikon Ts2R).
4.12 Analysis of cell apoptosis
The reported procedures with minor modification was employed for the detection
of percentage of apoptotic cells38. In brief, apoptotic cells were quantitated by flow
cytometry with annexin V–FITC/PI Detection Kit (Nanjing Jiancheng Bioengineering
Institute). MCF-7/PTX cells were seeded in a 6-well plate and cultured overnight.
Next, the cells were incubated with different concentrations of conjugates, or vehicle
for 12 h and were later processed according to the manufacturer’s instructions, and
then, analyzed using the flow cytometer. (Beckman Coulter Accuri C6).
4.13 Cellular Colocalization.
Mitochondrial membrane potential was used to determine the influence of
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compounds FA-P3-PTX and FA-P7-PTX on mitochondrial membrane, relying on the
change of dye JC-1 red to green44. Concisely, MCF-7/PTX cells were seeded in 6 ×
104 cells in glass bottom cell culture dishes and incubated overnight and then treated
with 3 µM of PTX, FA-P3-PTX and FA-P7-PTX for 12 h and stained with JC-1
according to the protocol of Mitochondrial Membrane Potential Assay Kit (Beyotime
Biotechnology). The change of fluorescence was visualized using confocal
microscopy (Olympus IX81).
4.14 Western Blot Analysis of Caspase-3, Cytochrome-C and Folate receptor.
The western blot procedure was conducted based on the protocol reported
formerly43. MCF-7/PTX cells were seeded in a 10 cm dish. The cells were then
treated with 3 µM of FA-P3-PTX or FA-P7-PTX for 2 h. The mediums containing
conjugates was discarded, and the cells were washed with cold PBS three times.
Cellular proteins were extracted in the RIPA lysis buffer (Beyotime) containing 1 mM
PMSF. The cell lysate was boiled in loading buffer for 10 min, and protein
concentration was quantified by BCA method following the procedures of the kit. 40
ug of cell lysate was resolved on gradient Bis-Tris acrylamide gels, and then
transferred to PVDF membrane which were then blocked by incubation for 2 h at
room temperature with 5% bovine serum albumin (BSA) in in TBST buffer (10 mM
Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) to block nonspecific binding.
Subsequently, the membranes were incubated with primary antibodies (CASP3
antibody, CYCS antibody, folate receptor antibody and GADPH antibody) at 4 °C
overnight. Next，the PVDF membranes were washed three times with TBST buffer
and incubated with HRP-conjugated secondary antibody at room temperature for 2 h.
After washing three times, the protein bands were displayed using Tanon High sig
ECL western blotting and a luminescent image analyzer (Tanon 5500). GADPH was
used to confirm equal loading in each lane in the samples prepared from cell lysates.
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4.15 Tubulin Polymerization Assay
BK011P-Tubulin Polymerization Assay Kit was used to determine that
compounds influence on Tubulin Polymerization. Briefly, tubulin (2 mg/mL) in buffer
complement with 1mMGTP and 15% glycerol was utilized. Then, PTX and
FA-P7-PTX were tested at 3 µM final concentration (n = 3 replicates, single
preparation). The changes in the fluorescence intensity (λex =370 nm, λem= 445 nm)
were tested by kinetic reading at 37℃ using a PerkinElmer EnSpire multimode plate
reader.
4.16 In vitro plasma stability
The stability of the conjugates was examined in plasma collected from Wistar
rats, as previously described49. Compounds were measured using an initial
concentration of 500 ug/mL in rat plasma at 37 °C. At 0.5, 1, 2, 3, 4, 8, 12 and 24 h
time points, 100 uL of mixture was extracted and then 400 uL of acetonitrile was
added. After vortex, the mixture was centrifuged at 10000 × g and supernatant were
collected and analyzed using UPLC/MS.
4.17 Animal Model
ICR mice (male, 18−22 g, Certificate number: NO.201822692) were purchased
from the Comparative Medical Center of Yangzhou University (Jiangsu, China). All
the animals involved were treated in accordance with protocols approved by the
ethical committee of China Pharmaceutical University. All animal experimental
protocols adhered to the Guide for the Care and Use of Laboratory Animals published
by the National Institutes of Health (NIH publication 85-23, revised 1986). H22 cells
(5 × 106 cells/ml) were inoculated into the abdomen of male ICR mice, after 1 week,
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