Self-assembling peptide-etoposide nanofibers for overcoming multidrug resistance

Article abstract of DOI:10.1039/d0cc06387h

We developed a new strategy to overcome the MDR of etoposide using self-assembling nanofibers. Compared with the original etoposide, the inhibitory activity of Nap-GFFpYK-etoposide1/2 against murine Lewis lung cancer or breast cancer cells was increased 10 times, and 20 times on these cells with artificially overexpressed MDR1. Our method to synthesize and separate etoposide isomers provides a new strategy for the modification of this drug. This journal is

Full text of DOI:10.1039/d0cc06387h

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Self-assembling peptide–etoposide nanofibers
for overcoming multidrug resistance†
Cite this:DOI: 10.1039/d0cc06387h
a
a
a
a
b
a
Li-Song Zhang, Li-Xin Yan, Shan Gao, Hui Long, Zhen Xi, Lu-Yuan Li and
a
Received 23rd September 2020,
Accepted 6th November 2020
Zhi-Song Zhang
*
DOI: 10.1039/d0cc06387h
rsc.li/chemcomm
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,13
We developed a new strategy to overcome the MDR of etoposide that activates MDR.
The use of biocompatible and readily
using self-assembling nanofibers. Compared with the original etopo- degradable supramolecular hydrogels in drug delivery has
side, the inhibitory activity of Nap-GFFpYK-etoposide1/2 against attracted extensive research interest because of their potential
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4,15
murine Lewis lung cancer or breast cancer cells was increased to fulfill these purposes.
0 times, and 20 times on these cells with artificially overexpressed In this study, we propose a strategy based on the self-
MDR1. Our method to synthesize and separate etoposide isomers assembly of peptides covalently linked to etoposide, with the
1
provides a new strategy for the modification of this drug.
purposes of enhancing the water solubility, facilitating cell
absorption efficiency and raising the intracellular retention
Etoposide is a semi-synthetic derivative of podophyllotoxins time (Scheme 1). The self-assembling prodrug consists of two
1
,2
and is used as a first-line drug for cancer chemotherapy. It parts: a peptide Nap-GFFpYK motif with enzymatic self-
inhibits the function of DNA topoisomerase II by capturing assembly properties and an etoposide ester motif which under-
topological II–DNA complexes, promoting the cleavage of DNA goes ester bond hydrolysis catalyzed by carboxylesterase. We
double strands and preventing cell cycle progression, thus show here that the new etoposide delivery system has the
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,4
inhibiting cell growth. However, poor water solubility and following advantages: first, the Nap-GFFpYK peptide increases
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,5
bioavailability severely limit the clinical utility. Additionally,
the development of multidrug resistance (MDR), partially arising
from a high level of efflux transporter proteins, contributes
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,7
significantly to severely lowered clinical efficacy of etoposide.
Enhancing solubility and raising intracellular retention are
therefore important towards alleviating cancer cell multidrug
resistance to these drugs.
Increasing the retention time of cancer drugs both in the
circulation and inside the target cells using nanocarriers have
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been shown to be a viable approach to enhance their efficacy.
Commonly used nanocarriers include liposomes, polymer
conjugates, micelles, dendrimers, and carbon-based and metal
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nanoparticles. The nanocarriers can facilitate the entering of
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0–12
the compounds into the cells through effective endocytosis.
Furthermore, the nanocarriers are capable of acting as a slow
release apparatus inside the cell so that the local concentration
of the drug may remain below the threshold of the mechanisms
a
State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and
Tianjin Key Laboratory of Molecular Drug Research, NanKai University,
Tianjin 300350, China. E-mail: zzs@nankai.edu.cn
b
State Key Laboratory of Elemento-Organic Chemistry and College of Chemistry,
Nankai University, 94 Weijin Road, Tianjin, 300071, China
Scheme 1 Plausible mechanisms of Nap-GFFpYK-etoposide1 (NFE1) and
Nap-GFFpYK-etoposide2 (NFE2). MDR1: multidrug resistance-1; ALP:
alkaline phosphatase; and CES: carboxylesterase.
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0cc06387h
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the water solubility and speeds up their cellular uptake, shown in Fig. S5 (ESI†). Following purification by HPLC, the
thus markedly enhancing the bioavailability. Second, the compounds were confirmed using NMR spectroscopy and HR-
Nap-GFFpYK-etoposide1/2 nanofibers are able to prevent the MS analysis (Fig. S6A-1–S6P-3, ESI†).
drugs from MDR-mediated efflux, giving rise to effective
concentrations of the drugs inside the cell.
We evaluated the water solubility of the prodrugs NFE1/2 at
pH = 7.4. The original etoposide at 0.1 mmol L forms a cloudy
ꢀ
1
The chemical structures of the Nap-GFFpYK-etoposide1/2 emulsion in PBS, whereas the solution of the NFE1/2 is clear
NFE1/2) hydrogel molecules are shown in Scheme 1. The and transparent, even when the concentration increased by
(
ꢀ
1
peptide Nap(7ACC)-GFFpYK was synthesized using standard 100 times to 10 mmol L (Fig. S7, ESI†). The reason for the
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6
solid-phase synthesis methods (Fig. S1, ESI†). The two hydro- improved solubility is likely attributed to the phosphate group
xyl groups on the opposite side of the glucopyranose ring on the self-assembling peptide, which allows the modified
0 0
(
1 and 2 ) in the etoposide structure can be reacted with molecule to exist in anionic form in PBS at pH = 7.4. We then
succinic anhydride to obtain two isomers of etoposide–succinic determined the ability of the NFE1/2 and AFE1/2 to self-
2
acid (Fig. S2, ESI†). The isomers of etoposide succinic acid (1E assemble when catalyzed by alkaline phosphatase, an enzyme
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and 2E) were isolated using HPLC. The activated ester formed reportedly enriched on the surface of cancer cells. When the
ꢀ
1
from the carboxyl group in 1E/2E and N-hydroxysuccinimide solution of each of the prodrug molecules (1 mmol L in PBS,
ꢀ
1
(NHS) is condensed with the epsilon amino group on the lysine pH = 7.4) with 2.0 U mL
of alkaline phosphatase was
residue on Nap(7ACC)-GFFpYK, giving rise to four etoposide subjected to transmission electron microscopy (TEM) analysis,
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self-assembly molecules (NFE1/2 and AFE1/2) (Fig. 1A and three-dimensional topography of hydrogelic nanofiber struc-
Fig. S3, ESI†). The two active esters of etoposide (E1 and E2) tures was observed, with a uniform width of about 10 nm
were reacted with 7ACC-NH2 to obtain etoposide fluorescent (Fig. 1B and Fig. S3, ESI†). The self-assembled hydrogel
derivatives without self-assembly properties (AE1/2) (Fig. S4, remains stable for more than 14 days at 37 1C.
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ESI†). The chemical synthetic route of ALP inhibitor (DQB) is
We used AFE1/2 molecules to analyze the cell absorption
efficiency, with fluorescent AE1/2 as the control. After treating
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1
LLC and MCF-7 cells with 20 mmol L molecules for 4 hours,
we examined the cells using a confocal laser scanning micro-
scope (CLSM). As shown in Fig. 2A and Fig. S8 (ESI†), a strong
blue fluorescence was observed in the cell cytosol of AFE1/2
groups. However, for the un-nanofibered AE1/2 groups, we can
only collect very weak fluorescence. After culturing MCF-7 cells
with a series of concentrations of AE1/2 and AFE1/2 for 4 hours,
we collected and washed the cells with PBS and then lysed and
checked the fluorescence intensity using a fluorescence spec-
trophotometer (Fig. 2B). The intracellular fluorescence inten-
sity is positively correlated with the drug concentration, and the
AFE1/2 group with self-assembly characteristics has stronger
fluorescence intensity than that of the AE1/2 group (Fig. 2B).
The efficiency of AFE1/2 entering the cell is significantly
reduced when an ALP inhibitor or ALP siRNA is added
(Fig. S9–S11, ESI†). These findings suggest that the modifica-
tion of the drug compounds with the nanofiber allows highly
effective uptake by the cancer cells.
To simulate the release profile of etoposide by intracellular
carboxylesterase from NFE1/2, we analyzed the release curve in
tumor cell lysate by HPLC. The release rate reached 90% at
60 min and exceeded 98% after 96 min (Fig. S12, ESI†). HPLC
detection also revealed that NFE1/2 release etoposide under the
catalysis of carboxylesterase in living cells (Fig. S13, ESI†).
We then determined the improvement in the anticancer
activities of the etoposide-nanofibers on two human breast
cancer cell lines (MCF-7 and MDA-MB-231) and two mouse
cancer cell lines (breast cancer 4T1 and Lewis lung cancer LLC).
We found that, compared to etoposide, the IC50 values of NFE1/
ꢀ
1
2
decreased by more than 10 times from more than 20 mmol L
Fig. 1 (A) Synthetic routes of Nap-GFFpYK-etoposide1 and Nap-GFFpYK-
ꢀ1
to about 2 mmol L (Fig. 3A and Fig. S15A, ESI,† for AFE1/2). No
significant differences in the activities were observed between
NFE1 and NFE2, likely reflecting the fact that the nanofiber
etoposide2. (B) TEM images of hydrogels formed by Nap-GFFpYK-
ꢀ1
etoposide1/2 with the addition of 2.0 U mL ALP at pH = 7.4. Scale bar:
00 nm.
1
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Fig. 3 (A) IC50 of etoposide and etoposide-nanofibers (NFE1/2) on cancer
ꢀ1
cell lines (48 h). (B) Apoptosis induced by the three drugs at 10 mmol L in
different cancer cell lines within 24 h. ***, P o 0.001; N.S., no-significance.
ꢀ1
activities of the original drug. It is plausible that the improve-
Fig. 2 (A) Cell uptake efficiency of AFE1/2 and AE1/2 (20 mmol L , 4 h) in
LLC cells. Scale bar = 10 mm. (B) Fluorescence emission spectra recorded for ment arises from two mechanisms: facilitated entrance of the
ꢀ1
AE1/2 and AFE1/2 in MCF-7 cells. Equal concentration dilution (20 mmol L ).
Time: 6 h. 7ACC: lex =408 nm, lem = 468 nm.
drug into the cells based on effective endocytosis of
the nanofibers, and controlled release of the etoposide from
the nanofibers catalyzed by carboxylesterase, slowing down
modifications and releasing mechanism of the two hydroxyl drug pump activated efflux, as reported previously by other
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sites were identical.
investigators.
Since multidrug resistance can in a significant part be
Next, we tested the antitumor activities of NFE1/2 in an LLC
attributed to the high levels of drug pumping activities of the cell-based mouse lung cancer model, and a 4T1 cell-based
cancer cells, we artificially overexpressed the MDR1 gene of the mouse breast cancer model. As shown in Fig. 4A–D
ABC pump family in LLC/4T1 cells and treated them with the (Fig. S16A–D, ESI†), compared with PBS or Nap-GFFpYK treated
nanofibers (Fig. S14, ESI†). We found that the IC50 value of the (F) control groups, tumors treated with etoposide exhibited
etoposide on the mock-transfected control cells was about 40%–50% reductions of growth rate, measured as tumor weight
ꢀ1
2
0 mmol L , whereas on the MDR1-overexpressing cells it as a function of time. Similar results were obtained in the
increased nearly 3-fold. Remarkably, the IC50 values of the etoposide + Nap-GFFpYK (E + F) group. Significantly, treat-
ꢀ
1
nanofibered drugs were 2–3 mmol L in both the control and ments with drugs NFE1/2 brought the inhibition rates to about
MDR1-overexpressing LLC/4T1 cells (Fig. 3A and Fig. S15A, 80%. These results indicate that the etoposide nanofibers
ESI†), representing a 20-fold enhancement of the drug activities dramatically improve the anticancer activities of the original
in highly drug-resistant cell lines. We evaluated the ability of etoposide.
NFE1/2 to induce apoptosis to these mouse cancer cells (Fig. 3B
The experiments were repeated with tumor models estab-
and Fig. S15B, ESI†). We found that overexpressing MDR1 led lished with MDR1 overexpressed LLC/4T1 cells (Fig. 4E–H and
to 5- to 10-fold decline of the sensitivity of the cancer cells to Fig. S16E–H, ESI†). Again, compared to PBS or Nap-GFFpYK,
etoposide, whereas treatment with the NFE1/2 led to apoptosis treatment with etoposide or mixtures of each of the etoposide
of about 90% of the cancer cells, regardless of the MDR1 with the Nap-GFFpYK, gave rise to 20–23% inhibition of tumor
overexpression, about a 5-fold increase compared to the growth rates, whereas treatment with the nanofibered drugs
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of etoposide either alone or in combination with the peptides
(Nap-GFFpYK) without conjugation. It is likely that the improve-
ment in water solubility resulted from the phosphoric acid group
on the peptidyl moiety, and the enhanced cellular uptake is due
to the effective endocytosis of the nanofibers. Additionally,
etoposide can be released from the nanofibers by the activity
of cytosolic carboxylesterase, constituting a slow release mecha-
nism that reduces the efflux of the drug caused by the ABCB
family of drug pumps.
This work was financial supported by the National Natural
Science Foundation of China (NSFC) projects (Grant no.
81672740, 81972687 and 81874167).
Conflicts of interest
There are no conflicts to declare.
Notes and references
1
Z. Wang, Z. Li, D. Zhang, L. Miao and G. Huang, Drug Delivery, 2015,
2, 79–85.
2
2
C. Che, G. Yang, C. Thiot, M. C. Lacoste, J. C. Currie, M. Demeule,
A. Regina, R. Beliveau and J. P. Castaigne, J. Med. Chem., 2010, 53,
2
814–2824.
3
4
5
6
7
E. G. Gibson, M. M. King, S. L. Mercer and J. E. Deweese, Chem. Res.
Toxicol., 2016, 29, 1541–1548.
Y. Pommier, E. Leo, H. Zhang and C. Marchand, Chem. Biol., 2010,
17, 421–433.
A. K. Mohanty, F. Dilnawaz, C. Mohanty and S. K. Sahoo,
Drug Delivery, 2010, 17, 330–342.
Z. Wang, Z. Wang, D. Liu, X. Yan, F. Wang, G. Niu, M. Yang and
X. Chen, Angew. Chem., 2014, 53, 1997–2001.
I. Landini, A. Lapucci, A. Pratesi, L. Massai, C. Napoli, G. Perrone,
P. Pinzani, L. Messori, E. Mini and S. Nobili, Oncotarget, 2017, 8,
9
6062–96078.
Fig. 4 Anti-tumor activity of Nap-GFFpYK-etoposide1/2 (NFE1/2) in LLC/
8
9
Y. Yuan, L. Wang, W. Du, Z. Ding, J. Zhang, T. Han, L. An, H. Zhang
and G. Liang, Angew. Chem., 2015, 54, 9700–9704.
J. L. Markman, A. Rekechenetskiy, E. Holler and J. Y. Ljubimova,
Adv. Drug Delivery Rev., 2013, 65, 1866–1879.
0 H. Wang, Z. Feng, Y. Wang, R. Zhou, Z. Yang and B. Xu, J. Am. Chem.
Soc., 2016, 138, 16046–16055.
1 Q. Wang, N. Jiang, B. Fu, F. Huang and J. Liu, Biomater. Sci., 2019, 7,
4
T1 cells and LLC/4T1 multi-drug resistant cells tumor model. (A, C, E and
G) Tumor volume curves during the curing process. (B, D, F and H) Excised
tumor weight after the treatment process. The doses of etoposide, F and
ꢀ1
NFE1/2 for peritumoral injection are 5, 7.72 and 13.41 mg kg (equimolar
1
1
1
1
concentration), respectively. **, P o 0.01; ***, P o 0.001.
4
888–4911.
2 D. Zheng, Y. Chen, S. Ai, R. Zhang, Z. Gao, C. Liang, L. Cao, Y. Chen,
Z. Hong, Y. Shi, L. Wang, X. Li and Z. Yang, Research, 2019, 4803624.
3 Q. Feng, J. Liu, X. Li, Q. Chen, J. Sun, X. Shi, B. Ding, H. Yu, Y. Li and
X. Jiang, Small, 2017, 13.
4 H. Wang, Z. Feng and B. Xu, Angew. Chem., 2019, 58, 10423–10432.
5 S. Kiran, Z. Hai, Z. Ding, L. Wang, Y. Liu, H. Zhang and G. Liang,
Chem. Commun., 2018, 54, 1853–1856.
(NFE1/2) led to 75%–79% inhibition of tumor growth. These
results indicate that NFE1/2 possess much improved anti-
cancer activities compared to etoposide against multidrug-
resistant cancer cells.
1
1
In conclusion, to improve the water solubility and cell absorp-
tion efficiency of etoposide, we successfully synthesized and 16 I. Coin, M. Beyermann and M. Bienert, Nat. Protoc., 2007, 2,
3
247–3256.
separated two self-assembling peptide–etoposide nanofiber iso-
mers Nap-GFFpYK-etoposide1/2 (NFE1/2) and evaluated their
1
7 J. Li, Y. Gao, Y. Kuang, J. Shi, X. Du, J. Zhou, H. Wang, Z. Yang and
B. Xu, J. Am. Chem. Soc., 2013, 135, 9907–9914.
anticancer activities in MDR1-overexpressing LLC and 4T1 can- 18 R. Dahl, E. A. Sergienko, Y. Su, Y. S. Mostofi, L. Yang, A. M. Simao,
S. Narisawa, B. Brown, A. Mangravita-Novo, M. Vicchiarelli,
L. H. Smith, W. C. O’Neill, J. L. Millan and N. D. Cosford, J. Med.
Chem., 2009, 52, 6919–6925.
cer cells and experimental tumor models established with these
cells. Our data show that the etoposide-conjugated nanofibers
have markedly improved anticancer efficacy compared with that 19 J. Gao, J. Zhan and Z. Yang, Adv. Mater., 2020, 32, e1805798.
Chem. Commun.
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