Self-Delivery Nanoparticles of Amphiphilic Acyclic Enediynes for Efficient Tumor Cell Suppression

Article abstract of DOI:10.1002/cjoc.201900034

Four acyclic maleimide-based enediyne compounds with different hydrophilicity were synthesized through Sonogashira reaction to reveal a self-delivery antitumor drug platform. As proved by ESR analysis, the enediyne compounds undergo Bergman-like cyclization and generate diradical intermediates at physiological temperature, which are able to induce DNA-cleavage through the abstraction of H atoms from the sugar-phosphate backbones. When the critical aggregation concentration is reached in water, the amphiphilic enediyne compounds self-assemble into nanoparticles and possess the self-delivery ability to be facilely admitted by tumor cells, resulting in greatly improved cytotoxicity (IC₅₀ down to 10 μmol·L^–1) and much higher tumor cell apoptosis rate (up to 86.6%) in comparison with either the hydrophilic or the lipophilic enediyne compound. The enhanced endocytosis of the amphiphilic enediyne compounds was further confirmed through confocal laser scanning microscopy analysis. The unveiled relationship between the hydrophilicity of enediyne drugs and their therapeutic efficacy will provide a guideline for the design of new self-delivery drugs employed in medicinal applications.

Full text of DOI:10.1002/cjoc.201900034

Accepted Article
Title: Self-delivery nanoparticles of amphiphilic acyclic enediynes for efficient
tumor cell suppression
Authors: Jing Li, Yuequn Wu, Lili Sun, Shuai Huang, Baojun Li, Yun Ding, Aiguo Hu*
This manuscript has been accepted and appears as an Accepted Article online.
This work may now be cited as: Chin. J. Chem. 2019, 37, 10.1002/cjoc.201900034.
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Self-delivery nanoparticles of amphiphilic acyclic enediynes for efficient
tumor cell suppression
Jing Lia, Yuequn Wub, Lili Sunb, Shuai Huanga, Baojun Lia, Yun Dinga, Aiguo Hu*,a
a Shanghai Key Laboratory of Advanced Polymeric Materials, School of Materials Science and Engineering, East China University of Science and Technology,
Shanghai 200237, China
b The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China
Cite this paper: Chin. J. Chem. 2019, 37, XXX—XXX. DOI: 10.1002/cjoc.201900XXX
Four acyclic maleimide-based enediyne compounds with different hydrophilicity were synthesized through Sonogashira reaction to reveal a self-delivery
antitumor drug platform. As proved by ESR analysis, the enediyne compounds undergo Bergman-like cyclization and generate diradical intermediates at
physiological temperature, which are able to induce DNA-cleavage through the abstraction of H atoms from the sugar-phosphate backbones. When the
critical aggregation concentration is reached in water, the amphiphilic enediyne compounds self-assemble into nanoparticles and possess the
self-delivery ability to be facilely admitted by tumor cells, resulted in greatly improved cytotoxicity (IC50 down to 10μM) and much higher tumor cell
apoptosis rate (up to 86.6%) in comparison with either the hydrophilic or the lipophilic enediyne compound. The enhanced endocytosis of the
amphiphilic enediyne compounds was further confirmed through confocal laser scanning microscopy analysis. The unveiled relationship between the
hydrophilicity of enediyne drugs and their therapeutic efficacy will provide a guideline for the design of new self-delivery drugs employed in medicinal
applications.
developing new anticancer drugs, the enediyne family remains
very small because of the limitation on the extraction and
Introduction
total-synthesis technique, with only 11 structurally characterized
members known to date15. Broadening the enediyne family
Cancer is a complex disease causing severe pain on the
patients and ranked as the leading cause of human death. Cancer
is considered as the single most important barrier to increasing
life expectancy in all the countries in the 21st century according to
the GLOBOCAN results in 2018.1 In recent years, various
therapeutics have been developed for cancer treatment including
chemotherapy, immunotherapy, radiation therapy and surgical
excision2,3. Among these therapeutics, the chemotherapy, which
relies on the high cytotoxicity of chemotherapeutic agents plays
an important role. It is therefore critical to explore and identify
new chemotherapeutic agents to achieve more efficient
chemotherapy and curing of cancer.
The majority of chemotherapeutic agents are extracted from
nature resources and were approved by the United States Food
and Drug Administration (USFDA) historically,4,5 like paclitaxel,
camptothecin and their analogs. Since the late 1980s, a series of
natural occurring compounds have attracted great research
attentions for their extremely high cytotoxicity.6,7 These
through designing their non-natural analogues is highly important.
Many enediyne with various structural features were synthesized
and their biological properties were evaluated.18 In our previous
work, we found that the onset temperatures of a thermal
Bergman cyclization reaction was drastically lowered when a
maleimide moiety was introduced at the ene position.19 Following
this line, we have synthesized several acyclic enediyne antibiotics
with maleimide structure to achieve moderate to high cytotoxicity
towards tumor cells.20-22
Direct administration of small molecular drugs suffers from
low bioavailability, nonspecific cytotoxicity, severe side effects,
rapid drug elimination, and severe multidrug resistance (MDR).23
To this end, nanoscale drug-carriers including liposomes,
polymeric micelles, inorganic nano-frameworks have been
employed on drug delivery.24-28 These nanoscale drug delivery
systems show higher therapeutic efficacy and lower side effects
than free drugs due to the enhanced permeability and retention
(EPR) effect.29 However, the drug-carriers themselves have no
therapeutic effect, and the unpredicted degradation of the
carriers might cause side-effects.30,31 The drug self-delivery
systems (DSDSs), in which active drugs exhibit nanoscale
characteristic to realize intracellular delivery by themselves have
been rapidly developed to address these issues.32-34 The DSDSs
hold the following features: (i) accumulation in tumors due to the
EPR effect; (ii) excellent drug loading capacities (up to 100% for
pure nanodrugs); (iii) no carrier-induced toxicity and
immunogenicity.35-40
compounds, to name
a
few, calicheamicin,8 dynemicins,9
C-1027,10 possess the enediyne structure which are readily
triggered in vivo to generate diradicals through Bergman or
Bergman-like cyclization.11 The highly reactive diradicals can
abstract the H atoms from the DNA’s sugar-phosphate backbones
to cause DNA cleavage and finally kill the cancer cells. Much
research progress has been made with the natural enediyne
antibiotics,12-15 and recently the USFDA has approved two
antibody drug conjugates, Mylotarg® and Besponsa®, with
calicheamicin as the “warhead”.16,17 Despite their high potential in
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differences between this version and the Version of Record. Please cite this article as doi:
10.1002/cjoc.201900034
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Herein, we report four maleimide-based enediyne compounds
cell culturing media or blood stream.
with different hydrophilicity. Among which, two of them exhibit
the potential as DSDSs. The two hydroxyl groups endow them
hydrophilicity, while the rest parts endow them lipophilicity.
When the concentrations of these two enediyne compounds
reach the critical aggregation concentration (CAC), self-assembled
nanoparticles with size around 100 nm were formed, which would
accumulate in tumor tissues through EPR effect and enter the
tumor cells by receptor-mediated endocytosis41,42. Encouragingly,
the enediyne compounds with DSDS feature show much higher
cytotoxicity than their hydrophilic or lipophilic counterparts,
suggesting a simple yet efficient drug design strategy.
The self-assembled nanoparticles (NPs) of EDY A and B were
prepared by slowly adding their THF solutions into water,
followed by removal of THF through evaporation. The DLS curves
(Figure 2A and 2B) of the assemblies show the formation of EDY
NPs with narrow size distributions. The average hydrodynamic
diameter of EDY A NPs and EDY B NPs are 97.5 nm and 129 nm,
respectively. TEM images (Figure 2C, 2D) show that the NPs are
spherical micelles with average diameter of 80 nm for EDY A, and
100 nm for EDY B. The slight difference of the size measured with
DLS and TEM is due to the shrinkage of nanoparticles in a drying
state during TEM sample preparation.
Enediyne compounds undergo Bergman or Bergman-like
cycloaromatization to generate diradical intermediates, endowing
them DNA-cleavage ability and cytotoxicity. To verify this
transformation, the structural change of the enediyne compounds,
in particular, the reaction of the triple bonds was characterized
with FT-IR and Raman spectroscopies. EDY A was chosen as the
representative compound for this study as all four compounds
share the same enediyne core structure. The characteristic peak
of the stretching of triple bond in EDY A is observed at 2211 cm-1.
This peak disappeared completely after EDY A was kept in
methanol at 37ºC for 2 days, while the other peaks almost
unchanged (Figure S11).
Results and Discussion
The four enediynes (EDY) (Fig. 1) were synthesized from
3,4-diiodomaleimides and terminal alkynes through Sonogashira
reaction (ESI). The chemical structure of EDY A-C were verified by
1H NMR, 13C NMR and HR-MS spectroscopies (ESI), while the EDY
D is referred to our previous work.43 The molecular structure of
the EDY A and B were carefully designed to endow them
amphiphilicity to meet the criteria of DSDSs, while the EDY C was
designed as a hydrophilic control and EDY D was used as a
hydrophobic control. The physicochemical parameters of these
enediynes are listed in Table S1. All of the enediyne compounds
are likely to exhibit good absorption or permeation to cancer cells
according to the Lipinski’s Rule of Five (Ro5, MWT ≤ 500, log P ≤ 5,
H-bond donors ≤ 5, H-bond acceptors ≤ 10).44
HO
HO
O
O
N
N
O
O
HO
HO
A
B
O
HO
HO
O
N
N
OH
O
O
O
D
C
Figure 1 Chemical structures of enediyne compounds.
The critical aggregation concentration (CAC) of two
amphiphilic enediynes were measured by using Nile Red as a
fluorescent probe.45 The fluorescence emission of Nile Red
increases drastically when it is transferred from an aqueous
solution to a hydrophobic environment, indicating the formation
of micellar (or micelle-like) assembly.46-48 Figure S10 shows that
the CAC values of the EDY A and EDY B are 19 μM and 7 μM,
respectively. The lower CAC value of EDY B is related to its higher
LogP value (Table S1), which is beneficial to promote the
formation of self-assembly for DSDSs. The hydrophilic EDY C is
freely soluble in aqueous solutions, however, it might encounter
difficulty when admitted by tumor cells as cell membranes are
lipophilic phospholipid bilayers. The lipophilic EDY D (LogP, 5.46)
might enter the tumor cells through penetration or diffusion, but
its bioavailability would be rather low due to its low solubility in
Figure 2 (A) (B) DLS curves of EDY A and EDY B NPs. (C) (D) TEM images of
EDY A and EDY B nanoparticles.
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Running title
Chin. J. Chem.
2h
12h
2D
5D
3490
3500
3510
3520
3530
3540
3550
G
Figure 3 (A) ESR spectra of the EDY A-PBN adduct in methanol at 37 ℃.
Figure 4 Cleavage of DNA by EDY A at different concentrations for 72 h at
37 ºC. Lane 1: DNA (4 μL) in TE buffer (2 μL, pH 7.6); lane 2: DNA (4 μL) +
DMSO (2 μL); Lane 3: DNA (4 μL) + EDY A (200 mM) in DMSO (2 μL); lane 4:
DNA (4 μL) + EDY A (100 mM) in DMSO (2 μL) ; lane 5: DNA (4 μL) + EDY
A(50 mM) in DMSO (2 μL)
Electron spin resonance (ESR) spectroscopy analysis was used
to verify the radical nature of the cycloaromatization. The highly
reactive carbon radicals generated from the reaction were
trapped by radical-trapping agents and turn into stable free
radicals for ESR study.49 Usuki et al. conducted a spin-trapping
experiment on calicheamicin to confirm the generation and the
evolution of radical species in ethanol solution.50 Regarding to this,
phenyl tert-butyl nitrone (PBN) was used for this experiment (PBN
itself shows no ESR signal, Figure S12A). As shown in Figure 3, ESR
spectrum of the mixture of EDY A and PBN in methanol exhibits
triplet signals51 with hyperfine coupling of 1.5 mT. The minor
triplets in the spectrum might come from the radical species
formed from the H-abstraction of the solvent molecules by the
highly reactive diradical intermediates followed by radical addition
with PBN. Interestingly, the intensity of the recorded ESR signals
increased in a time dependent manner in 5 days, suggesting that
the generation of radical species from EDY A would last for a long
time. Similarly, ESR spectrum of the mixture of EDY B and PBN in
DMSO exhibits triplet ESR signals (Figure S12B).
DNA cleavage experiments were conducted to further prove
the ability of the diradicals to abstract H atoms from the DNA’s
sugar-phosphate backbones. Two kinds of supercoiled plasmid
DNA were used for this study by analysing the conversion of DNA
from native supercoiled (Form I) to circular relaxed form (Form II,
single-strand cleavage).6 The supercoiled plasmid DNA was
incubated with EDY A at 37 ºC for 72 h and then subjected to
agarose gel electrophoresis (Figure 4). Free DNA or DNA treated
with DMSO were set as control group. The supercoiled DNA are
significantly converted to Form II in the presence of EDY A (lane 3,
4, 5) in a concentration dependent manner, while almost no
change was found for the control group (lane 1, 2). All the above
results indicate that the EDY are able to generate diradical
through cycloaromatization at physiological temperature to cause
DNA cleavage, endowing them the potential as antitumor agents.
Chin. J. Chem. 2019, 37, XXX－XXX
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(50 μM) at 37 °C for the predetermined time intervals (0.5 h, 1.5 h,
2.5 h and 3.5 h) and then subjected for flow cytometry analysis.
The fluorescence of EDY A was monitored with a 375 nm laser and
the cells without any treatment were taken as a control. As shown
in Figure 5, the fluorescence intensity of EDY A NPs in Hela cells
steadily increases with the extension of culture time.
Figure 5 The relative geometrical mean fluorescence intensities of Hela
cells incubated with the EDY A nanoparticles for different time and then
analyzed with flow cytometry. Insert: representative flow cytometry
histogram profiles of Hela cells cultured with EDY A nanoparticles for 3.5 h,
the untreated cells are used as a control.
Figure 6 CLSM images of Hela cells treated with a) EDY A at the
concentration of 5 μM b) EDY A Nps at the concentration of 50 μM and c)
EDY C at the concentration of 50 μM for 24 h. Cell nuclei are stained with
PI. The scale bar represents 50 μm.
It is generally acknowledged that the nanoparticular drugs
with size in the range of tens to hundreds of nanometer could be
internalized into cells through receptor-mediated endocytosis41
while the small molecular drugs mainly go through diffusion.
Whether the EDY compounds could be effectively transported
into tumor cells is a crucial aspect for their therapeutic efficacy. To
this end, confocal laser scanning microscopy (CLSM) and flow
cytometry technique were used to evaluate the cellular uptake of
the EDY compounds, and Hela cells were chosen as model cells.
Taking advantages of the blue fluorescent emission of these
enediyne compounds (Figure S13), they were directly used as the
fluorescence probes for the cell internalization analysis.
For CLSM study, HeLa cells were respectively treated with EDY
A (5μM, below CAC), EDY A NPs (50 μM, above CAC), EDY C (50
μM) and cultured for 24 h before observation. Propidium iodide
(PI) solution was used to stain the nuclei. As shown in Figure 6a,
the small molecular EDY A was found in the cancer cells, proving
the successful molecular design of the EDY molecule according to
the Lipinski’s Rule of Five (Ro5). Interestingly, when the
concentration of EDY A is higher than the CAC, both cytoplasm
and nuclei of the cells exhibit intense blue fluorescence,
demonstrating that EDY A NPs were efficiently internalized by
cancer cells in 24 h through endocytosis. The much brighter
fluorescence in Figure 6b than that in Figure 6a indicates that the
endocytosis of nanoparticular drugs is more efficient than the
diffusion or permeation of the small molecule drugs. In a sharp
contrast, even at a high concentration, the blue fluorescence of
EDY C is almost invisible in HeLa cells (Fig. 6c), indicating that the
highly hydrophilic drug had encountered difficulty to permeate
through the lipophilic cytomembrane. Flow cytometry analysis
was performed to further confirm the gradual internalization of
the EDY A NPs. Hela cells were firstly incubated with EDY A NPs
The proliferation inhibition of all four enediyne compounds
against a model cancer lines, HeLa cells, was evaluated by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide
(MTT) assay. The HeLa cells were firstly cultured with the
enediyne compounds at concentrations ranging from 0.9 to 60
μM and then respectively treated with MTT and characterized
with fluorescence spectroscopy. The cells treated with 0.1%
DMSO was used as a control. The cell proliferation results are
shown in Figure 7. After incubation for 48 h, EDY C almost shows
no cytotoxicity within the concentration range, which is in
consistent with the cell internalization result. EDY A starts to show
the proliferation inhibition of Hela cells when the concentration is
below the CAC value (19 μM). When the concentration is higher
than CAC value, EDY A would self-assemble into nanoparticles and
shows profound inhibition of the proliferation of Hela cells (over
80%). The therapeutic efficacy of EDY B is more dependent on its
concentration. When the concentration of EDY B is lower than the
CAC value (7 μM), the cytotoxicity towards HeLa cells is rather low.
However, when the concentration of EDY B is higher than the CAC
value, the cytotoxicity of the formed EDY B NPs is greatly
improved. The IC50 concentrations of EDY A (11.2 μM) and EDY B
(10 μM) are lower than that of their lipophilic analogue EDY D
(22.06 μM) and hydrophilic analogue EDY C (>100 μM), proving
that the DSDSs would greatly enhance the bioavailability of the
enediyne compounds and their therapeutic efficiency. Indeed, the
cytotoxicity of both EDY A and EDY B are comparable with many
clinically used antitumor agents such as doxorubicin and
cisplatin.52,53
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Running title
Chin. J. Chem.
EDY A Nps
EDY B Nps
EDY C
120
100
80
60
40
20
0
EDY D
#
#
#
#
#
**
**
##
**
##
***
**
**
##
##
***
***
Control
2
4
8
16
32
64
Concentration(μM)
Figure 8 (A) Flow cytometry analysis for apoptosis of Hela cells induced by
EDY A, B Nps and EDY C, D at the concentration of 30 μM for 24 h. Lower
left, living cells; lower right, early apoptotic cells; upper right, late
apoptotic cells; upper left, necrotic cells. Inserted numbers in the profiles
indicate the percentage of the cells present in this area. (B) Ratio of
apoptotic Hela cells based on the results of flow cytometry measurements.
Values represent mean ± SD (n = 3). A significant increase in the apoptosis
rate compared with the EDY C-treated cells is denoted with “**” (P < 0.01)
and “***” (P < 0.001), and a significant increase compared with EDY
D-treated cells is denoted with “#” (P < 0.01) and “##” (P < 0.001).
Figure 7 In vitro cytotoxicity study on Hela cells incubated with different
concentrations of EDY A, B Nps and EDY C, D for 24h determined by MTT
assay. Data are shown as means ± SD (n = 3). A significant decrease
compared with EDY C-treated cells is denoted by “*” (P < 0.05), “**” (P <
0.01) and “***” (P < 0.001). A significant decrease compared with EDY
D-treated cells is denoted by “#” (P < 0.05), “##” (P < 0.01) and “###” (P <
0.001)
Generally, driving tumor cells into apoptosis pathway is a
principal mechanism of most cytotoxic drugs to show their
chemotherapeutic ability.54 To confirm the fact that the induced
death of cancer cells incubated with enediyne compounds is
mainly through cell apoptosis, fluorescein isothiocyante
(FITC)-Annexin V/PI apoptosis detection kit was applied to qualify
the ratio of apoptosis cells. HeLa cells were respectively treated
with the enediyne compounds (30 μM) for 24 h, followed by
staining with FITC-Annexin V/PI. The cells without any treatment
were applied as a control. As shown in Figure 8, the apoptotic
percentages induced by EDY A-D are 64.8%, 86.6%, 20.8%, and
49.4%, respectively, while the necrotic cells were not significant in
all groups. The significant higher apoptosis rate induced by the
nanoparticular enediyne compounds (both above their CAC)
further corroborate the high efficiency of the amphiphilic DSDSs
to show great potential in chemotherapy and cancer curing.
Conclusions
We have designed and synthesized four acyclic enediyne
compounds, which are able to generate active diradicals and lead
to DNA cleavage through Bergman cyclization or Bergman-like
reaction. The molecular amphiphilic nature of two of the
enediynes endow them ability to self-assemble into nanoparticles
in water and to be self-delivered into tumor tissues through the
EPR effect with a high drug loading capacity (100%). After being
internalized into the HeLa cells through endocytosis, the
self-assembled enediyne nanoparticles would be swallowed by
the lysosome and disassembled into free enediyne, which further
cause the DNA-cleavage and induce tumor cell death through
apoptosis pathway. In comparison, both the hydrophilic and
lipophilic enediyne compounds have encountered difficulties to
be delivered into tumor cells and resulted in deteriorated
cytotoxicity. This study clearly shows that by sophisticated
adjusting the hydrophilic/lipophilic balance of a small molecular
drug, the therapeutic efficacy could be greatly enhanced,
provides a simple yet reasonable guideline for the development
of new chemotherapeutic agents with self-delivery feature.
Supporting Information
The supporting information for this article is available on the
WWW under https://doi.org/10.1002/cjoc.2018xxxxx.
Acknowledgement
The financial supports from National Natural Science
Chin. J. Chem. 2019, 37, XXX－XXX
© 2019 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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First author et al.
Report
Foundation of China (21871080, 21503078, 21474027), the
Fundamental Research Funds for the Central Universities
(22221818014), and Shanghai Leading Academic Discipline Project
(B502) are greatly acknowledged. AH thanks the “Eastern Scholar
Professorship” support from Shanghai Local Government. AH
thanks Prof. Runhui Liu for his kindly advice in cell experiments.
[13] Chang, C.-Y.; Yan, X.; Crnovcic, I.; Annaval, T.; Chang, C.; Nocek, B.;
Rudolf, J. D.; Yang, D.; Hindra; Babnigg, G.; Joachimiak, A.; Phillips, G.
N.; Shen, B. Resistance to Enediyne Antitumor Antibiotics by
Sequestration. Cell Chem. Biol. 2018, 25, 1075-+.
[14] Li, L.; Hu, L.; Zhao, C.-y.; Zhang, S.-h.; Wang, R.; Li, Y.; Shao, R.-g.;
Zhen, Y.-s. The Recombinant and Reconstituted Novel Albumin–
Lidamycin Conjugate Shows Lasting Tumor Imaging and Intensively
Enhanced Therapeutic Efficacy. Bioconjugate Chem. 2018, 29,
3104-3112.
[15] Yan, X. H.; Ge, H. M.; Huang, T. T.; Hindra; Yang, D.; Teng, Q. H.;
Crnovcic, I.; Li, X. L.; Rudolf, J. D.; Lohman, J. R.; Gansemans, Y.; Zhu,
X. C.; Huang, Y.; Zhao, L. X.; Jiang, Y.; Van Nieuwerburgh, F.; Rader, C.;
Duan, Y. W.; Shen, B. Strain Prioritization and Genome Mining for
Enediyne Natural Products. Mbio 2016, 7.
References
[1] Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R. L.; Torre, L. A.; Jemal,
A. Global cancer statistics 2018: GLOBOCAN estimates of incidence
and mortality worldwide for 36 cancers in 185 countries. CA Cancer J.
Clin. 2018, 68, 394-424.
[2] Torre, L. A.; Bray, F.; Siegel, R. L.; Ferlay, J.; Lortet-Tieulent, J.; Jemal,
A. Global cancer statistics, 2012. CA Cancer J. Clin. 2015, 65, 87-108.
[3] Xu, Z.; Shi, X.; Hou, M.; Xue, P.; Gao, Y.-E.; Liu, S.; Kang, Y.
Disassembly of amphiphilic small molecular prodrug with
fluorescence switch induced by pH and folic acid receptors for
targeted delivery and controlled release. Colloids Surf., B 2017, 150,
50-58.
[4] Qazi, A. K.; Siddiqui, J. A.; Jahan, R.; Chaudhary, S.; Walker, L. A.;
Sayed, Z.; Jones, D. T.; Batra, S. K.; Macha, M. A. Emerging
therapeutic potential of graviola and its constituents in cancers.
Carcinogenesis 2018, 39, 522-533.
[5] Newman, D. J. Natural Products as Leads to Potential Drugs: An Old
Process or the New Hope for Drug Discovery? J. Med. Chem. 2008, 51,
2589-2599.
[6] Kar, M.; Basak, A. Design, Synthesis, and Biological Activity of
Unnatural Enediynes and Related Analogues Equipped with
pH-Dependent or Phototriggering Devices. Chem. Rev. 2007, 107,
2861-2890.
[7] Zein, N.; Sinha, A. M.; McGahren, W. J.; Ellestad, G. A. Calicheamicin
gamma 1I: an antitumor antibiotic that cleaves double-stranded DNA
site specifically. Science 1988, 240, 1198-1201.
[8] Lee, M. D.; Manning, J. K.; Williams, D. R.; Kuck, N. A.; Testa, R. T.;
Borders, D. B. Calicheamicins, a novel family of antitumor antibiotics.
3. Isolation, purification and characterization of calicheamicins beta
1Br, gamma 1Br, alpha 2I, alpha 3I, beta 1I, gamma 1I and delta 1I. J.
Antibiot. 1989, 42, 1070-1087.
[9] Konishi, M.; Ohkuma, H.; Matsumoto, K.; Saitoh, K.; Miyaki, T.; Oki,
T.; Kawaguchi, H. Dynemicins, new antibiotics with the 1,
5-diyn-3-ene and anthraquinone subunit. I. Productin, isolation and
physico-chemical properties. J. Antibiot. 1991, 44, 1300-1305.
[10] Hirama, M.; Akiyama, K.; Tanaka, T.; Noda, T.; Iida, K.-i.; Sato, I.;
Hanaishi, R.; Fukuda-Ishisaka, S.; Ishiguro, M.; Otani, T.; Leet, J. E.
Paramagnetic Enediyne Antibiotic C-1027:ꢀ Spin Identification and
Characterization of Radical Species. J. Am. Chem. Soc. 2000, 122,
720-721.
[11] Nicolaou, K. C.; Sorensen, E. J.; Discordia, R.; Hwang, C.-K.; Bergman,
R. G.; Minto, R. E.; Bharucha, K. N. Ten-Membered Ring Enediynes
with Remarkable Chemical and Biological Profiles. Angew. Chem. Int.
Ed. Eng. 1992, 31, 1044-1046.
[16] Thomas, A.; Teicher, B. A.; Hassan, R. Antibody–drug conjugates for
cancer therapy. Lancet Oncol. 2016, 17, e254-e262.
[17] Bross, P. F.; Beitz, J.; Chen, G.; Chen, X. H.; Duffy, E.; Kieffer, L.; Roy,
S.; Sridhara, R.; Rahman, A.; Williams, G.; Pazdur, R. Approval
Summary：Gemtuzumab Ozogamicin in Relapsed Acute Myeloid
Leukemia. Clin. Cancer Res. 2001, 7, 1490-1496.
[18] Romeo, R.; Giofre, S. V.; Chiacchio, M. A. Synthesis and Biological
Activity of Unnatural Enediynes. Curr. Med. Chem. 2017, 24,
3433-3484.
[19] Sun, S.; Zhu, C.; Song, D.; Li, F.; Hu, A. Preparation of conjugated
polyphenylenes from maleimide-based enediynes through
thermal-triggered Bergman cyclization polymerization. Polym. Chem.
2014, 5, 1241-1247.
[20] Song, D.; Tian, Y.; Huang, S.; Li, B.; Yuan, Y.; Hu, A. An acyclic
enediyne with a furyl tethering group for efficient inhibition of tumor
cell viability. J. Mater. Chem. B 2015, 3, 8584-8588.
[21] Song, D.; Sun, S.; Tian, Y.; Huang, S.; Ding, Y.; Yuan, Y.; Hu, A.
Maleimide-based acyclic enediyne for efficient DNA-cleavage and
tumor cell suppression. J. Mater. Chem. B 2015, 3, 3195-3200.
[22] Li, J.; Li, B.; Sun, L.; Duan, B.; Huang, S.; Yuan, Y.; Ding, Y.; Hu, A.
Self-delivery nanoparticles of an amphiphilic irinotecan–enediyne
conjugate for cancer combination chemotherapy. J. Mater. Chem. B
2019, 7, 103-111.
[23] Huttunen, K. M.; Raunio, H.; Rautio, J. Prodrugs—from Serendipity
to Rational Design. Pharmacol. Rev. 2011, 63, 750-771.
[24] Tarn, D.; Ashley, C. E.; Xue, M.; Carnes, E. C.; Zink, J. I.; Brinker, C. J.
Mesoporous Silica Nanoparticle Nanocarriers: Biofunctionality and
Biocompatibility. Acc. Chem. Res. 2013, 46, 792-801.
[25] Zhang, J.; Yuan, Z.-F.; Wang, Y.; Chen, W.-H.; Luo, G.-F.; Cheng, S.-X.;
Zhuo, R.-X.; Zhang, X.-Z. Multifunctional Envelope-Type Mesoporous
Silica Nanoparticles for Tumor-Triggered Targeting Drug Delivery. J.
Am. Chem. Soc. 2013, 135, 5068-5073.
[26] Wang, W.; Despanie, J.; Shi, P.; Edman, M. C.; Lin, Y.-A.; Cui, H.;
Heur, M.; Fini, M. E.; Hamm-Alvarez, S. F.; MacKay, J. A.
Lacritin-mediated regeneration of the corneal epithelia by protein
polymer nanoparticles. J. Mater. Chem. B 2014, 2, 8131-8141.
[27] Sun, T.; Zhang, Y. S.; Pang, B.; Hyun, D. C.; Yang, M.; Xia, Y.
Engineered Nanoparticles for Drug Delivery in Cancer Therapy.
Angew. Chem. Int. Ed. 2014, 53, 12320-12364.
[12] Wang, R.; Li, L.; Zhang, S.; Li, Y.; Wang, X.; Miao, Q.; Zhen, Y. A novel
enediyne‐integrated antibody–drug conjugate shows promising
antitumor efficacy against CD30+ lymphomas. Mol. Oncol. 2018, 12,
339-355.
[28] Hubbell, J. A.; Chilkoti, A. Nanomaterials for Drug Delivery. Science
2012, 337, 303-305.
[29] Maeda, H. Toward a full understanding of the EPR effect in primary
www.cjc.wiley-vch.de
© 2019 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2019, 37, XXX－XXX
This article is protected by copyright. All rights reserved.

Running title
Chin. J. Chem.
and metastatic tumors as well as issues related to its heterogeneity.
Adv. Drug Del. Rev. 2015, 91, 3-6.
[43] Huang, S.; Li, J.; Li, B.; Hu, A. Design, synthesis and cytotoxicity
study of a novel acyclic enediyne J. East Chin. Univ. Sci. & Tech. 2017,
43, 1-7.
[30] Yu, D.; Peng, P.; Dharap, S. S.; Wang, Y.; Mehlig, M.; Chandna, P.;
Zhao, H.; Filpula, D.; Yang, K.; Borowski, V.; Borchard, G.; Zhang, Z.;
Minko, T. Antitumor activity of poly(ethylene glycol)–camptothecin
conjugate: The inhibition of tumor growth in vivo. J. Controlled
Release 2005, 110, 90-102.
[31] Greenwald, R. B.; Choe, Y. H.; McGuire, J.; Conover, C. D. Effective
drug delivery by PEGylated drug conjugates. Adv. Drug Del. Rev. 2003,
55, 217-250.
[32] He, D.; Zhang, W.; Deng, H.; Huo, S.; Wang, Y.-F.; Gong, N.; Deng, L.;
Liang, X.-J.; Dong, A. Self-assembling nanowires of an amphiphilic
camptothecin prodrug derived from homologous derivative
conjugation. Chem. Commun. 2016, 52, 14145-14148.
[33] Fumagalli, G.; Marucci, C.; Christodoulou, M. S.; Stella, B.; Dosio, F.;
Passarella, D. Self-assembly drug conjugates for anticancer
treatment. Drug Discov. Today 2016, 21, 1321-1329.
[44] Lipinski, C. A. Lead- and drug-like compounds: the rule-of-five
revolution. Drug Discovery Today: Technol. 2004, 1, 337-341.
[45] Shen, Y.; Jin, E.; Zhang, B.; Murphy, C. J.; Sui, M.; Zhao, J.; Wang, J.;
Tang, J.; Fan, M.; Van Kirk, E.; Murdoch, W. J. Prodrugs Forming High
Drug Loading Multifunctional Nanocapsules for Intracellular Cancer
Drug Delivery. J. Am. Chem. Soc. 2010, 132, 4259-4265.
[46] Shi, C.; Guo, D.; Xiao, K.; Wang, X.; Wang, L.; Luo, J. A drug-specific
nanocarrier design for efficient anticancer therapy. Nature Commun.
2015, 6, 7449.
[47] Harnoy, A. J.; Rosenbaum, I.; Tirosh, E.; Ebenstein, Y.; Shaharabani,
R.; Beck, R.; Amir, R. J. Enzyme-Responsive Amphiphilic PEG-Dendron
Hybrids and Their Assembly into Smart Micellar Nanocarriers. J. Am.
Chem. Soc. 2014, 136, 7531-7534.
[48] Edwardson, T. G. W.; Carneiro, K. M. M.; McLaughlin, C. K.; Serpell,
C. J.; Sleiman, H. F. Site-specific positioning of dendritic alkyl chains
on DNA cages enables their geometry-dependent self-assembly.
Nature Chem. 2013, 5, 868.
[34] Duan, X.; Chen, H.; Fan, L.; Kong, J. Drug Self-Assembled Delivery
System with Dual Responsiveness for Cancer Chemotherapy. ACS
Biomater. Sci. Eng. 2016, 2, 2347-2354.
[35] Qin, S.-Y.; Zhang, A.-Q.; Cheng, S.-X.; Rong, L.; Zhang, X.-Z. Drug
self-delivery systems for cancer therapy. Biomaterials 2017, 112,
234-247.
[49] Mifsud, N.; Mellon, V.; Perera, K. P. U.; Smith, D. W.; Echegoyen, L.
In situ EPR spectroscopy of aromatic diyne cyclopolymerization. J.
Org. Chem. 2004, 69, 6124-6127.
[36] Wang, Y.; Huang, P.; Hu, M.; Huang, W.; Zhu, X.; Yan, D.
[50] Usuki, T.; Kawai, M.; Nakanishi, K.; Ellestad, G. A. Calicheamicin
gamma(I)(1) and phenyl tert-butyl nitrone (PBN): observation of a
kinetic isotope effect by an ESR study. Chem. Commun. 2010, 46,
737-739.
[51] Luckhurst, G. R. Alternating linewidths. A novel relaxation process
in the electron resonance of biradicals. Mol. Phys. 1966, 10, 543-550.
[52] Dhar, S.; Lippard, S. J. Mitaplatin, a potent fusion of cisplatin and
the orphan drug dichloroacetate. Proc. Natl. Acad. Sci. 2009, 106,
22199-22204.
Self-Delivery
Nanoparticles
of
Amphiphilic
Methotrexate-Gemcitabine Prodrug for Synergistic Combination
Chemotherapy via Effect of Deoxyribonucleotide Pools. Bioconjugate
Chem. 2016, 27, 2722-2733.
[37] Mou, Q.; Ma, Y.; Zhu, X.; Yan, D. A small molecule nanodrug
consisting of amphiphilic targeting ligand–chemotherapy drug
conjugate for targeted cancer therapy. J. Controlled Release 2016,
230, 34-44.
[38] Huang, P.; Ao, J.; Zhou, L.; Su, Y.; Huang, W.; Zhu, X.; Yan, D. Facile
Approach To Construct Ternary Cocktail Nanoparticles for Cancer
Combination Therapy. Bioconjugate Chem. 2016, 27, 1564-1568.
[39] Zhang, T.; Huang, P.; Shi, L.; Su, Y.; Zhou, L.; Zhu, X.; Yan, D.
Self-Assembled Nanoparticles of Amphiphilic Twin Drug from
Floxuridine and Bendamustine for Cancer Therapy. Mol.
Pharmaceutics 2015, 12, 2328-2336.
[53] He, H. Y.; Zhao, J. N.; Jia, R.; Zhao, Y. L.; Yang, S. Y.; Yu, L. T.; Yang, L.
Novel Pyrazolo[3,4-d] pyrimidine Derivatives as Potential Antitumor
Agents: Exploratory Synthesis, Preliminary Structure-Activity
Relationships, and in Vitro Biological Evaluation. Molecules 2011, 16,
10685-10694.
[54] Hengartner, M. O. The biochemistry of apoptosis. Nature 2000, 407,
770.
[40] Huang, P.; Wang, D.; Su, Y.; Huang, W.; Zhou, Y.; Cui, D.; Zhu, X.; Yan,
D. Combination of small molecule prodrug and nanodrug delivery:
amphiphilic drug-drug conjugate for cancer therapy. J. Am. Chem.
Soc. 2014, 136, 11748-11756.
[41] Gao, H. J.; Shi, W. D.; Freund, L. B. Mechanics of receptor-mediated
endocytosis. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 9469-9474.
[42] Bareford, L. A.; Swaan, P. W. Endocytic mechanisms for targeted
drug delivery. Adv. Drug Del. Rev. 2007, 59, 748-758.
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Manuscript revised: XXXX, 2019
Manuscript accepted: XXXX, 2019
Accepted manuscript online: XXXX, 2019
Version of record online: XXXX, 2019
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Self-delivery nanoparticles of amphiphilic
acyclic enediynes for efficient tumor cell
suppression
Jing Li, Yuequn Wu, Lili Sun, Shuai Huang, Baojun
Li, Yun Ding, Aiguo Hu*
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© 2019 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2019, 37, XXX－XXX
This article is protected by copyright. All rights reserved.