Dual stimuli-responsive multi-drug delivery system for the individually controlled release of anti-cancer drugs

Article abstract of DOI:10.1039/c4cc08831j

A dual stimuli-responsive multi-drug delivery system was developed for "cancer cocktail therapy". Upon UV irradiation, microcapsules could rapidly release the small-molecule drugs, and thereafter the macromolecular drugs would be released in the presence of MMP in the tumor cells. This system will find great potential as a novel chemotherapeutic combination for cancer treatment.

Full text of DOI:10.1039/c4cc08831j

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: W. Xiao, X. Zeng,
H. Lin, K. Han, H. Jia and X. Zhang, Chem. Commun., 2014, DOI: 10.1039/C4CC08831J.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/chemcomm

Page 1 of 5
ChemComm
Dynamic Article Links ►
Chem Comm
View Article Online
DOI: 10.1039/C4CC08831J
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
ARTICLE TYPE
Dual Stimuli-responsive Multi-drug Delivery System for Individual
Controlled Release of Anti-cancer Drugs
Wang Xiao,‡ Xuan Zeng,‡ Hang Lin, Kai Han, Hui-Zhen Jia, and Xian-Zheng Zhang*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
A dual stimuli-responsive multi-drug delivery system was
developed for "cancer cocktail therapy". Upon the UV
irradiation, microcapsules can rapidly release the small-
molecule drugs, and thereafter the macromolecular drugs
would be released in the presence of MMP in tumor cells.
delivery system more efficient, accurate and controllable.
1
0
This system will find great potential as
chemotherapeutic combination for cancer treatment.
a
novel
Now, there are more than eight million people worldwide died
from cancer each year. Although thousands of antiꢀcancer drugs
and various therapeutic drug/gene delivery systems have been
developed, up to this day there is still lack of a highly effective
cancer treatment. As tumor formation is a complex and
multifactorial process, combination anticancer therapy was used
instead of singleꢀagent therapy avoiding drug resistance.
Therefore, based on the tumor development and invasion of a
patient, an individually tailored "cocktail therapy" to combine
different types of treatments (chemotherapy, gene therapy,
1
2
2
3
3
4
4
5
0
5
0
5
0
5
50
Scheme 1 Schematic illustration of the coꢀdelivery system individually
release loaded drugs.
In this study, we developed a dualꢀdrug delivery system for
"cocktail therapy" by using dual stimuliꢀresponsive
radiotherapy, etc.) can enhance the efficiency and reduce the 55 microcapsules, which has the following features: 1) Dualꢀdrug
1
risk, which has become a trend in cancer treatment.
encapsulation. The LbL selfꢀassembled microcapsules prepared
5
However, clinical application of “cocktail therapy” is facing
three major challenges: design of efficient multidrug vectors,
individual controlled release of multiple drugs, and minimization
of toxic side effects. Moreover, there is a wide range of antiꢀ 60 respectively; 2) Dual stimuliꢀresponse and individual controlled
cancer drugs could be employed, involves not only smallꢀ
molecule chemotherapy drugs but also bioꢀmacromolecular drugs
by hostꢀguest interactions have hollow sphere structures, so that
bioꢀmacromolecular drugs and smallꢀmolecule drugs could be
individually loaded in central cavities and multilayer shells
release. Upon the irradiation of 365 nm UV light, multilayered
shells can rapidly release the encapsulated smallꢀmolecule drugs.
On the other side, in tumor tissues with the high concentration of
MMP, the decomposition of microcapsule shells lead to the
(such as peptides, proteins and genes). Due to their significant
difference in molecular weight, it is difficult to load them
simultaneously in one vector and then release them separately. In 65 exposure of the cavities, and thereafter the macromolecular drugs
order to achieve synergistic effects through chemotherapeutic
combinations, wellꢀorganized multidrug release system, which
can provide "cocktail therapy" by controlling the release behavior
of each drug individually, is highly desired. Although many
would be released. These stimuliꢀresponsive approaches for the
controlled release of different drugs are independent without any
interference; 3) Cellular uptake shielding. As microcapsules will
3
not disassemble without particular stimulations, it is difficult for
efforts have been made in this regard, limitation of drug size and 70 normal cells to endocytose them due to their large particle sizes
2
individual controlled release was unsolved.
(about 5 ꢁm) and negative surface charges, which can prevent
normal tissues damage from antiꢀcancer therapy. Therefore, this
system can coꢀdeliver two different drugs, accurately target tumor
sites, and release each drug by UV irradiation and MMP
Various stimuliꢀresponsive drug vectors have been used to
trigger drug release at tumor site to enhance the cure effect. Since
matrix metalloproteinases (MMPs) are overexpressed in tumor
tissues, they can be used as internal stimuli in tumor targeting 75 respectively (Scheme 1).
3
drug delivery systems. Ultraviolet (UV) lights, due to their
controllable intensity, wavelength, irradiation range and exposure
time, have been employed as efficient external stimuli for
Microcapsules were fabricated through LbL selfꢀassembly
driven by hostꢀguest interaction between poly(aspartic acidꢀgraftꢀ
βꢀcyclodextrin) (PASPꢀgꢀβꢀCD) layer and poly(acrylic acidꢀgraftꢀ
4
medical treatment (especially for skin diseases). Therefore,
azobenzeneꢀgraftꢀprolineꢀleucineꢀglycineꢀvalineꢀarginineꢀ
6
design a suitable combination of these stimuli will make the 80 adamantane)
(PAAꢀgꢀAzoꢀgꢀPLGVRꢀAD)
layer.
Macꢀ
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1

ChemComm
Page 2 of 5
View Article Online
DOI: 10.1039/C4CC08831J
romolecular drugs were trapped in the hollow central cavity, and
cavity. Only the hostꢀguest interaction between αꢀCDꢀRhB and
smallꢀmolecule drugs modified by αꢀcyclodextrin (αꢀCD) were 60 PAAꢀgꢀAzoꢀgꢀPLGVRꢀAD was destroyed by UV irradiation,
loaded on layers through the hostꢀguest interaction between αꢀCD
and Azo. Moreover, the linkers between host and guest layers
which led to the release of αꢀCDꢀRhB. Above results indicated
that the release of Dex5000ꢀFITC in cavities of capsules in the
presence of MMP and the release of αꢀCDꢀRhB on the layers are
independent.
7
5
0
5
0
5
0
5
0
5
0
5
could be cut off due to PLGVR peptide hydrolysis caused by
8
MMP, which lead to gradual dissociation of capsules. As a
result, the entrapped macromolecular drugs could be released
from capsules. In contrast, αꢀCD modified smallꢀmolecule drugs
were still attached to PAAꢀgꢀAzo polymers as MMP has no effect
on the interaction between αꢀCD and Azo. And these smallꢀ
molecule drugs could not be taken up by neighboring cells even
PAAꢀgꢀAzo polymers were dissociated from capsules attributed
1
1
2
2
3
3
4
4
5
5
9
to the cellularꢀuptakeꢀshielding effect of negative charged PAA.
It is known that azobenzene which has trans and cis forms can be
10
reversibly switched to each other upon photoꢀirradiation, and
1
1
only transꢀazobenzene could be well recognized by αꢀCD.
Based on this mechanism, under UV irradiation, αꢀCD modified
smallꢀmolecule drugs could be released from layers and taken up
by neighboring cells. Moreover, UV irradiation has no effect on
PLGVR peptide and the interactions between βꢀCD and AD.
In order to investigate the encapsulation capability of the dual
drug delivery system, dextran5000ꢀfluorescein isothiocyanate
(Dex5000ꢀFITC) was used as a macromolecular model drug and
the αꢀCDꢀrhodamine B (αꢀCDꢀRhB) was used as a smallꢀ
molecule model drug. Five bilayers of PASPꢀgꢀβꢀCD/(PAAꢀgꢀ
AzoꢀgꢀPLGVRꢀAD&αꢀCDꢀRhB) were selfꢀassembled on the
poly(allylamine hydrochloride) (PAH) and Dex5000ꢀFITC
captured CaCO3 particles (Supporting Information). These
multiple coreꢀshell particles were added to ethylene
diaminetetraacetic acid (EDTA) solution and then washed with
water by centrifugation to obtain dual drug loaded hollow
microcapsules. Structure of these microcapsules was observed by
confocal laser scanning microscopy (CLSM). As shown in Fig.
S4, regular spherical microcapsules with
a diameter of
approximate 5 ꢁm were well dispersed. The CLSM images
exhibited that, after core removal, green fluorescent cavities (Fig.
S4A) and red fluorescent shell (Fig. S4B) coꢀexisted, which
means both Dex5000ꢀFITC and αꢀCDꢀRhB were loaded on
microcapsules successfully. The fluorescent intensity results were
accordant with CLSM images (Fig. S4D and Fig. S4E).
6
7
5
0
Fig. 1 Confocal microscopy images of microcapsules incubated in PBS
solution (A, D), MMP solution (B, E) and PBS solution under UV
irradiation (C, F) for 3 h. (AꢀC) green fluorescent images; (DꢀF) red
fluorescent images. The time interval between columns 1, 2, 3 and 4 is 1
h. (The scale bar is 3 µm).
To demonstrate the dual stimuliꢀresponse and individual conꢀ
trolled release of microcapsules, stimulusꢀinduced fluorescent
changes were observed by CLSM. As shown in Fig. 1, after
incubation in PBS solution for 3 h in the dark, green fluorescent
inner cavities (Fig. 1A) and red fluorescent shell (Fig. 1D) did not
exhibit significant changes, indicating that model drugs were not
released under this condition. However, after incubation in MMP
solution for 3 h, green fluorescence of microcapsules became
weakened (Fig. 1B) while red fluorescent shells did not show
significant changes (Fig. 1E). These results revealed that,
Dex5000ꢀFITC in the cavities was gradually released whereas αꢀ
CDꢀRhB was well entrapped in the multilayers, because only
PLGVR peptides were hydrolyzed by MMP. On the other hand,
after incubation in PBS solution under UV irradiation for 3 h, red
fluorescence intensity of shell was obviously weakened (Fig. 1F)
whereas green fluorescence inside microcapsules only exhibited a
slight change (Fig. 1C), suggesting that αꢀCDꢀRhB was released
from multilayers and most Dex5000ꢀFITC was still captured in the
The morphological changes of microcapsules in dual stimuliꢀ
responsive drug release process were further investigated by
TEM. As shown in Fig. 2, macromolecular drugs were
encapsulated by polymeric multilayer membrane. After
incubation in PBS solution, the membrane remains intact (Fig.
7
8
8
5
0
5
2
A and Fig. 2D), which means macromolecular drugs were still
entrapped inside microcapsules. However, after incubation in
MMP solution, small holes (10~200 nm in diameter) appeared in
the membrane (Fig. 2B and Fig. 2E) since PLGVR peptides were
hydrolyzed. On the other hand, after incubation in PBS solution
and under UV irradiation for 8 h, no holes could be observed on
the membrane (Fig. 2C and Fig. 2F) since UV irradiation had no
effect on PLGVR peptide as well as the interaction between βꢀCD
and AD. The TEM observation is consistent with the CLSM
results (Fig. 1C).
2
|
Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 3 of 5
ChemComm
View Article Online
DOI: 10.1039/C4CC08831J
2
2
3
0
5
0
FITC was also observed when the capsules were incubated in
PBS solution under UV irradiation. In contrast, Dex5000ꢀFITC
release rate was dramatically accelerated when the capsules were
incubated in MMP solution, and about 60% of the Dex5000ꢀFITC
was released in 12 h. Fig. S5B demonstrates the drug release
behaviors of αꢀCDꢀRhB. In a dark environment, αꢀCDꢀRhB was
released very slowly and less than 5% drug was released from the
microcapsules in 12 h no matter whether the MMP was present or
not. However, the release of αꢀCDꢀRhB was significantly
accelerated when the capsules were incubated in PBS solution
under UV irradiation, and more than 40% of the drug was
released from the microcapsules within 12 h. These results further
prove that release behaviors of two drugs can be controlled
separately by appropriate stimulation of MMP or UV irradiation,
i.e., one stimulus can only dramatically accelerate the release rate
Fig. 2 TEM images of microcapsules incubated in PBS solution (A, D),
MMP solution (B, E) and PBS solution under UV irradiation (C, F) for 24 35 of one particular drug and has no interference on the other one.
h. (The scale bars of A, B, C and D, E, F are1µm and 0.5 µm,
respectively).
In vitro endocytosis assay was carried out to illustrate the
cellular uptake shielding function of microcapsules. In this study,
5
SCCꢀ7 cells (squamous cell carcinoma) with highꢀlevel
8
expression of MMP was chosen as a model system. As shown in
4
4
5
5
6
6
0
5
0
5
0
5
Fig. 3A, both red and green fluorescences could hardly be
observed in cells after 5 h incubation in dark with microcapsules
and MMP inhibitor, indicating that microcapsules were not taken
up by SCCꢀ7 cells. Once cells were incubated in the dark with
capsules for 5 h without MMP inhibitor, green fluorescence could
be observed obviously in cells whereas the red fluorescence was
still invisible (Fig. 3B). This finding is attributed to the release
and cellular uptake of Dex5000ꢀFITC by MMP over expressed
SCCꢀ7 cells. But most of the αꢀCDꢀRhB was still encapsulated in
the multilayer shells under this condition. After 5 h incubation
with microcapsules and MMP inhibitor under UV irradiation,
bright red fluorescence could be seen in cells, whereas green
fluorescence was hardly detected (Fig. 3C). Clearly, UV
irradiation induced a rapid release of αꢀCDꢀRhB from multilayer
shells, and then drugs were taken up by cells quickly. Due to the
absence of MMP, Dex5000ꢀFITC was still entrapped in central
cavities of capsules. Moreover, after 5 h incubation under UV
irradiation without MMP inhibitor, both red and green
fluorescence could be observed clearly in Fig. 3D, because both
Dex5000ꢀFITC and αꢀCDꢀRhB were released from microcapsules.
The endocytosis experiments confirm that dual drugs can be
released separately from the cavities and shells of capsules with
the stimulation of MMP or UV irradiation, respectively.
In summary, we developed a dualꢀdrug delivery system for
"cancer cocktail therapy" by using dual stimuliꢀresponsive miꢀ
crocapsules. The LbL selfꢀassembled microcapsules prepared by
hostꢀguest interactions have hollow sphere structures, so that bioꢀ
macromolecular drugs and smallꢀmolecule drugs could be loaded
individually in cavities and multilayered shells. The
macromolecular drugs would be released in the presence of MMP
Fig. 3 Confocal microscopy images of SCCꢀ7cells incubated with
microcapsules in the presence of MMP inhibitor (A), in the absence of
MMP inhibitor (B), in the presence of MMP inhibitor under UV
irradiation (C) and in the absence of MMP inhibitor under UV
irradiation(D) for 5 h. A1, B1, C1, D1: green fluorescence images; A2,
1
0
B2, C2, D2: red fluorescent images;A3, B3, C3, D3:overlap of confocal 70 whereas the 365 nm UV irradiation would accelerate the release
fluorescence and bright field images. (The scale bar is 50 µm).
of small molecular drugs. Release behaviors of dual drugs can be
controlled separately by appropriate stimulation. This dual
stimuliꢀresponsive multidrug delivery system will find great
potential as a novel chemotherapeutic combination for cancer
treatment.
In vitro release under different stimuli was further carried out
to accurately evaluate the of drug release of microcapsules. As
presented in Fig. S5A, when incubated in PBS solution in dark
environment, a slow sustained release of Dex5000ꢀFITC was
observed, and only ~15% of Dex5000ꢀFITC was released from
1
5
75
Acknowledgement
microcapsules within 12 h. A similar release profile of Dex5000
ꢀ
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 3

ChemComm
Page 4 of 5
View Article Online
DOI: 10.1039/C4CC08831J
This work was financially supported by National Natural
Science Foundation of China (51125014, 51233003, 51303137),
the Ministry of Science and Technology of China
2006, 128, 2226; d) R. Breslow and S. D. Dong, Chem. Rev., 1998,
9
8, 1997; e) C. Dugave and L. Demange, Chem. Rev., 2003, 103,
475; f) A. Harada, Acc. Chem. Res., 2001, 34, 456.
2
(2011CB606202), the Ministry of Education of China
5
(20120141130003), the Natural Science Foundation of Hubei
Province of China (2013CFA003), and the Chinese Postdoctoral
Science Foundation (2012M521459, 2013M540598).
Notes and references
Key Laboratory of Biomedical Polymers of Ministry of Education &
Department of Chemistry, Wuhan University, Wuhan 430072, P. R.
China. Fax: +86 27 6875 4509; Tel.:+86 27 6875 5993;
E-mails: xz-zhang@whu.edu.cn (Z.X.Z.).
10
†
Electronic Supplementary Information (ESI) available: Materials and
methods, experimental details, drug release study, and cellular uptake
study. See DOI: 10.1039/b000000x/
15
‡
These authors contributed equally to this work.
1
.
a) Y. Zhao, B. G. Trewyn, I. I. Slowing and V. S. Y. Lin, J. Am.
Chem. Soc., 2009, 131, 8398; b) C. E. Ashley, E. C. Carnes, G. K.
Phillips, D. Padilla, P. N. Durfee, P. A. Brown, T. N. Hanna, J. Liu,
B. Phillips, M. B. Carter, N. J. Carroll, X. Jiang, D. R. Dunphy, C.
L. Willman, D. N. Petsev, D. G. Evans, A. N. Parikh, B.
Chackerian, W. Wharton, D. S. Peabody and C. J. Brinker, Nat.
Mater., 2011, 10, 389; c) M. B. Zheng, C. X. Yue, Y. F. Ma, P.
Gong, P. F. Zhao, C. F. Zheng, Z. H. Sheng, P. F. Zhang, Z. H.
Wang and L. T. Cai, ACS Nano, 2013, 7, 2056; d) N. Kolishetti, S.
Dhar, P. M. Valencia, L. Q. Lin, R. Karnik, S. J. Lippard, R.
Langer and O. C. Farokhzad, Proc. Natl. Acad. Sci. U.S.A., 2010,
20
25
30
35
40
45
50
55
60
65
1
07, 17939; e) T. Okuda, K. Tominaga and S. Kidoaki, J.
Controlled Release, 2010, 143, 258.
2. a) Z. J. Deng, S. W. Morton, E. BenꢀAkiva, E. C. Dreaden, K. E.
Shopsowitz and P. T. Hammond, ACS Nano, 2013, 7, 9571; b) C.
Wang, Z. X. Li, D. Cao, Y. L. Zhao, J. W. Gaines, O. A. Bozdemir,
M. W. Ambrogio, M. Frasconi, Y. Y. Botros, J. I. Zink and J. F.
Stoddart, Angew. Chem. Int. Ed., 2012, 51, 5460. c) R. Cheng, F.H.
Meng, C. Deng, H.A. Klok and Z.Y. Zhong, Biomaterials, 2013,
3
4, 3647.
3
4
5
.
.
.
J. Zhang, Z. F. Yuan, Y. Wang, W. H. Chen, G. F. Luo, S. X. Cheng,
R. X. Zhuo and X. Z. Zhang, J. Am. Chem. Soc., 2013, 131, 8398.
R.S. Stern, K.T. Nichols and L.H. Väkevä, N. Engl. J. Med., 1997,
336, 1041.
a) C. S. Peyratout and L. Dähne, Angew. Chem. Int. Ed., 2004, 43,
3
3
a) H. Liu, Y. F. Zhang, J. M. Hu, C. H. Li and S. Y. Liu, Macromol.
Chem. Phys., 2009, 210, 2125; b) J. Wang and M. Jiang, J. Am.
Chem. Soc., 2006, 128, 3703; c) J. X. Zhang and P. X. Ma, Angew.
Chem. Int. Ed., 2009, 48, 964.
a) Y. P. Wang, N. Ma, Z. Q. Wang and X. Zhang, Angew. Chem. Int.
Ed., 2007, 46, 2823; b) D. Tarn, D. P. Ferris, J. C. Barnes, M. W.
Ambrogio, J. F. Stoddart and J. I. Zink, Nanoscale, 2014, 6, 3335.
S. Lee, E. J. Cha, K. Park, S. Y. Lee, J. K. Hong, I. C. Sun, S. Y. Kim,
K. Choi, I. C. Kwon, K. Kim and C. H. Ahn, Angew. Chem., Int.
Ed., 2008, 47, 2804.
762; b) W. J. Tong and C. Y. Gao, J. Mater. Chem., 2008, 18,
799.
6
.
7
8
.
.
.
9
1
W. Xiao, W. H. Chen, X. D. Xu, C. Li, J. Zhang, R. X. Zhuo and X.
Z. Zhang, Adv. Mater., 2011, 23, 3526.
0. a) G. S. Kumar and D. C. Neckers, Chem. Rev., 1989, 89, 1915; b) P.
B. Wan, Y. G. Jiang, Y. P. Wang, Z. Q. Wang and X, Zhang, Chem.
Commun., 2008, 44, 5710; c) C. H. Tung and J. Q. Guan, J. Org.
Chem., 1996, 61, 9417; d) G. Pace, V. Ferri, G. Grave, M. Elbing,
C. von Hanisch, M. Zharnikov, M. Mayor, M. A. Rampi and P.
Samorì, Proc. Natl. Acad. Sci. U.S.A., 2007, 104, 9937.
1. a) Y. P. Wang, N. Ma, Z. Q. Wang and X. Zhang, Angew. Chem. Int.
Ed., 2007, 46, 2823; b) S. Tamesue, Y. Takashima, H. Yamaguchi,
S. Shinkai and A. Harada, Angew. Chem. Int. Ed., 2010, 49, 7461;
c) I. Tomatsu, A. Hashidzume and A. Harada, J. Am. Chem. Soc.,
1
4
|
Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 5 of 5
ChemComm
View Article Online
DOI: 10.1039/C4CC08831J
The table of contents
A dual stimuliꢀresponsive multiꢀdrug delivery system was developed for "cancer cocktail therapy".
Release behaviors of dual drugs can be controlled separately by appropriate stimulation.
5
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 5