Doxorubicin-loaded micelles with high drug-loading capacity and stability based on zwitterionic oligopeptides

Article abstract of DOI:10.1039/d0nj02785e

To simplify the preparation process and increase the drug-loading capacity of antitumor nanodrugs, doxorubicin-loaded micelles based on zwitterionic oligopeptides were fabricated through two step reactions in mild conditions. Zwitterionic oligopeptides Glu-Lys-Cys-Glu-Lys (EKCEK) were reacted with methacrylohydrazide (MAH) by click reaction, the product was further conjugated with doxorubicin (Dox) to form amphipathic molecules, and finally the amphipathic molecules self-assembled into doxorubicin-loaded micelles (EKCEK-Dox) in deionized water. TEM and DLS results showed that the EKCEK-Dox micelles were of the regular sphere, with the mean diameter approximately 70 nm. The drug-loading capacity of the prepared micelles was up to 44.6%, and the micelles were very stable in PBS solution. In vitro antitumor experiment showed that the EKCEK-Dox micelles had significant inhibition efficacy on MCF-7 cells, which was much better than the clinical antitumor drug (Doxil). The IC50 of the EKCEK-Dox micelles (5.6 μg Dox equiv. per mL) was significantly lower than Doxil (8.9 μg Dox equiv. per mL). Therefore, the drug delivery system based on zwitterionic oligopeptides will be a promising strategy for cancer chemotherapy.

Full text of DOI:10.1039/d0nj02785e

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Doxorubicin-loaded micelles with high drug-
loading capacity and stability based on
Cite this:DOI: 10.1039/d0nj02785e
zwitterionic oligopeptides†
Weili Xue, * Ashish Trital,
Sihang Liu
and Liangbo Xu
To simplify the preparation process and increase the drug-loading capacity of antitumor nanodrugs,
doxorubicin-loaded micelles based on zwitterionic oligopeptides were fabricated through two step
reactions in mild conditions. Zwitterionic oligopeptides Glu–Lys–Cys–Glu–Lys (EKCEK) were reacted
with methacrylohydrazide (MAH) by click reaction, the product was further conjugated with doxorubicin
(Dox) to form amphipathic molecules, and finally the amphipathic molecules self-assembled into
doxorubicin-loaded micelles (EKCEK–Dox) in deionized water. TEM and DLS results showed that the
EKCEK–Dox micelles were of the regular sphere, with the mean diameter approximately 70 nm. The
drug-loading capacity of the prepared micelles was up to 44.6%, and the micelles were very stable
in PBS solution. In vitro antitumor experiment showed that the EKCEK–Dox micelles had significant
inhibition efficacy on MCF-7 cells, which was much better than the clinical antitumor drug (Doxils).
The IC50 of the EKCEK–Dox micelles (5.6 mg Dox equiv. per mL) was significantly lower than Doxil
(8.9 mg Dox equiv. per mL). Therefore, the drug delivery system based on zwitterionic oligopeptides will
be a promising strategy for cancer chemotherapy.
Received 2nd June 2020,
Accepted 7th July 2020
DOI: 10.1039/d0nj02785e
rsc.li/njc
already used in the clinic, including liposomal doxorubicin,
merely slightly increased the survival rate of the patients.11–15
Introduction
In recent years, anticancer nanodrugs provide a better treatment
Polymeric vesicles are a kind of very stable carrier, which
strategy for human malignancies.1–4 Liposomal doxorubicin- are constructed from the self-assembly of amphiphilic block
Doxils is the first clinically approved nanodrug for treating copolymers.16–19 Compared with liposome, apart from stronger
various cancers, including ovarian cancer, multiple myeloma, stability, the surface of polymeric vesicles can be modified
Kaposi’s sarcoma and metastatic breast cancer.5,6 The success functionally, which makes polymeric vesicles increasingly
of liposomal doxorubicin can be ascribed to the following attractive.20–23 Therefore, many researchers have designed
reasons. Firstly, the amount of loaded doxorubicin (Dox) is various polymeric vesicles response to stimulus signals such
significantly increased through the pH-gradient preparation as pH, temperature, redox potentials, enzyme and magnetic
method. What’s more, the drug-loaded system can be trans- field.24–31 Moreover, researchers have developed some polymeric
mitted in blood circulation by stealth after the surface of the vesicles with tumor-targeting function by modifying the surface
drug carrier is PEGylated. Compared with free Dox, liposomal of polymeric vesicles with peptide, human serum albumin,
doxorubicin can efficiently prolong circulation time, improve antibody, folate and hyaluronic acid.32–35 In addition to various
biodistribution and therapeutic effect and reduce cardiotoxicity.7–9 signal response and tumor-targeting functions, the stability of a
However, liposomal doxorubicin still has its deficiencies such as drug carrier in blood circulation is the prerequisite for delivering
poor tumor selectivity and severe side effects like foot and mouth the drugs to the tumor site. Zwitterionic materials attract
disease and stomatitis.10 Although PEGylated drug carriers have researchers’ attention due to its excellent anti-nonspecific
broad prospects and significant advantages, only very few nano- protein absorption property, which makes zwitterionic materials
drugs are used in the clinic. Moreover, anticancer nanodrugs become promising drug-loaded materials.36–38 Uniform charge
distribution, low net charge density and strong hydratability are
the internal reasons for the non-fouling capacity of zwitterionic
Key Laboratory of Biomass Chemical Engineering of Ministry of Education,
materials.39
College of Chemical and Biological Engineering, Zhejiang University, Hangzhou,
Despite a large number of intricately designed drug delivery
Zhejiang 310027, China. E-mail: xueweili1028@zju.edu.cn
systems have been reported, it is the fact that only a few anticancer
nanodrugs are approved for the clinic. Besides ensuring
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0nj02785e
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Scheme 1 Illustration of preparing the EKCEK–Dox micelles.
therapeutic effect and safety, the complexity of preparation
method and the cost also play a vital role in the transformation
of nanodrugs into clinical antitumor medicines. For promoting
the development of anticancer nanodrugs, the novel doxorubicin-
loaded micelles based on Glu–Lys–Cys–Glu–Lys (EKCEK) were
introduced in this study (Scheme 1). The micelles were equipped
with stealth function under the protection of hydrophilic EKCEK
shells. In addition, EKCEK conjugated with Dox through hydra-
zone bond, which could be hydrolyzed in low pH conditions and
then released the loaded drugs quickly. More importantly, the
molecular weight of EKCEK is close to Dox molecules so that the
drug-loading capacity of the final product is very high. Therefore,
the doxorubicin-loaded micelles based on EKCEK will be an
excellent candidate for future antitumor nanodrugs.
Fig. 1 Morphology characterization, particle size distribution and zeta
potentials of the drug-loaded micelles prepared by EKCEK oligopeptides
(a) TEM image of the drug-loaded micelles, (b) partially enlarged TEM
image of the drug-loaded micelles, (c) size distribution of the drug-loaded
micelles measured by DLS, (d) zeta potentials of the drug-loaded micelles
at different pH values. Data are presented as mean Æ SD (n = 3).
Results and discussion
Synthesis of MAH and EKCEK–MAH
Before preparing the drug-loaded micelles based on zwitterionic
oligopeptides, MAH was synthesized, which acted as a bridge
connecting Dox and EKCEK. After that, EKCEK reacted with
MAH to form a drug-loading precursor EKCEK–MAH through
Michael addition reaction. 1H NMR spectra of synthesized MAH
and EKCEK–MAH were shown as Fig. S1 and S2 (ESI†).
Particle size and zeta potential characterization of EKCEK–Dox
micelles
Fig. 2 Particle size variation of EKCEK–Dox dissolved in PBS, 100% FBS
and 3 mg mLÀ1 Fg in PBS at 37 1C.
The morphology of drug-loaded micelles was characterized by
transmission electron microscopy (TEM). As shown in Fig. 1a, the
prepared micelles were of a regular sphere, and particle sizes of
the micelles were around 50–100 nm, which was beneficial for
blood circulation and enhanced penetration and retention (EPR)
effect. Fig. 1b showed the partially enlarged image of the drug-
loaded micelles and the particle size was uniform. It was shown
that the center color of the micelles was dark, whereas the edge
color was light, which demonstrated that hydrophobic Dox mole-
cules were successfully encapsulated by the hydrophilic EKCEK
shell. Particle size distribution measured by DLS was shown as
Fig. 1c, and it suggested the particle size of the most drug-loaded
micelles was at the range of 60–80 nm, which was consistent to
the results shown by TEM. Zeta potentials of EKCEK–Dox at
different pH values (pH 5–8) were characterized and the results
were shown as Fig. 1d. At pH 5.6–8, the surface of EKCEK–Dox was
negatively charged, and only when the pH was below 5.5, the
surface of EKCEK–Dox had a slightly positive charge.
micelles dissolved in PBS, 100% FBS and 3 mg mLÀ1 bovine
fibrinogen (Fg) in PBS within 96 h. As shown in Fig. 2, the
particle size of EKCEK–Dox in PBS was smallest and always stayed
around 50–80 nm within 96 h. When dissolved 3 mg mLÀ1 Fg in
PBS, the particle size of the EKCEK–Dox micelles increased
slightly, and the particle size of the micelles always stayed below
120 nm. Similarly, the stability of EKCEK–Dox in 100% FBS was
still very high, and the particle size fluctuated between 100 nm
and 120 nm. The increase of particles size should be due to the
presence of protein molecules in a complex medium. In short,
the drug-loaded micelles can keep stable in different protein
solutions, which will benefit to the safe transportation of the
micelles in blood circulation.
Drug-loading capacity and in vitro drug release of the
EKCEK–Dox micelles
Stability of EKCEK–Dox micelles
EKCEK–MAH conjugated with Dox by hydrazone bond and self-
Stability of the EKCEK–Dox micelles was characterized by assembled into stable drug-loaded micelles in which Dox
measuring the particle size variation of the drug-loaded formed a hydrophobic core and EKCEK formed a hydrophilic
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efficacy on MCF-7 cells, and the cell viability decreased drama-
tically with the concentration of Dox increasing. The IC50 of
free Dox was 4.8 mg mLÀ1, and almost all MCF-7 cells were
killed when the concentration of Dox reached 20 mg mLÀ1. By
contrast, the in vitro inhibition efficacy of the EKCEK–Dox
micelles was similar to free Dox, and much better than Doxil.
The IC50 of EKCEK–Dox was 5.6 mg Dox equiv. per mL, which
was significantly lower than Doxil (8.9 mg Dox equiv. per mL).
It is well known that free Dox is a small molecule so that it
can quickly diffuse into the tumor cells and kill them. Therefore,
it is understandable that free Dox has the highest inhibition
efficacy among the three groups. Doxil is a kind of nanodrug
coated with the polyethylene glycol (PEG) layer, which will impair
the internalization of Doxil by tumor cells.40 That may be the
main reason why the inhibition efficacy of Doxil on MCF-7 cells
is the lowest among the three groups. With respect to EKCEK–
Dox, high drug-loading capacity might promote the cellular
internalization of the micelles by tumor cells. Therefore, the
cellular uptake behavior and mechanism of the EKCEK–Dox
micelles by MCF-7 cells were further investigated.
Fig. 3 In vitro drug release profiles of the drug-loaded micelles dissolved
in pH 5.0 and 7.4 NaAc buffers respectively.
shell. According to the molecular weight of the final compound
EKCEK–Dox, theoretical drug-loading capacity was 44.16% and
the actually measured value was 44.6%. Because the hydrazone
bond can be hydrolyzed in acidic conditions, drug release of
EKCEK–Dox would be influenced by ambient pH conditions.
Therefore, the cumulative drug release of EKCEK–Dox dissolved
in pH 5.0 and 7.4 NaAc buffer was measured, and the result was
shown as Fig. 3.
In pH 7.4 conditions, the cumulative drug release of EKCEK–
Dox was always below 10% within 72 h, which demonstrated
that the drug-loaded micelles were very stable, and drug
leakage of EKCEK–Dox in physiological pH conditions was very
low. For that reason, the drug-loaded system can be circulated
in the blood safely. Compared with physiological pH condi-
tions, the cumulative drug release of EKCEK–Dox increased
remarkably when pH decreased to be 5.0, and nearly 40% of the
drugs were released in the first 24 h. After that, the drug release
of EKCEK–Dox increased slowly over time, and the total drug
release was close to 50% within 72 h.
Cellular uptake of the EKCEK–Dox micelles by MCF-7 cells
Hoechst 33 342 was used to stain the nuclei of MCF-7 cells so
that whether Dox molecules entered the nucleus or not could
be observed by an inverted fluorescence microscope. Fluores-
cence images of MCF-7 cells incubated with the micelles were
shown as Fig. 5.
At 2 h, all red fluorescence of Dox in three groups was not
obvious, which demonstrated that the amount of drugs endo-
cytosed by MCF-7 cells was low within 2 h. Among the three
groups, the intensity of red fluorescence of Doxil group was
the lowest, and the merged image indicated almost no Dox
molecules entered the nuclei. Dox fluorescence brightness of
EKCEK–Dox and free Dox groups was similar, indicating the
internalization capacity of EKCEK–Dox was comparable to free
Dox, much better than Doxil. In addition, free Dox is small
molecule so that it can be cleared quickly in blood circulation,
whereas the EKCEK–Dox micelles can accumulate in tumor site
through EPR effect. At 4 h, the difference between EKCEK–Dox
and Doxil was widened. The Dox fluorescence of MCF-7 cells
incubated with EKCEK–Dox was much brighter than incubated
with Doxil, suggesting the internalization process of EKCEK–
Dox by MCF-7 cells was accelerated over time, whereas the
internalization process of Doxil by MCF-7 cells was still very
slow. The difference of cellular internalization rate between
EKCEK–Dox and Doxil also demonstrated zwitterionic shell was
more beneficial for the internalization of nanodrugs by tumor
cells than the PEGylated one.
In vitro inhibition efficacy of the EKCEK–Dox micelles
on MCF-7 cells
The in vitro inhibition efficacy of the EKCEK–Dox micelles on
MCF-7 cells was evaluated by MTT assay, and the results were
shown as Fig. 4. Free Dox showed the highest inhibition
To sum up, free Dox was the fastest to be endocytosed by
MCF-7 cells and diffuse into the nuclei. Although endocytosis
rate of EKCEK–Dox was similar to free Dox, EKCEK–Dox failed
to deliver more Dox molecules into the nuclei. It can be
speculated that in the presence of a super-hydrophobic core,
the hydrazone bond cannot be hydrolyzed rapidly enough to
release Dox molecules so that there were not so much Dox
molecules in the nuclei within 4 h.
Fig. 4 In vitro inhibition efficacy of free Dox, Doxil and EKCEK–Dox on
MCF-7 cells.
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Fig. 6 Flow cytometry studies of MCF-7 cells following 4 h incubation
with the EKCEK–Dox micelles in the presence of cellular uptake inhibitors
like chlorpromazine, amiloride and genistein.
Experimental
Materials
Glu–Lys–Cys–Glu–Lys (EKCEK) (purity 99.98%) oligopeptides were
purchased from GL Biochem (Shanghai) Co., Ltd. Methacryloyl
chloride and tert-butyl carbazate (98 wt%) were purchased
from Aladdin-reagent (Shanghai, China) Co., Ltd. Doxorubicin
hydrochloride (DoxÁHCl) was purchased from Zhejiang Haizheng
pharmaceutical Co., Ltd. Sodium acetate (NaAc), acetic acid
(AcOH), anhydrous methanol, anhydrous sodium sulfate,
anhydrous tetrahydrofuran (THF) and triethylamine (TEA) were
purchased from Sinoreagent (Shanghai, China). All buffers used
in this study were filtrated through a 0.45 mm filter before
use. Human breast cancer cells (MCF-7) were obtained from
Shanghai academy of life sciences, Chinese academy of sciences.
Fig. 5 Fluorescence images of MCF-7 cells incubated with free Dox, Doxil
and EKCEK–Dox for 2 h and 4 h respectively (red represents natural
fluorescence color of Dox molecule; blue represents Hoechst 33 342
which was used to stain cell nuclei. Scale bar represents 20 mm).
Synthesis of methacrylohydrazide hydrobromide (MAHÁHBr)
MAHÁHBr was synthesized through two-step reactions, and the
synthesis procedure was shown in Fig. S3 (ESI†). Concrete
operations should refer to ESI.†
Endocytosis pathway of the EKCEK–Dox micelles by MCF-7
cells
The EKCEK–Dox micelles was co-incubated with MCF-7 cells
intervened with different kinds of endocytosis inhibitors for
4 h, and then the endocytosis pathway of EKCEK–Dox by MCF-7
cells was analyzed through flow cytometry (FCM) measurement.
As shown in Fig. 6, the red curve represented the result of a positive
control group in which no any endocytosis inhibitor was added,
and only EKCEK–Dox was co-incubated with MCF-7 cells. There-
fore, the endocytosis amount of EKCEK–Dox by MCF-7 cells was set
as 100%. Orange, green and blue curves represented FCM results of
MCF-7 cells intervened with genistein, chlorpromazine hydrochlor-
ide and amiloride hydrochloride respectively. The results suggested
that genistein and amiloride hydrochloride had few influences on
cellular uptake of EKCEK–Dox by MCF-7 cells in comparison to the
positive control group. The green curve suggested that Dox fluores-
cence intensity of MCF-7 cells intervened with chlorpromazine
hydrochloride decreased dramatically, proving cellular internaliza-
tion of the EKCEK–Dox micelles by MCF-7 cells were mostly
Preparation of drug-loaded micelles based on EKCEK
Synthesis route of the drug-loaded micelles based on EKCEK was
shown as Scheme 2. Concrete operations should refer to ESI.†
Particle size and zeta potential characterization of the EKCEK–
Dox micelles
The EKCEK–Dox micelles dissolved in deionized water were
used for TEM characterization. The particle size of EKCEK–Dox
dissolved in PBS was measured by dynamic light scattering
(DLS). Zeta potentials of EKCEK–Dox dissolved in Mcllvaine
Buffer with different pH were checked by Malvern Zetasizer ZS
(Malvern Instruments, UK). The concentration of all samples
used in the above characterization was 0.2 mg mLÀ1
.
Stability measurement of the EKCEK–Dox micelles
inhibited by chlorpromazine hydrochloride. Therefore, it could be The stability of the EKCEK–Dox micelles was evaluated
concluded that the endocytosis pathway of the EKCEK–Dox by monitoring the particle size variation of the micelles in
micelles by MCF-7 cells was clathrin-dependent.
different kinds of protein-contained solutions through DLS.
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gradient as 1.25 mg mLÀ1, 2.5 mg mLÀ1, 5 mg mLÀ1, 10 mg mLÀ1
and 20 mg mLÀ1 (calculated by Dox). Free Dox and Doxil with
corresponding concentrations were dissolved in RPMI-1640
medium and served as positive control groups. Only RPMI-
1640 medium without any drugs served as a negative control
group. After co-incubated with samples for 12 h, the medium
with drugs was removed and replaced by fresh RPMI-1640
medium containing 0.5 mg mLÀ1 MTT. After co-incubated with
MTT, the medium was replaced by DMSO solution. Finally, the
absorbance of each well was measured by an ELISA reader
(MK3, Thermo Co., USA) at 570 nm.
Cellular uptake and endocytosis mechanism of EKCEK–Dox
micelles
Scheme 2 Synthesis route of drug-loaded micelles based on EKCEK.
MCF-7 cells in the logarithmic phase were plated in a 24-well
plate by 1 Â 105 per well and cultured for 24 h. The RPMI-1640
medium was replaced by fresh medium containing EKCEK–
Dox, Doxil and free Dox, and the equivalent concentration
of Dox was 8 mg mLÀ1. After co-incubated with drugs for 2 h
and 4 h, the medium containing drugs was removed and corres-
ponding wells were washed by PBS for 2–3 times. Hoechst33342 in
PBS was added to stain the cell nucleus for 0.5 h. Finally, the cells
were fixed by 4% paraformaldehyde and placed on an inverted
fluorescence microscope to observe cellular uptake behavior.
Cellular uptake inhibitors such as chlorpromazine hydro-
chloride (30 mg mLÀ1), genistein (15 mg mLÀ1) and amiloride
hydrochloride (133 mg mLÀ1) were used to inhibit clathrin-
mediated endocytosis, caveolin-mediated endocytosis and macro-
pinocytosis, respectively. Before adding the durg-loaded micelles,
MCF-7 cells were cultured by RPMI-1640 medium containing
different inhibitors for 0.5 h. The fresh medium containing both
inhibitors and EKCEK–Dox (10 mg mLÀ1 of equiv. Dox) replaced
the above medium, and MCF-7 cells were cultured for another
4 h. Only EKCEK–Dox micelles without inhibitors served as a
control group. Eventually, the cells were digested by trypsin and
collected, and then the cellular uptake amount of EKCEK–Dox
micelles was measured by flow cytometer.
EKCEK–Dox lyophilized powders were respectively dissolved in
PBS, 100% fetal bovine serum (FBS) and 3 mg mLÀ1 bovine
fibrinogen (Fg) in PBS, and the concentration of EKCEK–Dox
was 0.2 mg mLÀ1. The samples in different solutions were
sealed and put in a constant temperature air shaker (37 1C).
Finally, the samples were taken out to check their particle size
variation at different time points.
Measurement of drug-loading capacity and drug release of the
EKCEK–Dox micelles
Firstly, standard DoxÁHCl solutions with different concentra-
tions were prepared and acidized by one drop of concentrated
HCl. The absorbance of the solutions was measured by UV
spectrophotometer at 485 nm so that the standard UV absorp-
tion curve of DoxÁHCl was established. 1 mg of EKCEK–Dox was
dissolved in 1 mL of acetic acid buffer (pH 3.0) and then shaken
in a thermostatic metal bath (50 1C) for 24 h. After that, the
above solution was diluted by 9 mL of deionized water acidized
by concentrated HCl. At last, the drug-loading capacity (DLC) of
EKCEK–Dox could be calculated by the following formula: DLC =
(mass of Dox in EKCEK–Dox/mass of EKCEK–Dox) Â 100%.
To simulate biosecurity of EKCEK–Dox in blood circulation
and the drug release rate in tumor cells, drug release behaviors
of EKCEK–Dox in pH 5.0/7.4 buffers were measured respectively.
EKCEK–Dox was dialyzed in pH 5.0 and pH 7.4 buffer solutions
and shaken in the constant temperature air bath shaker (37 1C).
At different time points, a certain amount of dialysate was taken
out and acidized by concentrated HCl to check the absorbance
of Dox at 485 nm. According to the prefabricated standard
UV absorption curve of DoxÁHCl, accumulative drug release of
EKCEK–Dox in pH 5.0/7.4 buffers could be calculated.
Conclusion
In this study, the doxorubicin-loaded micelles based on zwitter-
ionic oligopeptides EKCEK were successfully fabricated. Since
zwitterionic materials have excellent anti-nonspecific protein
adsorption property, zwitterionic oligopeptides EKCEK used as
drug-loading precursor can make the drug-loaded micelles
smoothly circulate in the blood and be nontoxic for the organ-
ism. The drug-loading capacity of EKCEK–Dox was up to 44.6%,
thus administration dosage can be decreased to some extent.
TEM and DLS characterization results showed the mean particle
In vitro inhibition efficacy of the EKCEK–Dox micelles on
MCF-7 cells
In vitro antitumor efficacy of EKCEK–Dox on MCF-7 cells size of the micelles was around 60–80 nm, and size distribution
was measured by MTT assay. MCF-7 cells were cultured by range was very narrow. In vitro cell experiment demonstrated the
RPMI-1640 medium containing 10% FBS with 5% CO2 at 37 1C. EKCEK–Dox micelles showed much better inhibition efficacy
MCF-7 cells in the logarithmic phase were plated in a 96-well and internalization capacity than Doxil. Therefore, the drug-
plate by 5 Â 103 per well. The EKCEK–Dox micelles were loaded system based on zwitterionic oligopeptides EKCEK can
dissolved in RPMI-1640 medium to make the concentration be a good candidate for delivery of antitumor drugs.
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19 D. E. Discher, V. Ortiz, G. Srinivas, M. L. Klein, Y. Kim,
D. Christian, S. Cai, P. Photos and F. Ahmed, Prog. Polym.
Sci., 2007, 32, 838–857.
Conflicts of interest
There are no conflicts to declare.
20 J. S. Lee and J. Feijen, J. Controlled Release, 2012, 161, 473–483.
21 E. Mabrouk, D. Cuvelier, F. Brochard-Wyart, P. Nassoy and
M.-H. Li, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 7294–7298.
22 L. Zhao, N. Li, K. Wang, C. Shi, L. Zhang and Y. Luan,
Biomaterials, 2014, 35, 1284–1301.
Acknowledgements
The work was supported by the National Natural Science
Foundation of China (21674092, 21975216 and 21474085).
23 L. Xu, V. Selin, A. Zhuk, J. F. Ankner and S. A. Sukhishvili,
ACS Macro Lett., 2013, 2, 865–868.
24 J. D. Robertson, G. Yealland, M. Avila-Olias, L. Chierico,
O. Bandmann, S. A. Renshaw and G. Battaglia, ACS Nano,
2014, 8, 4650–4661.
25 Y. Du, W. Chen, M. Zheng, F. Meng and Z. Zhong, Bio-
materials, 2012, 33, 7291–7299.
References
1 D. Peer, J. M. Karp, S. Hong, O. C. Farokhzad, R. Margalit
and R. Langer, Nat. Nanotechnol., 2007, 2, 751.
2 V. P. Torchilin, Nat. Rev. Drug Discovery, 2014, 13, 813–827.
3 R. Cheng, F. Meng, C. Deng, H.-A. Klok and Z. Zhong, 26 H. Sun, F. Meng, R. Cheng, C. Deng and Z. Zhong, Acta
Biomaterials, 2013, 34, 3647–3657. Biomater., 2014, 10, 2159–2168.
4 L. Xu, Z. Chu, H. Wang, L. Cai, Z. Tu, H. Liu, C. Zhu, H. Shi, 27 T. Thambi, V. Deepagan, H. Ko, D. S. Lee and J. H. Park,
D. Pan and J. Pan, ACS Appl. Bio Mater., 2019, 2, 3429–3438.
5 Y. C. Barenholz, J. Controlled Release, 2012, 160, 117–134.
6 T. M. Allen and P. R. Cullis, Adv. Drug Delivery Rev., 2013, 65,
36–48.
J. Mater. Chem., 2012, 22, 22028–22036.
28 F. Liu, V. Kozlovskaya, S. Medipelli, B. Xue, F. Ahmad,
M. Saeed, D. Cropek and E. Kharlampieva, Chem. Mater.,
2015, 27, 7945–7956.
7 S. H. Medina, G. Tiruchinapally, M. V. Chevliakov, Y. Y. 29 C.-Y. Chen, T. H. Kim, W.-C. Wu, C.-M. Huang, H. Wei,
Durmaz, R. N. Stender, W. D. Ensminger, D. S. Shewach and
C. W. Mount, Y. Tian, S.-H. Jang, S. H. Pun and A. K.-Y. Jen,
M. E. ElSayed, Adv. Healthcare Mater., 2013, 2, 1337–1350.
Biomaterials, 2013, 34, 4501–4509.
8 L. M. Kaminskas, V. M. McLeod, B. D. Kelly, C. Cullinane, 30 L. Xu, H. Wang, Z. Chu, L. Cai, H. Shi, C. Zhu, D. Pan, J. Pan,
G. Sberna, M. Williamson, B. J. Boyd, D. J. Owen and
C. J. Porter, Mol. Pharmaceutics, 2012, 9, 422–432.
9 H. He, Y. Li, X.-R. Jia, J. Du, X. Ying, W.-L. Lu, J.-N. Lou and
Y. Wei, Biomaterials, 2011, 32, 478–487.
10 M. E. Davis, Z. Chen and D. M. Shin, Nanoscience and
technology: a collection of reviews from nature journals, World
Scientific, 2010, pp. 239–250.
11 A. Wicki, D. Witzigmann, V. Balasubramanian and J. Huwyler,
J. Controlled Release, 2015, 200, 138–157.
12 V. J. Venditto and F. C. Szoka Jr, Adv. Drug Delivery Rev.,
2013, 65, 80–88.
X. Fei and Y. Lei, ACS Appl. Polym. Mater., 2020, 2, 741–750.
31 Y. Lu, A. Zhuk, L. Xu, X. Liang, E. Kharlampieva and
S. A. Sukhishvili, Soft Matter, 2013, 9, 5464–5472.
32 J. V. Georgieva, R. P. Brinkhuis, K. Stojanov, C. A. Weijers,
H. Zuilhof, F. P. Rutjes, D. Hoekstra, J. C. van Hest and
I. S. Zuhorn, Angew. Chem., Int. Ed., 2012, 51, 8339–8342.
33 H. Gao, S. Zhang, S. Cao, Z. Yang, Z. Pang and X. Jiang, Mol.
Pharmaceutics, 2014, 11, 2755–2763.
34 L. Pourtau, H. Oliveira, J. Thevenot, Y. Wan, A. R. Brisson,
O. Sandre, S. Miraux, E. Thiaudiere and S. Lecommandoux,
Adv. Healthcare Mater., 2013, 2, 1420–1424.
13 R. K. Jain and T. Stylianopoulos, Nat. Rev. Clin. Oncol., 2010, 35 B. Zhang, S. Wan, X. Peng, M. Zhao, S. Li, Y. Pu and B. He,
7, 653. J. Mater. Chem. B, 2020, 8, 3939–3948.
14 H. Cabral and K. Kataoka, J. Controlled Release, 2014, 190, 36 G. Ma, W. Lin, Z. Wang, J. Zhang, H. Qian, L. Xu, Z. Yuan
465–476. and S. Chen, J. Mater. Chem. B, 2016, 4, 5256–5264.
15 Y. Min, J. M. Caster, M. J. Eblan and A. Z. Wang, Chem. Rev., 37 G. Ma, W. Lin, Z. Yuan, J. Wu, H. Qian, L. Xu and S. Chen,
2015, 115, 11147–11190. J. Mater. Chem. B, 2017, 5, 935–943.
16 D. E. Discher and A. Eisenberg, Science, 2002, 297, 967–973. 38 Z. Wang, G. Ma, J. Zhang, Z. Yuan, L. Wang, M. Bernards
17 J. Gaitzsch, D. Appelhans, L. Wang, G. Battaglia and B. Voit,
Angew. Chem., Int. Ed., 2012, 51, 4448–4451.
18 K. K. Upadhyay, A. N. Bhatt, A. K. Mishra, B. S. Dwarakanath,
and S. Chen, Biomaterials, 2015, 62, 116–127.
39 S. Chen, J. Zheng, L. Li and S. Jiang, J. Am. Chem. Soc., 2005,
127, 14473–14478.
S. Jain, C. Schatz, J.-F. Le Meins, A. Farooque, G. Chandraiah 40 M. O. Oyewumi, R. A. Yokel, M. Jay, T. Coakley and
and A. K. Jain, Biomaterials, 2010, 31, 2882–2892. R. J. Mumper, J. Controlled Release, 2004, 95, 613–626.
New J. Chem.
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