Redox-Responsive Biomimetic Polymeric Micelle for Simultaneous Anticancer Drug Delivery and Aggregation-Induced Emission Active Imaging

Article abstract of DOI:10.1021/acs.bioconjchem.8b00119

Intelligent polymeric micelles have been developed as potential nanoplatforms for efficient drug delivery and diagnosis. Herein, we successfully prepared redox-sensitive polymeric micelles combined aggregation-induced emission (AIE) imaging as an outstanding anticancer drug carrier system for simultaneous chemotherapy and bioimaging. The amphiphilic copolymer TPE-SS-PLAsp-b-PMPC could self-assemble into spherical micelles, and these biomimetic micelles exhibited great biocompatibility and remarkable ability in antiprotein adsorption, showing great potential for biomedical application. Anticancer drug doxorubicin (DOX) could be encapsulated during the self-assembly process, and these drug-loaded micelles showed intelligent drug release and improved antitumor efficacy due to the quick disassembly in response to high levels of glutathione (GSH) in the environment. Moreover, the intracellular DOX release could be traced through the fluorescent imaging of these AIE micelles. As expected, the in vivo antitumor study exhibited that these DOX-carried micelles showed better antitumor efficacy and less adverse effects than that of free DOX. These results strongly indicated that this smart biomimetic micelle system would be a prominent candidate for chemotherapy and bioimaging.

Full text of DOI:10.1021/acs.bioconjchem.8b00119

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Article
Redox-Responsive Biomimetic Polymeric Micelle for Simultaneous Anticancer
Drug Delivery and Aggregation Induced Emission (AIE) Active Imaging
Jun Hu, Weihua Zhuang, Boxuan Ma, Xin Su, Tao Yu, Gaocan Li, Yanfei Hu, and Yunbing Wang
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00119 • Publication Date (Web): 02 May 2018
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Redoxꢀresponsive Biomimetic Polymeric Micelle for Simultaneous Anticancer
Drug Delivery and Aggregation Induced Emission (AIE) Active imaging
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Jun Hu , Weihua Zhuang , Boxuan Ma, Xin Su, Tao Yu, Gaocan Li, Yanfei Hu*,
Yunbing Wang*
National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road,
Chengdu 610064, China
1
These authors (Jun Hu and Weihua Zhuang) contributed equally to the work
Eꢀmail: huyanfei@scu.cn and Eꢀmail: yunbing.wang@scu.edu.cn.
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ABSTRACT
Intelligent polymeric micelles have been developed as potential nanoplatforms for
efficient drug delivery and diagnosis. Herein, we successfully prepared
redoxꢀsensitive polymeric micelles combined aggregation induced emission (AIE)
imaging as an outstanding anticancer drug carrier system for simultaneous
chemotherapy and bioimaging. The amphiphilic copolymer TPEꢀSSꢀPLAspꢀbꢀPMPC
could selfꢀassemble into spherical micelles and these biomimetic micelles exhibited
great biocompatibility and remarkable ability in antiꢀprotein adsorption, showing
great potential for biomedical application. Anticancer drug doxorubicin (DOX) could
be encapsulated during the selfꢀassemble process, and these drugꢀloaded micelles
showed intelligent drug release and improved antitumor efficacy due to the quick
disassembly in response to high level of glutathione (GSH) environment. Moreover,
the intracellular DOX release could be traced through the fluorescent imaging of these
AIE micelles. As expected, the in vivo antitumor study exhibited that these
DOXꢀcarried micelles showed better antitumor efficacy and less adverse effects than
that of free DOX. These results strongly indicated that this smart biomimetic micelle
system to be a prominent candidate for chemotherapy and bioimaging.
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INTRODUCTION
Nanoparticles have been extensively employed as efficient drug carriers for cancer
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therapy with notable reduced side effects and improved therapeutic efficacy .
Wellꢀdesigned polymeric nanoparticles with a size of 10ꢀ200 nm can spontaneously
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accumulate in tumor tissue during the longꢀcirculation in vivo due to the enhanced
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ꢀ6
permeability and retention (EPR) effect , which has greatly facilitated the
development of nanomedicine. Polymeric micelles, selfꢀassembling from amphiphilic
block copolymers, have been successfully used for the delivery of waterꢀinsoluble
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antitumor drugs, and several polymeric micelles have been reached to clinical trials .
However, the distribution of micelles in vivo is difficult to trace and the lack of
intelligent drug release would reduce the therapeutic efficacy of antitumor drugs.
As a desired polymeric micelle, it should not only deliver antitumor drugs to the
targeted site, but also trace the distribution of drug carriers in vivo. However,
conventional micelles are usually invisible, which makes it difficult to monitor the
track of drug carriers. Therefore, development of nanocarrier with highꢀefficiency
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fluorescence is urgent needed for drug delivery systems . However, when traditional
fluorescence agents are encapsulated into the core of nanoparticles, the wellꢀknown
phenomenon, aggregation caused quenching (ACQ), leads to the reduction of
fluorescence due to the “πꢀπ stacking” of the rigid planar structures, which limit their
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further application in biomedical . In 2001, Tang′s group first reported the
aggregationꢀinduced emission (AIE) effect of a new class of fluorescent molecule
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instead exhibiting strong luminescence in aggregated state . This special property is
widely utilized to overcome the defect of ACQ effect and make nanocarriers available
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for simultaneous cancer diagnostic and therapy
.
Ideally, drugꢀloaded micelles are expected to rapid release the cargos to ensure
efficient therapy effect after they accumulate in tumor sites. Therefore, intelligent
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micelles with stimuliꢀsensitive features (e.g., redox, temperature, pH, and specific
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enzyme response)
are developed for target siteꢀtriggered drugs release in
responding to the specific intracellular and extracellular environment of tumor tissues
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. Disulfideꢀbased redox responsiveness is widely used owing to the obvious
difference in GSH concentration between the reducing intracellular space
(approximately 2ꢀ10 mM) and mildly oxidizing in extracellular space (approximately
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2ꢀ20 µM) of tumor cells
. Disulfide linkage will be quickly cleaved at high
concentration of GSH in the intracellular environment of tumor cells, while keeping
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stable at a low concentration of GSH in the extracellular environment . Taking
advantage of this characteristic, disulfide linkage contained polymeric micelles have
been developed for controlled delivery and intelligent release of anticancer drugs,
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resulting in enhanced antitumor efficacy .
The biocompatibility and stability during blood circulation of polymeric micelles
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are significant to drug delivery systems for increasing the accumulation of drugs . In
this context, functional and biological compatible shell of the polymeric micelles will
prevent micelles from interaction with cells and proteins in the blood and avoid rapid
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renal clearance by reticuloendothelial system (RES)
. Numerous hydrophilic
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polymers with great biocompatibility are selected as shellꢀforming segments .
Among these polymers, hydrophilic poly (ethylene glycol) (PEG) is widely used as
the hydrophilic shell due to its ability to form tight association with water molecules
via hydrogen bond, giving “stealth” properties to micelles by generating a hydrating
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layer
. Apart from conventional PEG, zwitterionic phosphorylcholine based
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polymers are often used for engineering the hydrophilic shell of micelles for their
excellent biocompatibility, high hydrophily, superior biomimetic properties and
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splendid resistance to protein adsorption
. Phosphorylcholine based polymers, for
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instance, poly (2ꢀmethacryloyloxyethyl phosphorylcholine) (PMPC) has been
successful introduced to several nanocarriers as shellꢀbuilding blocks for drug
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delivery
. Due to its cell membraneꢀmimetic structure, PMPC is supposed to
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enhance cellular uptake of drugꢀloaded micelles .
In this work, we reported redoxꢀsensitive polymeric micelles with AIE property for
intelligent drug delivery and activated bioimaging (Scheme 1). Disulfide linkage
contained TPEꢀconjugated poly (aspartic acid)ꢀblockꢀpoly (2ꢀmethacryloyloxyethyl
phosphorylcholine) (TPEꢀSSꢀPLAspꢀbꢀPMPC) has been successful synthesized.
TPEꢀSSꢀPLAspꢀbꢀPMPC could selfꢀassemble into coreꢀshell structure with a diameter
of 87.3 nm with fairly narrow size distribution. DOX is encapsulated into the core of
micelles via hydrophobic interaction and πꢀπ stacking with benzene rings of TPE
during the selfꢀassembly of the copolymer. The TPE groups inside the core of
micelles endowed these micelles unique AIE property, which could be used for
bioimaging. Furthermore, the stability, drug release behavior, cellular uptake, in vitro
cytotoxicity and in vivo antitumor efficacy of these DOXꢀloaded
TPEꢀSSꢀPLAspꢀbꢀPMPC micelles were systematically studied and the results were
relatively attractive and inspiring.
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Scheme 1. Illustration of DOXꢀloaded TPEꢀSSꢀPLAspꢀbꢀPMPC micelles,
GSHꢀtriggered drug release and AIE cell imaging.
RESULTS AND DISCUSSIONS
Synthesis of TPEꢀSSꢀPLAspꢀbꢀPMPC
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The C NMR spectra of TPEꢀNO
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was recorded in the Figure S1 and the high
resolution mass spectrum (HRꢀMS) was further adopted for the structural
confirmation of TPEꢀ NO2. The detailed synthetic route of TPEꢀSSꢀPLAspꢀbꢀPMPC
was shown in Scheme 2. First, ring opening polymerization (ROP) of BLAꢀNCA was
performed using Nꢀ(2ꢀaminoethyl)ꢀ2ꢀbromoꢀ2ꢀmethylpropanamide as functional
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initiator to obtain PBLAspꢀBr macroinitiator. The H NMR spectrum of PBLAspꢀBr
(Figure S2) proved the successful synthesis of PBLAspꢀBr. The degree of
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polymerization (DP) of PBLAspꢀBr block was 18 as calculated from the integral ratio
of the peaks at δ 1.80 ppm of methyl and δ 4.62 ppm of methyne. The molecular
weight of the PBLAspꢀBr was 4700 with a polymer dispersity index (PDI) of 1.39
measured by GPC using DMF as eluent (Figure S3). Second, PBLAspꢀbꢀPMPC was
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obtained by ATRP of MPC using PBLAspꢀBr as macroinitiator. The H NMR
spectrum result of PBLAspꢀbꢀPMPC copolymer was showed in Figure S4, peaks at
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.32 ppm were assigned to benzene groups of PBLAsp block, the characteristic peaks
at δ 3.3 ppm and peaks from 3.6ꢀ4.3 ppm were assigned to methyl and methylene
groups of PMPC block. The DP of PMPC block was calculated to be 57 based on the
integral ratio of the peaks at δ3.70 ppm of methylene group of PMPC block to that at
δ7.32 ppm of benzene group from PBLAsp block. Third, benzyl groups of
PBLAspꢀbꢀPMPC were replaced by cystamines, and the total disappear of peaks of
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benzyl groups and methylenes (δ 5.12 ppm) in H NMR spectrum of
NH ꢀSSꢀPLAspꢀbꢀPMPC (Figure S5) demonstrated the successful synthesis of
2
NH
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ꢀSSꢀPLAspꢀbꢀPMPC. Finally, TPE was conjugated to NH
2
ꢀSSꢀPLAspꢀbꢀPMPC
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to
obtain
TPEꢀSSꢀPLAspꢀbꢀPMPC.
The
H
NMR spectrum of
TPEꢀSSꢀPLAspꢀbꢀPMPC was shown in Figure 1, peaks of TPE were found at δ
.90ꢀ7.50 ppm, and the characteristic peaks of PMPC were unabridged comparing to
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PBLAspꢀbꢀPMPC, indicating the successful synthesis of TPEꢀSSꢀPLAspꢀbꢀPMPC.
The grafting degree of TPE was 16 as calculated from the integration ratio of peaks at
δ 6.90ꢀ7.50 ppm of TPE to peaks at δ 3.71 ppm of PMPC blocks. Therefore, the
copolymer named TPE16ꢀSSꢀPLAsp18ꢀbꢀPMPC57 has been synthesized with the
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molecular weight of 26200 g/mol.
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Scheme 2. Synthetic route of TPEꢀSSꢀPLAspꢀbꢀPMPC.
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Figure 1. H NMR spectrum of TPEꢀSSꢀPLAspꢀbꢀPMPC in CD
1:1, V/V).
Preparation and Characterization of TPEꢀSSꢀPLAspꢀbꢀPMPC Micelles
3 6
OD and DMSOꢀd
(
The amphiphilic copolymer TPEꢀSSꢀPLAspꢀbꢀPMPC could selfꢀassemble into
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coreꢀshell micelles, which could be confirmed by the H NMR result in D O. As
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shown in Figure S6, the peaks of TPEꢀSSꢀPLAsp blocks were totally disappeared,
while the peaks of PMPC blocks could be observed. Besides, UV absorption spectra
of TPEꢀSSꢀPLAspꢀbꢀPMPC in different solvents were further applied to support the
formulation of nanoparticles, levelꢀoff tails could be observed in the UVꢀVis spectrum
of TPEꢀSSꢀPLAspꢀbꢀPMPC in H
2
O in Figure S7, which were commonly observed in
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nanoparticle suspensions and due to the light scattering effect of the aggregates
.
DOX was encapsulated into the core of TPEꢀSSꢀPLAspꢀbꢀPMPC micelles due to the
hydrophobic interaction and πꢀπ stacking between DOX and TPE. As shown in Figure
2A, the particle size of TPEꢀSSꢀPLAspꢀbꢀPMPC blank micelles was 87.3 nm with a
PDI of 0.146 and the size of DOXꢀloaded micelles was 123.1 nm with a PDI of 0.135.
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The increase of size indicated the successful encapsulation of DOX in micelles. The
uniform spherical morphology of micelles was further observed by TEM (Figure 2E).
The particle size of micelles in TEM image was smaller than that of DLS, which was
due to the dehydration of the polymeric micelles during sample preparation for TEM.
The DLC and DLE of TPEꢀSSꢀPLAspꢀbꢀPMPC micelles were 5.6 % and 29.6 % as
determined by UV spectroscopy, respectively.
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Preparetion and Characterization of TPEꢀSSꢀPLAspꢀbꢀPMPC Polymeric
Micelles
To investigate the in vitro stability of polymeric micelles, blank micelles and
DOXꢀloaded micelles were incubated in PBS with continuous oscillation for 48 h, and
DOXꢀloaded micelles with 10% and 50% FBS (volume fraction) added were also
studied under the same condition. As shown in Figure 2B, both blank micelles and
DOXꢀloaded micelles exhibited excellent stability after incubation for 48 h and these
DOXꢀloaded micelles also showed excellent antiꢀprotein adhesion due to the
biomimetic hydrophilic PMPC shell. The results in Figure 2B also demonstrated that
these micelles would be stable during long circulation in vivo.
The micellar structure was supposed to be disintegrated in a medium contained
high concentration of GSH due to the cleavage of disulfide bonds. DOXꢀloaded
micelles were incubated with 10 mM GSH and DLS was employed to monitor the
size changes of micelles. As shown in Figure 2C and Figure 2D, the DLS results of
TPEꢀSSꢀPLAspꢀbꢀPMPC micelles changed from unimodal peak to multimodal peaks
with the obviously increased particle size, which indicated the destruction of micellar
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structure and the formation of aggregates. The redoxꢀresponse behavior of these
micelles was further investigated by TEM. As shown in Figure 2F, obvious
heterogeneous spherical micelles were observed after incubation in 10 mM GSH for 8
h, which was in accord with Figure 2C and Figure 2D.
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AIE behavior of TPEꢀNO
THF and water with different water fraction. As shown in Figure 3A, the fluorescence
intensity (FL) of TPEꢀNO was gradually enhanced along with the increase of water
fraction, suggesting the formulation the aggregates of TPEꢀNO , which restricted the
free motions of TPEꢀNO and exhibited strong fluorescence AIE active fluorescent
2
was studied by fluorescence spectra in the mixture of
2
2
2
.
emission of micelles was investigated subsequently. TPE was restricted in the
hydrophobic core during the formulation of micelles, and as expected, those micelles
exhibited excellent fluorescent property in aqueous solutions, which would be suitable
for bioimaging. As shown in Figure 3B, TPEꢀSSꢀPLAspꢀbꢀPMPC micelles exhibited
strong fluorescence in water, while the FL intensity would significantly reduce when
the copolymers were dissolved in DMF solution (the water fraction decreased), owing
to the disassembly of micelles, which set TPE groups free and the AIE effect was
reduced. Moreover, FL spectra of DOXꢀloaded TPEꢀSSꢀPLAspꢀbꢀPMPC micelle were
also studied. As shown in Figure 3C, the FL intensity of both TPE and DOX were
decreased compared to that of the same concentration of blank
TPEꢀSSꢀPLAspꢀbꢀPMPC micelle and free DOX, respectively. Moreover, with the
increase of loaded DOX, the FL intensity of TPE was decreased with slightly
emission peak blue shifting (Figure 3D), demonstrating the fluorescence resonance
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energy transfer (FRET) effect between TPE and encapsulated drug DOX might be
46
existed .
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Figure 2. The particle size of blank micelle and DOXꢀloaded micelles (A). Particle
size changes of micelles after different incubation time in different solution (B).
Changes of size distribution and variation of TPEꢀSSꢀPLAspꢀbꢀPMPC micelles
incubated with 10 mM GSH (C) and (D). TEM image of DOXꢀloaded micelles (E).
TEM image of DOXꢀloaded micelles treated with 10 mM GSH for 8 h (F).
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Figure 3. A) FL intensity of TPEꢀ NO
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in mixture of THF/water with different fraction
330 nm). B) FL intensity of
of water (40 ꢁM TPEꢀNO
2
,
λ
ex
=
TPEꢀSSꢀPLAspꢀbꢀPMPC (40 ꢁM TPE) in mixture of DMF/water with different
fraction of water. C) FL spectra of TPEꢀSSꢀPLAspꢀbꢀPMPC micelles (40 ꢁM TPE),
DOXꢀloaded TPEꢀSSꢀPLAspꢀbꢀPMPC micelles (40 ꢁM TPE, 2 ꢁM DOX) and free
DOX (2 ꢁM). D) Fluorescence of DOXꢀloaded TPEꢀSSꢀPLAspꢀbꢀPMPC micelles
with different DOX loading content (λex = 330 nm).
In Vitro Drug Release
Antitumor drug was supposed to rapidly release as soon as drugꢀloaded nanocarriers
reached tumor tissue. The GSH triggered drug release of DOXꢀloaded
TPEꢀSSꢀPLAspꢀbꢀPMPC micelles was performed in PBS buff saline solution with 10
mM GSH or without GSH. As shown in Figure 4, approximately 30% of drug was
released in the medium without GSH after 48 h. On the contrary, more than 90 % of
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DOX was released in 48 h. Thus, the release of DOX was much quicker in medium
contained 10 mM GSH with more than 90% of DOX was released in 48 h, which
would enhance antitumor efficacy. In addition, drug release at medium containing 10%
FBS was also studied, the result was shown in Figure S8, which was similar with the
result in the medium without FBS as shown in Figure 4.
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Figure 4. In vitro drug release of DOXꢀloaded polymeric micelles in medium
contained 0 or 10 mM GSH
In vitro Cytotoxicity and Anticancer Activity
The biocompatiability of polymeric micelle was performed with HeLa and 4T1 cells
by MTT assay. As shown in Figure S9, both HeLa cells and 4T1 cells showed great
cell viability even the concentration of polymeric micelles increased to 200 µg/mL,
showing excellent biocompatiability of TPEꢀSSꢀPLAspꢀbꢀPMPC copolymers, which
could be attributed to the biomimetic zwitterionic PMPC shell of the micelles.
Anticancer efficiency of free DOX and DOXꢀloaded micelles were investigated with
various concentrations against HeLa and 4T1 cells for 24 h and 48 h. As shown in
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Figure 5, DOXꢀloaded micelles showed lower inhibition efficiency than that of free
DOX, which was due to the high initial inhibition efficiency of free DOX with full
exposure to cells in 24 h. However, the tumor cells growth inhibition efficacy of
drugꢀloaded micelles with more than 90% drug release after 48 h (Figure 4) was
similar with that of free DOX, which indicated the great antitumor efficacy of
DOXꢀloaded TPEꢀSSꢀPLAspꢀbꢀPMPC micelles.
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Figure 5. In vitro cytotoxicity of DOXꢀloaded micelles and free DOX against HeLa
cells and 4T1 cells after incubation for 24 h and 48 h.
Cellular Imaging of TPEꢀSSꢀPLAspꢀbꢀPMPC Micelles
The excellent biocompatibility of blank TPEꢀSSꢀPLAspꢀbꢀPMPC micelles made it
suitable for bioimaging. The AIE active cellular imaging of TPEꢀSSꢀPLAspꢀbꢀPMPC
micelles was investigated by CLSM. 4T1 cells were cultured with blank
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TPEꢀSSꢀPLAspꢀbꢀPMPC micelles with a final concentration of TPE at 40 ꢁM for 2 h
and 4 h and then observed by CLSM. Clear blue fluorescence could be seen due to the
AIE character. As shown in Figure 6, the blue fluorescence of TPE groups was mainly
located in the cytoplasm region of 4T1 cells, and the blue fluorescence intensity of
TPE was significantly enhanced with the incubation time prelonged, suggesting the
efficiently internalized of TPEꢀSSꢀPLAspꢀbꢀPMPC micelles by cells. Additionally,
CLSM imaging of HeLa cells was further performed to confirm the bioimaging ability
of TPEꢀSSꢀPLAspꢀbꢀPMPC micelles (Figure S10), which also exhibited great cellular
imaging ability. Therefore, TPEꢀSSꢀPLAspꢀbꢀPMPC micelles would be suitable for
bioimaging.
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Figure 6. CLSM imaging of 4T1 cells after coꢀculture with TPEꢀSSꢀPLAspꢀbꢀPMPC
micelles for 2 and 4 h.
Cellular Uptake and Intracellular Drug Release
Efficient internalization was significant to drug carriers. The cellular uptake and
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intracellular trace of DOXꢀloaded micelles were investigated by CLSM. As shown in
Figure 7, both red fluorescent of DOX and blue fluorescent of TPE could be easily
found in cytoplasm after incubation for 1 h, and the fluorescent became stronger with
the incubation time increasing to 3 h and 5 h, which indicated that the cellular uptake
of micelles was increased with time went on. Furthermore, obvious red fluorescence
of DOX could be observed in the nuclei region after incubation for 7 h, which
indicated the intracellular drug release from DOXꢀloaded micelles and subsequently
delivered to the cell nuclei, suggesting the great AIE feature of
TPEꢀSSꢀPLAspꢀbꢀPMPC could be utilized to monitor the intracellular DOX delivery.
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Figure 7. CLSM image of 4T1 cells cocultured with DOXꢀloaded
TPEꢀSSꢀPLAspꢀbꢀPMPC micelles for 1 h, 3 h, 5 h and 7 h. (scale bar 20 µm).
In Vivo Antitumor Activity
BALB/c mice bearing 4T1 breast cancer tumors were used to evaluate the in vivo
antiꢀtumor efficacy. Free DOX and DOXꢀloaded TPEꢀSSꢀPLAspꢀbꢀPMPC micelles
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were allowed to inject intravenously to mice with the dose of 5 mg DOX/kg body
weight, and the same volume of saline solution was administered as the blank control.
All the mice were alive before they were sacrificed and their body weights and tumor
volumes were recorded continuously during the experimental period. The changes of
tumor volumes were shown in Figure 8A, the tumor volumes of control group
administered with saline solution increased rapidly, while both the free DOX treated
and DOXꢀloaded TPEꢀSSꢀPLAspꢀbꢀPMPC micelles treated groups exhibited effective
inhibition in tumor growth compared to the control. More clearly, after 20 days, the
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average tumor volumes of control, free DOX and DOXꢀmicelle groups were ~1700,
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00 and 350 mm , respectively (*p < 0.001, **p < 0.05). In addition, the tumor
volumes of DOXꢀloaded micelles group were smaller than that of free DOX,
indicating the better antitumor efficacy of DOXꢀloaded TPEꢀSSꢀPLAspꢀbꢀPMPC
micelles than free DOX. Meanwhile, the body weight change of mice was used to
reflect the DOXꢀrelated systemic toxicity. As shown in Figure 8B, a significant body
loss was observed when the mice were treated with free DOX, showing serious
systemic toxicity to the mice. While the body weight changes of mice did not show
distinct difference in DOXꢀloaded micelles treatment group and the control group,
indicating the limited side effects of DOXꢀloaded TPEꢀSSꢀPLAspꢀbꢀPMPC micelles.
After a treatment cycle, the tumors were harvested and the picture of tumors was
showed in Figure 8C, which obviously confirmed the great antitumor efficacy of
DOXꢀloaded micelles. The tumor inhibition rates were further calculated after the
mice were sacrificed (Figure 8D), which further confirmed the better antitumor
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efficacy of DOXꢀloaded micelles compared to free DOX. The great antitumor effect
and lower drugꢀrelated toxicity confirmed the DOXꢀloaded micelle would be a good
candidate for tumor therapy.
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Figure 8. The in vivo antitumor activity of saline, free DOX and DOXꢀloaded
TPEꢀSSꢀPLAspꢀbꢀPMPC micelles. Volume changes of tumors after injection, data
were presented as mean ± SD (n = 6). *P < 0.001; **P < 0.05 (A); Body weight
changes of mice after injection (the arrows represented the injection time points) (B);
Image of tumors after treatment for three weeks with different formulations (C); The
inhibition rate of free DOX and DOXꢀloaded micelles treated groups compared with
the control group (**p < 0.05) (D).
EX Vivo Optical Image Study
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To investigate the tumorꢀtargeting ability of DOXꢀloaded TPEꢀSSꢀPLAspꢀbꢀPMPC
micelles in tumorꢀbearing mice, the ex vivo imaging of major organs (heart, liver,
spleen, lung and kidney) and tumors treated by DOXꢀloaded micelles at different time
intervals were performed by fluorescence imaging system (CRi, Inc.). As the result
shown in Figure 9A, the major organs and tumor of control group did not show
fluorescence, while the kidney, liver and tumor of mouse treated with DOXꢀloaded
micelles showed strong fluorescence. Strong DOX fluorescence in tumor could be
obvious observed at 12 h after intravenous injection of DOXꢀloaded micelles.
Meanwhile, the signal of DOX was increased with time went on (Figure 9B), which
was most evident at 48 h after treated with DOXꢀloaded micelles, indicating the
longꢀcirculating and continuous accumulation of micelles in vivo and effective remain
of these DOXꢀloaded micelles. The fluorescence of DOX was also found in livers and
kidneys, due to the metabolism. These results indicated that these DOXꢀloaded
TPEꢀSSꢀPLAspꢀbꢀPMPC micelles could rapid accumulate in tumor tissue via EPR
effect and effective retention to inhibit tumor growth.
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Figure 9. A) EX vivo imaging and biodistribution studies. DOX fluorescence images
of tumor and organs at different time post injection of DOXꢀloaded micelle; B)
Average signals collected from the major organs (heart, liver, spleen, lung and kidney)
and tumors in tumorꢀbearing mice at different time points (n = 3).
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Histological and Immunohistochemical Analyses
After 21 days, major organs and tumors from mice were collected and observed with
hematoxylin and eosin (H&E) staining. As shown in Figure 10, similar with control
group, no significant organs damages were found in DOXꢀloaded micelles treated
mice. However, free DOX showed more obvious damage to major organs compared
with control group and DOXꢀloaded micelles. Moreover, multifocal tumor metastases
were observed in livers of mice treated with free DOX and saline. Mice treated with
DOXꢀloaded micelles showed very few metastatic lesions. Therefore, these
DOXꢀloaded TPEꢀSSꢀPLAspꢀbꢀPMPC micelles would help to reduce the side effects
of DOX and control tumor metastasis.
Immunohistochemical study was further used to evaluate the antitumor effects of
DOXꢀloaded micelles and free DOX. CD31 was used to mark endothelial cells in
blood vessels to reflect tumor angiogenesis and tumor microvessel density (MVD),
which were closely associated with tumor growth, invasion and metastasis. As shown
in Figure 11b, mice treated with DOXꢀloaded TPEꢀSSꢀPLAspꢀbꢀPMPC micelles
showed a significant lower MVD value than that of salineꢀtreated mice and
DOXꢀtreated mice (*p < 0.001, **p < 0.05), indicating the DOXꢀloaded micelle
would inhibit angiogenesis as well as inhibit tumor growth. The growth of tumor was
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i
closely related to the proliferative cells in tumor. K ꢀ67 was a specific nuclear protein
of proliferative cells, which was used to label the proliferative cells. As shown in
Figure 10, mice treated with DOXꢀloaded micelles exhibited significant lower
expression of Kiꢀ67 than control and free DOX treated mice (*p < 0.001, **p < 0.05),
which was in accordance with in vivo antitumor studies. Therefore, DOXꢀloaded
TPEꢀSSꢀPLAspꢀbꢀPMPC micelles could limit the proliferation of tumor cells and
improved antitumor efficacy.
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TUNEL was used to confirm why tumor growth was inhibited in free DOX
treated mice and DOXꢀloaded micelles treated mice. As shown in Figure 11, only
small a number of apoptotic cells were observed in saline treated mice. However,
DOXꢀloaded micelles had the highest ratio of apoptosis, with more than 70% of
apoptotic cells was observed, indicating these DOXꢀloaded micelles would contribute
to induce tumor apoptosis.
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Figure 10. H&E staining assays for tumors, hearts, livers, spleens, lungs and kidneys
after treatment with different administrations over 21 days (all tissues: 200×). The
arrows indicated the damaged region of tissues.
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Bioconjugate Chemistry
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Figure 11. Histological and immunohistochemical analyses (Kiꢀ67, CD31 and
TUNEL) of tumor sections (× 400). The Kiꢀ67 density in each image was calculated
by Kiꢀ67ꢀpositive area to total area, the CD31ꢀpositive area was expressed in
endothelial cell pack and count capillary number by reading each section (MVD) and
the apoptotic index was calculated as the ratio of the apoptotic cell number to the total
tumor cell number in each field of view. Data were presented as mean ± SD (n = 6, *p
<
0.001, **p < 0.05).
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MATERIALS AND METHODS
MATERIALS
ꢀmethacryloyloxyethyl phosphorylcholine (MPC) was purchased from Nanjing
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Natural Science and Technology Institute and used without further purification.
Bisꢀ(2ꢀaminoethyl)disulfide dihydrochloride and Doxorubicin hydrochloride
(DOX
·HCl) were supplied by Sun Chemical Technology. (Shanghai, China) Co., Ltd.
γꢀbenzylꢀLꢀaspartateꢀNꢀcarboxyanhydride (BLAꢀNCA) was synthesized as the
39
reported literatures . Nꢀ(2ꢀaminoethyl)ꢀ2ꢀbromoꢀ2ꢀmethylpropanamide was acquired
3
7
according to previous work . N, Nꢀ Dimethylformamide (DMF) was refluxed with
calcium hydride (CaH ) for 3 h and purified by vacuum distillation. 3ꢀ(4,
5ꢀDimethylꢀthiazolꢀ2ꢀyl)ꢀ2, 5ꢀdiphenyl tetrazolium bromide (MTT), cuprous bromide
CuBr), 2,2ꢀBipyridine (bpy) and other reagents were purchased from BestꢀReagent
2
(
(Chengdu, China) and used as received.
Synthesis of Poly (γꢀBenzylꢀʟꢀAspartate) Macroinitiator (PBLAspꢀBr)
PBLAspꢀBr was synthesized by ringꢀopening polymerization of BLAꢀNCA monomer
with Nꢀ(2ꢀaminoethyl)ꢀ2ꢀbromoꢀ2ꢀmethylpropanamide as initiator. Under an
atmosphere of argon, BLAꢀNCA (5 g, 19 mmol) and initiator (0.2091 g, 1 mmol)
were dissolved in 50 mL dry DMF in a dry flask. After stirring at room temperature
for 3 days, the resulting solution was concentrated and poured into excess amount of
cold diethyl ether. The precipitates were washed with diethyl ether for several times.
The resultant PBLAspꢀBr was dried under vacuum for 24 h.
Synthesis of Poly (γꢀBenzylꢀʟꢀAspartate)ꢀblockꢀPoly (2ꢀMethacryloyloxyethyl
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Phosphorylcholine) (PBLAspꢀbꢀPMPC) Copolymer
The PBLAspꢀbꢀPMPC copolymer was synthesised by ATRP of MPC with
PBLAspꢀBr as macroinitiator and CuBr/bpy as catalyst. Briefly, PBLAspꢀBr initiator
(0.9 g, 0.184 mmol), MPC (1.8 g, 6.102 mmol) and 20 mL of solvent (DMSO/MeOH,
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1/1, v/v) were added to a dry flask. After four cycles of freezeꢀpumpꢀthaw procedure,
CuBr (26.5 mg, 0.184 mmol) and bpy (57.4 mg, 0.368 mmol) were added into the
solution under the protection of argon and the reaction was allowed to perform at 40
o
C for 2 days. The resultant crude product was purified by passing a neutral aluminum
oxide column with the eluent of dimethyl formamide /methanol (1/2, v/v),
concentrated, and precipitated into excess of cold ethyl ether. PBLAspꢀbꢀPMPC was
dried under vacuum for 24 h.
Preparation of Pure Pystamine
The pure cystamine was obtained by dehydrochloration of cystamine dihydrochloride
41
according to literature . Typically, cystamine dihydrochloride (4.5 g, 0.02 mol) was
2
dissolved in 10 mL water in a 100 mL flask, then diethyl ether (Et O, 30 mL) and
tetrahydrofuran (THF, 15 mL) were added. After cooling down in ice bath, 40%
aqueous NaOH solution (40 g, 1 mol) was slowly added to the mixture solution. The
oil layer was collected and the residue of aqueous layer was additionally extracted
with a mixture of Et O (20 mL) and THF (10 mL). The combined organic phase was
2
dried over NaOH and filtrated, the solution was removed by rotary evaporator to
obtain pure cystamine.
Synthesis of Cystamineꢀconjugated Coly (Aspartate Acid)ꢀblockꢀPoly
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(
2ꢀMethacryloyloxyethyl
Phosphorylcholine)
Copolymer
(H NꢀSSꢀPLAspꢀ ꢀPMPC)
b
2
PBLAspꢀbꢀPMPC (1 g, 0.08 mmol) was dissolved in 30 mL solution of
DMSO/MeOH (1/1, v/v) at room temperature, and cystamine (1.3 g, 8 mmol) was
added under the protection of argon. The solution was stirred for 36 h, concentrated
by rotary evaporator and transferred into a dialysis bag (MWCO = 3500) and dialyzed
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against RO (pH ~ 5) water for 48 h. H
2
NꢀSSꢀPLAspꢀbꢀPMPC was obtained by
lyophilizing.
Synthesis of TPEꢀconjugated TPEꢀSSꢀPLAspꢀ
b
ꢀPMPC
) was prepared according to
OD): 155.27, 150.86, 149.01, 145.59,
43.32, 143.23, 142.29, 141.83, 139.53, 132.55, 131.30, 131.23 127.83, 127.69,
4
ꢀnitrophenyl chloroformateꢀactivated TPE (TPEꢀNO
2
4
0 13
previous work . C NMR (100 MHz, CD
3
1
+
126.69, 126.61, 125.39, 121.75, 119.85. High resolution ESIꢀMS: [M + Na + H]
calcd as 537.55, found 537.54.
2 2
PMPCꢀbꢀPLAspꢀSSꢀNH (0.4 g, 0.033 mmol) and TPEꢀNO (0.4 g, 0.781 mmol)
were dissolved in a mixture of DMF (12 mL) and MeOH (10 mL) at room
temperature. 200 µL of TEA (1.446 mmol) were added under the protection of argon.
The mixture was stirred for 36 h and subsequently allowed to concentrate by rotary
evaporator, the resulting mixture was taken into a dialysis bag and dialyzed (MWCO
= 3500) against RO water for 48 h and dried under vacuum to obtain the pure product.
Preparation of DOXꢀloaded Polymeric Micelles
The polymeric micelles were prepared by solvent exchange method. In brief,
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TPEꢀconjugated copolymer (10 mg) and DOX (2 mg) (dehydrochlorinated as
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previous work ) were dissolved in a mixture of DMF/MeOH solvent (2 mL, v/v, 1/1).
The mixture solution was added dropwise to 5 mL of RO water under strong stirring.
After 30 min, the mixture was transferred into a dialysis bag (MWCO = 3500) and
dialyzed against deionized water for 24 h at room temperature to remove the organic
solvent and unloaded DOX. The solution was filtered through a 0.22 µm filter
membrane and diluted to 1 mg/mL (copolymers). The drug loading efficiency (DLE)
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and drug loading content (DLC) were evaluated by previous method . Blank
polymeric micelles were prepared as the same way without the addition of DOX.
Redoxꢀsensitive Behavior and In Vitro Drug Release
The redoxꢀresponsive behavior of TPEꢀSSꢀPLAspꢀbꢀPMPC polymeric micelles was
investigated with Dynamic Light Scattering (DLS) by monitoring the changes of
particle sizes in the presence of GSH. Particularly, TPEꢀSSꢀPLAspꢀbꢀPMPC micelles
o
were incubated with 10 mM GSH at 37 C, and the size changes of micelles were
measured by DLS at preselected time intervals (0 h, 1 h, 2 h and 6 h). In addition, the
influence of GSH on micellar structure was further investigated by TEM after
incubation with 10 mM for 6 h. The in vitro release behavior of DOX was evaluated
o
by dialysis method at 37 C. In brief, 2 mL of DOXꢀloaded micelles (1 mg/mL) was
transferred into a dialysis bag (MWCO = 3500) with 20 mL of PBS solution (pH 7.4)
contained 0 or 10 mM GSH as release medium, respectively. The whole procedure of
drug release was kept in the dark with constant shaking. At predetermined time
interval, 2mL of release medium was taken out to measure the amount of released
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