Dual-Responsive Doxorubicin-Conjugated Polymeric Micelles with Aggregation-Induced Emission Active Bioimaging and Charge Conversion for Cancer Therapy

Article abstract of DOI:10.1021/acs.bioconjchem.8b00671

In recent years, intelligent polymeric micelles with multifunctions are in urgent demand for cancer diagnosis and therapy. Herein, pH and redox dual-responsive prodrug micelles with aggregation-induced emission (AIE) active cellular imaging and charge conversion have been prepared for combined chemotherapy and bioimaging based on a novel doxorubicin-conjugated amphiphilic PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA-PMPC copolymer. The doxorubicin is conjugated via a pH cleavable imine linkage and can be packed in the hydrophobic core along with the glutathione (GSH)-sensitive disulfide bond. The DOX-conjugated inner core is sealed with a pH-responsive PAEMA as the "gate", which would rapidly open in the acidic condition, following the drug release and charge conversion-mediated acceleration of endocytosis. After an efficient internalization, the disulfide bond can be cleaved by the high concentration of GSH causing the further accelerated drug release. Meanwhile, intracellular drug delivery can be traced due to the AIE behavior of micelles. Moreover, great tumor inhibition in vitro and in vivo has been demonstrated for these DOX-conjugated micelles. This smart prodrug micelle system would be a desirable drug carrier for cancer therapy and bioimaging.

Full text of DOI:10.1021/acs.bioconjchem.8b00671

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Article
Dual-responsive doxorubicin-conjugated polymeric
micelles with aggregation-induced emission active
bioimaging and charge conversion for cancer therapy
Xin Su, Boxuan Ma, Jun Hu, Tao Yu, Weihua Zhuang, Li Yang, Gaocan Li, and Yunbing Wang
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/
acs.bioconjchem.8b00671 • Publication Date (Web): 08 Nov 2018
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Dual-Responsive Doxorubicin-Conjugated Polymeric Micelles with Aggregation-
Induced Emission Active Bioimaging and Charge Conversion for Cancer Therapy
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Xin Su1, Boxuan Ma1, Jun Hu, Tao Yu, Weihua Zhuang*, Li Yang, Gaocan Li and
Yunbing Wang*
National Engineering Research Center for Biomaterials, Sichuan University, 29
Wangjiang Road, Chengdu 610064, China;
Xin Su and Boxuan Ma contributed equally to this work.
Corresponding authors.
E-mail: weihua-zhuang@qq.com (Weihua Zhuang)
E-mail: yunbing.wang@scu.edu.cn (Yunbing Wang)
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ABSTRACT
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In recent years, intelligent polymeric micelles with multi-functions are in urgent
demand for cancer diagnosis and therapy. Herein, pH and redox dual-responsive
prodrug micelles with aggregation-induced emission (AIE) active cellular imaging and
charge conversion have been prepared for combined chemotherapy and bioimaging
based on a novel doxorubicin-conjugated amphiphilic PMPC-PAEMA-P (TPE-co-
HD)-ss-P (TPE-co-HD)-PAEMA-PMPC copolymer. The doxorubicin is conjugated via
a pH cleavable imine linkage and can be packed in the hydrophobic core along with the
glutathione (GSH)-sensitive disulfide bond. The DOX-conjugated inner core is sealed
with a pH-responsive PAEMA as the “gate”, which would rapidly open in the acidic
condition, following the drug release and charge conversion-mediated acceleration of
endocytosis. After an efficient internalization, the disulfide bond can be cleaved bythe
high concentration of GSH causing the further accelerated drug release. Meanwhile,
intracellular drug delivery can be traced due to the AIE behavior of micelles. Moreover,
great tumor inhibition in vitro and in vivo has been demonstrated for these DOX-
conjugated micelles. This smart prodrug micelle system would be a desirable drug
carrier for cancer therapy and bioimaging.
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INTRODUCTION
In the past decades, cancer has been one of the world’s most devastating diseases with
about 14.1 million new cases every year1. Currently, radiotherapy2 ，chemotherapy3
and surgical intervention4 are still the main strategies for cancer therapy in clinic. Anti-
cancer drugs for chemotherapy can inhibit tumor growth but also accompany with
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multiple side effects to main organs5. Compared to normal tissues, more structural
damage was found in tumor blood vessels6, where the micelles with a size under 200
nm can aggregate via enhanced permeability and retention (EPR) effect7. Thus, a
number of polymeric micelles have been developed to improve anti-cancer efficiency
and reduce the side effects by loading hydrophobic drugs in the core of the micelles via
EPR effect and releasing the cargos in target sites8.
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However, numerous problems occur during the process of circulation, accumulation,
internalization and drug release, which would limit target-site drug delivery9. For
example, hydrophobic drugs are often encapsulated into the core of micelles via
hydrophobic effect, which can easily leak out during blood circulation, leading to
reduced drug accumulation at the target-site10. Meanwhile, on account of providing a
good hemocompatibility and great stability during circulation, the shells of micelles are
usually composed of strong hydrophilic polymer and the nanoparticles are often
electronegative, which can also reduce micellar endocytosis11. What’s more, making
drugs and nanoparticles visible in vivo are also in urgent demand for cancer therapy and
diagnosis12.
In order to reduce the leakage of drugs during the transportation in blood stream,
prodrug offers an effective strategy to develop nano-carriers with neglectable drug
leakage13-15. Environment-responsive prodrugs have been widely developed, which are
stable in physiological environment but rapidly release the cargos at tumor targets due
to the specific tumor microenvironment, such as redox-environment, lower pH and
specific enzymes. Disulfide bond is often utilized for engineering nanocarriers
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responsive to high level of glutathione (GSH) 16,17with accelerated drug release and
enhanced anti-tumor effect due to the extremely higher intracellular concentration of
GSH (2 - 10 mM) in tumor cells, which is much higher than that in the extracellular
environment (2-20 μM)18. Acidic pH values are also found in different sites of tumor
tissue19, such as extracellular matrix (pH 6.5-7.2), endosomes (5.0-6.5) and lysosomes
(4.5-5.0)20-22. Hence, pH-sensitive structures, for instance, imine, acetal and orthoester
have been developed for drug delivery, which can be broken under acid condition. In
addition, polymers with pH-triggered hydrophilic-hydrophobic conversion such as poly
(2-(tetramethyleneimino) ethyl methacrylate) (PTEMA) 23, poly (2-(dibutylamino)
ethyl methacrylate) (PDEMA) 24 and poly (2-azepane ethyl methacrylate) (PAEMA)25
have been introduced into pH-responsive drug delivery systems, which also show
charge conversion feature via protonation effect and can consequently turn into
electropositive in acid condition, resulting in enhanced cellular internalization by
combining with electronegative cytomembrane26.
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Besides intelligent drug release, ideal nanoparticles should also be visible so as to
monitor the biodistribution of nanoparticles and trace the intracellular drug delivery.
Fluorescent dyes are widely used to trace nanoparticles. However, conventional dyes
are often limited by the aggregation-caused quenching (ACQ) effect27. Since 2001, a
series of fluorescent molecules with aggregation-induced emission (AIE) effect have
been developed, which emit strong fluorescence in the aggregated state28.
Tetraphenylethene (TPE), a typical molecule with AIE effect29, exhibits significant AIE
phenomenon in virtue of the restriction of their intramolecular rotations in the
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aggregation state and has been diffusely used as a fluorescent bioprobe in biosensing
and imaging30. Therefore, TPE can be introduced into polymeric micelles as the
fluorescent bioprobe to trace the biodistribution of micelles.
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In this work, we have successfully synthesized a novel DOX-conjugated multiblock
copolymer PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA- PMPC with
pH and redox dual responsiveness and AIE active bioimaging. Poly (2-
methacryloyloxyethyl phosphorylcholine) (PMPC), known as an excellent hydrophilic
polymer with zwitterionic phosphorylcholine30, was selected as the hydrophilic
segment owing to its high hydration by ionic solvation, which could avoid rapid
clearance by the reticuloendothelial system (RES) 31. It was expected that PMPC-
PAEMA-P(TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA-PMPC prodrug copolymer could
self-assemble into core-shell structural micelles with TPE inside the core, which
endowed these micelles with great bioimaging ability (Scheme 1). In addition, under
tumor acidic conditions, the pH-controlled “gate” PAEMA would open due to the
hydrophilic transformation, allowing accelerated drug release and charge convention
with enhanced endocytosis of micelles. Furthermore, the high level of GSH in tumor
cells would promote micellar structure transformation with further accelerated drug
release. Therefore, these multifunctional micelles were anticipated to improve
antitumor efficacy with reduced side effects. The cellular imaging, intracellular drug
delivery, ex vivo drug biodistribution, in vitro and in vivo antitumor assay were further
carried out in detail.
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Scheme 1. Illustration of DOX-conjugated PMPC-PAEMA-P (TPE-co-HD)-ss-P
(TPE-co-HD)-PAEMA-PMPC polymeric micelles with pH and redox responsive drug
release, charge conversion and AIE bioimaging.
RESULTS AND DISCUSSION
Synthesis of PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA-PMPC
PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA-PMPC multiblock
copolymer was synthesized step-by-step via reversible addition-fragmentation chain
transfer polymerization (RAFT) with CTA-ss-CTA as the chain transfer agent. The
exhaustive synthetic route was shown in Figure 1.
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Figure 1. Synthetic route of PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-
PAEMA-PMPC.
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The chemical structures of the copolymer in each step were characterized using 1H
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NMR spectroscopy. The H NMR spectra in Figure S1, Figure S2 and Figure S3
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confirmed the successful preparations of CTA-ss-CTA, HD-MA and TPE-MA,
respectively. Afterwards, P (TPE-co-HD)-ss-P (TPE-co-HD) was obtained via RAFT
polymerization of HD-MA and TPE-MA using CTA-ss-CTAas the chain transfer agent.
The degree of polymerization (DP) of PTPE was calculated to be 6 based on the integral
ratio of the peaks at δ 7.35-6.65 ppm (PTPE, b, 19H) and δ 8.0-7.34 ppm (CTA-ss-CTA,
a, 5H) (Figure S4), while the DP of PHD-Boc was calculated to be 2 (c, 4H; d, 18H).
The molecular weight distribution of P (TPE-co-HD)-ss-P (TPE-co-HD) was
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determined to be 1.31 by GPC (Figure S5). As shown in Figure S6 for the H NMR
result of PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA, the characteristic peaks
of PAEMA (b, 4H; c, 8H) were found at δ 4.03 ppm and δ 2.70 ppm, and the DP of
PAEMA was calculated to be 20. Furthermore, the molecular weight distribution of
PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA was determined to be 1.29 by
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GPC (Figure S7). The H NMR spectrum of PMPC-PAEMA-P (TPE-co-HD)-ss-P
(TPE-co-HD)-PAEMA-PMPC was shown in Figure S8. The characteristic peak of TPE
(a, δ 7.2-6.45 ppm) and the characteristic peak of PMPC (b, δ 4.24 ppm; c, δ 4.15 ppm;
d, δ 4.00 ppm; e, δ 3.71 ppm) could be easily found, suggesting the successful synthesis
of the copolymer. The DP of PMPC was calculated to be 40 based on the integral ratio
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of the peak a (PTPE, 19H) and peak c (-CH2-CH2-PO4 , 2H). Therefore, the PMPC-
PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA-PMPC could be defined as
PMPC20-PAEMA10-P (TPE3-co-HD1)-ss-P (TPE3-co-HD1)-PAEMA10-PMPC20 with a
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molecular weight of 19000 g/mol. After that, the copolymer was treated with TFA, and
the t-Butyloxy carbonyl (Boc) group (a, δ 1.40 ppm) was disappeared in Figure S9,
revealing the successful remove of the Boc group. Subsequently, the exposed amino
groups could be used as the functional groups to conjugate the DOX via a pH-sensitive
imine linkage. As shown in Figure 2, the characteristic peak of DOX could be easily
found (h, δ 8.01-7.59 ppm; j, δ 2.32-2.08 ppm). What’s more, the 13C NMR and FT-IR
spectrums of final product were also provided in Figure S10 and S11.
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Figure 2. 1H NMR spectrum of DOX-conjugated PMPC-PAEMA-P (TPE-co-HD)-ss-
P (TPE-co-HD)-PAEMA-PMPC in CD3OD and DMSO-d6 (1:1, V/V).
Preparation and the Dual-Response of DOX-Conjugated Polymeric Micelles
The dual-responsive micelles with AIE imaging and charge conversion could be formed
by self-assembling from the PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-
PAEMA-PMPC copolymer with the PMPC as hydrophilic shells and the other segments
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as the hydrophobic cores. The particle size of blank micelles was 141.2 nm with a
narrow size distribution of 0.153 (Figure 3A). After DOX was conjugated to the HD
segments, the particle size of drug-loaded micelles was 121.0 nm with a distribution of
0.227. Moreover, the morphology of DOX-conjugated micelles was further observed
by TEM (Figure 3G), which was well-defined spherical particles and the particle size
measure by TEM was about 90 nm. The size measured by TEM was smaller than the
DLS result owing to the shrinkage of the micellar shell during the sample preparation.
Then, drug loading content (DLC) and drug loading efficacy (DLE) were calculated to
be 4.0 % and 20.9 % measured by UV, which was in keeping with the result based on
1H NMR result. The CMC for PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-
PAEMA-PMPC prodrug micelles was determined to be 7.56 μg/mL, which provided a
powerful supply for prodrug micellar stability after the high concentration of micelles
diluted by blood in human body (Figure S12).
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When the micelles accumulated in the tumor tissue by EPR effect, the drug-loaded
micelles would first response to the acidic extracellular environment. PAEMA,serving
as a pH-sensitive “gate”, was hydrophobic at pH higher than 6.8 and in a state of closed,
which could protect the cargos from leakage during blood circulation. However, when
pH was lower than 6.8, this PAEMA “gate” would rapidly open and changed from
hydrophobic to hydrophilic, allowing drug release and charge convention. What’s more,
the imine linkage between DOX and HD segments would be cleaved resulting in DOX
release. As shown in Figure 3B, the particle size significantly decreased to about 74 nm
after incubated at pH 6.0 for 6 h with larger size distribution attributed to the stretchof
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PAEMA and HD block after becominghydrophilic and the hydrophobic core would be
shrunk, which was further proved by TEM image in Figure 3H. In addition, the
protonation of PAEMAtogether with the exposed amino groups in HD segments would
lead to the charge conversion. When incubated at pH 7.4, the micelles maintained a
stable negative zeta potential at a range of -0.5 mV to -2 mV (Figure 3C). However,
when at pH 6.0, the zeta potential of these micelles rapidly increased, which reached to
+5, +8 and +10 mV after 1, 4 and 6 h, respectively. This ingenious charge conversion
feature of the micelles could result in a strong binding force between cytomembrane
and the micelles, which enhanced the endocytosis of these prodrug micelles.
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Figure 3. (A) The particle size of blank and drug-loaded micelles measured by DLS.
(B) Size variation of prodrug micelles at pH 6.0. (C) Zeta potential variation of prodrug
micelles at different pH over time measured by DLS. (D) Size variation of prodrug
micelles in 10 mM GSH. (E) Size variation of prodrug micelles at pH 6.0 and 10 mM
GSH. (F) Variation trend of particle size at different pH and different concentrationsof
GSH. TEM image of prodrug micelles in pH 7.4 (G), pH 6.0 (H), 10 mM GSH (I) and
10mM GSH at pH 6.0 (J).
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On the other hand, the disulfide bond was introduced to these micelles, which could
be broken by the intracellular high concentration of GSH. As shown in Figure 3D, when
incubated in medium contained 10 mM GSH, the size distribution quickly changed to
multimodal, suggesting the breaking of disulfide bond. Meanwhile, TEM image in
Figure 3I showed obviously reduced particle size, which confirmed the cleavage of
disulfide bond and reconstruction of micellar structure. Interestingly, when micelles
were incubated in medium contained 10 mM GSH at pH 6.0, the micellar distribution
changed to unimodal after 6 h (Figure 3E), and the particle size decreased to about 63
nm (Figure 3F) due to the total cleavage of disulfide bond and formation of new
micellar structure, which was further confirmed by TEM image in Figure 3J. Therefore,
the environment of acid and redox would synergistically accelerate micellar structural
reorganization, which was hopeful to release drug intelligently.
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Figure 4. In vitro drug release of produrg micelles in different pH and different
concentrations of GSH. Scale bars were 50 nm.
In Vitro Drug Release Behavior
The drug release behavior of DOX-conjugated prodrug micelles was investigated at 37
oC in different pH and different concentrations of GSH. As shown in Figure 4E, less
than 35% of the drug was released from the micelles after 48 h at physiological pH
(7.4), which indicated a great stability of these prodrug micelles when transported in
blood stream. In addition, only about 50% of the cargos were released in the medium
containing 10 mM GSH. However, when at pH 6.0, the PAEMA transformed to
hydrophilic, which provided a channel for drug ejectment and the conjugated DOX was
set free due to the cleavage of imine linkage. As a result, about 80% of the cargos were
released after 48 h at pH 6.0. Furthermore, following the pH-sensitivity, the destruction
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of the disulfide bond triggered by intracellular redox environment of tumor cells would
further accelerate drug release due to the transformation of micellar structure and more
than 90% of the drug was released after incubation at pH 6.0 and 10 mM GSH for 12
h. Therefore, this step-by-step dual-response to the extracellular pH and intracellular
GSH would lead to a smart drug release, resulting in better antitumor effect.
AIE Behavior and Cellular Imaging
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Aside from the drug release behavior, the ability of tracing the intracellular drug
distribution of these micelles was equally important. As shown in Figure 5A, these
micelles exhibited strong fluorescence in pure water, while the FL intensity reduced
with the increase of THF fraction owing to the good solubility of the TPE groups in
THF, which changed from aggregation state to dissolved state and led to the
fluorescence quenching. Furthermore, the cellular bioimaging ability of these micelles
was investigated by confocal laser scanning microscopy (CLSM). Blue fluorescence of
TPE was clearly observed in cytoplasm after incubation with the micelles for 2 h
(Figure 5 B). With the extension of time, the blue fluorescence intensity of TPE was
significantly enhanced, indicating more micelles were internalized by cells, which
might be ascribed to the charge conversion of PAEMA and PHD segments. Thus, these
fluorescence micelles would be practical for bioimaging.
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Figure 5. (A) FL spectrum of the micelles in different solvent systems. (B) CLSM
images of 4T1 cells after co-culture with blank micelles for 2 h, 4 h and 6 h. Scale bars
were 10 μm.
Intracellular Drug Tracking and In Vitro Tumor Inhibition
Owing to the AIE feature of the TPE groups, the intracellular drug release of these
prodrug micelles could be traced by fluorescence imaging. DOX-conjugated micelles
were incubated with 4T1 cells for 2 h, 4 h, and 6 h, and then observed by CLSM. As
shown in Figure 6A, the blue fluorescence of TPE was clearly visible in the cytoplasm
after 2 h, where the red fluorescence of DOX was observed overlapping, indicating that
DOX had not escaped from the micelles yet. With prolonged incubation time, more red
fluorescence was separated from the blue fluorescence. When the incubation time was
increased to 6 h, the red fluorescence of DOX could be observed in the cell nucleus,
which was distinguished with the blue fluorescence of TPE. The statistic of Pearson’s
correlation coefficient (Rr) was further calculated from CLSM result (Figure S13),
which demonstrated an increasingly obvious separation between DOX and TPE-
conjugated micelles, suggesting the released drug could efficiently escape from
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micelles and result in an efficient tumor inhibition.
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Figure 6. (A) CLSM image of 4T1 cells co-cultured with DOX-conjugated micelles
for 2 h, 4 h and 6 h. Scale bars were 10 μm; (B) In vitro cytotoxicity of DOX-conjugated
micelles and free DOX against 4T1; (C) HeLa cells after incubation of 48 h.
Before conjugating DOX, the blank micelles exhibited excellent biocompatibility
against 4T1 cells and HeLa cells investigated by MTT assays (Figure S14), where the
relative cell viability of both two cells was around 100% even the concentration of
blank micelles up to 200 μg/mL. On the other hand, the DOX-conjugated micelles
performed strong power in killing both 4T1 and HeLa cells (Figure 6B, Figure 6C and
Figure S15). As shown in Figure 6B and 6C, 75% of 4T1 cells and 80% of HeLa cells
were exterminated after 48 h at a DOX concentration of 10 μg/mL. The cellular growth
inhibition ability of DOX-loaded micelles was evaluated by IC50 (half maximal
inhibitory concentration), and the IC50 values of DOX-conjugated micelles were 1.98
and 0.57 μg/mLfor 4T1 cells and HeLa cells, respectively. The IC50 values of free DOX
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were 0.68 and 0.27 μg/mL for 4T1 cells and HeLa cells, respectively. The antitumor
effect of the prodrug micelles was similar with that of free DOX, indicating the
outstanding in vitro tumor inhibition efficiency of these DOX-conjugated micelles.
In Vivo Anti-Tumor Activity and Ex Vivo Optical Imaging
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To evaluate the anti-cancer efficiency of DOX-conjugated micelles, a xenograft tumor
model was established on mice followed by intravenous injection with free DOX,
DOX-loaded conjugated micelles and saline as the control. As shown in Figure 7A, on
the 21st day after first injection, the tumor volume of saline treated group reached about
1400 mm3, while the group treated with free DOX and DOX-conjugated micelles
exhibited effective tumor growth inhibition with tumor volumes of about 510 and 350
mm3 (*p < 0.05), respectively. The great antitumor efficacy of these DOX-conjugated
micelles could be attributed to their charge conversion and dual-responsive drug release.
In addition, body weight was measured to evaluate the toxicity of drug to mice. As
shown in Figure 7B, severe body weight loss was detected in mice treated with free
DOX. By comparison, the body weight of the DOX-conjugated micelles treated group
showed a slight rise as well as the control group, suggesting a significant lower toxicity
of the DOX-conjugated micelles. Owing to the improved antitumor efficacy and
decreased side effects, these micelles could be considered as a potential choice for
cancer therapy.
The biodistribution and accumulation of DOX-conjugated micelles in different
organs directly influenced the micellar antitumor effect along with the side effects. The
ex vivo optical imaging of major organs and tumors at different time was shown in
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Figure 7C, where the control group showed scarcely any fluorescent emission inmajor
organs or tumor. However, the group treated with DOX-conjugated micelles showed
clear fluorescence of DOX in livers, kidneys and tumors. The fluorescent intensity at
tumors was increased gradually over time, suggesting that DOX-conjugated micelles
could efficiently accumulate in tumor issues, which could be further proved by the
fluorescence intensity of DOX average signal in Figure 7D. The efficient accumulation
of DOX-conjugated micelles would help to enhance antitumor efficacy and reduce the
side effects. Besides, the fluorescent emission could also be detected in other organs,
which was on account of the body circulation of the DOX-conjugated micelles.
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Figure 7. The variation of tumor volume (A) and body weight (B) after injection with
saline solution, free DOX and DOX-conjugated micelles over 21 days (*p < 0.05); (C)
Ex vivo fluorescent images of organs and tumors after tumor-bearing mice were injected
with DOX-conjugated micelles at predetermined time; (D) Fluorescence intensity of
heart, liver, spleen, lung, kidney and tumor were calculated based on fluorescence
signals of DOX (n = 6).
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Histological and Immunohistochemical Analysis
In order to further investigate the organ toxicity and antitumor efficacy of DOX-
conjugated micelles, the histological tissue slides of tumor, heart, liver, spleen, lung and
kidney of the mice were performed as shown in Figure S16. Significantly visible
metastases could be observed in major organs of control group but rarely seen in the
organs treated with DOX-conjugated micelles. Moreover, free DOX treated group
showed more focal necrosis and inflammation in major organs (liver, spleen and kidney)
compared with that of DOX-conjugated micelles treated group, indicating that the
prodrug micelles could actually reduce the side effect of DOX and showed outstanding
organ compatibility. In addition, a certain degree of tissue necrosis was observed in
tumor tissues treated with both free DOX and prodrug micelles, where the prodrug
micelles exhibited much higher tumor inhibition effect.
To accurately characterize the tumor inhibition, the growth, invasion and metastasis
of solid tumors were estimated by immunohistochemical analysis (Figure 8A). The Ki-
67 protein was a cellular marker for proliferation and the expression of Ki-67 was
significantly reduced in DOX-conjugated micelles treated group in contrast with free
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DOX and control group (Figure 8A and Figure 8B), indicating a lower cancer cell
propagation of micelles treated mice. What’s more, CD31 was used primarily to
evaluate the degree of tumor angiogenesis and the number of angiogenesis was rarely
observed in the tumor tissue treated with DOX-conjugated micelles compared with free
DOX and control group as shown in Figure 8C. It was illustrated that the DOX-
conjugated micelles could significantly inhibit the tumor growth by reducing tumor
vasculogenesis. Last but not least, TUNEL was a method for detecting apoptotic DNA
fragmentation, which could be used to reflect antitumor effect, which was used to
identify and quantify apoptotic cells (Figure 8D). Compared with the saline group
(9.9%), 57.5% of apoptotic cells were observed for DOX-conjugated micelles and 47.7%
for free DOX, suggesting DOX-conjugated micelles possessed the best antitumor effect
according to above-mentioned systematic evaluation. The DOX-conjugated micelles
could effectively inhibit angiogenesis, restrain tumor cell proliferation and encourage
apoptosis, resulting in high-efficiency in tumor inhibition.
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Figure 8. (A) Immunohistochemical (IHC) analysis of the tumor (Ki-67, CD31 and
TUNEL); The scale bars were 500 μm; (B) The Ki-67 density in each image was
calculated by Ki-67-positive area/total area (**p < 0.01); (C) Brown area of CD31
images indicated CD31-positive (**p < 0.01); (D) TUNEL-positive staining and the
apoptotic indices were measured as the ratio of apoptotic cells to the total tumor cells
in each microscopic field view (**p < 0.01).
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CONCLUSION
In this work, intelligent multifunctional DOX-conjugated micelles with dual-response,
charge conversion and bioimaging feature have been prepared for efficient cancer
therapy and bioimaging. These prodrug micelles can maintain stable at physiological
environment, but immediately respond to the specific tumor microenvironment. The
acidic environment of tumor tissue would trigger PAEMA changing from hydrophobic
to hydrophilic and open a channel for drug release, along with the charge convention to
enhance endocytosis. Moreover, high concentration of GSH in tumor cells would
accelerate micellar structure transformation and further accelerate drug release so asto
obtain desirable antitumor efficacy. Furthermore, the intracellular drug delivery can be
traced by the AIE fluorophore TPE. The efficient accumulation in tumor targets,
excellent antitumor in vivo and obvious reduction of side effects make these DOX-
conjugated micelle system highly attractive as a drug carrier for cancer therapy and
bioimaging. This kind of smart multifunctional polymeric micelle would promote the
development of nanocarriers for theranostic and offer new strategy for designing novel
nanoparticles for nanomedicine.
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MATERIALS AND METHODS
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Materials
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Doxorubicin hydrochloride (DOX•HCl) and 3-(4, 5-Dimethyl-thiazol-2-yl)-2, 5-
diphenyl tetrazolium bromide (MTT) were purchased from Chengdu Best Reagent Co.,
LTD (Chengdu, China). 2-methacryloyloxyethyl phosphorylcholine (MPC) was
purchased from Nanjing Natural Science and Technology Institute. 4-(1, 2, 2-
triphenylvinyl) phenol (TPE-OH) was synthesized according to previously literature32.
4-Cyanopentanoic acid dithiobenzoate (CTA) was obtained from Sigma-Aldrich.
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Methacryloyl
chloride,
N,
N’-dicyclohexylcarbodiimide
(DCC),
4-
(dimethylamino)pyridine (DMAP), 2,2’-azobis(isobutyronitrile) (AIBN), di-tert-butyl
dicarbonate, trifluoroacetic acid (TFA) and all other reagents and solvents were
purchased from Chengdu KeLong Chemical Reagent Company (Chengdu, China) and
used as received.
Synthesis of CTA-ss-CTA
CTA-ss-CTA was synthesized through esterification between CTA and bis (2-
hydroxyethyl) disulfide. Under argon (Ar) atmosphere, CTA (0.60 g, 2.15 mmol) was
dissolved in dry dichloromethane, and then bis (2-hydroxyethyl) disulfide (0.17g, 1.07
mmol), DCC (0.45 g, 2.15 mmol) and DMAP (52.53 mg, 0.43 mmol) were added.The
mixed solution was stirred for 24 h at room temperature. The product CTA-ss-CTAwas
purified by chromatography on silica gel (dichloromethane : ethyl acetate = 5 : 1). The
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yield of CTA-ss-CTA was 65.3 %. H NMR (400 MHz, CDCl3): 7.36-7.94 (m, 10H,
C6H5), 4.41-4.35 (t, 4H, -CH2CH2OCOCH2), 2.97-2.89 (t, 4H, -SCH2CH2), 2.78-2.59
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(m, 4H, -CH2CH2CNSCH3) ， 2.49-2.40 (m, 4H, -COCH2CH2), 1.94 (s, 6H, -
CH2CNSCH3).
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Synthesis of HD-MA
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1, 6-diamino-hexan (HD) (4.00 g, 34.42 mmol) and triethylamine (TEA) (5 mL, 35.97
mmol) were dissolved in methanol, and the methanol solution of di-tert-butyl
dicarbonate (7.51 g, 26.70 mmol) was dropwise added to the HD solution. After 12 h,
the reaction mixture was concentrated and re-dissolved in DCM, followed by extraction
with 1 M HCl aq, and the obtained aqueous phase was alkalinized by NaHCO3 aq (satd.)
and extracted with DCM. The product Boc-HD-NH2 was purified by removing the
solvent under reduced pressure (yield 70.2 %).
Under Ar atmosphere, the Boc-HD-NH2 (1.90 g, 8.78 mmol) and TEA (1.80 mL,
12.95 mmol) were dissolved in 100 mL tetrahydrofuran (THF), then methacryloyl
chloride in 10 mL THF (850 μL, 8.78 mmol) was added dropwise. The mixed solution
was stirred overnight at room temperature. After filtration and concentration, the
product was dissolved with DCM and purified by column chromatography
(dichloromethane : methanol = 20 : 1). The yield of HD-MA was 82.6 %. 1H NMR (400
MHz, CDCl3): 5.67 (s, H, -COCH2CH3), 5.31 (s, H, -COCH2CH3), 3.11 (m, 4H, -
NHCH2), 1.44 (s, 9H, -COOC(CH3)3).
Synthesis of TPE-MA
TPE-OH (2.70 g, 7.75 mmol) and TEA (1.35 mL, 9.71 mmol) were dissolved in 100
mL THF, and methacryloyl chloride (750 μL, 7.75 mmol) in 10 mL THF was added
dropwise. The mixed solution was stirred overnight at room temperature. After
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filtration and concentration, the product was purified by column chromatography
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(dichloromethane : n-hexane = 1 : 1). The yield of TPE-MA was 67.2 %. H NMR
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(400 MHz, CDCl3): 7.17-6.78 (m, 19H, -COOC6H4CC6H5C(C6H5)2), 6.29 (s, H, -
COCH2CH3), 5.71 (s, H, -COCH2CH3).
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Synthesis of P (TPE-co-HD)-ss-P (TPE-co-HD)
CTA-ss-CTA (162 mg, 0.24 mmol), AIBN (32 mg, 0.192 mmol), TPE-MA (0.60 g, 1.44
mmol) and HD-MA (0.28 g, 0.98 mmol) were dissolved in a schlenk flask with 10 mL
THF. The flask was sealed and degassed with three cycles of freeze-pump-thaw
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procedure. The reaction was carried out at 70 C with stirring overnight, followed by
dialysis (MWCO = 1000) against deionized water for 24 h. The product was obtained
by freeze drying and the yield was 90.7 %.
Synthesis of PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA
AEMA was synthesized according to previously literature33. Briefly, N-(2-
hydroxyethyl) hexamethyleneimine (4.00 g, 27.9 mmol) and TEA(7.60 mL, 55.8 mmol)
were dissolved in 150 mL THF, and methacryloyl chloride (4.60 mL, 44.6 mmol) in 20
mLTHF was added dropwise under the protection of Ar. The mixed solution was stirred
for 24 h at room temperature. After filtration and concentration, AEMA was purified
by column chromatography (ethyl acetate : n-hexane = 1 : 4).
P (TPE-co-HD)-ss-P (TPE-co-HD) (0.30 g, 0.1 mmol), AIBN (8.21 mg, 0.05 mmol)
and AEMA (0.36 g, 1.69 mmol) were dissolved in a schlenk flask with 5 mL THF.
The flask was sealed and degassed with three cycles of freeze-pump-thaw procedure,
and the resulting solution was stirred at 70 oC for 24 h. PAEMA-P (TPE-co-HD)-ss-P
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(TPE-co-HD)-PAEMA was purified by dialyzing (MWCO = 3500) against deionized
water for 2 days. Then the pink product was gained after freeze drying and the yield
was 93.5%.
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Synthesis of PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA-PMPC
Briefly, AIBN (4.92 mg, 0.03 mmol), PAEMA-P(TPE-co-HD)-ss-P(TPE-co-HD)-
PAEMA (0.5 g, 0.1 mmol) and MPC (1 g, 3.39 mmol) were dissolved in 10 mL of
solvent (THF/MeOH, 1:1, v/v) in a schlenk flask. After three cycles of freeze pump-
thaw procedure, the mixture was allowed to stir at 70 oC overnight. Then, the reaction
solution was dialyzed against deionized water (MWCO = 3500) for 2 days, and the
product was obtained by freeze drying. Afterwards, the resulting copolymer (1.40 g,
0.074 mmol) was dissolved in solvent (THF/MeOH, 1:1, v/v), and 3 mLTFAwas added
to abandon Boc group at room temperature for 24 h, followed by neutralization with
NaHCO3 aq (satd.) and dialysis (MWCO = 3500) against deionized water for 24 h. The
final product was obtained by freeze drying and the yield was 91.9%.
Preparation of DOX-Conjugated Prodrug Polymeric Micelles
4 mg DOX, 5 μL TEA and 20 mg PMPC-PAEMA-P(TPE-co-HD)-ss-P(TPE-co-HD)-
PAEMA-PMPC copolymer were dissolved in 2 mL dissolvent (THF/MeOH, 1/1, v/v).
The mixture was stirred at room temperature for 24 h. Then the solution was added
dropwise to 10 mL of deionized water (pH = 7.4) under vigorous stirring. After 1 h, the
solution was transferred into a dialysis bag (MWCO = 3500) and dialyzed against
deionized water (pH = 7.4) for 2 days. Then the solution was filtered with a 220 nm
filter membrane. The blank micelles were prepared in the same way without the
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addition of DOX. The drug loading content (DLC) and drug loading efficacy (DLE)
were measured with UV spectroscopy at 480 nm, which were calculated as the
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following equations.
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Weight of DOX in prodrug micelles
DLC(%)=
DLE(%)=
× 100%
× 100%
Weight of prodrug micelles
Weight of DOX in prodrug micelles
Weight of the feeding DOX
pH and Redox Sensitive Behavior
Dynamic light scattering (DLS) measurements was used to investigate the pH and redox
sensitive behavior of the polymeric micelles by monitoring the size and zeta potential
variation of micelles in PBS solutions with different pH and different concentrations of
GSH. The particle size and zeta potential of the micelles was measured at preselected
time intervals (0 h, 0.5 h, 1 h, 2 h, 4 h and 6 h). What’s more, the morphology of micelles
was further investigated by TEM after incubation in different pH and concentration of
GSH for 6 h.
The AIE Behavior of TPE Groups
In order to cognize the fluorescence behavior of TPE groups, THF with different
volume fraction was added to polymeric micelles solution with the sameconcentration
of copolymer and the AIE behavior of the samples were investigated by fluorescence
spectra with exciting of 330 nm.
In Vitro Drug Release
The DOX released from drug-conjugated micelles was measured under different
mediums at 37 °C. Typically, 1 mL solution of DOX-conjugated micelles (1 mg/mL)
was added into a dialysis tube (MWCO = 3500) at 37 oC in PBS (20 mL) with different
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pH or different concentrations of GSH, which was performed with incessantly shaking
for 48h in the dark. At selected time interval, 2 mL solution of release medium was
taken out and another 2 mL fresh solution was added. The amount of released DOX
was measured by UV−vis spectrometer at 480 nm.
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Characterization
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The H NMR spectra was recorded on a spectrometer operating at 400 MHz (Bruker
AMX-400). The particle size and the zeta potential of the micelles were analyzed with
a Malvern Zetasizer Nano ZS at room temperature. Transmission electron microscopy
(TEM) measurements were operated on a Hitachi H-600 transmission electron
microscope with an accelerating voltage of 75 KV and the samples were dyed with 2%
phosphotungstic acid before measurement. The molecular weight distribution of
synthetic products was determined by gel permeation chromatography (GPC) (Agilent
1260) using THF as the eluent at a flow rate of 1 mL/min at 40oC.
In Vitro Cytotoxicity Assays
The cytotoxicity of blank and DOX-conjugated PMPC-PAEMA-P (TPE-co-HD)-ss-P
(TPE-co-HD)-PAEMA-PMPC polymeric micelles was evaluated by MTT assays.
Human cervical cancer (HeLa) cells and mouse breast cancer (4T1) cells were seeded
in 96-well plates (5000 cells per well) and incubated with DMEM and RPMI-1640
medium containing 10% (v/v) fetal bovine serum and 1% (w/v) penicillin-streptomycin
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at 37 C in 5% CO2, 95% humidified atmosphere for 24 h. The culture medium was
removed and new medium contained different concentrations of the blank micelles, free
DOX and DOX-conjugated micelles were added and the cells were incubated for
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another 24 or 48 h. Then 20 µL MTT solution (5 mg/mL) was added. After 4 h, the
medium was removed and 200 µL DMSO was added to dissolve the formazan. The
absorbance of solutions was detected at 490 nm on a Bio-Rad microplate reader.
Cellular Uptake and Intracellular Drug Release
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The cellular uptake of DOX-conjugated polymeric micelles was evaluated by confocal
laser scanning microscopy (CLSM). 4T1 cells were seeded in glass dishes (1×10 4 per
dish) and incubated for 24 h. Then DOX-conjugated micelles were added with the
ultimate DOX concentration of 10 μg/mL. The cells were allowed to incubated for 2 h,
4 h and 6 h, then the culture medium was removed and washed with PBS for 3 times
before observing with CLSM. The DOX was excitated at 488 nm and the TPE was
excited at 405 nm to trace the intracellular drug delivery.
Ex Vivo Imaging Study
Animal studies were approved by the Sichuan Provincial Committee for Experimental
Animal Management and scrupulously abide by the Guiding Principles in the Care and
Use of Animals of the American Physiological Society. BALB/c mice (female, 18-20
g) were purchased from West China Experimental Animal Center of Sichuan University
(China) and housed in SPF class animal facility at a controlled temperature of 20-22 oC,
relative humidity of 50-60 % and 12 h light-dark cycles with access to commercial rat
pellet diet and deionized water ad libitum.
Animal xenograft tumor model was established by subcutaneous injecting 4T1
suspension cells with the amount of 1×106 per mouse. The site of injection was at the
right back of mice closed to the hind limbs. When the tumor volume reached about 300
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Bioconjugate Chemistry
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mm3, the mice were injected with DOX-conjugated micelles at a dose of 5 mg DOX/kg
body weight via the tail vein. After 6 h, 12 h, 24 h and 48 h, the mice were sacrificed
and the major organs (heart, liver, lung, spleen and kidney) were harvested as well as
the tumors. After washed with PBS for three times, the tumors and organs were
performed with ex vivo fluorescence imaging using a CRI Maestro Imaging System
(Cambridge Research & Instrumentation, Inc., USA).
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In Vivo Antitumor Efficacy
4T1 tumor-bearing mice (BALB/c) were used for investigating the antitumor efficacy
of DOX-conjugated micelles. When the tumor volume approximately reached to 100
mm3, the tumor-bearing mice were randomly divided into 3 groups and mice were
injected with saline solution, free DOX and DOX-conjugated micelles at selected time
points (day 0, 4, 8 and 12). The free DOX and DOX-conjugated micelles were injected
at a dose of 5 mg DOX/kg body weight. The body weight and tumor volume of the
mice were measured every two days. The tumor volume was calculated by the equation:
tumor volume (mm3) = length ×width2/2. At the 21st day, all the mice were sacrificed.
The specimens of mean organs (heart, liver, spleen, lung and kidney) were excised from
each group, washed by PBS, fixed with 4% formaldehyde and after dehydrating with
gradient ethanol, organs and tumor were embedded in paraffin blocks to prepare the
tissue sections of 5 μm. Sections were stained with hematoxylin and eosin (H&E) for
histopathological evaluation. Moreover, for tumor tissue, CD3134, Ki-6735 and
TUNEL36 were analyzed according to literatures. We selected one representative
paraffin wax block from each group. Consecutive 4 mm thick sections were cut from
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