Fabrication of Polymeric Micelles with Aggregation-Induced Emission and Forster Resonance Energy Transfer for Anticancer Drug Delivery

Article abstract of DOI:10.1021/acs.bioconjchem.7b00274

With the aim of obtaining effective cancer therapy with simultaneous cellular imaging, dynamic drug-release monitoring, and chemotherapeutic treatment, a polymeric micelle with aggregation-induced emission (AIE) imaging and a Forster resonance energy tran

Full text of DOI:10.1021/acs.bioconjchem.7b00274

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Article
Fabrication of Polymeric Micelles with AIE
and FRET for Anticancer Drug Delivery
Na Hao, Changzhen Sun, Zhengfei Wu, Long Xu, Wenxia Gao, Jun Cao, Li Li, and Bin He
Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 01 Jun 2017
Downloaded from http://pubs.acs.org on June 2, 2017
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Fabrication of Polymeric Micelles with AIE and
FRET for Anticancer Drug Delivery
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a
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b
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a
Na Hao, Changzhen Sun, Zhengfei Wu, Long Xu, Wenxia Gao, * Jun Cao, Li Li *, Bin He
a
National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064,
China
b
College of Chemistry and Materials Engineering, Wenzhou University, Wenzhou 325027, China
Corresponding authors:
Wenxia Gao
Tel/Fax:+86ꢀ577ꢀ88368280
Email: wenxiag@wzu.edu.cn
Li Li
Tel: +86ꢀ28ꢀ8547 0770
Fax: +86ꢀ28ꢀ8541 2848
Email: li_li@scu.edu.cn
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ABSTRACT
With the aim to obtain effective cancer therapy of simultaneous cellular imaging, dynamic drug release
monitoring and chemotherapeutic treatment, a polymeric micelle with aggregationꢀinduced emission (AIE)
imaging and forster resonance energy transfer (FRET) effect was fabricated as the drug carrier. Amphiphilic
conjugate of MALꢀPEGꢀTripp bearing AIE molecules were synthesized and selfꢀassembled into micelles to
load anticancer drug doxorubicin (DOX). Spherical DOXꢀloaded micelles with the mean size of 106 nm were
obtained with good physiological stability (CMC, 12.5 ꢁg/mL), high drug loading capacity (10.4%) and
encapsulation efficiency (86%). The cellular uptake behavior of DOXꢀloaded MALꢀPEGꢀTripp micelles was
visible for highꢀquality intracellular imaging due to the AIE property. The delivery of DOX from the
drugꢀloaded micelles was dynamic monitored by the FRET effect between the DOX and MALꢀPEGꢀTripp.
Both in vitro (IC50, 2.36 ꢁg/mL) and in vivo anticancer activity tests revealed that the DOXꢀloaded
MALꢀPEGꢀTripp micelles exhibited promising therapeutic efficacy to cancer with low systematic toxicity. In
summary, this micelle provided an effective way to fabricate novel nanoplatform for intracellular imaging, drug
delivery tracing and chemotherapy.
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KEYWORDS
polymeric micelle, AIE imaging, FRET effect, chemotherapy
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INTRODUCTION
Multifunctional nanocarrier has attracted more and more attention for its effective cancer therapeutic effect.
The theranostic nanoparticles integrated with diagnostic imaging and therapeutic capability have exhibited great
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potential for tumor therapy by realizing imagingꢀguided drug delivery and tumor treatment. In order to obtain
the imaging guider, intracellular tracing nanomedicine based on fluorescent tagging has became an important
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method. Conventional fluoresent dyes such as fluorescein isothiocyanate (FITC), rhodamine and quantum dots
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are commonly used as fluorescent imaging materials.
However, the drawbacks such as instability in
modification, hydrolysis and detachment of dyes during the phagocytic process impede the performance for
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bioimaging. Quantum dots based biosensors and fluoresent inorganic dyes have relatively strong and stable
fluorescence, they suffer from the toxicity to the cell due to their nonbiodegradation and intrinsic heavy metal
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toxicity.
Moreover, traditional fluorogens further suffer from aggregation caused quenching (ACQ) when
incorporated in nanoparticles with high loading content, which greatly reduces the fluorescence intensity and
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impedes the imaging effect.
Therefore, the novel category of fluorophores with aggregation induced
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emission (AIE) effect has attracted great interest since it was reported in 2001.
The AIE probes have been
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employed for cell imaging.
The nanoꢀaggregates can stay inside the live cells for long time without
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fluorescence reduction to enable longꢀterm cell tracing for the whole biological event.
Taking advantage of
this unique AIE property, we fabricated an AIEꢀactive polymeric micelle as drug carrier with the fluorescent
imaging in this study.
Although the cellular uptake of polymeric micelles could be intracellular traced by the AIE effect in the
nanoꢀdrug carrier, the drug release behavior of the nanoparticles is still unknown. With the expectation to
investigate the intracellular drug release from nanoparticles, we further introduce the forster resonance energy
transfer (FRET) effect between the AIE group and antitumor drug of doxorubicin (DOX) as FRET is a dynamic
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process monitoring for specific imaging systems.
In this study, we use the molecular interaction between the
AIE group and DOX to detect drug release using the mechanism that the FRET effect between the AIE group
and DOX would disappear when DOX was released from nanoparticles. A polymeric micelle with the capacity
for simultaneous cellular imaging, dynamic drug release monitoring, and chemotherapy was fabricated for
effective cancer therapy (Figure 1) .
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Figure 1. Schematic diagram of the selfꢀassembly of polymeric micelles with AIE imaging and FRET effect for
anticancer drug delivery.
RESULTS AND DISCUSSION
Preparation and characterization of polymeric micelles
This study aimed to fabricate a novel polymeric micelles with AIE effect for cell imaging and FRET effect for
monitoring anticancer drug delivery of doxorubicin (Figure 1). We expected that the synthetic polymer
molecule could selfꢀassemble into micelles in which the hydrophobic AIE molecule core was used not only for
cell imaging but also for loading hydrophobic anticancer drugs, and the hydrophilic PEG moiety could serve as a
micelle shell with a long blood circulation time. The amphiphile of MALꢀPEGꢀTripp was designed and
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synthesized as shown in Scheme 1. The H NMR spectra of TrippꢀCOOH and MALꢀPEGꢀTripp were shown in
Figure 2. The chemical shifts of the protons in TrippꢀCOOH were located at 6.50 ppm (1), 7.03 ꢀ7.24 ppm (2ꢀ5),
7.85 ppm (6), 13.12 ppm (7), respectively. As for MALꢀPEGꢀTripp, except for the characteristic chemical shifts
of TrippꢀCOOH, the peaks at 3.30ꢀ3.81 ppm (8, 10) were assigned to the protons of OCH CH in PEG, and the
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other two protons of COCH CH and CH CH N in 1HꢀPyrroleꢀ1ꢀpropanoicacid were appeared at 2.40ꢀ2.60 ppm
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(
11), and 4.61 ppm (12), respectively. The peak at 13.12 ppm assigned to the terminal carboxyl disappeared after
esterification. The conjugate was further confirmed by molecular weight of MALDIꢀTOFꢀMS analysis (Figure
S1), the molecular weight of MALꢀPEG was rightꢀshifted after conjugation.
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Figure 2. The H NMR spectra of TrippꢀCOOH and MALꢀPEGꢀTripp with DMSOꢀd as solvent
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The size distribution and morphology of the micelles were tested by DLS and SEM. Both blank and DOXꢀloaded
MALꢀPEGꢀTripp micelles were well dispersed in aqueous media. The mean size of blank MALꢀPEGꢀTripp
micelles was about 91 nm (Figure 3A) and that of DOXꢀloaded MALꢀPEGꢀTripp micelles was about 106 nm
(
Figure 3B). Both blank and drugꢀloaded micelles were spherical in SEM images and the size was consistent
with DLS result. The relative small mean size was highly suitable for nanoparticles to evade scavenging by the
mononuclear phagocyte system (MPS) and passive targeting via enhanced permeability and retention (EPR)
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effect. The critical aggregation concentration (CMC) for the micelles was also investigated to evaluate the
stability of micelles. Since the amphiphiles of MALꢀPEGꢀTripp possessed the AIE property when they
selfꢀassembled into micelles in aqueous solution, here we used this mechanism to test the CMC of micelles, the
tested CMC was 12.5 ꢁg/mL (Figure S2). It implied that the MALꢀPEGꢀTripp micelles were very stable when
highly diluted, which was necessary for intravenous application. Furthermore, the DOXꢀloaded
MALꢀPEGꢀTripp micelles had high drugꢀloading capacity (DLC) of 10.4% and high drug encapsulation
efficiency (DEE) of 86%.
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Figure 3. DLS and SEM images of (A) blank MALꢀPEGꢀTripp micelles and (B) DOXꢀloaded MALꢀPEGꢀTripp
micelles micelle.
AIE imaging and FRET effect of the micelles
Since the TrippꢀCOOH groups as the hydrophobic moieties in the amphiphile MALꢀPEGꢀTripp were trapped
into the micellar cores during the micellar formation, the aggregation state of TrippꢀCOOH molecules would
excite the AIE behavior. The AIE behavior of MALꢀPEGꢀTripp could be evaluated by fluorescence assay. With
the same concentration and excitation condition, MALꢀPEGꢀTripp dissolved in DMSO showed weak
fluorescent, the fluorescent intensity increased dramatically with adding water to the DMSO solution (Figure
4A). Interestingly, when the water fraction (fw) reached 99 vol%, the fluorescent intensity of MALꢀPEGꢀTripp
in water was nearly 52ꢀfold stronger than that in 100% DMSO (Figure 4B). This result indicated the AIE effect
of MALꢀPEGꢀTripp micelles, which was further used for cell imaging.
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Figure 4. The fluorescent spectra of micelles, (A) fluorescent spectra of MALꢀPEGꢀTripp in DMSO/water
mixtures with different fractions of water (fw), insert image showing fluorescence of MALꢀPEGꢀTripp solutions
dissolved in pure DMSO (1) and 99% water (2); (B) plot of fluorescent intensity of MALꢀPEGꢀTripp versus
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water content of DMSO/water mixture, I= fluorescent intensity of MALꢀPEGꢀTripp in mixed solution, I =
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fluorescent intensity of MALꢀPEGꢀTripp in pure DMSO (λex= 330 nm).
Besides the AIE effect of the MALꢀPEGꢀTripp micelles, DOXꢀloaded MALꢀPEGꢀTripp micelles also showed
FRET effect. A phenomenon was found when blank MALꢀPEGꢀTripp micelles and DOXꢀloaded
MALꢀPEGꢀTripp micelles were excited at the same wavelength of 330 nm. Two emission peaks were obtained
for the DOXꢀloaded MALꢀPEGꢀTripp micelles excited at 330 nm, one was around 470 nm assigned to the Tripp
group in blank MALꢀPEGꢀTripp micelles, and the other around 580 nm was assigned to DOX (Figure 5A). This
result demonstrated the Tripp group was firstly excited at 330 nm and gave the emission wavelength of 470 nm,
The emission of 470 nm was further acted as the excitation wavelength for DOX, which demonstrated the FRET
effect occurred between Tripp and DOX. This phenomenon was further explained by the absorption and
emission spectra of MALꢀPEGꢀTripp and DOX (Figure 5B). The existence of FRET phenomenon might also be
related to the πꢀπ stacking between DOX and the AIE group of Tripp (Figure S3), a significant fluorescence
quenching and red shift in UV spectra occurred when the DOX was encapsulated in the micelles .
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The FRET phenomenon was further used to keep tracking drug release process of DOX from the micelles. The
DOX release profiles of the DOXꢀloaded MALꢀPEGꢀTripp micelles at different pH were investigated (Figure
5C). The amounts of released DOX at different predetermined time points were measured by fluorescence
detector (λex= 480 nm, λem= 550 nm). The curves exhibited a biphasic pattern including an early burst release
and followed sustaining release. The burst release was the rapid transfer of DOX adsorbed to the surface of
micelles, and the sustaining release was the diffusion of DOX from micellar cores. The accumulated release rates
of DOXꢀloaded MALꢀPEGꢀTripp micelles was about 28% at pH 7.4 and 67% at pH 5.5 in the first 24 h,
respectively. And finally the drug release from DOXꢀloaded MALꢀPEGꢀTripp micelles was much higher at pH
5.5 (90%) than that at pH 7.4 (35%). The DOXꢀloaded MALꢀPEGꢀTripp micelles exhibited faster release in
lower pH environment such as in lysosomes. During the process of drug release, the fluorescence of
DOXꢀloaded MALꢀPEGꢀTripp micelles was detected (Figure 5D). The progressive decline in emission at
wavelength of 500 nmꢀ700 nm (emission of acceptor DOX) was in accompany with the gradually growth in
emission at wavelength of 470 nm (emission of donor MALꢀPEGꢀTripp) with the extension of release time. This
result demonstrated the FRET effect between the DOX and MALꢀPEGꢀTripp was weakened gradually till it
disappeared with the release of DOX from the drug loaded micelles. The delivery of DOX could be dynamic
monitored by the FRET effect.
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Figure 5. Fluorescence spectra of blank micelles and DOXꢀloaded MALꢀPEGꢀTripp micelles (λex= 330 nm),
inset: photograph showing fluorescence of blank micelles (1), DOXꢀloaded micelles (2), and DOX (3) under UV
irradiation at 365 nm (A); the absorption and emission spectra of MALꢀPEGꢀTripp and DOX (B); the release
profiles of DOXꢀloaded MALꢀPEGꢀTripp micelles at pH 5.5 and 7.4, means ± SD (n =3) (C); the fluorescence
spectra of DOXꢀloaded MALꢀPEGꢀTripp micelles in PBS (pH 5.5) at different time during DOX releasing
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λex= 330 nm) (D).
Stability of DOX-loaded micelles
Furthermore, the stability of DOXꢀloaded MALꢀPEGꢀTripp micelles was examined in serumꢀsupplemented
medium by monitoring the change of MALꢀPEGꢀTripp and DOX fluorescent intensity. If the fluorescent
intensity of MALꢀPEGꢀTripp decreased or disappeared, it indicated that the DOXꢀloaded MALꢀPEGꢀTripp
micelles disassembled. Alternatively, once DOX released from DOXꢀloaded MALꢀPEGꢀTripp micelles, the
fluorescence intensity of DOX would increase because it was not affected by the ACQ effect and the
fluorescence intensity of the MALꢀPEGꢀTripp would also increase as the emission from MALꢀPEGꢀTripp was
no longer transferred to DOX via FRET. As shown in Figure S4, after incubated with serum for 24 h, the
fluorescence intensity of MALꢀPEGꢀTripp was almost not changed and the fluorescence intensity of DOX was
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slightly enhanced, we considered that this might be due to a small amount of DOX release from the DOXꢀ loaded
micelle. The results illustrated that DOXꢀloaded MALꢀPEGꢀTripp micelles were stable in serum.
In vitro anticancer activity study
The cytotoxicity of blank MALꢀPEGꢀTripp micelle was evaluated with 4T1 breast cancer cells and C2C12 cells
via CCKꢀ8 assay (Figure S5). The result indicated that the micelles were nonꢀtoxic after incubated for 48 h even
when the concentration increased to 1mg/mL. The DOXꢀloaded MALꢀPEGꢀTripp micelles were incubated with
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4T1 cells at different drug concentrations to study the in vitro anticancer activities. The intracellular localization
and distribution of DOXꢀloaded MALꢀPEGꢀTripp micelles were investigated using confocal fluorescence
microscopy (CLSM). As shown in Figure 6, the blue fluorescence of both blank and DOXꢀloaded
MALꢀPEGꢀTripp micelles were all clearly observed in the cytoplasm, which indicated the accumulation of
MALꢀPEGꢀTripp molecules in the cytoplasm. In the images of DOX•HCl samples, red fluorescence was
observed in both cytoplasm and nuclei, it was attributed to the excellent solubility of DOX•HCl in aqueous
medium, DOX•HCl was internalized in the cells and rapidly reached nuclei via passive diffusion mechanism.
However, for the DOXꢀloaded MALꢀPEGꢀTripp micelles, most of the red fluorescence of DOX was
concentrated in cytoplasm, only weak read fluorescence was observed in nuclei. The fluorescence intensity of
both DOX and MALꢀPEGꢀTripp increased for 3 h incubation comparing to 1 h incubation, which implied that
more drug loaded micelles were internalized into cells and a sustained release of DOX from the DOXꢀloaded
MALꢀPEGꢀTripp micelles was happened. This result demonstrated that the DOXꢀloaded MALꢀPEGꢀTripp
micelles could be internalized through an endocytosis process.
Figure 6. The confocal laser scanning microscopy images of 4T1 cells incubated with blank MALꢀPEGꢀTripp
micelles (A), DOX•HCl (B), and DOXꢀloaded MALꢀPEGꢀTripp micelles (C) for 1 h and 3 h, the 1, 2, 3 and 4
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represented the images of bright field, fluorescence channel of DOX, MALꢀPEGꢀTripp, and overlay, the scale
bar was 20 mm.
In order to further investigate the intracellular distribution of the micelles after cellular uptake, staining of
lysosomes with Lysotracker Green was observed by CLSM. As shown in Figure 7, the blue fluorescence from
blank MALꢀPEGꢀTripp micelles and DOXꢀloaded MALꢀPEGꢀTripp micelles was mostly coꢀlocalized with the
green fluorescence of lysosomes, which indicated that MALꢀPEGꢀTripp remained in the cytoplasm as
luminescent micelles. Meanwhile, for DOX loaded micellesꢀtreated cells, we found that the strong red
fluorescence signal was observed in the cytoplasm and weak red fluorescence was observed in the cell nucleus,
this indicated that most of the DOX was still in the lysosomes and only a small amount of DOX was delivered
into cells nucleus.
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Figure 7. The confocal laser scanning microscopy images of spatiotemporal distributions of blank
MALꢀPEGꢀTripp micelles (A), DOX (B), and DOXꢀloaded MALꢀPEGꢀTripp micelles (C) coꢀlocalized with
lysosomes in 4T1 cells for 3 h, the 1, 2, 3, 4 and 5 represented the images of bright field, fluorescence channel of
DOX, MALꢀPEGꢀTripp, Lysotracker Green and overlay, the scale bar was 20 mm.
To investigate the existence of FRET phenomenon in the cell, the images of CLSM were taken by using three
separate channels to collect the emission of MALꢀPEGꢀTripp (donor), DOX (acceptor) and FRET channel. A
405 nm laser was used to excite the MALꢀPEGꢀTripp in the donor channel and the emission was collected from
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475 to 495 nm (blue fluorescence). The acceptor channel was excited at 488 nm for the DOX and the emission
was collected from 550 to 625 nm (red fluorescence). The FRET channel was excited at 405 nm for the
MALꢀPEGꢀTripp donor, and the emission of DOX excited by energy transfer from donor was also collected
from 550 to 625 nm. As shown in Figure S6, the strong blue fluorescence of blank MALꢀPEGꢀTripp micelles in
the cytoplasm of cells in the donor channel (A1), no red fluorescence was observed in the FRET channel and
acceptor channel because of the absence of red emitters (A2, A3). Interestingly, we found that the blue
fluorescence from DOXꢀloaded MALꢀPEGꢀTripp micelles was slightly quenched (B1), meanwhile, the red
fluorescence in the FRET channel (B2) and the acceptor channel (B3) were observed. These results suggested
that the efficient FRET from the MALꢀPEGꢀTripp donor to the DOX acceptor was happened after the cellular
internalization of DOXꢀloaded MALꢀPEGꢀTripp micelles. For the free DOX, however, there was no blue
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fluorescence was observed and the red fluorescence was only observed in the acceptor channel (C3).
The quantitative cellular uptake and drug release abilities of DOXꢀloaded MALꢀPEGꢀTripp micelles were
further illustrated in flow cytometry. 4T1 breast cancer cells were used as a negative control and only
autofluorescence of cells was observed. The free DOX and DOXꢀloaded MALꢀPEGꢀTripp micelles containing
the same concentration DOX (10 ꢁg/mL) were coꢀcultured with 4T1 cells for 1h and 3h. As presented in Figure
8A, the fluorescence intensity of DOX·HCl group was higher than that of DOXꢀloaded MALꢀPEGꢀTripp
micelles group for 1 h incubation. The fluorescence intensities of both groups increased with longer incubation
time, which suggested more DOXꢀloaded MALꢀPEGꢀTripp micelles accumulated in the cytosol. The result was
in agreement with that of CLSM.
Moreover, the IC50 (half maximal inhibitory concentration) values of DOX·HCl (0.41ꢁg/mL), DOXꢀloaded
MALꢀPEGꢀTripp micelles (2.36 ꢁg/mL) to 4T1 cells were investigated (Figure 8B). Compared to free
DOX·HCl group, the lower anticancer activity of DOXꢀloaded MALꢀPEGꢀTripp micelles was owed to the slow
release of DOX from micelles and the cellular uptake via endocytosis, which retarded the diffusion and
aggregation of DOX from the cytoplasm to the nuclei. DOX·HCl was a waterꢀsoluble molecule, which diffused
much faster into cells to kill cells efficiently and resulted in lower IC50.
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Figure 8. The flow cytometry histogram profiles of 4T1 cells incubated with DOX·HCl and DOXꢀloaded
MALꢀPEGꢀTripp micelles for 1h and 3 h, the DOX concentration was 10 ꢁg/mL (A); the IC50 of the
DOXꢀloaded MALꢀPEGꢀTripp micelles against 4T1 cells, the incubation time was 48 h, means ± SD (n = 3) (B).
In vivo anticancer activity investigation
The in vivo anticancer activity study was carried out in breast cancerꢀbearing BALB/c mice. DOX·HCl (5
mg/kg) and DOXꢀloaded MALꢀPEGꢀTripp micelles (DOX, 5 mg/kg) were administrated via tail vein injection
for 4 times and the saline was used as control. The changes of tumor sizes and body weight treated with
DOXꢀloaded MALꢀPEGꢀTripp micelles, DOX·HCl and saline were presented in Figure 9.
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Time (day)
Time (day)
Figure 9. The in vivo anticancer activity of DOXꢀloaded micelles, (A) the volumes of tumors treated with
DOXꢀloaded MALꢀPEGꢀTripp micelles, DOX·HCl and saline; p values at 21 days: DOXꢀloaded
MALꢀPEGꢀTripp micelles, p=0.0062; DOX·HCl, p=0.0247, P < 0.05 meant significant difference; (B) body
weights of the mice treated with DOXꢀloaded MALꢀPEGꢀTripp micelles, DOX·HCl and saline.
After 21days administration, the saline group showed almost no inhibition effect since the tumor volume
increased rapidly. The DOX·HCl group exhibited the most efficient anticancer activity in vivo. And it is satisfied
that the inhibition efficiency of the DOXꢀloaded MALꢀPEGꢀTripp micelles group was near to the free
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DOX·HCl group (Figure 9A). The body weight of the mice treated with DOX·HCl decreased 20% after 12d.
The mice not only suffered from substantial weight loss and weakness after injections, but also suffered from
erythema on the mice, which indicated the severe systemic toxicity of DOX·HCl (Figure 9B). The body weight
of the mice treated with DOXꢀloaded MALꢀPEGꢀTripp micelles did not show significant difference from that of
saline group, which suggested DOXꢀloaded MALꢀPEGꢀTripp micelles did not exhibited marked side effect.
The histological tissues slides of tumor, heart, liver, spleen, lung and kidney of the tumorꢀbearing mice treated
with DOXꢀloaded MALꢀPEGꢀTripp micelles, DOX·HCl, and saline were presented in Figure 10. The
DOXꢀloaded MALꢀPEGꢀTripp micelles and DOXꢂHCl group exhibited much better tumor inhibition effect, as
tumor necrosis with hemorrhage appeared in most areas. While the free DOXꢂHCl group led to serious heart
toxicity as necrosis (heart image in DOXꢂHCl group) with acute inflammatory cells filtration in epicardium and
congestion between myocardial cells. Simultaneously, little necrosis area in the center of neoplastic cells was
observed in saline group (tumor image). White pulp atrophy and congestion occurred in spleen (spleen images).
Lobular pneumonia was observed in lung in DOXꢂHCl and saline group, and less inflammation in micelles
group. Kidney glomerular swelling shrinked or disappeared (kidney images in DOXꢂHCl and saline group). All
the organs of heart, liver, spleen, lung and kidney in micellar formulation group were normal in the histological
tissue slides, indicating less toxicity of micellar formulation to organs.
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Figure 10. Histological analysis of different organs in tumor bearing mice with DOXꢀloaded MALꢀPEGꢀTripp
micelles, DOX·HCl, and saline. Each group of BALB/c mice (male, n = 10) was intravenously administered four
times at a threeꢀday interval at a dose of 5 mg/kg (DOX).
Immunohistochemical analysis of CD31, Ki-67 and TUNEL
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Invasion and metastasis always accompanied with angiogenesis in the process of solid tumors growth. The
CD31 and Kiꢀ67 antigen staining were further performed to assess the antitumor efficacy of DOXꢀloaded
MALꢀPEGꢀTripp micelles on tumor cell proliferation, and the terminal deoxynucleotidyl transferaseꢀmediated
dUTP nick end labeling (TUNEL) assay was employed to detect apoptotic programmed cell death (Figure 11).
About 47% of apoptotic cells were obtained for the DOX/MALꢀPEGꢀTripp micelles group and 54% for
DOX·HCl group, both them were significantly different with the saline group (11%). The CD31 and Kiꢀ67
immunohistochemical staining studies showed that the number of MVD and Kiꢀ67ꢀpositive cells administrated
with DOXꢂHCl actively inhibited the proliferation and induced the apoptosis of tumor cells.
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DOX loaded micelle
Figure 11. The TUNEL, Kiꢀ67 and CD31 immunohistochemical (IHC) staining of tumor tissues (× 400). The
brown areas indicated TUNELꢀpositive, CD31ꢀpositive or Kiꢀ67 positive staining. The apoptotic index was
calculated as the ratio of the apoptotic cell number to the total tumor cell number in each field of view. The CD31
positive area was expressed in endothelial cell pack and count capillary number by reading each section (MVD).
The Kiꢀ67 density in each image was calculated by Kiꢀ67ꢀpositive area to total area. Data was presented as mean
±
SD (n = 6) (*: P<0.05;**: P<0.01;***: P<0.001). The dose of DOX was 5 mg/kg.
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CONCLUSION
This study devoted to obtain a polymeric micelle for cancer therapy with AIE effect for cell imaging and FRET
for monitoring anticancer drug delivery. AIE molecule of TrippꢀCOOH was selected to fabricate the lyophobic
micellar core for imaging, as well as generate FRET effect with doxorubicin (DOX) to detect the drug release.
The results demonstrated this DOXꢀloaded MALꢀPEGꢀTripp micelle exhibited not only outstanding cellular
tracing, but also excellent therapeutic efficacy and low side effects in cancer chemotherapy. This study
provided an effective way to fabricate a polymeric micelle with the capacity for simultaneous cellular imaging,
dynamic drug release monitoring, and chemotherapeutic treatment for effective therapy.
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Materials and methods
Materials
Poly(ethylene glycol) (PEG, Mw = 2000g/mol), 1Hꢀpyrroleꢀ1ꢀpropanoicacid (MAL) and deuteron dimethyl
sulphoxide (DMSOꢀd6) were purchased from SigmaꢀAldrich Co. Ltd. Phenylacetylene, methyl
4
1
ꢀaminobenzoate,
1ꢀethylꢀ(3ꢀdimethyllaminopropyl)
carbodiie
hydrochlide
(EDC·HCl)
and
ꢀhydroxyꢀbenzotriazole monohydrate (HOBt) were purchased from GL Biochem. Ltd. (Shanghai, China).
Tetramethylethylenediamine, and N, Nꢀdiisopropylethylamine (DIEA) were purchased from Asta Tech
Biopharm. Co. Ltd (Chengdu, China). Doxorubicin hydrochloride (DOX·HCl, Shanghai Yingxuan Chempharm
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Co. Ltd. China) was deprotonated according to the method previously reported. Diethyl ether and CH Cl were
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2
purified before use and all other solvents were used as received. All solvents were obtained from Chengdu
Kelong Chemical Co. (China). Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial
Institute (RPMI) 1640 medium, fetal bovine serum (FBS), and cell counting kitꢀ8 (CCKꢀ8) were used for
cytotoxicity test.
Characterizations
1
H NMR was performed on Bruker (400 MHz) in DMSOꢀd with tetramethylsilane as the internal standard. The
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molecular weights were measured by matrix assisted laser desorption/ionization timeꢀofꢀflight mass
spectroscopy (MALDIꢀTOFꢀMS). The mean size and size distribution of the micelles were carried out using a
dynamic light scattering (DLS) spectrometer (Malvern ZetasizerNano ZS). The morphology of the micelles was
measured by transmission electron microscopy (TEM, JEMꢀ100CXꢀJEOL). The samples were prepared by
dropping the freshly micelle solution onto a copper grid coated with carbon film and dried overnight at room
temperature before observation. The samples were negatively stained with 1% uranyl acetate aqueous solution.
The AIE properties and FRET phenomenon were determined by UVꢀevisabsorption (Specord 200 PLUS) and
Fluorescence spectra (HITACHI Fꢀ700).
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Synthesis of MAL-PEG-Tripp
The synthetic process of the MALꢀPEGꢀTripp was presented in Scheme 1.
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Scheme 1. The synthesis of MAL ꢀPEGꢀTripp.
Synthesis of Tripp-COOH
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Compound 1 of 1, 4ꢀdiphenylbutaꢀ1, 3ꢀdiyne was firstly synthesized according to the previous literature. Then
compound 1 (2.0 g, 1.0 mmol), copper (I) chloride (0.12 g, 1.0 mmol) and methyl 4ꢀaminobenzoate (3.01 g, 20.0
mmol) were added to a 250 mL bottomꢀround flask stirred under argon atmosphere for 12 h at 115 °C. The crude
product of compound 2 (TrippꢀCOOCH ) was purified by silica column chromatography with the eluent
3
(petroleum ether/dichloromethane = 2/1). Then the above purified product of compound 2 (0.28 g, 0.8 mmol)
and NaOH (0.2 g, 5.0 mmol) were dissolved in 200 mL of methanol and stirred at 65 °C for 15 h, the crude
product was precipitated in a large amount of CH Cl , the white solid precipitate was washed by acetone and
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chloroform for three times to obtain compound 3 (TrippꢀCOONa). Compound 3 was added to HCl (6 M)
solution with stirring at room temperature for overnight and the product was washed by water several times to
obtain compound 4 (TrippꢀCOOH).
Synthesis of MAL-PEG
PEG2k (10.0 g) and compound of MAL (1.0 g) were dissolved in 200 mL of the anhydrous CH Cl in an ice bath
2
2
under nitrogen atmosphere. A solution of DCC (2.06 g, 10.0 mmol) and DMAP (0.061 g, 0.5 mmol) in CH Cl2
2
(60 mL) was added dropwise into the mixture. The mixture was stirred at room temperature for 48 h. The white
solid dicyclohexylurea (DCU) precipitate was removed by filtration, the filtrate was concentrated and
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precipitated into large amount of cold diethyl ether. This purification procedure was repeated for several times to
obtain compound 5 (MALꢀPEG).
Synthesis of MAL-PEG-Tripp
Compound 4 (0.41 g, 1.2 mmol), compound 5 (2.0 g, 1.0 mmol), EDC·HCl (0.27 g, 1.4 mmol) and HOBt (0.19
g, 1.4 mmol) were dissolved in anhydrous dichloromethane, DIEA was injected into this solution and stirred at
room temperature for 48 h. The solution was concentrated by evaporated under vacuum and precipitated in a
large amount of cold diethyl ether for three times. After that, the product was dialyzed with a molecular weight
cutoff of 1000 Da and freezeꢀdried for further purification to obtain compound 6 (MALꢀPEGꢀTripp).
Critical micelle concentration (CMC)
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A simple method was developed to determine the CMC by using the aggregationꢀinduced emission of the
amphiphiles, the emergence of the blue fluorescence of MALꢀPEGꢀTripp appeared when the formation of
micelles in double distilled water. The solution with different concentration of MALꢀPEGꢀTripp (from 0.49 to
100 ꢁg/mL) in double distilled water was processed by ultrasound for 0.5 h, the CMC of the amphiphiles was
measured by fluorescence spectra. The fluorescence absorbance values at a wavelength of 480 nm were collected
for the calculation of critical micelle concentration.
Preparation of drug-loaded micelles
Amphiphile (MALꢀPEGꢀTripp, 10 mg) and DOX (2.5 mg) were dissolved in 1 mL of DMSO. This mixture
solution was sonicated for 1 h, and then dropped into 10 mL deionized water with vigorous stirring overnight.
The solution was dialyzed against deionized water at 4 °C for 12 h in a dialysis tubing (Spectra/Por MWCO =
1000). The outer phase was replaced with fresh deionized water every 2 h. The solution in the tubing was
lyophilized after centrifugation. The whole procedure was performed in the dark.
The content of encapsulated DOX was determined by UVꢀVis measurement (λmax= 480 nm) in DMSO using
calibration curve obtained from DOX/DMSO solutions with different DOX concentrations. Drug loading
content (DLC) and drug loading efficiency (DLE) were calculated according to the following formulas:
DLC (%) = (weight of drug in micelle/weight of drugꢀloaded micelle)× 100%
DLE (%) = (weight of drug in micelle/weight of drug in feeding)× 100%
Drug release profile
DOXꢀloaded MALꢀPEGꢀTripp micelles were dispersed in PBS (1 mL, ionic strength = 0.01 M, pH = 7.4/5.5).
The mixture was transferred into dialysis membrane tubing (Spectra/Por MWCO = 1000), the tubings were
immersed in vials containing 25 mL of PBS solution. The vials were put in a shaking bed and the release profiles
were tested at 37 °C. The volume of medium was kept constant by adding fresh medium after each sampling at
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prescribed time intervals. The released DOX was detected by a fluorescence detector (λ =480 nm, λ =550 nm).
ex
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The release experiments were conducted in triplicate under sink conditions, the mean value was presented.
Cytotoxicity assessment
4T1 cells were cultured in RPMI 1640 media, C2C12 cells were cultured in DMEM medium. The media were
supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin at 37 °C in a humidified atmosphere
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with 5% CO . The cells were harvested with 0.02% EDTA and 0.025% trypsin and rinsed. The cytotoxicity of
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with 4×10 cells per well in 100 ꢁL of medium for 24 h incubation before the tests. After 24 h incubation, the
medium was removed and replaced with 100 ꢁL of medium containing different concentrations of blank
micelles. Then the cells were incubated for an extra 48 h. The culture medium was removed and the wells were
rinsed with PBS (pH=7.4). 100 ꢁL of Cell Counting Kitꢀ8 assay (CCKꢀ8, Dojindo, Japan, volume fraction 10%)
solution was added to each well. After incubated for 2 h, the absorbance was measured in a Thermo Scientific
MK3 (Thermo fisher; US) at the wavelength of 450 nm.
Cellular uptake
Confocal laser scanning microscopy (CLSM) and flow cytometry was employed to examine the cellular uptake
of DOX·HCl and DOXꢀloaded MALꢀPEGꢀTripp micelles. 4T1 cells at a logarithm phase were seeded on 35 mm
4
ꢀ1
diameter glass dishes at a cell density of 1×10 mL . After incubating for 24 h, DOXꢀloaded MALꢀPEGꢀTripp
ꢀ1
micelles were dissolved in each culture medium until the final DOX concentration was 10 ꢁg mL , The mixture
was added into the each glass dishes with the medium removed. After incubated for 1 h and 3 h, the culture
medium was removed and the dishes were rinsed with PBS (pH = 7.4). DOX was excited at 480 nm with
emission at 590 nm.
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ꢀ1
For the flow cytometry tests, 4T1 cells were seeded in 6ꢀwell plates at a density of 2×10 mL cells per well and
ꢀ1
incubated for 24 h. The cells were treated with DOX·HCl and s at the same DOX concentration (10 ꢁg mL ) for
1 h and 3 h, respectively. The culture medium was eliminated, the cells were washed with PBS for three times
and harvested by trypsinization. The cells were resuspended in PBS after centrifugation (1000 rpm, 5 min) and
the fluorescence intensity was measured (λ =480 nm; λ =590 nm) on a BD FACS Calibur flow cytometer
ex
em
(
Beckton Dickinson).
In vitro anticancer activity
The anticancer activity of drug loaded micelles was evaluated in vitro with 4T1 cells. Cells were harvested and
4
ꢀ1
seeded in 96ꢀwell plates with 1×10 mL cells per well with 100 ꢁL medium for 24 h. DOX·HCl and
DOXꢀloaded MALꢀPEGꢀTripp micelles in culture mediums were added to the plates with different DOX
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concentrations (from 0.001 to 100 mg/mL) and incubated for 48 h. The cell viability was measured by CCKꢀ8
assay.
In vivo antitumor activity study
All the animal experiments were performed according to the institutional and NIH guidelines for care and use of
research animals. Male BALB/c mice (body weight: 21ꢀ24 g) were purchased from West China Experimental
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Animal Culture Center of Sichuan University. 5×10 4T1 cells were injected into right flank subcutaneously of
BALB/c mice. The mice were randomly divided into 3 groups when the inoculated tumor volume reached about
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100 mm , and then injected intravenously via the tail vein with a DOX dose of 5 mg/kg body weight for four
times at a 3ꢀday interval. The tumor volume and body weight were monitored at prescribed time intervals. The
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tumor volume was calculated by the flowing formula: V(mm )=1/2×ab , where a and b presented the largest and
smallest diameter of the tumor tissue respectively. All mice were sacrificed on day 21.
The heart, liver, spleen, lung, kidney and tumor were excised from each group and fixed in 4% formaldehyde for
histological examination. The representative tissues were processed for routine histopathological procedures.
Paraffin embedded specimens were cut into 5 mm sections and stained with hematoxylin and eosin (H&E) for
histopathological evaluation.
Immunohistochemical analysis of CD31, Ki-67 and TUNEL
Streptavidine peroxidase methods were specially designed for Immunohistochemical (IHC) reactions and other
immune detection. The paraffinꢀembedded tumor sections were deparaffinaged, rehydrated and incubated
overnight at 4 °C with primary monoclonal antibody against CD31 or Kiꢀ67(1:200) (British abcamꢀ(Shanghai)
trading Co., LTD). Biotinylated goat antirabbit antibodies were used as secondary antibodies at 1:200 for 30 min
at room temperature and the egg protein reagent marked with horseradish peroxidase was added. The data of
immunohistochemistry staining was obtained as described. IHC images were taken by the Motic Images
Advanced software (Motic China Group CO., LTD.), the positiveꢀstained integrated optical density (IOD) of the
CD31 and kiꢀ67 was scaled with each image by ImageꢀPro Plus 6.0 software (Media Cybernetics, Bethesda,
MD). The microvessel density (MVD) was counted by reading each section which referenced Weidner method
for quantitative analysis and the CD31 positive area was expressed in endothelial cell pack. In addition, the kiꢀ67
density in each photograph was calculated as the ratio of kiꢀ67ꢀpositive area to total area. For terminal
deoxynucleotidyl transferase mediated UTP nick end labeling (TUNEL) assay, the dewaxed and rehydrated
tumor tissue sections were incubated with proteinase K at 37 °C for 15 min, rinsed with PBS twice. The TUNEL
assay was performed on in situ cell death detection kitꢀPOD (Roche Group, Switzerland) to evaluate apoptotic
cells. Positive TUNEL staining was visualized by optical microscopy and the apoptotic index was formulated as
the ratio of apoptotic cell number to total tumor cell number in each microscope field.
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Statistical analysis
All data are presented as means±S.D. Statistical significance (p < 0.05) was evaluated by using Student tꢀtest
when experiment groups were compared.
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AUTHOR INFORMATION
Corresponding Author
W.X. Gao (wenxiag@wzu.edu.cn); L. Li (li_li@scu.edu.cn).
ACKNOWLEDGMENT
The authors thank for the financial support of National Science Foundation of China (No. 51573111, 21672164,
81371666), Program for Changjiang Scholars and Innovative Research Team in University (IRTꢀ15R48),
Ministry of Education of China (No. 20130181110038), and Natural Science Foundation of Zhejiang Province
(LY15B020001).
SUPPORTING INFORMATION
Supporting information is available free of charge via the Internet at http://pubs.acs.org.
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Table of Contents (TOC)
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