An Amphiphilic Micromolecule Self-Assembles into Vesicles for Visualized and Targeted Drug Delivery

Article abstract of DOI:10.1021/acsmedchemlett.0c00212

Described here is the first example of the construction of multifunctional drug delivery systems by employing an amphiphilic micromolecule. The intrinsic aggregation-induced emissive and tumor-targeting amphiphilic conjugate of β-d-galactose with tetraphe

Full text of DOI:10.1021/acsmedchemlett.0c00212

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Letter
An amphiphilic micromolecule self-assembles into
vesicles for visualized and targeted drug delivery
Weiwei Ma, Jingjing Bi, Hao Wu, and Guisheng Zhang
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00212 • Publication Date (Web): 20 Jul 2020
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An amphiphilic micromolecule self-assembles into vesicles for vis-
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Weiwei Ma,a,b Jingjing Bi,*a Hao Wu,a Guisheng Zhang*a
a Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Collaborative Innovation Center of Henan
Province for Green Manufacturing of Fine Chemicals, Henan Key Laboratory of Organic Functional Molecule and Drug In-
novation, School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, Henan 453007, China.
b College of Pharmacy, Xinxiang Medical University, China.
*Fax: (+86)-373-332-5250; E-mail: zgs@htu.cn or bijingjing2008@126.com
ABSTRACT: Described here is the first example for constructing multifunctional drug delivery systems by employing an am-
phiphilic micromolecule. The intrinsic aggregation-induced emissive and tumor-targeting amphiphilic conjugate of β-D-galactose
with tetraphenylethene (TPE-Gal), in which the hydrophobic TPE moiety spontaneously acts as the imaging chromophore and hy-
drophilic Gal moiety spontaneously as the targeting ligand and galactosidase trigger, can self-assemble into fluorescent vesicles that
can efficiently load both water-soluble and -insoluble anticancer drugs. In vitro and in vivo evaluations revealed that the pH/β-D-
galactosidase dual responsive doxorubicin-loaded vesicles TPE-Gal@DOX exhibited good targeting effect and higher anti-tumor
efficacy than free doxorubicin. H&E staining analysis displayed remarkable necroses and weak cell proliferation in the tumor area,
and no toxicity to major organs, indicating the superior targeting anti-tumor therapeutic efficacy of TPE-Gal@DOX.
KEYWORDS. -D-galactose-modified tetraphenylethenes, aggregation-induced emission, fluorescent vesicles, cell imagimg, tar-
geted drug delivery.
Chemotherapy is an indispensable cancer-treatment method.
However, the poor pharmacokinetics and lack of tumor-target-
ing of the conventional chemotherapeutic drugs limited their
clinic uses.1-3 Drug delivery systems (DDSs) are essential for
improving the effectiveness of chemotherapy.4-8 Over the past
years, amphiphilic block copolymers self-assembled into lipo-
somes, micelles, or vesicles in selective solvents as DDSs in the
use of cancer-treatment have attracted much attention.9-11 Re-
cent researches concerning DDSs stressed on multi-functional-
ization that can achieve targeted delivery and triggered the re-
lease of the drugs inside targeted cells, as well as the real-time
monitoring of drug localization.12-16 The general multi-function-
alization method was to physically wrap or chemically integrate
a fluorescent dye and a targeting unit into the self-assembled
skeletons to construct a visualized and targeted DDS. However,
with such functionalizations, the physical and chemical proper-
ties of DDSs may be changed; the behavior of labelled DDSs in
endocytosis or phagocytosis may be different from that of unla-
beled DDSs; fluorescent dyes may be hydrolyzed and dissoci-
ated from DDS during phagocytosis;17 Especially, the toxicity
and aggregation caused quenching (ACQ) characteristics of tra-
ditional fluorescent dyes seriously limit their applications.18
Since the first reported aggregation-induced emission (AIE)
phenomenon by Tang et al.,19 fluorescent nano-particles based
on AIE active tetraphenylethene (TPE) chromophores20 have
emerged in bioimaging and drug delivery.21-26 These TPE-based
copolymers or coordination polymer nanoparticles used for
target drug delivery can efficiently address ACQ drawbacks of
traditional fluorescent-labelled multi-functional DDS.
It is noteworthy that almost all of the known nanoparticle
DDSs of liposomes, micelles, and vesicles were constructed by
the self-assemblies of amphiphilic polymers. However, the pol-
ymeric DDSs have some manufacturing-related problems like
low drug entrapment and stability problems due to the sensitive
degradation of the polymer main chain or the spontaneous es-
cape of the nanoparticles.27-28 Therefore, if multi-functional
DDSs were self-assembled by amphiphilic micromolecules
with intrinsic AIE and targeting characteristics (instead of
wrapping or integrating a fluorescent dye and a targeting unit
into polymeric skeletons), it would be a new generation of
multi-functional DDSs overcoming most of the drawbacks of
the traditional multi-functional DDSs. Herein, we present an in-
trinsic AIE and tumor-targeting amphiphilic micromolecule, a
conjugate of tetraphenylethene with β-D-galactose (TPE-Gal),
which can facilely self-assemble into the pH/β-D-galactosidase
dual responsive vesicles for cancer cell imaging, targeted drug
delivery and chemotherapy (Scheme 1). The hydrophobic TPE
moiety of the novel amphiphile spontaneously acts as the imag-
ing chromophore, and Gal moiety spontaneously as the target-
ing ligand and galactosidase trigger.
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between TEM and DLS can be attributed to the technique of
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measurement conditions. The zeta potential (Figure S3) of TPE-
Gal vesicles was determined to be -25 ± 2.3 mV. It has been
well known that 150 nm nanoparticles with slightly negative
charge have the good stability under the physiological condi-
tions and tend to accumulate in tumor more efficiently.38 The
TPE-Gal vesicles was stable in water，PBS and plasma of mice
over multiple weeks without significant aggregation (Figure
S4).
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Scheme 1. The synthesis of TPE-Gal and TPE-Gal vesicles
generating for visualized and targeted drug delivery.
Galectin-1, a 14-kD laminin-binding lectin that widely ex-
pressed in and around the membrane of various solid tumors
cells,29-30 can bind to β-galactosides through a rigorous carbo-
hydrate recognition domain.31 Meanwhile, β-galactosidase, a
lysosomal enzyme that is known to be upregulated in various
cancer subtypes, can be utilized to activate galactoside-contain-
ing DDSs.32-36 Based on the AIE behavior of hydrophobic TPE
and the unique tumor-targeting ability of hydrophilic β-galac-
tose, a rational design for developing novel visualized and tar-
get anticancer DDSs has emerged: The conjugates of TPE with
β-D-galactose self-assemble into uniform and robust vesicles to
load anticancer drugs. To be our best knowledge that employing
amphiphilic micromolecules in construction of DDSs has not
been reported.
Figure 1. TEM photograph of TPE-Gal vesicles (A); Cumu-
lated release profiles of DOX from TPE-Gal@DOX in different
media (B); Cell viabilities of TPE-Gal@DOX and free DOX to
HepG2 cells (C) and normal L02 cells (D).
Water-soluble doxorubicin (DOX) and water-insoluble
paclitaxel (PTX) were chosen as model drugs to investigate the
loading capacity of TPE-Gal vesicles. TPE-Gal vesicles show a
decent DOX loading capacity with drug-loading contents (DLC)
of 15.4 wt%, which corresponded to encapsulation efficiency
(EE) of 88.5%. Furthermore, TEM images (Figure S5) have
clearly shown the morphology of the DOX-loaded vesicles
TPE-Gal@DOX was spherical and had good dispersion with a
diameter of 165.2 ± 8.31 nm (Figure S6) and zeta potential of -
17.1 ± 4.4 mV (Figure S7). Similarly, TPE-Gal@PTX also
showed good PTX loading capacity with DLC of 7.6 wt% and
EE of 82.3%.
The synthesis of TPE-Gal was illustrated in Scheme 1. The
reaction of 4-hydroxybenzophenone with 1,6-dibromohexane
in the presence of K2CO3 afforded 2, from which the TPE de-
rivatives 3 were obtained through the McMurry reaction.37 Fur-
ther treatment of 3 with excess NaN3 gave azido compounds 4.
Finally, TPE-Gal was synthesized using classic CuAAC reac-
tion between 4 and propargyl 2,3,4,6-tetra-O-acetyl-β-D-galac-
toside 1 followed by the global deacetylation with Na-
OMe/MeOH.
UV-Vis absorption (Figure S8), IR spectra (Figure S9) and
fluorescence spectra (Figure S10) of different samples demon-
strated the successful fabrication of the TPE-Gal@DOX. More-
over, the distribution state of DOX in TPE-Gal@DOX was de-
tected by DSC methods (Figure S11). The DSC thermogram of
DOX shows one endothermic peak at 218 oC, while it cannot be
observed for TPE-Gal@DOX, indicating that DOX disperses in
vesicles with an amorphous state instead of crystalline state.
This result has also strongly proved the successful fabrication
of the TPE-Gal@DOX.
TPE-Gal exhibited extremely weak fluorescence in DMSO
(10 μM) and the maximum peaks generally located at 390 nm.
However, with the addition of water, the fluorescence intensity
dramatically increased, and the maximum emission was broad-
ened and red-shifted to 473 nm (Figure. S1). When the water
fraction reached to 90%, the fluorescence intensity of TPE-Gal
was nearly 50-fold stronger than in pure DMSO, indicating the
AIE characteristics of TPE-Gal. The morphology of TPE-Gal
vesicles was observed by TEM (Figure 1A) and exhibited a hol-
low spherical shape with the particle-size of about 120 nm. In
addition, the dynamic light scattering (DLS) assay (Figure S2)
demonstrated that the average hydrodynamic size and the poly-
dispersity index of TPE-Gal vesicles are 157.4 ± 7.69 nm and
0.074 respectively, disclosing that the vesicles have a relatively
homogenous size and well dispersibility in aqueous media with-
out significant aggregation. The difference in particle-size
For an ideal DDS, it is critical to release the drug triggered
by the stimuli associated with the specific microenvironmental
changes. Under intracellular-mimicking high expression of ga-
lactosidase conditions (in the presence of 100U β-galacto-
sidase), DOX can be rapidly released from TPE-Gal@DOX
(Figure 1B), in which the cumulative DOX releases are ca. 25%
and 50% in 2 h and 12 h, respectively. In contrast, less than 10%
drug release is observed in 12 h for TPE-Gal@DOX in the ab-
sence of galactosidase. Moreover, the fluorescence change
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could be detected with the naked eye under UV light (365 nm;
Figure S12). It’s well known that the microenvironment of tu-
mor tissues is more acidic than normal tissues. Then, the con-
trollable DOX release under different pH was carried out. As
shown in Figure 1B, the release of DOX from TPE-Gal@DOX
was pH- and time-dependent. Consequently, the pH/β-galacto-
sidase dual-responsive release behavior of TPE-Gal@DOX
provides a theoretical basis for safe and efficient tumor chemo-
therapy.
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The determination of hemolytic properties is a common bio-
compatibility test in drug carrier development. After 4 h of in-
cubation, there was no obvious hemolysis under different con-
centrations of TPE-Gal, even at high dosage (Figure S13, S14).
It caused only 4.78 ± 1.04% hemolysis in fresh human blood at
1000 g/mL concentration, suggesting that TPE-Gals possess
high hemocompatibility and low toxicity. The MTT assay
showed that the cytotoxicity of TPE-Gal vesicles to HepG2
cells was similar to controls (Figure S15), the cells retained 85%
viability even at a maximum concentration of 100 g/mL. TPE-
Gal@DOX and free DOX had almost the same anti-prolifera-
tive effects on of cancer cells at the same concentration of DOX
(Figure 1C). Moreover, compared to free DOX, the TPE-
Gal@DOX showed a significantly low cytotoxicity to normal
L02 cells (Figure 1D). These results indicated that the vesicles
formulation had the capability of delivering DOX molecules
into tumor cells, causing the intracellular release and leading to
cytotoxicity.
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Figure 2. CLSM images of the cellular uptake and controlled
release behaviors of HepG2 cells and L02 cells (A); CLSM im-
ages of HepG2 cells in TPE-Gal vesicles (B).
Encouraged by the exciting results in vitro evaluations, the in
vivo antitumor efficacy and tumor-targeting effect of TPE-
Gal@DOX were evaluated in tumor-bearing mice by giving
drugs intravenously. As shown in Figure 3A, the average tumor
volume in the mice treated with saline solely and the blank TPE-
Gal vesicles increased rapidly. In strong comparison, the tumor
volume in the animals treated with free DOX increased slowly.
To our delight, TPE-Gal@DOX exhibited obviously stronger
inhibition effect on tumor growth compared with the free DOX
group (Figure S16), which might attribute to the enhanced cel-
lular uptake of TPE-Gal@DOX by targeted delivery and pH/β-
galactosidase-sensitive drug release at tumor sites. The biodis-
tribution of DOX clearly expressed the amount of drug in each
tissue (Figure S17). According to the results, distribution of
TPE-Gal@DOX was significantly lower in heart, spleen, lung
and kidney compared to that of free DOX, implying that DOX
can be gradually eliminated in major organs with the time ex-
tending. Notably, TPE-Gal@DOX achieved the highest con-
centration in the tumor by comparison to free DOX. Photo-
graphs of tumors on day 21 after the treatment also showed that
TPE-Gal@DOX inhibited the tumor growth much better than
free DOX (Figure 3C). In addition, as shown in Figure 3B, TPE-
Gal@DOX showed no obvious influence on the bodyweight of
mice, implying good biocompatibility of this delivery system.
The histological examination of the tumors and main organs
were evaluated after 21 days of treatment. As shown in Figure
3D, the H&E staining of sectioned tumor tissues showed that
increased apoptosis of tumor cells was observed in the groups
of TPE-Gal@DOX compared with tissues in other groups, fur-
ther indicating the superior antitumor therapeutic efficacy of
TPE-Gal@DOX. Furthermore, the TPE-Gal@DOX groups
showed no apparent morphological difference among the saline
groups in heart, liver, spleen, lung, or kidney, proving that TPE-
Gal@DOX caused no harm to the mice (Figure S18). All of the
results implied that TPE-Gal vesicle is a very promising tumor-
targeting drug carrier for cancer chemotherapy.
The cellular uptake of TPE-Gal@DOX was evaluated by
confocal laser scanning microscopy (CLSM). As shown in Fig-
ure 2A, in CLSM images using the blue (TPE) and red (DOX)
fluorescent channels, the fluorescence intensity steadily in-
creased over time, suggesting the efficient uptakes of vesicles
by HepG2 cells and the successful drug release from the TPE-
Gal@DOX in a time-dependent manner. After treated with
TPE-Gal@DOX for 0.5 h, HepG2 cells exhibited distinct fluo-
rescence, each cell showed a region of particularly high fluores-
cence intensity where both TPE and DOX were gathered. We
hypothesized that this region might correspond to lysosomes,
where the releasing of DOX from the TPE-Gal@DOX led to
the fluorescence of both TPE and DOX. “After two hours of
incubation, the fluorescence of TPE only appeared in lysosomes,
but not in the nucleus, indicating that the vesicles just performs
its delivery function and does not influence the biological fate
of the drug. In the DOX fluorescence channel, the DOXs were
released from vesicles in the lysosome and translocated into the
cell nucleus. However, compared to HepG2 cells, similar TEP
fluorescence but weak DOX fluorescence has been observed at
the same time for L02 cells. These phenomena illustrated that
the TPE-Gal vesicles can be taken efficiently by cells for imag-
ing, but only exhibit a sustained drug-releasing pattern in tumor
cells. Furthermore, the blue fluorescence of TPE from TPE-Gal
vesicles mostly colocalized with the “green” fluorescence of
lysotracker near the nucleus (Figure 2B), indicating the indeed
colocalization of TPE-Gal with lysosomes.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
We thank the NSFC (U1604285 and 21877206), PCSIRT
(IRT1061), the 111 Project (D17007).
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ABBREVIATIONS
TPE, tetraphenylethene; Gal, β-D-galactose; DOX, doxorubicin;
PTX, paclitaxel; DDS, drug delivery system; ACQ, aggregation
caused quenching; AIE, aggregation-induced emission; DMSO, di-
methyl sulfoxide; DLS, dynamic light scattering; TEM, transmis-
sion electron microscope; DLC, drug-loading contents; EE, encap-
sulation efficiency; DSC, differential scanning calorimetry; UV-
Vis, ultraviolet and visible spectrophotometry; IR, infrared radia-
tion; CLSM, confocal laser scanning microscopy; H&E, hematox-
ylin and eosin.
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Figure 3. Growth curves of the relative tumor volume (A); Bod-
yweight of tumor-bearing mice (B); Photographs of tumors ex-
cised on day 21 after treatment (C); H&E staining of tumor (D).
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