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Figure 1. Typical (a) TEM and (b) SEM images of HBCCD−ADA supramolecular assembly. (c) DLS and (d) zeta potential experiment results of
HBCCD−ADA supramolecular assembly in deionized aqueous solution.
(2) the controlled binding and release of pDNA was easily were characterized by transmission electron microscopy
achieved via ester hydrolyze into “zwitterionic” structure;29−31 (TEM), scanning electron microscopy (SEM), dynamic light-
and (3) anticancer drugs, such as mitoxantrone (MTZ), could scattering (DLS), and zeta potential measurements. The TEM
be readily integrated into the water-soluble supramolecular image in Figure 1a showed that HBCCD−ADA supra-
assembly of HBCCD−adamantane (ADA) by the π−π stacking molecular assembly have a spherical morphology with a
interaction between the coronene surface and the aromatic ring diameter of ca. 300 nm. The SEM image (Figure 1b) also
of drug molecule MTZ. Therefore, this water-soluble and showed the 280−310 nm homogeneous spherical nano-
biocompatibility multifunctional nanostructure is taken into particles. Moreover, DLS measurement indicated that the
account as a novel strategy for improving cancer treatment. hydrodynamic diameter of HBCCD−ADA assembly was ca.
465 nm with a narrow distribution (Figure 1c), which was in
2. RESULTS AND DISCUSSION accordance with the results in microscopic images. The zeta
Benefiting from the strong host−guest inclusion complexation potential of HBCCD−ADA was measured as ca. +14.8 mV
between ADA and β-CD,32 the fluorescent supramolecular (Figure 1d), suggesting that the surface charge of assembly was
assembly was prepared by simply mixing the aqueous solutions positive charged, which is beneficial to promote the electro-
of ADA and HBCCD together. The structural and morpho- static interaction between HBCCD−ADA supramolecular
logical features of the HBCCD−ADA supramolecular assembly assembly and pDNA.
11982 DOI: 10.1021/acsomega.9b01436
ACS Omega 2019, 4, 11981−11987
ACS Omega Article
inhibitory concentrations (IC50) of MTZ and MTZ@ biochemical inhibitors, that is, NaN3/2-deoxyglucose (NaN3/
HBCCD−ADA toward HCT-116 human colon cancer cells DOG) was utilized to inhibit the energy-dependent pathway
were measured as 2.2236 and 2.04 μM by the MTT assay, and sucrose was used to perturb clathrin-mediated endocy-
which indicated the enhanced anticancer activity of MTZ@ tosis.37,38 As shown in Figure S11, compared with control, the
HBCCD−ADA assembly. In addition, after a 24 h incubation, cells treated with inhibitors exhibited dramatic decrease in the
the MTZ@HBCCD−ADA assembly exhibited satisfactory cellular uptake of assembly. Thus, we deduced that the
malignant cell inhibition effect toward the HCT-116 cell line internalization of assembly might be via multiple endocytic
with a relative cellular viability of 43.7% (Figure S7a), which pathways.
was slightly lower than that of commercial anticancer drug
MTZ (49.1%); this might be attributed to the assembly that 3. CONCLUSIONS
was preferably internalized in HCT-116 cells through the
endocytosis. The side effects of MTZ@HBCCD−ADA In conclusion, we successfully constructed a fluorescent
assembly were also tested by using NIH3T3 mouse embryonic supramolecular assembly composed of doubly positively
fibroblast cells. As shown in Figure S7b, MTZ@HBCCD− charged ADA with β-CD modified HBCCD as a multifunc-
ADA assembly gave a relative cellular viability as 56.5%, which tional supramolecular platform for controlled DNA condensa-
was slightly higher than that with free MTZ. Moreover, it is also tion, cell imaging, and drug delivery via host−guest inclusion
noteworthy that HBCCD−ADA assembly was nontoxic to all complexation of ADA with β-CD and further π-stacking of
the examined cell lines because of its good biocompatibility. coronene with anticancer drug MTZ. After the ester group in
These results indicated that the supramolecular assembly could ADA was hydrolyzed to a negative carboxyl, the positively
facilitate the selective and accumulation of MTZ in cancer cells, charged quaternary ammine strand converted into a zwitterion
which holds great promise to present a safe and effective tool structure, which realized the controlled binding and release of
for cancer therapy. Similar results were also observed at 48 h of pDNA. Moreover, cytotoxicity experiments indicated that the
incubation (Figure S8). Afterward, we investigated the assembly displayed a slightly higher inhibition effect and a
intracellular fluorescence imaging ability of the conjugated lower toxicity than free drug. Besides, because of the coronene
supramolecular assembly. As shown in Figure 4, HCT-116 cells intrinsic fluorescence properties, it has potential application as
cell imaging agent. Considering the facile synergetic interaction
in the design of macrocycle-based assembly, we envision that
the constructed multifunctional assembly can be further
functionalized easily by chemical modification of other guest
molecules, thus providing us a facile modular approach for
supramolecular cancer therapy.
4. EXPERIMENTAL SECTION
4.1. General Methods. All chemicals were of reagent grade
Figure 4. Confocal fluorescence images of HCT-116 cells incubated unless noted. HBCCD was synthesized according to our
with HBCCD−ADA for 24 h. The nucleus was stained by 4′,6- reported method,25 and ADA was synthesized according to the
diamidino-2-phenylindole dihydrochloride hydrate (DAPI). reported method.30 NIH3T3 mouse embryonic fibroblast cell
line and HCT-116 human colon cancer cell line were obtained
gave a green fluorescence in cytoplasm after a 24 h incubation from China Infrastructure of Cell Line Resource. The
with HBCCD−ADA fluorescence supramolecular assembly. absorbance spectra were recorded in a conventional quartz
Besides, late endosomes and lysosomes were stained with cell (light path 10 mm) by using a UV/vis spectrophotometer
Lysotracker Red for colocalizing assembly.37 As shown in (U-2900, Hitachi). Steady-state fluorescence emission spectra
Figure S9, high colocalization was observed for HBCCD−ADA were recorded in a conventional quartz cell (10 × 10 × 45 mm)
assembly, suggesting that the assembly was successfully on a fluorescence spectrometer (Fluorolog-MAX 4, Horiba).
internalized into cells, which might have a potential application TEM images were acquired by an FEI Tecnai G2 F20
value in cancer diagnosis. transmission electron microscope. The samples were prepared
Moreover, the real-time cellular uptake of assembly was by placing a drop of solution onto a carbon-coated copper grid
evaluated by confocal laser scanning microscopy during the and air-dried. The SEM images were recorded on a JSM-6700F
incubation from 2 to 6 h. As shown in Figure S10, green scanning electronic microscope. The DLS and zeta potentials
fluorescence can be observed at 2 h, and the green fluorescent were recorded on Malvern Zetasizer Nano ZS90 (Malvern
intensities increased with time, indicating that the assembly has Instruments Ltd., Worcestershire, UK). Gel electrophoresis
entered the cells. This phenomenon validated that the assembly experiments were measured on a 1% (w/v) agarose gel at 60 V
could act as a fluorescence probe to monitor the distribution, for 45 min and photographed using a UV transilluminator and
accumulation, and real-time drug release. After verifying the a WD-9415B gel documentation system (Beijing Liuyi
internalization of HBCCD−ADA assembly, we continued to Instrument Factory, P. R. China). Confocal laser scanning
investigate the pathway of cellular internalization with confocal microscopy was performed on a fluorescence inverted micro-
laser scanning microscopy. First, we examined the cellular scope (Zeiss LSM 800).
uptake of assembly at 4 and 37 °C (Figure S11). At the lower 4.2. Preparation of HBCCD−ADA Supramolecular
temperature, the cellular uptake ability weakened, indicating an Assemblies. ADA was dissolved in 10 mL of deionized
energy-dependent process in the cellular internalization. In water to prepare a stock solution with the concentration of 2
order to further understand which of the energy-dependent mM, and HBCCD was dissolved in 5 mL of dimethyl sulfoxide
processes involved in the internalization of assembly, we to obtain a stock solution with the [CD] concentration of 4
performed the cellular uptake treated with two types of mM. The stock solution ADA was diluted 20 folds to 0.1 mM.
11984 DOI: 10.1021/acsomega.9b01436
ACS Omega 2019, 4, 11981−11987
ACS Omega Article
Upon sonication, the HBCCD solution (0.5 mL) was slowly for 24 h. The cells incubated with HBCCD−ADA for 24 h and
added into the diluted ADA solution (19.5 mL) over 5 min. washed with PBS buffer for three times. Then, the cells were
The resulting HBCCD−ADA solution was stored at 4 °C. fixed with 4% paraformaldehyde for 15 min. After that, the cell
4.3. Agarose Gel Electrophoresis Experiments. The nuclei were stained with DAPI (1 μg/mL) for 5 min and
controlled condensation ability of assembly to pDNA was observed by a confocal laser scanning microscope. In order to
measured by evaluating the electrophoretic mobility on the evaluate the location of assembly in cells, late endosomes and
agarose gel at different N/P ratios. The gel electrophoresis lysosomes were stained with Lysotracker Red at 200 nM for 30
experiments were performed in TAE (0.04 M Tris, 0.02 M min after incubation with cells.
acetic acid, and 2.0 mM EDTA) buffer at 25 °C. After samples 4.8. Cellular Uptake of the HBCCD−ADA Supra-
loading and electrophoresis process, pDNA bands were stained molecular Assembly. HCT-116 cells were cultured in
in Gen Green solution and were photographed using UV light confocal dish (5 × 104 cells/mL, 1.5 mL per well) for 24 h.
at 302 nm. The cells incubated with HBCCD−ADA for 2, 4, and 6 h.
4.4. MTZ Loading on the HBCCD−ADA Supra- Then, the cells were washed with PBS buffer for three times
molecular Assembly. The solution of MTZ (0.65 mg) in and fixed with 4% paraformaldehyde for 15 min. After that, the
0.5 mL of deionized water was dropwise added to 10 mL of cell nuclei were stained with DAPI (1 μg/mL) for 5 min and
above prepared aqueous solution of HBCCD−ADA assembly, observed by a confocal laser scanning microscope.
and the obtained mixture was stirred overnight at room 4.9. Inhibition Studies of Endocytosis. HCT-116 cells
temperature in dark for 12 h. The resulting solution was were cultured in confocal dish (5 × 104 cells/mL, 1.5 mL per
dialyzed against deionized water for 2 h, and the deionized well) for 24 h. The cells were separately incubated with 0.1%
water was replaced every 0.5 h. Then, the mixture was freeze- NaN3/50 mM 2-deoxy-glucose for 1 h and 225 mM sucrose for
dried. The drug loading efficiency and encapsulation of MTZ 30 min or 4 °C for 1 h. Then, they are washed with PBS buffer
onto HBCCD−ADA were estimated by the following for three times and further incubated with HBCCD−ADA
equations assembly for 2 h at 37 °C. The cells were washed with PBS
buffer for three times and fixed with 4% paraformaldehyde for
Drug loading efficiency (%) 15 min. After that, the cell nuclei were stained with DAPI (1
μg/mL) for 5 min and observed by a confocal laser scanning
= 100 mMTZ in HBCCD − ADA /mHBCCD − ADA
microscope.
■
the medium were supplemented with 10% fetal bovine serum.
NIH3T3 cells and HCT-116 cells were seeded in 96-well plates
(5 × 104 cells/mL, 100 μL per well) for 24 h; then, the cells AUTHOR INFORMATION
were incubated with MTZ, MTZ@HBCCD−ADA, HBCCD− Corresponding Authors
ADA, HBCCD, and ADA for 24 and 48 h, respectively. The *E-mail: shengxl@iccas.ac.cn (X.S.).
relative cellular viability of the cells was determined by the *E-mail: guozeyu2010@imau.edu.cn (Z.G.).
MTT assay. ORCID
Furthermore, the IC50 values of MTZ and MTZ@ Xianliang Sheng: 0000-0003-2055-672X
HBCCD−ADA toward HCT-116 cell line were evaluated by
Author Contributions
MTT assay. HCT-116 cells were seeded in 96-well plates (5 × ∥
Y.-H.Z. and J.Y. are equal contributors.
104 cells/mL, 100 μL per well) for 24 h, and then the cells were
incubated with MTZ and MTZ@HBCCD−ADA at different Notes
The authors declare no competing financial interest.
■
concentrations for 48 h. The anticancer activities were
determined with MTT assay. All the data were presented as
the mean ± standard deviation. ACKNOWLEDGMENTS
4.7. Fluorescent Confocal Imaging. HCT-116 cells were We thank the Inner Mongolia Autonomous Region Natural
cultured in confocal dish (5 × 104 cells/mL, 1.5 mL per well) Science Fund Project (2017BS0206), the Program for Young
11985 DOI: 10.1021/acsomega.9b01436
ACS Omega 2019, 4, 11981−11987
ACS Omega Article
Talents of Science and Technology in Universities of Inner (20) Hu, X.-Y.; Wang, L. Two sides to supramolecular drug delivery
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