Amphipathic β-cyclodextrin nanocarriers serve as intelligent delivery platform for anticancer drug

Article abstract of DOI:10.1016/j.colsurfb.2019.05.011

A novel glutathione-responsive (GSH-responsive)star-like amphiphilic polymer (C₁₂H₂₅)₁₄-β-CD-(S-S-mPEG)₇ (denoted as CCSP)was designed for efficient antitumor drug delivery. The amphiphilic β-cyclodextrin (β-CD)self-polymerize in water to form a sphere with a diameter of 40–50 nm. The secondary hydroxyl groups of β-CD were modified by dodecyl to form a hydrophobic core and the primary hydroxyl groups of β-CD were decorated with PEG through disulfide bond to form a hydrophilic shell. Since the hydrophobic cavity of β-CD was maintained, the hydrophobic core formed by dodecyl as well as cavity of β-CD provided CCSP with a loading content as high as 39.6 wt%. Importantly, DOX@CCSP exhibited low drug leakage and negligible cytotoxicity in non-reductive physiological environment, while it showed rapid release and high cytotoxicity in reductive tumorous environment via the breakage of disulfide bond. In view of the above-mentioned advantages of DOX@CCSP nanocarriers such as high loading content, proper size, favorable stimulus-response release performance and low leakage, it is believed that CCSP may offer great potential to be used as an intelligent nanocarrier for anticancer drug delivery.

Full text of DOI:10.1016/j.colsurfb.2019.05.011

ColloidsandSurfacesB:Biointerfaces180(2019)429–440
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces
journal homepage: www.elsevier.com/locate/colsurfb
Amphipathic β-cyclodextrin nanocarriers serve as intelligent delivery
platform for anticancer drug
Hui Liu, Jian Chen, Xiufang Li, Zhiwei Deng, Peiru Gao, Jianbin Li, Tao Ren, Ling Huang,
Yanjing Yang⁎, Shian Zhong⁎
College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, 410083, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A novel glutathione-responsive (GSH-responsive) star-like amphiphilic polymer (C12H25)14-β-CD-(S-S-mPEG)7
Antitumor drug
Amphiphilic β-CD
GSH-responsiveness
Drug loading
(denoted as CCSP) was designed for efficient antitumor drug delivery. The amphiphilic β-cyclodextrin (β-CD)
self-polymerize in water to form a sphere with a diameter of 40–50 nm. The secondary hydroxyl groups of β-CD
were modified by dodecyl to form a hydrophobic core and the primary hydroxyl groups of β-CD were decorated
with PEG through disulfide bond to form a hydrophilic shell. Since the hydrophobic cavity of β-CD was main-
tained, the hydrophobic core formed by dodecyl as well as cavity of β-CD provided CCSP with a loading content
as high as 39.6 wt%. Importantly, DOX@CCSP exhibited low drug leakage and negligible cytotoxicity in non-
reductive physiological environment, while it showed rapid release and high cytotoxicity in reductive tumorous
environment via the breakage of disulfide bond. In view of the above-mentioned advantages of DOX@CCSP
nanocarriers such as high loading content, proper size, favorable stimulus-response release performance and low
leakage, it is believed that CCSP may offer great potential to be used as an intelligent nanocarrier for anticancer
drug delivery.
1. Introduction
responsive release in tumor tissues is another critical aspect that needs
to be addressed.
Today, cancer represents a disease with the highest mortality rate
[1]. However, currently available clinical chemotherapeutic agents are
subjected to common shortcomings including high toxicity, short half-
live and low selectivity [2]. Therefore, it is particularly important to
develop new technologies to improve anticancer efficiently. With the
development of nanotechnology, nanocarriers have been gaining more
and more attention in the scientific community partially due to the
large surface area, appropriate size and unique structure [3–5]. These
characteristics made it particularly suitable for serving as drug carrier
with the advantages of prolonging blood circulation [6], and accumu-
lation in tumor tissues benefiting from the enhanced permeability and
retention effect (EPR effect) [7,8]. However,severe side effects are
mainly caused by no selectivity of anticancer drug between tumor tis-
sues and healthy tissues. Moreover, premature drug leakage from drug
carriers is often observed during blood circulation in the body [9,10],
and drug loading is also a critical aspect when drug delivery systems
(DDS) was designed [11]. Therefore, drug leakage during blood circu-
lation need to be decreased and the drug loading content need to be
improved when DDS was used in vivo. Moreover, the stimulus-
There are various differences in the microenvironment between
tumor cells and normal cells, such as temperature [12,13], pH [14,15]
and content of reduced glutathione (GSH) [16,17]. These differences
provide a theoretical basis for the design of stimulus-responsive nano-
carriers. β-CD represents a type of natural compound which proves to
be nontoxic, biocompatible and biodegradable. There is a hydrophobic
cavity and a hydrophilic shell in β-CD [18], which renders the material
suitable to be used as drug carrier. Moreover, There are 21 hydroxyl
groups at two ends of β-CD, which make it easy to modify [19,20].
However, poor water solubility and low drug loading content of β-CD
limit its further development. At present, polyethylene glycol (PEG) is
most commonly used as hydrophilic material to decorate particulate
surfaces since its advantages: increasing water solubility, avoiding re-
cognition by cells of the mononuclear phagocyte system (MPS) [21],
prolonging blood circulation and nontoxicity. Based on the above case,
β-CD was modifiedwith hydrophilic (PEG) and hydrophobic (dodecyl)
groups to form amphipathic nanocarriers for loading chemotherapy
drugs. Then, water solubility as well as the drug loading content could
be increased, in addition, the stimulus-responsive bonds were
⁎ Corresponding authors.
E-mail addresses: yangyanjing@csu.edu.cn (Y. Yang), zhongshian@aliyun.com (S. Zhong).
https://doi.org/10.1016/j.colsurfb.2019.05.011
Received 6 January 2019; Received in revised form 17 April 2019; Accepted 7 May 2019
Availableonline08May2019
0927-7765/©2019ElsevierB.V.Allrightsreserved.

H. Liu, et al.
ColloidsandSurfacesB:Biointerfaces180(2019)429–440
introduced into the DDS to endow the material with a stimulus-re-
sponsive control release behavior.
(Shanghai, China). Fetal bovine serum (FBS), Dulbecco’s modified eagle
medium (DMEM), and Phosphate buffered saline (PBS) were purchased
from Beyotime Biotechnology Co. Ltd (Hunan, China). N,N-
Dimethylformamide (DMF), dimethylsulfoxide (DMSO) and di-
chloromethane (DCM) were distilled over calcium hydride (CaH2) to
remove any residual moisture before use. Other reagents were com-
mercially available and usedwithout further purification.
In general, amphipathic β-CD is constructed by host-guest interac-
tion in the previous studies [22–24]. Whereas amphipathic β-CD was
synthesized by chemical bonding so as to retain the nature cavity of β-
CD and increase the drug loading content. Herein, a novel amphiphilic
star-like copolymer as DDS based on β-CD derivatives was prepared in
this work. First, the primary hydroxyl groups of β-CD were protected.
Then, the secondary hydroxyl groups of β-CD were modified with
bromododecane to form hydrophobic ends. Next, bromine was grafted
into the primary hydroxyl groups of β-CD upon addition of Br2. Sulfy-
dryl was then introduced into the primary hydroxyl groups of β-CD and
the final product was reacted with PEG-SH to form CCSP. It is note-
worthy that amphiphilic β-CD could self-polymerize in water to form
spherical polymers. The dodecyl groups acted as a hydrophobic core
and polyethylene glycol groups served as hydrophilic shell, where the
cavity of β-CD and the hydrophobic cavity after self-assembly provided
a suitable area to entrap DOX with a drug loading content as high as
39.6 wt%. Disulfide bonds are known to be reduction-sensitive che-
mical bonds and it was demonstrated that DOX could be released from
DOX@CCSP by disulfide bond dissociation in a reductive tumor en-
vironment. It showed that DOX@CCSP could selectively kill cancerous
SKOV3 cells but showed low toxicity towards normal HEK 293T cells.
Moreover, because DOX@CCSP could selectively accumulate and re-
lease in the tumor tissues, it demonstrated a beneficial anticancer effect
with low toxicity to normal tissues in nude mice (Fig. 1).
2.2. Characterization
1H NMR spectra were operated on a 400 MHz or 500 MHz NMR
spectrometer (Bruker, Germany) and the internal standard was tetra-
methylsilane (TMS). The average size of micelles was obtained using
dynamic light scattering (DLS) through Zetasizer Nano-ZS from
Malvern Instruments equipped with a 633 nm He-Ne laser using back-
scattering detection. The molecular weight and mass distribution of
CCSP and CSP were determined by a Perkin-Elmer Series-200 gel per-
meation chromatograph (GPC) instrument equipped with PLgel 5 μm
Mixed 2D columns. Fourier transform infrared spectrometric (FTIR)
analysis was carried out on a Nicolet iS5 Fourier transform infrared
spectrometer (USA). Morphology images were obtained using trans-
mission electron microscopy (TEM) and energy dispersive spectro-
metric (EDS) data of the synthetic micelles were obtained on a JEOL-
2100F instrument (Japan). Fluorescence images were obtained on an
Operetta High-Content Imaging System (PerkinElmer). The flow cyt-
ometer used throughout the studies was purchased from Beckman
Coulter Co. Ltd(USA). The DOX contents in tissues were detected using
high performance liquid chromatography (HPLC) (Agilent 1260, USA).
Biodistribution analysis of DOX in nude mice was obtained using an
IVIS Lumina II multispectral imaging system (Caliper Life Sciences,
USA).
2. Experimental section
2.1. Materials and reagents
Chemical reagents including 1-bromododecane, anhydrous pyr-
idine, methoxypolyethylene glycol (mPEG, Mn = 2 kDa), β-cyclodextrin
(β-CD), tert-butyldimethylsilyl chloride (TBDMS-Cl), triphenylpho-
sphine (Ph3P), DL-1,4-dithiothreitol (DTT), and doxorubicin hydro-
chloride (DOX) were purchased from Energy Chemical Technology Co.
Ltd (Shanghai, China) or Aladdin Chemical Technology Co. Ltd
2.3. Synthesis of per-6-tert-butyldimethysilyl-β-cyclodextrins [β-CD-
(OTBDMS)7]
To ensure the successful synthesis of amphiphilic β-CD, hydroxyl
groups in the primary face of β-CD featuring high reactivity, had to be
Fig. 1. (A) Schematic illustration highlighting the preparation of amphipathic DOX-loaded β-CD material; (B) Controlled release of DOX in blood circulation; (C)
Controlled release of DOX in tumor cells.
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H. Liu, et al.
ColloidsandSurfacesB:Biointerfaces180(2019)429–440
protected. Tert-butyldimethylsilyl chloride, a widely used protective
group for primary hydroxyl groups, was selected to protect the hy-
droxyl groups [25,26]. The preparation method for β-CD-(OTBDMS)7
comprise the following steps, firstly, dry β-CD (9 g, 7.93 mmol) was
dissolved in anhydrous pyridine (100 mL) with constant stirring under
argon atmosphere. And the resulting mixture was cooled to 0 °C before
tert-butyldimethylsilyl chloride (14.5 g, 96.2 mmol) was added drop-
wise over 3.5 h. The reaction was continued for another 3 h at 0 °C
before it was warmed to room temperature for a further 24 h. Then, the
pyridine was removed under reduced pressure to provide a milky white
powder. The powder was then dissolved in dichloromethane and
treated with KHSO4 (aqueous solution) to remove the remaining pyr-
idine. The organic phase was then washed with saturated NaCl aqueous
solution and deionized water, dried over anhydrous Na2SO4. Di-
chloromethane was removed in vacuo and per-6-tert-butyldimethysilyl-
β-CD (yield: 86.3%) was obtained via recrystallization from MeOH/
CHCl3 (95:5, V/V).
under reduced pressure and NaOH (0.13 g, 3.25 mmol, aqueous solu-
tion) was added. The reaction system was then refluxed for 1.5 h before
allowed to cool down to room temperature. The pH of the resulting
suspension was adjusted with KHSO4 solution until pH < 7, extracted
with dichloromethane, and dried over anhydrous Na2SO4. Di-
chloromethane was removed and the crude product was purified by
glucan gel chromatography with MeOH as an eluant to obtain a light
yellow product (2.1 g, yield: 74.0%).
2.7. Synthesis of amphiphilic β-cyclodextrins [(C12H25
)
14-β-CD-(S-S-
mPEG) abbreviated as CCSP]
7
Synthetic (C12H25)14-β-CD-(SH)7 (0.20 g, 0.055 mmol) and synthetic
mPEG-SH (0.78 g, 0.39 mmol, see Supporting information section) were
dispersed in DMSO (10.0 mL) and H2O2 (0.4 mL, 30%) was added
dropwise. The reaction proceeded at room temperature for 24 h.
Deionized water (50 mL) was added dropwise with gentle stirring for
24 h. The mixture was then dialyzed (MWCO 3500) against deionized
water for 3 days and lyophilized. A fluffy white product (0.45 g, yield:
45.9%) was obtained.
2.4. Synthesis of per-[6-(tert-butyldimethylsilyl)-6-deoxy] -per-2,3-
dodecyl-β-cyclodextrin [(C12H25)14-β-CD-(OTBDMS)7]
(1) NaH (0.5 g, 20.83 mmol) was placed in a three-neck flask. Then,
anhydrous hexane was added to wash the extra kerosene on the NaH
solid. (2) Anhydrous THF/DMF (1:1, V/V, 80 mL) was added and the
suspension was cooled to 0 °C before β-CD-(OTBDMS)7 (1 g, 0.52 mmol)
in anhydrous THF/DMF (1:1, V/V, 20 mL) was added by slow dripping.
(3) The reaction was allowed to proceed for 0.5 h at 0 °C before warmed
to room temperature for a further 24 h. (4) 1-Bromododecane (7.3 mL,
15.44 mmol) was added dropwise. (5) The reaction was allowed to
proceed in the dark for 4 days. (6) Additional NaH (0.5 g, 20.83 mmol)
was introduced carefully with cooling. (7) Steps (3), (4), and (5) were
repeated once. (8) MeOH was added at 0 °C to quench excess NaH until
no more molecular hydrogen (bubbles) was produced. (10) The solvent
mixture was removed under reduced pressure and the crude product
was dissolved in dichloromethane. The dichloromethane layer was
washed with deionized water until the aqueous phase became neutral
and the organic phase was then dried over anhydrous Na2SO4.
Dichloromethane was removed to obtain a gray solid which was pur-
ified by glucan gel chromatography with MeOH as an eluant. A yel-
lowish oily substance (1.19 g, yield: 54.1%) was obtained.
2.8. Synthesis of amphiphilic β-cyclodextrins [β-CD-(S-S-mPEG)7
abbreviated as CSP] as control materials
The synthetic procedure to produce β-CD-(S-S-mPEG)7 included
iodination of hydroxyl groups in the primary face of β-CD, thiolation
and thiol oxidation procedures were described in the
SupportingInformation section.
2.9. Synthesis of amphiphilic β-cyclodextrin nanocarriers loaded
doxorubicin [CCSP@DOX or CSP@DOX] and assessment of drug loading
performance
The model drug DOX·HCl (20 mg) was dispersed in DMSO (2 mL),
and trimethylamine (10.64 μL) was added to neutralize HCl. The color
of the solution slowly turned deep purple from the initial red after the
mixture was stirred in the dark overnight. CCSP or CSP (20 mg) was
added and the resulting mixture was allowed to stir for 3 h. Then,
deionized water (10 mL) was added dropwise, followed by gentle stir-
ring for another 24 h. Then, the mixture was dialyzed (MWCO 3500)
against deionized water for 3 days and the mixture was lyophilized. In
doing so, a fluffy red product DOX@CCSP (19 mg) or DOX@CSP
(15 mg) was obtained.
2.5. Synthesis of per-(6-bromo-6-deoxy)-per-2,3-dodecyl-β-cyclodextrin
[(C12H25)14-β-CD-Br7]
The reaction was carried out according to a procedure reported in
the literature [27]. In a general synthesis and under argon atmosphere,
PPh3 (0.67 g, 2.55 mmol) was dissolved in dry dichloromethane
(10 mL). After the reaction system was cooled to 0 °C, Br2 (2.52 mL,
1 mol/L Br2-CH2Cl2) was added by slow dripping. Then, per-[6-(tert-
butyldimethylsilyl)-6-deoxy]-per-2,3-dodecyl-β-CD (1.44 g, 0.34 mmol,
dissolved in 4 mL dry dichloromethane) was added and the reaction
proceeded at room temperature for 36 h. The dichloromethane layer
was washed by saturated NaHCO3 aqueous solution and deionized
water. The organic phase was combined and dried over anhydrous
Na2SO4. Dichloromethane was removed and the residue was purified by
glucan gel chromatography with MeOH as an eluant to obtain a yellow
substance (0.91 g, yield: 68.3%).
To determine the loading content of DOX@CCSP or DOX@CSP, the
DOX@CCSP or DOX@CSP (1 mg) was dissolved in MeOH (5 mL) with
ultrasound aid and a UV spectrum was recorded at 490 nm. The loading
content (LC%) of DOX was measured using the following equation:
quality of drug in nanocarriers
quality of drug and nanocarriers
LC% =
× 100
The reductive environment in tumor tissue was simulated by addi-
tion of dithiothreonol (DTT). The in vitro release behavior of
DOX@CCSP and DOX@CSP was investigated with or without 10 mM
DTT. Typically, DOX@CCSP or DOX@CSP (10 mg) was dissolved under
simulative physiological conditions in phosphate buffer (PBS, 2 mL)
with or without 10 mM DTT. The solutions were then transferred to a
dialysis bag (MWCO 3500). The drug release started once the dialysis
bag was placed into a vial in which 8 mL of corresponding PBS were
added. The vials were placed onto shaking tables and the temperature
was maintained at 37 °C. At certain time intervals, 200 μL of dialysate
was taken for DOX content analysis using a UV–vis spectrophotometer
at 490 nm. The cumulative release rate of the drug from the loaded-
nanocarriers was measured as the mass ratio of released drug compared
to the loaded drug.
2.6. Synthesis of per-(6-thiol-6-doexy)-per-2,3-dodecyl-β-cyclodextrin
[(C12H25)14-β-CD-(SH)7]
The reaction was carried out according to a procedure reported in
the literature [28]. In a general synthesis and under argon atmosphere,
synthetic (C12H25)14-β-CD-Br7 (3.10 g, 0.79 mmol) and thiourea (0.93 g,
12.20 mmol) were dissolved in dry DMF (40 mL) and the reaction
proceeded at 70 °C for 24 h. Afterwards, half of the DMF was removed
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ColloidsandSurfacesB:Biointerfaces180(2019)429–440
2.10. Hemolysis and biocompatibility assay
2.12. In vivo antitumor efficiency of CCSP and CSP
In order to further evaluate the biocompatibility of nanocarriers and
hemolytic toxicity of nanocarriers and loaded nanocarriers, hemolysis
and biocompatibility assays were carried out. To obtain red blood cells
(RBCs), fresh goat blood was diluted in PBS (20:1, v/v) and centrifuged
at 1500 rpm for 10 min. The supernatant was removed subsequently
and the process was repeated until the supernatant became colourless.
Afterwards, 10% v/v of the RBC suspension in PBS was obtained.
100 μL of various concentrations of DOX, nanocarriers and DOX@ na-
nocarriers were blended with 100 μL of the RBCs suspension on a 96-
well plate which was later incubated in a cell incubator for 1.5 h.
Meanwhile, 1% of Triton X-100 was used for complete hemolysis as
positive control and PBS was used as negative control. After incubation,
the supernatant was moved to a homologous location of another 96-
well plate after centrifugation at 1500 rpm for 10 min. The absorbance
Skov3 and HEK293T cells were cultivated on a 96-well plate (5000
cells per well) and incubated for 24 h. Afterwards, the nutrient medium
was updated with fresh nutrient medium containing varying con-
centrations of DOX, DOX@CCSP or DOX@CSP (equivalent DOX con-
centration). After another 36 h of incubation, CCK-8 assay (10 μL) so-
lution was added to each well. The absorbance at 450 nm was recorded
using a microplate reader after another 2 h of incubation.
2.13. In vivo antitumor efficiency
Female BALB/c nude mice (18
3 g, 4 weeks old) were purchased
from Hunan Silaike Experimental Animal Co. Ltd. The approval number
of the mice was SYXK 2016-0002. The nude mice were fed at the de-
partment of laboratory animals, Central South University. All animal
experiments were carried out in accordance with regulations of the
Institutional Committee for the Care and Use of Laboratory Animals in
cancer research.
at 540 nm was determined using
a microplate reader (Thermo
Scientific™ Multiskan™ FC). The hemolysis rate was measured using the
following equation:
ODsample − ODnegative
Where 1% of Triton X-100 was used for complete hemolysis as positive
control and PBS was used as negative control.
Skov3 and HEK293T cells were cultured using RPMI-1640 and
DMEM supplemented with 10% FBS at 37 °C with 5% CO2 in a humi-
dified incubator, respectively. The biocompatibility assay was de-
termined using a CCK-8 assay. Firstly, Skov3 and HEK293T cells were
cultivated in a 96-well plate (8000 cells per well) and incubated for
24 h. Afterwards, the culture medium was replaced with new medium
containing varying concentrations of CCSP and CSP micelles. Then, the
cells were incubated for another 24 h and a CCK-8 assay (10 μL) was
introduced. Simultaneously, a CCK-8 assay (10 μL) with 90 μL nutrient
solution was introduced into the blank wells without cells as a control
group. After the cells were incubated for 2 h, the absorbance at 450 nm
was measured through a microplate reader. The cell viability rate was
control
2.13.1. Tumor formation in nude mice
Hemolysis% =
× 100
SKOV3 cells (200 μL, 5 × 106 cells ml−1 in PBS) were injected
subcutaneously under the front legs of nude mice for tumor formation.
The body weight and tumor volume were recorded every three days
thereafter. The tumor volume was obtained through the formula as
follows: V = (length × [width]2)/2, where length and width stand for
the longest and shortest diameter, respectively. When the tumor volume
reached about 50 mm3 in size, the mice were randomly divided into
four groups (n = 6) for subsequent antitumor efficiency assays. Two
animals were used for in vivo imaging studies.
ODpositive
− ODnegative
control
control
2.13.2. In vivo imaging study
1,1-Dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine
iodide
(Dir), a kind of NIR fluorescent dyes, was used to study the distribution
of CCSP and CSP in vivo. CCSP or CSP loaded with Dir (Dir@CCSP or
Dir@CSP) were synthesized similar to the method described for the
synthesis of DOX@CCSP. Two mice were injected via tail vein injection
with Dir@CCSP and Dir@CSP, respectively. After the mice were nar-
cotized with isoflurane, the fluorescence intensity and distributions
were recorded using a multi spectral imaging system. Finally, the mice
were dissected, and fluorescence images of the main organs as well as
the tumors were recorded using a multi spectral imaging system.
calculated as follows (data were expressed as average
SD (n = 6)):
ODsample − ODcontrol
Cell viability rate =
× 100
ODblank − ODcontrol
Where sample, control and blank refer to the cells treated with varying
concentrations of CCSP and CSP micelles, the untreated cells and cul-
ture medium without cells.
2.13.3. In vivo antitumor efficiency assay
The four groups of mice were administered with 200 μL saline so-
lution (as the blank control), free DOX, DOX@CCSP, or DOX@CSP
every three days, respectively. Before injection, the same amount of
DOX in each solution was confirmed in each injection except for the
blank control. Before injection, the body weight and tumor volume
were recorded to assess the antitumor effect and any potential side
effects of the treatments.
2.11. Cellular uptake of CCSP and CSP
The loading process of Coumarin-6 (C6) in CCSP or CSP was carried
out following a similar procedure as described for Dox loading. Skov3
and HEK293T cells were cultivated on a 96-well plate (8000 cells per
well) and incubated for 24 h. In order to further explore the controlled
release performance towards glutathione (GSH, an antioxidant specifi-
cally elevated in tumor tissue), the cells were pretreated with BSO
(250 μM, inhibitor of GSH) and GSH (5 mM) before addition of
C6@CCSP and C6@CSP. Then, the cell nuclei and GSH were stained
with Hoechst 33342 (C1025, Beyotime) and thioltracker (T10095,
Thermo Fisher Scientific) for 30 min, respectively. Fluorescence images
were recorded by an Operetta High-Content Imaging System. A flow
cytometer was used to quantify the cellular-uptake of CCSP and CSP.
SKOV3 and HEK293T cells were cultivated on a 6-well plate. Then,
C6@CCSP and C6@CSP along with BSO and GSH were added to the cell
culture similarly to the method described above. For another 2 h, the
cells were collected and the fluorescence intensity of C6 was measured
by flow cytometer.
2.13.4. Distribution of DOX in nude mice
To explore the distribution of DOX in main tissues of nude mice, the
mice were dissected after about three weeks of treatment. Then, the
tumor tissues and major organs were weighed, grinded, extracted by
chloroform, evaporated by vacuum-rotary, dissolved in chromato-
graphic MeOH, ultrafiltered, and subjected to HPLC analysis. The cor-
responding drug was dissolved in 1 mL of the mobile phase (0.01 M
KH2PO4: acetonitrile: acetic acid = 44:55:0.2 (V:V:V)) for subsequent
HPLC analysis. The flow rate was 1 mL/min and the detection wave-
length was set to 490 nm. The column parameters were: Agilent SB-
C18, 4.6 × 150 mm diameter, 5 μm packing size.
2.13.5. Histological analysis with ematoxylin and eosin staining (H&E)
Samples of major organs and tumor were fixed in 4%
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ColloidsandSurfacesB:Biointerfaces180(2019)429–440
Scheme 1. Detailed synthetic procedure of CCSP and CSP.
paraformaldehyde, dehydrated, embedded in paraffin, cut into slices of
5 μm, dewaxed, HE dyed, and analyzed using a fluorescence micro-
scope.
disappeared, providing evidence for the successful iodination reaction
to form compound 6. As shown in Fig. S19, the appearance of a new
peak at 2.14 ppm, corresponding to a thiol group, confirmed that iodide
was successfully replaced with thiol to form compound 7. As shown in
Fig. S20, an intense signal corresponding to PEG at about 3.45 ppm
appeared and the thiol peak at 2.14 disappeared, providing evidence for
the successful synthesis of compound 8. The 13C NMR spectroscopy are
consistent to a series of compound (see Supporting information sec-
tion).
2.13.6. Terminal deoxynucleotidyl transferase biotin-d UTP nick end
labeling (TUNEL) assay
TUNEL is a method to detect DNA apoptosis [26]. The detection
method was employed to evaluate the anticancer effects of DOX,
DOX@CCSP and DOX@CSP. This experiment was carried out using a
TUNEL in situ Cell Death Detection Kit (Roche Diagnostics, Mannheim,
Germany). Briefly, the tumor was fixed, embedded in paraffin, installed
on glass slides, dewaxed, rehydrated, treated with proteinase K, dyed
with TUNEL, and analyzed using a fluorescence microscope.
Fig. 2A shows the FTIR spectra of a sequence of prepared products.
After protection of the primary hydroxyl groups in β-CD with tert-bu-
tyldimethylsilyl (Fig. 2A(b)), two new peaks at 1496 and 1360 cm−1
appeared, corresponding to eCe(CH3)3 bound to Si, as well as a new
peak corresponding Sie(CH3)2 could be observed. The finding provided
evidence for the successful introduction of tert-butyldimethylsilyl. As
3. Results and discussion
shown in Fig. 2A(c), the enhanced signals at 2823, 2924, 1463 cm−1
exhibiting bonds stretching and deformation vibrations of e(CH2)n
,
,
−
3.1. Characterization of a series of synthetic intermediates
could be detected. This finding corresponded to the successful func-
tionalization of the dodecyl group. As shown in Fig. 2A(d), after bro-
mination, the peak at 1255 cm−1 disappeared, providing further evi-
The preparation routes of CSSP and CSP are illustrated in Scheme 1.
The compounds 1–8 were characterized using 1H NMR and 13C NMR
spectroscopy (see Supporting information section) and FTIR. As shown
in Figs. S3 and S5 (1H NMR spectrum of compound 1), two new peaks at
0.01, 0.84 ppm appeared and the peak at 4.45 ppm disappeared, which
verified the successful functionalization of tert-butyldimethylsilyl on
the primary hydroxyl groups. For compound 2 (Fig. S6), a new peak at
1.21 ppm, corresponding to the dodecyl group, appeared. Furthermore,
the peak at 5.77 ppm, corresponding to the second hydroxyl groups of
β-CD, disappeared. As shown in Fig. S8, the disappearance of the peak
at 0.01 ppm and the reduction of the peak at 0.84 ppm provided evi-
dence for the successful synthesis of compound 3. As shown in Fig. S10,
the appearance of a peak at 2.10 ppm, corresponding to eSH, con-
firmed that the bromide group was successfully replaced with thiol to
form compound 4. As shown in Fig. S16, an intense signal corre-
sponding to PEG appeared at about 3.66 ppm and the thiol signal at
2.10 ppm disappeared, which provided evidence for the successful
formation of compound 5. Furthermore, as shown in Fig. S18, the peak
at 4.45 ppm, corresponding to the primary hydroxyl groups of β-CD,
dence for the successful synthesis of (C12H25 14
appearance of corresponding to -SH
new peak at 2565 cm−1
(Fig. 2A(e)), indicated a successful synthesis of (C12H25 14-β-CD-(SH)7.
) -β-CD-Br7. Next, the
a
,
)
As shown in curves (f) and (g), the peak at 2565 cm-−1, corresponding
to -SH, disappeared and a peak at 1116 cm−1 corresponding to an
asymmetric stretching vibration of CeOeC increased. This finding
shows that PEG was successfully linked to β-CD via formation of a
disulfide bond.
As shown in Fig. 2B, the SEM images show that CCSP exhibited a
regularly spherical morphology, with a homogeneous diameter of about
40–50 nm and a distinct border. CSP (Fig. 2C) featured a spherical
morphology, with a diameter of about 30–40 nm. The DLS (see Figs.
S24–S27) showed that CCSP in PBS and cellular media had a diameter
of about 200 nm. And CCSP had no obvious change after 10 days, which
proved its good stability to some extent. However, the distribution of
CSP had a great change especially in cellular media, which may result
from the instability and bonding with proteins for CSP in cellular
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Fig. 2. (A) FTIR spectrum of a sequence of products (a: β-CD; b: β-CD-(OTBDMS)7; c: (C12H25)14-β-CD-(OTBDMS)7; d: (C12H25)14-β-CD-Br7; e: (C12H25)14-β-CD-(SH)7;
f: (C12H25)14-β-CD-(S-S-PEG-OCH3)7 and g: β-CD-(S-S-PEG-OCH3)7). TEM images of (B) CCSP and (C) CSP.
media. Furthermore, It showed that CCSP and CSP could self-assemble
in water. For CCSP, the hydrophobic core was formed by dodecyl
groups and polyethylene glycol served as hydrophilic shell. For CSP, the
hydrophobic core was formed by β-CD (the water solubility of β-CD was
poorer than that of PEG) and PEG served as hydrophilic shell. More-
over, the energy spectra of CCSP and CSP, as shown in Figs. S22 and
S23, provided further evidence for the successful synthesis of CCSP and
CSP. The GPC measurement was conducted to obtain the relative mo-
lecular weights of CCSP and CSP (Fig. S28), which showed that the MW
(weight-average molecular weight) were 16108 Da (PDI = 1.171) and
14021 Da (PDI = 1.017), respectively. It could prove the successful
synthesis of CCSP and CSP to a certain degree.
3.2. Drug loading and drug release studies
According to UV spectrometry results obtained, the loading ratio of
DOX in CCSP and CSP was 39.6 wt% and 21.4 wt%, respectively. To
further explore the GSH-responsive release of DOX from DOX@CCSP
and DOX@CSP, dithiothreitol (DTT) was used to simulate the in-
tracellular environment in cancer. As shown in Fig. 3, a low cumulative
release rate of DOX from DOX@CCSP in PBS could be observed without
DTT (curve d), and only 23% of DOX was released after 103 h. On the
contrary, the cumulative release rate of DOX from DOX@CCSP in PBS
with DTT reached over 50% within 9 h (curve a). This finding was
mainly due to the rapid cleavage of disulfide bonds and the disassembly
of CCSP in the reductive microenvironment, whereas the disulfide
bonds proved to be relatively stable in a non-reductive environment. On
the contrary, the cumulative release rate of DOX from DOX@CSP in PBS
with DTT (curve b) was similar to that of DOX@CCSP with DTT (curve
Fig. 3. DOX release curves from DOX-loaded CD nanoparticles in different
media (a: DOX release curves from DOX@CCSP in PBS with DTT; b: DOX re-
lease curves from DOX@CSP in PBS with DTT; c: DOX release curves from
DOX@CSP in PBS without DTT; d: DOX release curves from DOX@CCSP in PBS
without DTT).
a). However, the cumulative release rate of DOX from DOX@CSP in PBS
without DTT (curve c) was found to be increased compared to that of
DOX@CCSP without DTT (curve d). This latter finding may be attrib-
uted to drug leakage from DOX@CSP in this non-reductive environ-
ment. As expected, DOX@CCSP exhibited an excellent and fast release
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Fig. 4. (A) Viabilities of HEK293T and SKOV3 cells incubated with CCSP or CSP at different concentrations for 24 h, respectively; (B) Hemolytic test of CSP、
DOX@CSP、CCSP or DOX@CCSP.
performance in a reductive environment and, moreover, showed low
leakage in simulated blood circulation.
HEK293T cells, which were treated with C6@CCSP or C6@CSP in the
absence of BSO or GSH. This latter finding indicated that C6 could be
released from C6@CCSP or C6@CSP, specifically in SKOV3 cells com-
pared to HEK293T cells. This further demonstrated the high potency of
DOX@CCSP to find use as a potential anticancer drug. In conclusion, C6
was found to be released both from C6@CCSP and C6@CSP in SKOV3
cells selectively, while only negligible C6 was found to be leaked from
C6@CCSP in HEK293T cells. Therefore, CCSP proved to be more sui-
table than CSP as a carrier for a hydrophobic anticancer drug.
Flow cytometry was employed to quantify the cellular uptake of
C6@CCSP and C6@CSP and the release of C6. The corresponding re-
sults were shown in Fig. 5B. We showed that the fluorescence intensity
could be significantly enhanced when increasing the GSH content, and
the fluorescence intensity in SKOV3 cells was far stronger than that in
HEK293T cells without BSO or GSH. This latter finding was consistent
with the results obtained from fluorescence images and the results
further verified that C6 could be released from C6@CCSP in SKOV3
cells selectively. Taken in concert, DOX@CCSP indeed showed char-
acteristics promising to enhance the anticancer effect with reduced side
effects towards normal cells.
3.3. Biocompatibility in HEK293T and SKOV3 cells and hemolytic analysis
In order to estimate the biocompatibility of CCSP and CSP, the vi-
abilities of HEK293T and SKOV3 cells cultured with CCSP or CSP at
different concentrations and incubation for 24 h were measured. As
shown in Fig. 4(A), even when the concentration of CCSP or CSP was up
to 500 μg/mL, the viabilities of HEK293T and SKOV3 cells remained
over 85%. This results explain that the prepared CCSP and CSP ex-
hibited low toxicity to both HEK293T and SKOV3 cells and may
therefore be considered promising to find use as carrier materials in
clinical practice.
As shown in Fig. 4(B), the hemolytic toxicity of the carrier materials
and the DOX-loaded system were below 2%. When the concentration
reached up to 800 μg/mL, the hemolysis rate of CCSP or DOX@CCSP
was below 0.8%, far below the international standard (5%), while the
hemolysis rate of DOX@CSP was higher than that of DOX@CCSP, re-
sulting from drug leakage from DOX@CSP without stimulation. The
results indicate that CCSP exhibited hardly any hemolytic toxicity to
RBC and could therefore be classified suitable for the use as a carrier
material in clinical applications.
3.5. Cytotoxicity studies
It is significant to assess the cytotoxicity of DOX@CCSP and
DOX@CSP. Through a CCK-8 assay, the half-maximal inhibitory con-
centration (IC50) was employed as a means to assess the DOX cyto-
toxicity to normal HEK293T cells and cancerous SKOV3 cells. As shown
in Fig. 6, free DOX exhibited significant toxicity to both HEK293T and
SKOV3 cells, with an IC50 value of ˜0.8 μg/mL and 1.0 μg/mL, respec-
tively. DOX@CCSP and DOX@CSP also exhibited a strong cytotoxicity
to SKOV3 cells, but showed weak cytotoxicity towards HEK293T cells.
The IC50 value of DOX@CCSP and DOX@CSP at an equivalent DOX
concentration was 2.0 μg/mL and 1.0 μg/mL against SKOV3 cells and
over 12 μg/mL and ˜12 μg/mL against HEK293T cells, respectively.
When the concentration of DOX was 5.0 μg/mL, the observed viability
of SKOV3 cells and HEK293T cells was about 32%, 80% upon treatment
with DOX@CCSP and 40%, 68% after treatment with DOX@CSP, re-
3.4. Intracellular drug release of C6@CCSP and C6@CSP
In order to analyze the cellular uptake of CCSP and CSP and the
intracellular release of the drug, a green fluorescence probe-C6 was
used to label CCSP and CSP. Moreover, Hoechst 33258 (blue) was
employed to stain the nuclei, and a thioltracker (purple) was used to
track GSH and thiol groups resulting from disulfide cleavage and dis-
sociation on CCSP and CSP. As shown in Fig. 5A, an intense green and
bright purple fluorescence could be observed in SKOV3 cells treated
with C6@CCSP or C6@CSP without BSO or GSH. This latter finding
verified that C6 could be released from C6@CCSP or C6@CSP in SKOV3
cells. When GSH was added to the system, the green and purple
fluorescence of the SKOV3 cells increased, indicating that the release of
C6 increased with the increase of intracellular GSH content. After BSO
was added, the green and purple fluorescence of SKOV3 cells was found
to be reduced due to the BSO inhibiting secretion of GSH. Meanwhile,
after BSO was added, the green and purple fluorescence of the SKOV3
cells after incubation with CCSP was found to be weaker than that of
CSP, resulting from the less C6 leaked from C6@CCSP in this non-re-
ductive environment. As for the HEK293T cells, a very weak green
fluorescence and a weak purple fluorescence could be observed in the
spectively. Therefore, at
a
DOX concentration of 5.0 μg/mL,
DOX@CCSP exhibited high cytotoxicity towards SKOV3 cells and
showed no obvious damaging effects towards HEK293T cells. However,
DOX@CSP showed a lower efficacy and more severe side effects com-
pared to DOX@CCSP which may be a result of the DOX leakage from
DOX@CSP under normal physiological conditions. The cytotoxicity
results demonstrated the excellent anticancer efficacy and low side ef-
fects of DOX@CCSP, most likely resulting from the high loading
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Fig. 5. (A) Fluorescence images of SKOV3 and HEK293T cells after incubation with C6@CCSP and C6@CSP for 1 h and 3 h, respectively; (B) Cellular uptake and
median fluorescence intensity of C6@CCSP and C6@CSP in SKOV3 and HEK293T cells by flow cytometry.
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Fig. 6. Cytotoxicity studies for HEK293T and SKOV3 cells cultured with DOX, DOX@CSP and DOX@CCSP at different concentrations for 24 h (*p < 0.05,**p <
0.01).
Fig. 7. Biodistribution of Dir@CCSP and Dir@CSP in mice bearing SKOV3 tumors for different time periods and biodistribution in major tissues and tumor at 24 h.
capacity, superior stimuli-responsive release properties, low leakage
and suitable particle size.
SKOV3 cells and the nuclei of SKOV3 cells were found to be damaged to
varying degrees. Due to the poor adhesion of 293T cells, dead cells
would fall out, therefore most remaining 293T cells were found to be
undamaged, and the decrease of 293T cells would result from in-
creasing cytotoxicity. As shown in Figs. S32–S34, at a DOX concentra-
tion of 5 μg/mL, DOX@CCSP exhibited low cytotoxicity towards 293T
cells. These results demonstrated the excellent anticancer performance
To further study the cellular uptake and cytotoxicity properties, the
cells were incubated with DOX@CCSP, DOX@CSP and DOX for 36 h
and exhibited fluorescence. As shown in Figs. S29–S31, when the
concentration of DOX was 5 μg/mL, DOX@CCSP, DOX@CSP and DOX
exhibited a strong red fluorescence and high cytotoxicity toward
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ColloidsandSurfacesB:Biointerfaces180(2019)429–440
Fig. 8. (A) Photographs of mice after treatment with saline, DOX, DOX@CSP, DOX@CCSP for 27 days. (B) Body weight changes of mice during treatment with saline,
DOX, DOX@CSP or DOX@CCSP. The body weights were normalized to the initial values (*P < 0.05 and **P < 0.01). (C) Photographs of tumor after treatment
with saline, DOX, DOX@CSP or DOX@CCSP for 27 days. (D) Relative tumor volume changes of tumors in mice during the treatment with saline, DOX, DOX@CSP or
DOX@CCSP. (*P < 0.05 and **P < 0.01). (E) Concentration of DOX in tumor tissues and major organs in mice after treatment by HPCL. (*P < 0.05 and
**P < 0.01 versus free DOX group, #P < 0.05 and ##P < 0.01 versus DOX@CSP group).
and low side effects of DOX@CCSP, consistent with the results de-
scribed above.
the fluorescence of Dir@CCSP and Dir@CSP was accumulated in the
tumor sites with time due to the EPR effect, favourable stimuli-re-
sponsive properties and suitable particle size. After 24 h of imaging, the
tumors and main tissues were extracted for imaging. The results ob-
tained indicate that strong fluorescence could only be detected in tumor
tissue, while no obvious significant fluorescence could be detected in
the main tissues. However, the fluorescence intensity of Dir@CCSP in
the main tissues was weaker than that of Dir@CSP, mainly due to the
3.6. In vivo biodistribution
Two mice were injected with Dir@CCSP and Dir@CSP via tail vein
injection, respectively. Then, the mice were imaged via an IVIS Lumina
II multispectral imaging system for 3, 6 and 24 h. As shown in Fig. 7,
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ColloidsandSurfacesB:Biointerfaces180(2019)429–440
drug leakage of Dir@CSP in blood circulation. The suitable size and
favourable stimuli-responsive properties led to accumulation and re-
lease of Dir@CCSP in tumor tissues while the low drug leakage of CCSP
may benefit to decrease side-effect in normal tissues.
dodecyl acted as hydrophobic core and the polyethylene glycol served
as hydrophilic shell, where the cavity of β-CD and the hydrophobic
cavity of the self-assembled material provided a suitable area to load
DOX, with a drug loading content as high as 39.6 wt%. Drug-release
analysis in vitro under simulated tumor environments provided evi-
dence for the excellent performance for drug controlled-release. The
results obtained from cellular experiments demonstrated that CCSP
were biocompatible and DOX@CCSP could selectively destroy can-
cerous SKOV3 cells, however, exhibited low toxicity towards normal
HEK293T cells. Moreover, the experimental results in vivo showed that
DOX@CCSP could accumulate and release in tumorous tissue and re-
vealed a suitable anticancer effect and very low side effects in normal
tissues of tumor-bearing nude mice. Benefiting from the advantages of
DOX@CCSP nanocarriers such as proper size, high loading content,
favourable stimulus-response release performance and low drug
leakage, it is believed that DOX@CCSP may potentially be used as an
intelligent nanocarrier system for efficient anticancer drug delivery.
3.7. Antitumor efficacy of DOX@CCSP and DOX@CSP in vivo
The mice were divided into four groups. And each group of animals
were injected with saline, free DOX, DOX@CSP and DOX@CSSP, re-
spectively. As shown in Fig. 8, the relative body weights and the tumor
sizes were recorded. Mice in the saline group exhibited an obvious
decrease in body weight and a distinct increase in tumor size over time.
The loss in body weight mainly resulted from the fact that the tumors
were too large for the mice to bear. As for the mice in the other three
groups, the tumor sizes were significantly smaller compared with those
in the saline group, particularly in the DOX@CCSP groups. This latter
finding provides more evidence for the outstanding anticancer efficacy
of DOX@CCSP due to the EPR effect and favourable stimuli-responsive
properties. However, the body weights in the DOX group decreased
over time and the mice were skinny after therapy for 27 days due to the
severe side effects of DOX. The body weights in DOX@CCSP group
increased gradually due to the low side effect resulting from the low
DOX leakage in normal tissues. As described above, DOX@CCSP
showed excellent anticancer efficacy and reduced toxicity to normal
tissues in vivo.
The concentration of DOX in tissues and tumors were quantified by
HPLC. As shown in Fig. 8E, the DOX concentration in tumor tissues for
the DOX@CCSP group was 2.2 times greater than that of the free DOX
group. Meanwhile, in the DOX@CCSP group, the DOX concentration in
tumor tissues was more than twice of that in normal tissues. Here,
DOX@CCSP mainly benefited from the EPR effect and superior stimuli-
responsive release properties. Furthermore, in the free DOX group, the
DOX concentrations in normal tissues were higher than the con-
centration in tumor tissues. This latter finding was the main reason
causing significant toxicity to normal tissues.
Author contributions
S.Z. and H.L. designed the study; H.L., J.C., X.L., Z.D. and J.L. car-
ried out the synthetic route and characterization of the corresponding
intermediates and amphiphilic β-CD copolymer; H.L. and T.R. per-
formed the drug release studies; H.L., L.H., and Z.D. accomplished the
cell experiments; H.L, J.C. and X.L. accomplished the animal experi-
ments; Y.Y. provided significant advice for article writing; H.L. ac-
complished the manuscript. All authors have approved the final article.
Acknowledgments
The authors declare no competing financial interest. This study was
funded by the National Natural Science Foundation of China (Grant No.
21576295), the Open-End Fund for the Valuable, Precision Instruments
of Central South University (CSUZC201830) and the Fundamental
Research Funds for the Central Universities of Central South University
(2017zzts175 and 2018zzts371).
Histopathological analysis was performed to assess potential da-
mages to normal tissues and tumor tissues caused by DOX, DOX@CCSP
or DOX@CSP. As shown in Fig. S34-A, compared to the free DOX group,
no obvious damages were observed in the main tissues of the heart,
liver, spleen, lung and kidney in the DOX@CCSP and DOX@CSP
groups. Particularly for the DOX@CCSP, the damages to normal tissues
were similar to the saline group due to the EPR effect, superior stimuli-
responsive release properties and negligible drug leakage. The tumor
tissues after treatment with DOX@CCSP exhibited significant damages
compared to the free DOX group. This latter finding was most likely due
to the accumulation of DOX in tumor tissues, resulting in an improved
anticancer efficacy. Moreover, a TUNEL assay was carried out to detect
tumor cell apoptosis, as shown in Fig. S34-B. Green fluorescence in-
dicated the varying levels of cell apoptosis. As shown here, compared to
the free DOX group, a stronger green fluorescence could be observed in
tumor tissue after treatment with DOX@CCSP and DOX@CSP com-
pared to that treated with free DOX. DOX@CCSP in particular exhibited
a superior anticancer efficacy. Taken in concert, the list of results
confirmed that the DOX@CCSP system featured an outstanding antic-
ancer effect and low side effects.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.05.011.
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