Smart GSH/pH dual-bioresponsive degradable nanosponges based on β-CD-appended hyper-cross-linked polymer for triggered intracellular anticancer drug delivery

Article abstract of DOI:10.1016/j.jddst.2021.102650

Efficient accumulation and on-demand intracellular drug release in the desired site are a crucial issue in developing ideal drug delivery systems (DDSs). Glutathione (GSH)/pH dual-bioresponsive degradable Nanosponges were developed based on β-CD-appended hyper-cross-linked polymer by one-pot polymerization of acryloyl-6- ethylenediamine-6-deoxy-β-Cyclodextrin (β-CD-NH-ACy), acrylic acid (AA) and N,N-bis(acryloyl)- cystamine (BACy) as cross-linker to deliver doxorubcin (DOX) and investigated for GSH/pH triggered DOX release, in which the massive carboxyl and amino groups, and disulfide bonds were used as pH and GSH bioresponsive fragments, respectively. In the proposed DDSs, DOX was readily incorporated into the three dimensional networks of the Nanosponges either as inclusion complexes or as non-inclusion complexes, with a high drug loading capacity of 22.6%. In vitro release studies suggested that the Nanosponges exhibited GSH/pH triggered disintegration and drug release performance, in which DOX release was significantly accelerated in acidic (pH5.0) and cytosolic reduction (10 mM GSH) conditions, with ~77.0% of DOX release. The morphology changes of DOX@Nanosponges in releasing media (pH5.0, 10 mM GSH) were further studied by TEM. Confocal microscopy observation demonstrated that DOX was delivered and released into cytoplasm and nucleus of A549 cells in 7 h incubation with DOX@Nanosponges. MTT assays manifested that the Nanosponges exhibited low cytotoxicity up to a concentration of 1000 μg/mL and DOX@Nanosponges had high anti-tumor activity. These findings demonstrated that the dual-bioresponsive Nanosponges may function as a promising platform for targeted delivery and intracellular drug controlled release in tumor therapy.

Full text of DOI:10.1016/j.jddst.2021.102650

Journal of Drug Delivery Science and Technology 64 (2021) 102650
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology
journal homepage: www.elsevier.com/locate/jddst
Research paper
Smart GSH/pH dual-bioresponsive degradable nanosponges based on
β-CD-appended hyper-cross-linked polymer for triggered intracellular
anticancer drug delivery
Yutong Daia, Qingman Lib, Shurong Zhanga, Shan Shia, Yang Lia, Xudong Zhaoa, Liping Zhoua,
,
*
Xin Wanga, Yijian Zhua, Wei Lia
a Department of Medicinal Chemistry, School of Pharmacy, Chongqing Medical University, Chongqing, 400016, China
b The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Efficient accumulation and on-demand intracellular drug release in the desired site are a crucial issue in
developing ideal drug delivery systems (DDSs). Glutathione (GSH)/pH dual-bioresponsive degradable Nano-
sponges were developed based on β-CD-appended hyper-cross-linked polymer by one-pot polymerization of
acryloyl-6- ethylenediamine-6-deoxy-β-Cyclodextrin (β-CD-NH-ACy), acrylic acid (AA) and N,N-bis(acryloyl)-
cystamine (BACy) as cross-linker to deliver doxorubcin (DOX) and investigated for GSH/pH triggered DOX
release, in which the massive carboxyl and amino groups, and disulfide bonds were used as pH and GSH bio-
responsive fragments, respectively. In the proposed DDSs, DOX was readily incorporated into the three dimen-
sional networks of the Nanosponges either as inclusion complexes or as non-inclusion complexes, with a high
drug loading capacity of 22.6%. In vitro release studies suggested that the Nanosponges exhibited GSH/pH
triggered disintegration and drug release performance, in which DOX release was significantly accelerated in
acidic (pH5.0) and cytosolic reduction (10 mM GSH) conditions, with ~77.0% of DOX release. The morphology
changes of DOX@Nanosponges in releasing media (pH5.0, 10 mM GSH) were further studied by TEM. Confocal
microscopy observation demonstrated that DOX was delivered and released into cytoplasm and nucleus of A549
cells in 7 h incubation with DOX@Nanosponges. MTT assays manifested that the Nanosponges exhibited low
Nanosponges
Dual-bioresponsive
β-Cyclodextrin
Controlled release
Hyper-cross-linked polymer
cytotoxicity up to a concentration of 1000 μg/mL and DOX@Nanosponges had high anti-tumor activity. These
findings demonstrated that the dual-bioresponsive Nanosponges may function as a promising platform for tar-
geted delivery and intracellular drug controlled release in tumor therapy.
1. Introduction
agents [4]. This situation is driving a rapid increase in the demand for
developing the novel therapeutic strategies for effective cancer treat-
Cancer has become one of the most dreaded diseases and the single
most important barrier to increase life expectancy in both developing
and developed countries [1]. Nowadays, the most common cancer
treatment approaches are surgery, chemotherapy, radiation therapy and
immunotherapy. Among them, surgery is the best treatment strategy for
most solid tumors without metastasis. In spite of advances in surgical
techniques, the residual tumor cells will increase the risk of cancer
recurrence and metastasis [2]. The combination of chemotherapy and to
a limited extent radiotherapy, has been the last resort to control cancer
[3]. However, most of the conventional cancer chemotherapy is far from
successful, mainly due to the lack of tumor selectivity of anticancer
ments, and the application of nanotechnology on cancer is anticipated to
provide significant improvements in diagnosis and therapy of the dis-
ease. Therefore, tumor specific drug delivery systems (DDSs), which can
selectively deliver anticancer drugs to the desired site by passive as well
as active mechanisms and achieve controlled and predictable release of
the drugs, have been intensely developed to improve the therapeutic
efficacy of chemotherapeutics and simultaneously minimize the toxic
side effects [5,6].
In the last decades, various DDSs including liposomes, polymer mi-
celles, inorganic nanoparticles, viral vectors, dendrimers etc., have been
explored for realizing tumor-selective delivery. Liposomes have often
* Corresponding author.
E-mail address: li_wei@cqmu.edu.cn (W. Li).
https://doi.org/10.1016/j.jddst.2021.102650
Received 8 December 2020; Received in revised form 31 May 2021; Accepted 3 June 2021
Available online 7 June 2021
1773-2247/© 2021 Elsevier B.V. All rights reserved.

Y. Dai et al.
Journal of Drug Delivery Science and Technology 64 (2021) 102650
Scheme 1. Schematic illustration of the synthesis of Nanosponges and DOX@Nanosponges.
been used to deliver various drug modalities, but with relatively low
efficiency and storage stability [7,8]. Inorganic nanoparticles, such as
iron oxide, silica, gold, quantum dots, etc., are also attractive family of
DDSs [9–11]. But they are non-biodegradable and difficult to load or
conjugate with macromolecules. Polymeric micelles can fulfill the re-
quirements for selective drug delivery [12], but in vivo instability, low
cargo capacity and special storage conditions are problematic [13,14].
Viral vectors are effective for various active ingredients delivery as well
as can be genetically or chemically conjugated to specific ligands to
achieve tissue target, but have some issues such as easy of proteolytic
degradation and potential safety concerns [15,16]. Dendrimers are
employed for both the therapeutic and diagnostic applications [17], but
it is difficulty in controlling the multi-step fabrication and purification
process because numerous reaction steps take place at the same time in
each growing step [18,19]. The above-mentioned strategies have shown
promise, but other dosage forms using different strategies should also be
developed for drastic improvements in the delivery of anticancer drug
into the desired site.
for proteins and other macromolecules, such as enzymes, DNA and oli-
gonucleotides, which can increase the cellular internalization, prevent
degradation and improve their bioavailability [20]. In the case of
Nanosponges-loaded enzymes, their activity and efficiency are even
enhanced in terms operative temperature and pH range [20]. Notably,
Nanosponges are safe and biodegradable, display negligible toxicity on
cell cultures, and are well-tolerated upon injection in experimental an-
imals [24,25]. So Nanosponges are extensively used for delivering
anticancer drugs [26], such as camptothecin [27], doxorubicin (DOX)
[25,28], and paclitaxel [29]. Cytotoxicity studies suggested that
camptothecin-loaded in Nanosponges was more than 20 times as effec-
tive than the drug alone and reduced by 70% the growth of prostate
cancer cells in mouse xenograft models [27,30]. Furthermore, these
Nanosponges can selectively extravasate in tumor tissue by the
enhanced permeability and retention (EPR) effect, thereby achieving
targeted delivery of drug inside the tumor tissue [28–30]. Although
Nanosponges have already shown a great likelihood to be a targeting
drug carrier for anti-cancer drugs, they have not yet been targeted to
achieve controlled drug release to a desired cell or organ. pH and redox
potential are often selected as pathological “triggers”, and used to
develop new bio-responsive Nanosponges [24,27,31,32]. pH sensitive
DDSs are usually designed to destabilize vehicles and release drugs in
tumor tissues which the extracellular pH tends to be significantly more
acidic (~ 6.5) than the pH of the blood (7.4), or in endosomal and
lysosomal compartments with pH values typically as low as ~ 5.0,
respectively [33]. In comparison, redox sensitive DDSs are developed
with the aim to disassemble and release drugs in response to the intra-
cellular glutathione (GSH) concentration, which is higher in tumor
Recently, a new type of cyclodextrin (CD) nano-vehicles are Nano-
sponges, which are composed of hyper-crosslinked
α, β, and γ-CD
polymers with porosity arising from CD nano-sized cavities and inter-
connecting voids, and possess innovative three dimensional (3D) mesh-
like networks by chemical cross-linking [20–22]. Compared to CD,
which can host various guest molecules with compatible geometry and
polarity, Nanosponges are capable of entrapping and storing both hy-
drophilic and lipophilic small molecule drugs by 3D scaffolds or
host-guest inclusion complex, and have been proposed as nanomedicine
strategy to address challenging issues of DDSs, such as solubility, sta-
bility, sustained and controlled release [21,22], and even reduce the side
effects of drugs [23]. Moreover, Nanosponges are also efficient carriers
compared to normal tissue (approximately 0.5–10 mM vs. 2–20
μM)
[33]. Significantly, chemoresistant cancer cells show even higher levels
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Y. Dai et al.
Journal of Drug Delivery Science and Technology 64 (2021) 102650
of GSH [34]. Previous reports have demonstrated that drug-loaded
GSH-Nanosponges exhibited remarkably higher effectiveness than the
drug alone both in vitro and in vivo, and could escape the efflux drug
pump, thus contributing to overcoming drug resistance [25,28]. Un-
fortunately, the Nanosponges still face some flaws including low loading
capacity (only about 12%) [25,31], time consuming and complex syn-
thetic procedures. Single-responsive DDSs can’t yet completely meet
demands of tumor-targeted delivery and controlled drug release to a
desired cell or organ. Therefore, dual stimuli-bioresponsive DDSs will be
a judicious choice for better anticancer therapy [35]. To the best of our
knowledge, there are few reports on GDH/pH dual-bioresponsive
Nanosponges based on β-CD as anticancer DDSs.
2.4. Synthesis of mono-(6-ethylenediamine-6-deoxy)-β-cyclodextrin
(β-CD-NH2)
In the reaction flask under N2 atmosphere, 9 mL ethylenediamine
was gradually added to the solution of Ts-CD (9.00 g 6.90 mmol) in
dimethylformamide (30 mL) with stirring at 70 ◦C. The reaction was
allowed to proceed for 7 h. Then acetone was added to precipitate the
products. The solid was collected and dried under vacuum, obtaining
β-CD-NH2 as white powder.
2.5. Synthesis of acryloyl-6-ethylenediamine-6-deoxy-β-cyclodextrin
(β-CD-NH- ACy)
In the present work, we reported the one-step synthesis of a novel
class of GSH/pH dual-bioresponsive degradable Nanosponges based on
β-CD by the free radical inverse-emulsion polymerization of acryloyl-6-
ethylenediamine-6-deoxy-β-CD and arcylic acid using N,N-bis(acryloyl)
cystamine as a cross-linker (Scheme 1), which was fully characterized
through physical-chemical data, drug loading and intracellular drug
release behaviors in cancer cells. The particle size and morphology and
the morphology changes of DDSs were tested by transmission electron
microscopy (TEM). In vitro drug release behavior in response to different
pH and GSH concentration, cell uptake, the cytotoxicity and anti-tumor
activity were also investigated.
Acryloyl chloride (1.75 g, 19.4 mmol) was added gradually to a so-
lution of β-CD-NH2 (2.23 g, 1.89 mmol) in 30 mL of NaHCO3 solution
with stirring in an ice bath under N2 atmosphere. After that, the reaction
was allowed to proceed for another 3 h. The white β-CD-NH2-ACy was
obtained by precipitation using acetone, and dried under vacuum.
2.6. Preparation of nanosponges
Nanosponges were prepared by the free radical inverse-emulsion
polymerization as described elsewhere [38]. The continuous phase
consists of toluene (w/w, 63%) and surfactant Span-40 (sorbitan
monopalmitate, 2%), and the dispersed phase was composed of water
(w/w, 34%) and various monomer including acrylic acid (AA, 672 mg,
9.330 mmol), β-CD-NH-ACy (580 mg, 0.473 mmol) and BACy (36.02
mg, 0.139 mmol). The dispersed phase was added dropwise to the
continuous phase with ultrasonication for 10 min at 15 ◦C. The emulsion
was purged with N2 to remove the dissolved O2 which otherwise can
scavenge free radicals required for polymerization. Subsequently, the
miniemulsion was heated to 40 ◦C, then the initiator ammonium per-
sulfate (APS) and 1% N,N,N′,N′-Tetramethylethylenediamine (TEMED)
were added and stirred for 5 h. After demulsification using ethanol,
Nanosponges were isolated and washed for several times by centrifu-
gation at 14, 000 rpm for 10 min. The collected Nanosponges were
freeze-dried for 72 h to remove residual solvent.
2. Materials and methods
2.1. Materials
Doxorubicin hydrochloride (DOX), acryloyl chloride, cystamine
dihydrochloride, glutathione and p-toluenesulfonyl chloride were pur-
chased from Aladdin Bio-Chem Technology Company (Shanghai,
China). β-cyclodextrin, acrylic acid, Span-40, ammonium persulfate, 3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
and ethylenediamine were obtained from Sigma-Aldrich (Shanghai,
China). All other chemicals and reagents used were analytical grade.
2.2. Preparation of disulfide crosslinker N,N-bis(acryloyl)cystamine
(BACy)
2.7. Fabrication of DOX-loaded nanosponges (DOX@Nanosponges)
BACy was synthesized as previously described [36]. Typically, to a
solution of cystamine⋅2HCl (2.82 g, 0.0125 mol) in deionized water (20
mL) was dropwise added a mixture solution of acryloyl chloride (2.26 g,
0.025 mol) in CH2Cl2 (3 mL) and NaOH (2.0 g, 0.05 mol) in deionized
water (5 mL) under stirring at 0 ◦C over 1 h. After that, the reaction was
allowed to proceed at room temperature for at least another 6 h. The
product was extracted twice with CH2Cl2 and the organic layer was
thoroughly dried with anhydrous Na2SO4. After remove of CH2Cl2 under
vacuum, the resulting BACy was purified by recrystallization using 1/2
vol ratio of ethyl acetate/heptane as solvent.
In order to load DOX in Nanosponges, to an aqueous suspension of
Nanosponges (50 mg) in 30 mL water was added a solution of DOX (15
mg) dissolved in 10 mL phosphate buffer saline (PBS) and stirred for 24
h in the dark at room temperature. The aqueous suspension was then
centrifuged at 18, 000 rpm for 20 min to separate the free drug. The
DOX-loaded Nanosponges (DOX@Nanosponges) were stored at 4 ◦C
until use. The concentration of DOX in the supernatant was determined
by Shimadzu UV-2600 UV–Vis spectrophotometer at 480 nm [36] to
calculate the drug loading capacity (DLC) and drug loading efficiency
(DLE) by the following formula:
2.3. Synthesis of 6-mono-(p-toluenesulfonyl)-β-cyclodextrin (Ts-CD)
initial weight of DOX ꢀ weight of DOX in supernatant
DLC (%) =
weight of DOX@Nanosponges
Ts-CD was synthesized by the tosylation reaction of the C6-hydroxyl
groups of β-CD as described previously [37]. Briefly, in the reaction flask
under N2 atmosphere, to a stirred solution of NaOH (0.1 mol/L) in water
(200 mL) was transferred β-CD (22.39 g, 19.70 mmol) at 0 ◦C in an ice
bath until the β-CD was dissolved completely, followed by adding a
solution of p-toluenesulfonyl chloride (TsCl, 5.51 g, 28.90 mmol) in
acetonitrile (15 mL). The reaction mixture was stirred continuously for
another 5 h under the same conditions. After remove of the unreacted
TsCl by filtration, the filtrate was neutralized and then kept at 4 ◦C
refrigerator for overnight. The Ts-CD was isolated as a white solid and
dried under vacuum.
× 100%
weight of DOX in Nanosponges
DLE (%) =
× 100%
initial weight of DOX
2.8. In vitro DOX release study
The in vitro release of DOX from DOX@Nanosponges was investi-
gated at 37 ◦C under six different conditions, i.e. (i) PBS (100 mM, pH
7.4), (ii) PBS (100 mM, pH 6.5), (iii) acetate buffer (100 mM, pH 5.0),
(iv) PBS containing 10 mM GSH (100 mM, pH 7.4), (v) PBS containing
10 mM GSH (100 mM, pH 6.5), and (vi) acetate buffer containing 10 mM
GSH (100 mM, pH 5.0). DOX@Nanosponges suspension was divided
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Y. Dai et al.
Journal of Drug Delivery Science and Technology 64 (2021) 102650
into six aliquots and immediately placed into dialysis tubes with a
MWCO of 12000–14000. The dialysis tube was immersed into 50 mL of
appropriate releasing medium and shaken at 120 rpm and 37 ◦C. At
regular intervals, 5.0 mL of the receiving phase was withdrawn and
replenished with an equal volume of fresh medium to maintain sink
conditions. To avoid oxidation of GSH, the release media were perfused
with N2 [39]. DOX concentration was determined by UV–Vis
spectrophotometer.
2.9. In vitro cytotoxicity assays
The cytotoxicity of free drug, empty and DOX@Nanosponges was
studied by MTT assay using A549 cells. Cells were seeded in 96-well
plates at a density of 1 × 104 cells per well and incubated in Dulbec-
co’s modified Eagle’s medium (DMEM) supplemented with 10% heat-
inactivated fetal bovine serum (FBS) at 37 ◦C in a 5% CO2 humidified
environment for 24 h. The growth medium was removed and replaced
Fig. 1. TEM images of Nanosponges (a) and DOX@Nanosponges (b).
3. Results and discussion
3.1. Fabrication of the nanosponges
by 180 μL of fresh DMEM medium. 20 μL of samples at different con-
centrations in PBS (10 mM, pH 7.4) were added. After 24, 48, and 72 h of
The purpose of the present study was to develop a novel one-pot
synthesis approach to produce GSH/pH dual-bioresponsive Nano-
sponges and investigate their intracellular drug release behaviors in
cancer cells. This one-pot procedure was based on the free radical
inverse-emulsion polymerization of monomer β-CD-NH-ACy and AA in
the presence of BACy cross-linker initiated by APS. The in-situ formation
of Nanosponges based on β-CD-appended hyper-cross-linked polymer
had 3D network structure (Scheme 1). Amino groups-containing β-CD-
NH-ACy and carboxyl groups-containing AA as comonomer were used to
impart the Nanosponges pH-responsiveness. Disulfide bond-containing
BACy was utilized to endow the Nanosponges GSH-responsiveness, in
which the disulfide bonds might be disintegrated in response to high
intracellular GSH concentration while affording good stability in plasma
with low GSH concentration [40]. The representative TEM image of the
obtained Nanosponges (Fig. 1a) and DOX@Nanosponges (Fig. 1b)
showed the spherical-like morphology. The average diameter of the
DOX@Nanosponges (190 ± 20 nm) were slightly smaller than the blank
ones (180 ± 23 nm), mainly owing to the encapsulated hydrophobic
DOX molecules, which decreased their swelling degree in neutral
aqueous media. The DLE and DLC were examined to be 90.3% and
22.56%, respectively. The DLC was higher than that of the previously
reported GSH-responsive cyclodextrin-nanosponges (12 wt %) [25,31],
and cyclodextrin-Calixarene Nanosponges (<20%) [32]. This may be
ascribed to the combined interactions, including the 3D network
providing the physical adsorption, the massive amino and carboxyl
groups offering chemical adsorption by electrostatic interaction of
hydrogen bond or slat bond, and β-CD forming inclusion complexes with
DOX, etc.
incubation at 37 ◦C, the medium was aspirated and replenished with
100 μL of fresh medium, and 20 μL of MTT solution (5 mg/mL in DMEM)
was added. The cells were incubated for another 4 h. After remover of
the MTT solution, 150 μL of dimethylsulfoxide was added to dissolve the
formed purple crystals. The absorbance was determined at 570 nm using
a microplate reader (Bio-Rad, model 550). Untreated control cells were
normalized to 100%. The relative cell viability was calculated by the
following equation:
ODsam ple
ODcontrol
ꢀ
ꢀ
OD
Cell viablity (%) =
blank × 100%
ODblank
2.10. In vitro uptake study
A549 cells were seeded in confocal dishes and grown in DMEM
medium containing 10% FBS overnight to make sure cells attached
completely. The cells were incubated with prescribed amounts of free
DOX or DOX@Nanosponges at 37 ◦C and 5% CO2. After incubation for 1,
3, 5 and 7 h, the culture medium was removed. The cells were rinsed
three times with PBS to remove the redundant DOX@Nanosponges, and
then fixed with 4.0% formaldehyde at 37 ◦C for 10 min. The cell nuclei
were stained by 4′,6-diamidino-2-phenylindole (DAPI) working solution
for 10 min. Fluorescence images of cells were obtained with a Zeiss LSM
510 confocal laser scanning microscope (Carl Zeiss, Oberkochen,
Germany).
2.11. Other instrumental methods
Fourier transform infrared spectroscopy (FT-IR) spectra of all sam-
ples were recorded on a PerkinElmer 1600 FTIR spectrometer (Perki-
3.2. Physicochemical characterization of nanosponges
nElmer, Italy) in the 4000-400 cmꢀ 1 range with a resolution of 4 cmꢀ 1
.
The dried samples were mixed with KBr to be compressed to a plate for
analysis. 1H nuclear magnetic resonance (1HNMR) spectra were recor-
ded on Bruker 500 MHz NMR spectrometer (Bruker Avance, USA) with
D2O as solvent. Powder X-ray diffraction (XRD) analysis were recorded
on XD-2 X-ray diffractometer persee (Beijing Persee instrument Co. Ltd,
China) with Cu Kα radiation (λ = 1.5405 Å) operating at 30 kV voltage
and 15 mA current density and using a scanning rate of 2◦/min up to
50◦. Transmission electron microscopy (TEM) micrographs were taken
on a Hitachi 7500 electron microscope.
The Nanosponges were first characterized by FTIR analysis. As can
be seen in Fig. 2A, Nanosponges (curve c) showed typical β-CD ab-
sorption features of the ring vibrations at 578, 708, 756, and 943 cmꢀ 1
,
,
the coupled C–O–C stretching/O–H bending vibrations at 1160 cmꢀ 1
the coupled C–O/C–C stretching/O–H bending vibrations at 1035 and
1092 cmꢀ 1, CH2 stretching vibrations at 2917 cmꢀ 1, C–H/O–H bending
vibrations at 1415 cmꢀ 1and O–H stretching vibrations at 3375 cmꢀ 1
[41,42]. This clearly confirmed that β-CD molecules were successfully
introduced into the proposed DDSs. The signals at 1646 and 1560 cmꢀ 1
were attributed to the typical carbonyl group stretching vibration
(amide I) and the N–H bending vibration (amide II) [36], while the
peaks at 1075 cmꢀ 1 and 514 cmꢀ 1 were corresponded to -S-S- stretching
vibration [40,43]. Furthermore, the signal at 1620 cmꢀ 1 (curve a and b)
2.12. Statistical analysis
Data were displayed as mean ± S.D (standard deviation). Statistical
data analyses were performed using one-way ANOVA (Origin Pro 9.0)
for n ≥ 3. The significant difference between the experimental and the
control group was set at different levels as p < 0.05.
–
which was assigned to the C C (sp2 carbon) stretching vibration dis-
–
appeared [40,44], indicating that the free radical polymerization reac-
tion took place. Additionally, the previously published reports had
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Y. Dai et al.
Journal of Drug Delivery Science and Technology 64 (2021) 102650
Fig. 2. FTIR spectra. A, β-CD-NH-ACy (a), BACy (b) and Nanosponges (c). B, free DOX (a), Nanosponges (b), physical mixture (c) and DOX@Nanosponges (d).
Fig. 3. 1HNMR spectrums of free DOX (a), blank Nanosponges (b) and DOX@nanosponges (c).
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Y. Dai et al.
Journal of Drug Delivery Science and Technology 64 (2021) 102650
Fig. 4. The XRD diffractograms of free DOX, blank Nanosponges, physical
Fig. 5. The in vitro cumulative release profiles of DOX@Nanosponges at
different pH values (7.4, 6.5 and 5.0) and different concentrations of GSH (0
and 10 mM).
mixtures of DOX and Nanosponges and DOX@Nanosponges.
demonstrated that multiple hydrogen bonds could be strong enough to
fabricate a complex structure such as layer-by-layer assemblies and su-
pramolecular dendrimers or D-networks [41,42,45]. Furthermore, when
hydrogen bonding is formed, the O–H stretching vibration peak could
exhibit typical red-shift [41,46]. Note that the O–H stretching vibration
signal at 3375 cmꢀ 1 for Nanosponges (bonded OH mode) exhibited
typical large red-shift compared with the free OH mode (located at about
3700 cmꢀ 1), demonstrating that there existed a strong hydrogen bond
between β-CD molecules and some O or N-containing groups of Nano-
sponges. The appearance of a new peak at about 1421 cmꢀ 1 clearly
revealed the formation of intermolecular hydrogen bonds between the
the molecule would interfere with drug encapsulation and release be-
haviors. Crystalline intense peaks of β-CD at 2θ of 4.7◦, 9.1◦, 10.7◦,
11.8◦, 14.9◦, 17.2◦, 19.8◦ and 23◦ [50] were not seen in the XRD pattern
of Nanosponges (Fig. 4), demonstrating that crystal structure of β-CD
was converted into amorphous owing to the polymerization reaction
and β-CD molecules distributed homogenously in Nanosponges without
the formation phase-separated crystal aggregates. While characteristic
intense signals of DOX were observed at 2θ of 12.35◦, 16.44◦, 18.76◦,
20.46◦, 22.50◦, 24.94◦, 26.14◦ and 30.06◦, indicative of crystalline
structure. As expected, the diffractogram of the physical mixture did not
reveal any deviation in the peak position with respect to those of DOX
and Nanosponges. Notably, after loading of crystalline DOX molecules
into amorphous Nanosponges, the signals corresponded to the DOX were
not observed in the XRD pattern of DOX@Nanosponges, which clearly
supported that DOX was an amorphous phase in Nanosponges and
formed the inclusion complex with β-CD. Consequently, it could also be
concluded that β-CD-appended hyper-cross-linked polymer was formed.
The appearance of new lines at 2θ of 45.10◦ and 56.25◦ may be due to
the presence of new solid crystalline phases which corresponded to a
host-guest complexes of the same nature [51].
–
–
–
–
C
O groups and the N–H groups (C O⋅⋅⋅H–N), owing to that the ab-
sorption peak is close to 1560 cmꢀ 1 (δNH) characteristic peak of the
hydrogen bonded urethane groups [47]. These results indicated that the
Nanosponges had been synthesized successfully by a free radical poly-
merization reaction. In addition, the FT-IR spectra of the DOX, blank
Nanosponges, the physical mixture of DOX and Nanosponges, and
DOX@nanosponges were shown in Fig. 2B. It was found that DOX (curve
a) showed a series of characteristic peaks at 2917, 1616, 1580, 1495,
1284, 1114 and 805 cmꢀ 1 [48], and the physical mixture spectrum
(curve c) was expected to be the sum of the DOX (curve a) and Nano-
sponges (curve b). Compared with DOX and the physical mixture, for
DOX@nanosponges (curve d) there were significant changes in the
characteristic absorption peaks of DOX; some signals disappeared and
some signals became less intense, particularly in the range of
1616–1495 cmꢀ 1 for DOX aromatic rings and 1284-805 cmꢀ 1, but it
retained all characteristic peaks from Nanosponges. The complete
disappearance or strong reduction of the DOX characteristic bands was
owing to the change in environment after the formation of the host-guest
complexes between DOX and β-CD or supramolecular encapsulation
between DOX and carrier networks. In addition, the global shape of the
broad peak at 3397 cmꢀ 1 for –OH group (curve d) was modified leading
to a decrease of the half-band width and the new peak at about 1421
cmꢀ 1 appeared when DOX loading occurred, revealing the formation of
intermolecular hydrogen bonds between DOX and carrier network
(Scheme 1). These strong interactions between DOX and Nanosponges
could be further demonstrated by the following 1HNMR analysis. The
1HNMR spectra of free DOX (Fig. 3a) and blank Nanosponges (Fig. 3b)
clearly exhibited a series of characteristic peaks. For DOX, all of char-
acteristic signals were attributed to the corresponding protons in the
DOX molecule [49]. However, for DOX@Nanosponges, it was noticed
that the H signals from ~7.1 to 7.7, at about 5.4, 4.15, 2.1 and 1.2 ppm
which were assigned to No. 3 and 4, 15, 19, 9 and 20 protons’ peak of
DOX became weak (Fig. 3c), implying that DOX was efficiently loaded
into Nanosponges.
3.3. In vitro DOX release and morphological changes of the Nanosponges
Controlled release of DOX in the acidic/redox environments of the
tumor is important to reduce side effect and enhance the drug
bioavailability. The in vitro release of DOX from DOX@Nanosponges
was investigated at 37 ◦C under six different conditions, including (i)
PBS (100 mM, pH 7.4), (ii) PBS (100 mM, pH 6.5), (iii) acetate buffer
(100 mM, pH 5.0), (iv) PBS containing 10 mM GSH (100 mM, pH 7.4),
(v) PBS containing 10 mM GSH (100 mM, pH 6.5), and (vi) acetate
buffer containing 10 mM GSH (100 mM, pH 5.0). There was no obvious
burst drug release in the six drug release profiles (Fig. 5). At physio-
logical pH (pH7.4) about 11.0% of DOX originally encapsulated within
Nanosponges was released, even after 96 h incubation. When pH
reduced to 6.5 and 5.0 mimicking the tumor extracellular and intra-
cellular microenvironment (endosomal compartments [34], about
19.0% and 31.0% of DOX was released in 96 h, respectively. Obviously,
the drug release ratio at the acidic medium was much higher than that in
the physiological medium, demonstrating the pH-triggered drug release
property. This phenomenon can be explained by these facts. At neutral
pH7.4, the ionized amino group of DOX (pKa of 8.3 [52] and
β-CD-NH-ACy units (-NH+3 ) and the deprotonated carboxyl group of AA
units (-COO-) were combined tightly by electrostatic interaction, leading
to the low release of DOX [53,54]. While on reaching acidic environ-
ments (especially pH5.0), most of the carboxyl groups of AA units were
in a non-ionized state (-COOH) and became hydrophobic [54], and the
P-XRD is a very useful technique to investigate the structural
arrangement within DDSs because the structural arrangement of inside
6

Y. Dai et al.
Journal of Drug Delivery Science and Technology 64 (2021) 102650
that the Nanosponges were disintegrated into water soluble fragments,
triggering the DOX rapid dissociation and release from the network
structure of Nanosponges, simultaneously accompanied by release of
DOX from the cavities of β-CD. The drug release mechanism was also
simulated with Korsmeyer-Peppas (Fig. S1) and Higuchi (Fig. S2)
models, and the values of release exponent (n), kinetic constant (k) and
regression coefficient (R2) were summarized in Table 1. R2 was often
used as criteria to evaluate the accuracy of these models. According to
the obtained R2 values the Korsmeyer-Peppas kinetic model exhibited a
better fit for the DOX release from DOX@Nanosponges, suggesting the
drug release mechanism could be better described with the
Korsmeyer-Peppas models, The n value from Korsmeyer-Peppas model,
consist of 0.5 < n < 1.0 for mass transfer following non-Fickian or
anomalous diffusion model and n ≤ 0.5 for Fickian diffusion mechanism.
The n values were less than 0.5 and Fickian diffusion was observed for
DOX release period. As for the Higuchi model, all the k values were near
or higher than 1, implying the drug release mechanism was governed by
Fickian diffusion. Even so, the DOX release from the Nanosponges was
very complicated and difficult to understand thoroughly.
Table 1
Fitted DOX release parameters with Korsmeyer-Peppas and Higuchi models.
Releasing media
Korsmeyer-Peppas
Higuchi
n
R2
k
R2
pH7.4
0.08233
0.34547
0.27232
0.26546
0.32405
0.31655
0.73772
0.91844
0.93199
0.90860
0.92956
0.95435
0.75334
3.88594
1.72083
4.75039
3.05392
7.33992
0.47029
0.86332
0.91895
0.80858
0.88101
0.90372
pH7.4 + 10 mM GSH
pH6.5
pH6.5 + 10 mM GSH
pH5.0
pH5.0 + 10 mM GSH
amino groups of β-CD-NH-ACy units and DOX were in a protonated state
(-NHþ3 ), which destroyed the interaction (electrostatic interaction or
hydrogen bond) between DOX and the Nanosponges in DDSs, caused the
internal mutual strong exclusion between the Nanosponges and DOX
leading to DOX@Nanosponges swell and more of the incorporated DOX
release [53,54]. Furthermore, a positive charge was attained by
accepting protons thereby promoting cell uptake of the DOX@Nano-
sponges. On the other hand, as for the pH-responsive Nanosponges, the
premature drug leakage at pH7.4 was about 11.0%, and a release ratio of
19.0% was obtained at pH6.5 because of its excellent responsiveness in
the desired pH range, implying that the DOX@Nanosponges could
effectively accommodate DOX before entering cancer cells. The char-
acter of the minimal drug leakage from the DOX@Nanosponges would
be of great significance for practical applications due to the low side
effects to normal tissues. The cumulative release was further boosted in
presence of 10 mM GSH (equal to the concentration of GSH intra the
cancer cells) [34] at pH7.4, 6.5 and 5.0, with ~40.0%, ~52.0% and
~77.0% of DOX released in 96 h, respectively, much higher than these
values without GSH, indicating the excellent reduction responsive trig-
gered drug release performance of the DOX@Nanosponges. In the
stimulated tumor intracellular microenvironment (pH5.0 and 10 mM
GSH), the disulfide bonds cleavage-induced the 3D worknets breakage
and lower pH-induced swelling of the DOX@Nanosponges resulted in
The incorporation of bioreducible disulfide bonds into the Nano-
sponges is expected to improve the biodegradation behavior of DDSs.
The morphological changes of Nanosponges during the course of DOX
release in releasing medium at pH 5.0 with 10 mM GSH were directly
observed by TEM. Obviously, the drug release process was characterized
by swelling and disintegration of the Nanosponges (Fig. 6). Compared
with the initial DOX@Nanosponges, the spherical Nanosponges became
irregular and loose after 12 h of incubation (Fig. 6b), and the compact
structure of 3D networks begin to disintegrate (Fig. 6c). With the in-
crease in the incubation time, the degree of disintegration of the
Nanosponges gradually increased over the next 72 h. By 96 h of incu-
bation, almost all of the Nanosponges were collapsed into small stripe
fragments (Fig. 6g). Interestingly, even up to 96 h of incubation, there
were still a few of Nanosponges which did not completely disintegrated
into fragments (Fig. 6h). This provided the favorable conditions for drug
Fig. 6. TEM images depicting the morphology of DOX@Nanosponges in releasing media at pH5.0 with 10 mM GSH, (a) images of initial DOX@Nanosponges before
incubation, (b), 12 h, (c), 24 h, (d), 36 h, (e), 48 h, (f) 72 h and (g) 96 h of incubation, respectively. (h) the higher magnification of the red boxed section in image (g).
(For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
7

Y. Dai et al.
Journal of Drug Delivery Science and Technology 64 (2021) 102650
Fig. 7. CLSM images of A549 cells after incubation with DOX@Nanosponges (10 mg DOX equiv/mL) for 1 h, 3 h, 5 h and 7 h, respectively. Scale bar: 10 μm.
Scheme 2. Illustration of pH/GSH dual-bioresponsive degradable Nanosponges for dually activated intracellular release of DOX.
8

Y. Dai et al.
Journal of Drug Delivery Science and Technology 64 (2021) 102650
Fig. 8. (a) Viabilities of A549 cells treated with Nanosponges for 24, 48 and 72 h, respectively, (b) DOX@Nanosponges and free DOX for 72 h.
sustained and controlled release.
4. Conclusions
We have demonstrated that the GSH/pH dual-bioresponsive
degradable Nanosponges based on β-CD-appended hyper-cross-linked
polymer efficiently deliver doxorubicin into tumor cells and respond
to endosomal pH as well as cytoplasmic GSH to enhance intracellular
doxorubicin release, leading to the improved anti-tumor activity. The
smart Nanosponges exhibit several interesting characteristics including
low degradable and cytotoxicity; decent drug loading capacity; suffi-
ciently stable with low drug release (only 11% doxorubicin release in 96
h) under physiological conditions (pH7.4, 37 ◦C); and fast and maximum
drug release triggered by acidic endosomal pH and cytoplasmic GSH so
on. All these characteristics demonstrate that the GSH/pH dual-
bioresponsive degradable Nanosponges can be employed as a potential
anticancer delivery system for future applications in cancer
chemotherapy.
3.4. Uptake and intracellular DOX release
The uptake of DOX into the tumor cell nucleus is crucial because DOX
has to intercalate with DNA to kill the tumor cells [39]. A549 cells
treated with DOX@Nanosponges were observed by CLSM to visualize
cellular uptake and track the intracellular localization and drug release
profiles. For the experiment, A549 cells were stained with DAPI to locate
the nucleus (blue) while DOX presented red fluorescence. A549 cells
were incubated for 1, 3, 5 and 7 h with DOX@Nanosponge. CLSM
observation was carried out to observe the combination of the red
fluorescence (DOX) and the blue fluorescence (DAPI) to confirm
whether the drugs enter the nucleus. As can be seen from Fig. 7, strong
DOX fluorescence was observed in the cell cytoplasm and a relatively
weak red fluorescence presented in the cell nucleus following 1 h in-
cubation with DOX@Nanosponges. However, the red fluorescence in-
tensity inside the cell nuclei increased gradually with the increase in
incubation time and reached a maximum at 7 h. It was proved that the
DOX@Nanosponges were internalized into the tumor cells by endocy-
tosis. Upon endocytosis, the DOX@Nanosponges were swollen due to
the protonation effect resulting in partial DOX release in endosomes, and
then the swollen Nanosponges escaped form endosomes following
further trafficking to cytoplasm where Nanosponges would rapidly
disintegrate due to disulfide bonds cleavage and DOX was completely
released in response to high intracellular GSH concentration (Scheme 2),
simultaneously accompanied by DOX release from the cavities of β-CD.
The released DOX was finally translocated to nucleus to exert cytotox-
icity against tumor cells.
Declaration of competing interest
The authors declare no competing financial interest.
Acknowledgment
The authors acknowledge the financial support from Chongqing
Municipality Education Commission (KJ1400212), the Municipal Sci-
ence and Technology Committee of Chongqing (CSTC2014jcy-jA0019),
and School of Pharmacy, Chongqing Medical University.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jddst.2021.102650.
3.5. Antitumor activity analysis
To determine the antitumor activity of the DOX@Nanosponges,
A549 cells were treated with different concentrations of DOX@Nano-
sponges, blank ones and free DOX for 72 h, and cell viability was eval-
uated by MTT assay. It was found that the Nanosponges was practically
non-toxic (cell viabilities > 90%) up to a tested concentration of 1000
Author statement
Yutong Dai: Methodology, Investigation, Data curation, Writing-
original draft. Qingman Li: Methodology, Investigation, Writing-
original draft. Shurong Zhang: Methodology, Investigation, Writing-
original draft. Shan Shi: Investigation. Yang Li: Investigation. Xudong
Zhao: Liping Zhou, Investigation. Xin Wang: Supervision. Yijian Zhu:
Supervision. Wei Li: Conceptualization, Writing - review & editing,
Project administration, Funding acquisition.
μ
g/mL in 72 h (Fig. 8a), confirming that the Nanosponges had good
biocompatibility. DOX@Nanosponges, however, exhibited significant
antitumor activity against A549 cells following 72 h incubation
(Fig. 8b). The half maximal inhibitory concentration (IC50) of DOX@-
Nanosponges was 1.201 μg DOX equiv/mL for A549 cells, which was
much higher than that of reported DOX-loaded degradable block
copolymer (27 g/mL [55], 15 g/mL [56] and 3.2 g/mL [57]. This
References
μ
μ
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greater inhibitory effect of DOX@Nanosponges was in agreement with
the CLSM observations that Nanosponges mediated fast targeted intra-
cellular drug release by intracellular reduction in the acid environment
to directly bind to the nucleus, thereby preventing loss of the DOX and
enhancing the antitumor activity.
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10