Research Article

Cite This: ACS Appl. Mater. Interfaces 2019, 11, 5851−5861 www.acsami.org

A CD44-Targeting Programmable Drug Delivery System for

Enhancing and Sensitizing Chemotherapy to Drug-Resistant Cancer
Min Zhang,†,‡,∥ Yi Ma,†,∥ Zhaohui Wang,† Zhihao Han,† Weidong Gao,† Qiumei Zhou,†
and Yueqing Gu*,†
†
State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Screening, Department of Biomedical Engineering,
School of Engineering, China Pharmaceutical University, Nanjing 210009, China
‡
Institute of Biomedical Materials and Engineering, College of Materials Sciences and Engineering, Qingdao University, Qingdao
266071, China
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*
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ABSTRACT: Programmable drug delivery systems hold great promise to enhance cancer
treatment. Herein, a programmable drug delivery system using a chondroitin sulfate (CS)-
based composite nanoparticle was developed for enhancing and sensitizing chemotherapy to
drug-resistant cancer. The nanoparticle was composed of a cross-linked CS hydrogel shell
and hydrophobic cores containing both free drugs and CS-linked prodrugs. Interestingly, the
nanoparticle could mediate tumor-specific CD44 targeting. After specific cellular uptake, the
payloads were suddenly released because of the decomposition of the CS shell, and the free
drug molecules with synergistic effects induced tumor-specific cytotoxicity rapidly.
Subsequently, the inner cores of the nanoparticles sustainedly release their cargos in drug-
resistant tumor cells to keep the effective drug concentration against the drug efflux mediated
by P-glycoprotein. CS dissociated from the outer shell and sensitized cancer cells to the
antitumor drugs through downregulation of Bcl-XL, an antiapoptosis protein. Such a
programmable drug delivery system with specific tumor-targeting and sensitized therapy is
promising for rational drug delivery and provides more versatility for controlled release in
biomedical applications.
KEYWORDS: Programmable sequential effect, CD44-targeting, Drug-resistant cancer, Sensitized therapy, Chondroitin sulfate

1. INTRODUCTION of drug and sustain an effective therapeutic concentration

Despite tremendous effort made in anticancer drug research
over the past decades, conventional drugs still face some
serious drawbacks in tumor therapy.1−3 Accordingly, drug
delivery systems have been explored because of their potential
of a more effective treatment.4−13 As widely known, ideal drug
delivery systems should satisfy the conditions of no leakage in
the delivery process and easy unloading of the cargos once they
arrived at the target sites.14 To meet these criteria, various
stimuli-responsive drug delivery systems were proposed.
Stimuli-responsive drug delivery systems are specialized
nanosized drug delivery carriers equipped with environmental
sensitive modalities within their structures.15,16 These drug
delivery systems can release their encapsulated cargos in
response to specific environmental stimuli, such as light,17
temperature,18 pH,19 enzyme,20 reactive oxygen species,21
ultrasound,22,23 and redox agents.24 However, most stimuli-
responsive drug delivery systems usually release their guests
simultaneously once attacked by a stimulus,25 lacking sustained
effective drug concentration against the drug efflux mediated
by P-glycoprotein, which greatly impedes their practical
application in drug-resistant cancers with drug efflux functions.
Therefore, a controlled drug delivery system with a program-
mable drug release function to quickly release a large amount
becomes an attractive alternative for drug-resistant cancers.
Antibodies, kinase inhibitors, endogenous molecules, and
lectin saccharides are widely used in target therapy. However,
some exogenous molecules might cause immune responses,
which lead to serious side effects. Endogenous molecules, such
as folic acid26,27 and chondroitin sulfate (CS),28 are
increasingly used as alternative tumor-targeting ligands because
of better biocompatibility. CS, a natural polysaccharide, has
universally acknowledged good druggability, for it has been
used as an anti-inflammatory drug in Europe.29 CS possesses a
strong affinity for CD44 receptors,30−32 which is highly
expressed on various types of cancer cells and determines
the tumorigenic and metastatic capacities of cancer cells.33,34
More importantly, as an anti-inflammatory drug, CS inhibits
the synthesis of the proinflammatory enzyme COX-2,35 which
directly regulates the phosphorylation of Akt and subsequently
decreases the levels of Bcl-XL, a Bcl-2 family member with
antiapoptotic functions.36 The downregulation of Bcl-2
increases sensitivity of tumor cells to chemotherapeutic
Received: November 18, 2018
Accepted: January 16, 2019
Published: January 16, 2019

© 2019 American Chemical Society 5851 DOI: 10.1021/acsami.8b19798

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Scheme 1. Schematic Illustration of the CS-Based Nanoparticle (CBN) as a Tumor-Targeting Programmable Nanosystem for
Enhancing and Sensitizing Chemotherapy to Drug-Resistant Cancer. (a) Fabrication of the tumor-targeting and
programmable drug delivery system. The system is composed of a hydrogel shell and drug-loaded cores. (b) The specific
cellular uptake and intracellular fate of CBN. After specific accumulated in tumor cells through CD44-mediated
internalization, the outer hydrogel can degrade and the free drugs with synergistic effects release out to induce cytotoxicity
rapidly. CS dissociated from hydrogels, causing Bcl-XL downregulation and increased chemotherapeutic response of drug-
resistant tumor cells. Simultaneously, the drug-loaded cores can provide a second sustained release and keep an effective
concentration for a longer period

drugs,37 which could further reverse cancer drug resistance.
Therefore, CS was not only an attractive candidate as a
targeting ligand for building the structure of the drug delivery
system but also an amplifier of the therapeutic efficacy.
Herein, we report a programmable sequential drug delivery
system using CS-based composite nanoparticles (CBN) to
enhance and sensitize chemotherapy to overcome drug-
resistant cancer. As illustrated in Scheme 1a, anticancer drug
paclitaxel (PTX) and sunitinib (Su) with synergetic effects
were used as therapeutic agents. PTX was connected with CS
to form CS-connected prodrug (CP) and self-assembled with
free drugs (PTX+Su) into smaller nanoparticles (CPNs).38−40
Then, the free drugs and drug-loaded CPN were encapsulated
into a cross-linked CS-based hydrogel shell to form this
programmable composite nanoparticle, PTX/Su@CBN. The
CS shells were cross-linked with the GSH-sensitive disulfide
bond, which enabled the shell to specifically degrade under
high GSH concentration in tumor cells after CD44-mediated
internalization. Then, free PTX and Su located in the CS shells
rapidly diffused into tumor cells and exerted synergetic effects,
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whereas the drug-loaded cores, CPNs, were also released out.
CPNs provided a second sustained drug release and
maintained an effective concentration in drug-resistant tumors
against the drug efflux mediated by P-glycoprotein. Meanwhile,
CS dissociated from the hydrogels, causing Bcl-XL down-
regulation and increasing sensitivity of tumor cells to
chemotherapeutic drugs, which could further improve the
therapeutic effect on drug-resistant cancer (Scheme 1b). This
study provided a strategy to prepare smart drug delivery
systems, which can achieve programmable drug release and
enhance treatment, and that is of great potential to realize
more rational drug administrations for the therapy of drug-
resistant cancer.
2. RESULTS
2.1. Synthesis and Characterization of CBN. The CS-
based nanoparticles were composed of hydrogel shells and
drug-loaded cores. The chemical structure of CP was
confirmed using FTIR and 1H NMR (Figure S1). CBN were
characterized by TEM (Figure 1a). The nanoparticles were
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Figure 1. Characterization of the nanosystem (CBN). (a) Morphology of CBN by TEM; (b) dynamic Light Scattering (DLS) results of CP, CBN,
and PTX/Su@CBN; (c) photographic images of CBN and (d) NIR dye, Cypate-loaded CBN (Cy@CBN) treated with different concentrations of
GSH; (e) TEM images of CBN treated with different concentrations of GSH; (f) drug-release profiles of PTX/Su@CBN in the presence of
different concentrations of GSH; (g) DLS results of CBN treated with 10 mM GSH after different times.

dispersed and spherical, and they had a negative surface ζ-
potential (Figure S2a). The ratio of PTX to Su in PTX/Su@
CBN was 1:40 (w/w). Further quantification found that 20.7%
PTX and 58.4% Su were distributed in the CS shell and 79.3%
PTX and 41.6% Su were in the inner core. After drug loading,
PTX/Su@CBN showed a smaller size than CBN (Figure 1b),
which was indicative that the addition of hydrophobic drugs to
the inner particles improved their assembly. Moreover, the
electric attraction between the drug and nanoparticles may also
contribute to a more compact structure. Both CBN and PTX/
Su@CBN were stable through long-term storage (Figure S2b).
As shown in Figure 1g, the particle size of CBN changed
into two diameter distributions in 10 mM GSH, for the CS
shells were degraded and the cores with smaller particles were
released out. After 12 h incubation with 10 mM GSH, the
nanoparticles were mostly degraded (Figure 1c), and the
hydrophobic dye loaded in CBN was released (Figure 1d).
TEM was employed to confirm the responsiveness of the CBN
to GSH (Figure 1e). The nanoparticles remained spherical
after incubation with 10 μM and 2 mM GSH. By contrast,
CBN disintegrated into irregularly shaped components in 10
mM GSH. The disassembly of CBN was a result of the
cleavage of the disulfide linkages in high GSH concentrations,
which led to the release of payloads from the interior of the
nanoparticles in high concentrations of GSH.
The programmable drug release of CBN was designed as,
first, rapid release after cell uptake and, second, sustained
release in the tumor cells. The release behaviors of two kinds of
drugs were investigated. During the initial 30 min, free drug
molecules loaded in CS shells had a burst release behavior in
the presence of 10 mM GSH (Figure 1f). About 53.8% PTX
and 59.9% Su were released. Then, a following sustained
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release was observed within 24 h. The two-stage drug release
profile could confirm the core−shell structure of CBN. Also,
such a release behavior may help maintain an effective drug
concentration in tumor cells against drug efflux, thus
enhancing the therapeutic effect on drug-resistant cancer. As
the GSH concentration in normal tissues is much lower than
that in the tumor environment,41 only about 5.9% PTX and 8%
Su were detected at 24 h under 10 μM GSH (plasma GSH
concentration), whereas about 17.8% PTX and 20.7% Su were
released in 2 mM GSH conditions (normal tissue GSH
concentration). The negligible drug leakage in low GSH
concentration promised low systemic toxicity in vivo. There-
fore, the proposed drug delivery system was supposed to
remain stable in the delivery process after intravenous
administration and release of anticancer agents specifically in
tumor in a programmed manner.
To further evaluate the stability of CBN in vivo, the
concentration of free drugs in blood was studied. Khoei et
al.42,43 investigated the in vivo behavior of drug-loaded
nanocarriers through measuring the mean plasma concen-
tration of free drug, which could clearly illuminate the
prolonged elimination half-life. As shown inFigure S3, both
of the free drugs displayed a one-compartment model, whereas
PTX/Su@CBN followed a multicompartment model, showing
a longer elimination time. More importantly, being constructed
as GSH-sensitive nanoparticles, PTX/Su@CBN showed little
drug release in blood, which further confirmed the stability of
nanoparticles in circulation.
2.2. In Vitro Targeting Ability. Targeting specificity of
CBN was evaluated on cancer cell lines MDA-MB-231,
HepG2, U87MG, and MCF-7 and the normal cell lines L02
and MSCs (normal primary human adipose-derived mesen-
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Figure 2. Uptake of Flu-loaded CBN (Flu@CBN) by cells expressing different levels of CD44. (a) Hyperspectral microscopy and fluorescence
images of different cells after 2 h incubation with Flu@CBN; (b) 3D reconstruction image of MDA-MB-231 cells treated with Flu@CBN; (c)
linear fitting between relative fluorescence intensity of Flu@CBN and CD44 expression (blue: Hoechst, nuclear dye; green: Flu loaded in CBN).

chymal stem cells). Fluorescein (Flu) was loaded into CBN

(Flu@CBN) for cell imaging, and hyperspectral microscopy
was used to image the intracellular CBN to exclude the
possibility that differences in Flu@CBN uptake affected the
fluorescence signal. As shown in Figure 2a, bright fluorescence
was observed in MDA-MB-231 cells, where Z-axis images
acquired at different depths (Figure 2b) indicate that CBN
accumulated in the cytoplasm. A gradual decrease in the
fluorescence signal was observed across tumor cell lines, which
is consistent with their CD44 expression levels (Figure 2c). Of
note, there was nearly no signal detected in normal cells MSCs
and L02, despite the high expression of CD44 on MSCs. The
low uptake of MSCs was possibly due to the different isoforms
of CD44 expressed on MSCs and tumor cells. Therefore,
further experiments were carried out to confirm the hypothesis.
Anti-CD44 antibody was incubated with MDA-MB-231 and
MSC cell lines to visualize CD44 expression. As shown in
Figure 3, both the cancerous and normal cells displayed bright
fluorescence after antibody staining, confirming abundant Figure 3. Uptake of Flu@CBN in CD44-overexpressing cells in 2D
CD44 expression on these cell lines. Next, the cellular uptake and 3D cultures. (a) Fluorescence images of cellular uptake using 2D-
of CBN was evaluated. An obvious signal was observed in cultured cells after incubation with Flu@CBN; (b) fluorescence
MDA-MB-231 cells, but not in MSCs after Flu@CBN images of cellular uptake using 3D-cultured spheres of cells after
incubation. A similar phenomenon was observed in both 3D incubation with Flu@CBN (red: anti-CD44 antibody; green: Flu@
spheres and 2D adherent cultures, indicating that CS CBN).
specifically binds to CD44 on cancer cells, but not on normal
cells. These results demonstrated that the CS-based nano- cells. As shown in Figure S4, the fluorescence signal of the
particles were internalized through CD44 expressed on CD44 antibody was almost obliterated in normal cells after
cancerous cells, but not on noncancerous cells, which could CD44s silencing, whereas the tumor cells retained bright
avoid unnecessary toxicity toward normal cells. It was further fluorescence, implying that normal cells that primarily express
speculated that the different targeting effects of CS were due to CD44s and CD44v may be the major isoform in tumor cells.
the variance of CD44 isoforms between normal and tumor To investigate the binding of CS with CD44 receptors after
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Figure 4. Tumor-targeting efficacy of Cy@CBN in vivo. (a) Live in vivo fluorescence images of tumor-bearing mice up to 72 h post-treatment with
Cy@CBN; (b) fluorescence imaging of tumors and other organs harvested from MDA-MB-231 tumor-bearing mice treated with Cy@CBN and
free Cy at 2, 6, and 12 h; (c) fluorescence imaging of sectioned tissues 12 h after Cy@CBN treatment; (d) linear fitting between the tumor/normal
tissue ratio of Cy@CBN at 12 h and relative CD44 expression of the specific cancer cell type.

gene silencing, the CS-Flu conjugate was synthesized. The
intracellular accumulation of CS-Flu was unchanged in MDA-
MB-231 cells following CD44s knockdown. By contrast,
silencing of total CD44 blocked the uptake of CS-Flu into
tumor cells. Under these conditions, uptake of CS-Flu was
negligible as proved by minimal fluorescence. Therefore, CS
binding was probably mediated by CD44v exclusively, which
may explain the tumor-specific recognition by CS.
2.3. In Vivo Targeting Ability. To evaluate the in vivo
tumor-targeting ability of CBN, an NIR fluorescent dye,
cypate, was encapsulated into CBN (Cy@CBN). As shown in
Figure 4a, the fluorescence signal appeared in the tumors at 4
h, peaked at 12 h, and remained until 72 h in all tumor models.
Meanwhile, there was no accumulation of the free NIR dye in
the tumors. Moreover, the fluorescence signal in the MDA-
MB-231 tumor was stronger than those in the HepG2, MCF7,
and U87MG tumors. Consistent with the in vitro results, the
fluorescence signals in the tumors were lower in relation to a
decrease in CD44 expression (Figure 4d). Fluorescence
imaging of different organs (Figure 4b) excised from tumor-
bearing mice revealed that, at 12 h post-injection, CBN had
accumulated only in tumor tissue and had been nearly cleared
from other organs. The NIR dye served as a control. Confocal
images of tissue sections confirm the accumulation of CBN in
tumors (Figure 4c), supporting that this nanosystem has an
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enhanced ability to target tumors and only has negligible
accumulation in normal tissues.
To confirm the specificity of CBN to tumor tissues, breast
tumor and normal tissues were excised from the tumor-bearing
mice and analyzed by laser confocal microscopy (Figure 5).
Although both normal and tumor tissues showed a certain
extent of CD44 expression, an obvious CBN signal was only
observed in tumor tissues, indicating that CBN could
specifically accumulate in CD44 overexpressed tumor tissues.
Along with the cell imaging results, these data supported that
the difference in CD44 isoforms on tumor and normal cells
might induce the different binding abilities.
2.4. In Vitro Antitumor Efficacy and Molecular
Mechanism. Cytotoxicity from PTX/Su@CBN and the free
drug combination (PTX+Su) was compared by performing cell
viability analysis on MDA-MB-231, HepG2, and MCF7 cell
lines. As shown in Figure S5, both of the two groups displayed
a dose-dependent increase in antitumor efficacy. The IC50
value of each group was calculated on the basis of the MTT
assay (Table S1). Notably, the IC50 value of PTX/Su@CBN
was the lowest in all groups, which can be attributed to their
superior tumor-targeting ability, programmable drug release,
and drug sensitization. Apoptosis of PTX-resistant cancer cells
(MDA-MB-231/Taxol) due to PTX, PTX+Su, and PTX/Su@
CBN was evaluated by flow cytometry. As shown in Figure
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Figure 5. Uptake of Flu@CBN and CD44 expression in tumors and
normal tissue (blue: Hoechst, nuclear dye; red: anti-CD44 antibody;
green: Flu@CBN).
6a−d, the PTX+Su-treated cells had a larger apoptotic
population (51.1%) than those treated with PTX alone
(14.5%), suggesting that combination therapy enhances the
apoptotic potential. Notably, 72.8% of PTX/Su@CBN-treated
cells were apoptotic, significantly enhancing the inhibition of
drug-resistant tumor cells. MTT assays further confirmed the
efficacy of PTX/Su@CBN against MDA-MB-231/Taxol
(Figure S6). More importantly, CS has been reported to
interfere with nuclear factor-κB (NF-κB) and decrease COX-2
levels, which subsequently leads to downregulation of Bcl-XL.
The effect of CS on COX-2 and Bcl-XL downregulation was
confirmed by Western blot (Figure 6e,f). This increased the
sensitivity of cancer cells to chemotherapeutic agents (Figure
6i) and caused enhanced anticancer toxicity by PTX/Su@
CBN. Meanwhile, decreased expressions of COX-2 and Bcl-XL
were observed following incubation with PTX/Su@CBN,
suggesting that the CS component of the nanosystem
effectively neutralizes the drug-stimulated antiapoptotic effects
mediated by Bcl-XL upregulation. ELISA assays and
immunofluorescence imaging were carried out to further
elucidate the influence of CS on the signaling pathway (Figure
6g,h). Furthermore, we detected other apoptotic-related
proteins, Bax and caspase-3. After incubation with CS, neither
of the proteins’ expressions changed (Figure S7). Consistent
with the result of flow cytometry (Figure S8), we speculate that
CS would not induce cell apoptosis under the working
concentration, which implied that CS could reverse cancer
drug resistance without any toxicity. All these results implied
that CS not only served as a hydrophilic moiety for the drug
delivery nanosystem but also contributed to tumor selectivity
and therapeutic efficacy enhancement.
2.5. Biodistribution and in Vivo Antitumor Efficacy of
PTX/Su@CBN. To measure the plasma concentrations of PTX
and Su, the structure of PTX/Su@CBN was destructed and
the released drugs were collected and extracted. As shown in
Figure S9a, PTX and Su entrapped in PTX/Su@CBN
exhibited longer blood circulation even at 12 h post-injection.
Meanwhile, free PTX and Su were cleared quickly from the
blood stream. The AUC and T1/2 of PTX/Su@CBN and free
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drugs were calculated using WinNonlin. As shown in Table S2,
PTX/Su@CBN exhibited a longer elimination half-life and a
larger area under the curve than free drugs. Furthermore, the
amounts of drugs in different tissues at 12 h post-injection
were measured (Figure S9b), and the drug (PTX and Su)
concentrations were nearly 5-fold higher in the tumor tissues
of the PTX/Su@CBN-treated group than the PTX+Su-treated
group. Meanwhile, the normal organs retained little to none of
the drugs following PTX/Su@CBN treatment with the
exception of minimal drug accumulation in the liver. This is
possibly due to the capture of nanoparticles by the
reticuloendothelial system. These results demonstrated that
PTX/Su@CBN greatly increased the tumor accumulation of
anticancer drugs, and it was expected to achieve a more potent
tumor inhibition effect in vivo.
Next, MDA-MB-231/Taxol tumor-bearing mice were used
to evaluate the antitumor efficacy of the PTX/Su@CBN with
free drugs (PTX+Su) at the same dose as the control. In
addition, a double dose of PTX+Su was also assessed as a high
dose control. As displayed in Figure 7, CBN exhibited superior
tumor-growth inhibition compared to that of the PTX because
of the tumor-targeting ability of CBN, resulting in a higher
accumulation of PTX. Notably, PTX/Su@CBN inhibited
tumor growth more potently than other treatments at the
same dose, which was due to specific tumor-targeting,
programmable drug release, and drug sensitivity enhancement
of PTX/Su@CBN. Histologic assays also revealed observable
tumor necrosis with decreased tumoral cellularity in PTX/Su@
CBN-treated tumors. Meanwhile, PTX/Su@CBN had a
similar antitumor efficacy to the high dose of PTX+Su,
indicating that PTX/Su@CBN can significantly inhibit tumors
at a relatively lower dose. Moreover, the administration of
PTX/Su@CBN caused notable downregulation of CD31, a
marker of blood vessel formation, indicating synergistic effects
between these two drugs when incorporated into one system.
Compared to the free solution of the two drugs, PTX/Su@
CBN reduced the systemic toxicity of these chemotherapeutic
reagents. As shown in Figure 7d, the free multidrugs caused
evident damage to spleen and kidney tissues, which was
notably decreased in PTX/Su@CBN-treated mice. Body
weight measurements demonstrate an improved quality of
life for the mice after treatment with PTX/Su@CBN, as there
was less weight loss in this treatment group than in free drug-
treated mice (Figure 7e). The enhanced antitumor properties
and reduced toxicity of the PTX/Su@CBN compared to PTX
+Su alone translated into significant improvements in the
survival of mice with MDA-MB-231/Taxol-derived xenogenic
breast tumors. The median survival time of each group [saline,
CBN, PTX, PTX+Su, PTX+Su (double dose)] is shown in
Table S3. The PTX/Su@CBN-treated group achieved a higher
survival rate of 70% on day 28, which also demonstrated a
better health condition during the therapy, confirming the
enhanced therapeutic efficacy and reduced toxicity of PTX/
Su@CBN. In summary, these results demonstrate the potential
translational applications of PTX/Su@CBN for treatment with
multidrugs using a delivery system that substantially reduces
systemic toxicity and enhances antitumor activity in drug-
resistant cancer.
3. CONCLUSIONS
In this study, we successfully synthesized a CD44-targeting
programmable drug delivery system for enhancing and
sensitizing chemotherapy to drug-resistant cancer. The
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Figure 6. In vitro anticancer activity of PTX/Su@CBN and the mechanism of CS sensitization of drug-resistant tumor cells to drugs. (a−d)
Apoptosis was analyzed using dual staining with Annexin V and PI. Cells in early apoptosis, late apoptosis, and necrosis are located in the lower
right, upper right, and upper left quadrants of the dot plot (Annexin V positive and PI negative, Annexin V positive and PI positive, and Annexin V
negative and PI positive), respectively; (e,f) Western blot analysis of COX-2 and Bcl-XL expression after cells were incubated with CS, PTX+Su,
and PTX/Su@CBN, and quantitative analysis of the grayscales of different groups; (g) ELISA for NF-κB, COX-2, Akt, and Bcl-XL in cells treated
with CS, PTX+Su, and PTX/Su@CBN; (h) immunofluorescence assay of COX-2 and Bcl-XL expression in CS-treated and control cells; (i)
schematic of intracellular fate of PTX/Su@CBN.

proposed nanosystem was composed of cross-linked CS
hydrogel shells and hydrophobic cores containing both the
free drug and CS-linked prodrugs. PTX and Su were used as
anticancer agents in this work, which also have synergistic
efficacy. The nanosystem can keep its structural integrity
without leakage of drugs in the drug delivery process. After
cellular uptake mediated by tumor specific CD44, the
proposed nanosystem can successfully achieve programmable
drug release. In the presence of high GSH concentration, the
outer shells of CBN degraded and the encapsulated free drugs
were rapidly released as the first burst release stage. The
disassembled CS increased the sensitivity of drug-resistant
tumor cells to anticancer agents and further enhanced the
treatment efficiency. Subsequently, the drug-loaded cores
released to provide the second sustained release stage, which
maintained the effective drug concentration in drug-resistant
tumor cells against the drug efflux. Such a well-designed
nanosystem with programmable drug release and tumor
specific targeting and sensitization of tumor cells to chemo-
therapeutics is promising to achieve a more effective treatment
in drug-resistant cancer. In addition, the nanosystem designed
in this study provides more versatility for loading different
drugs with synergistic efficacy and also provides a versatile
strategy for developing novel drug delivery systems for
biomedical applications.
4. METHODS
4.1. Materials. All cell lines were purchased from American Type
Culture Collection. CS and hyaluronic acid are provided by Yuanmu
Bioengineering Co., Ltd. Succinic anhydride, 1-(3-(dimethylamino)-
propyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysucci-
nimide (NHS), cystamine dihydrochloride, and ethanediamine were
purchased from Sigma-Aldrich. PTX and Su were purchased from
Meilun Biotechnology Co., Ltd. The other chemicals were purchased
from Sinopharm Chemical Reagent Co., Ltd. Cypate (MW: 689) was
prepared by our own research group following a protocol described by
Achilefu et al.44,45
4.2. Preparation of CBN. For synthesis of the hydrogel shell, CS
(240 mg) was activated by EDC/NHS catalyst systems, and then

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Figure 7. In vivo antitumor efficacy of PTX/Su@CBN. (a) Images of tumors collected from mice in different cohorts at day 28 post-treatment; (b)
survival rates over time of MDA-MB-231/Taxol tumor-bearing mice after different treatments; (c) tumor growth curves in response to different
treatments; (d) vascular marker CD31 expression in tumors and H&E-stained sections of tumors, spleens, kidneys; (e) changes in body weights of
mice following different treatments.
cystamine dihydrochloride (500 mg) was added and stirred for 12 h.
The obtained mixture was dialyzed against deionized water and then
lyophilized.
CPs were prepared through the connecting ligand CS and
anticancer drug PTX through ethanediamine. PTX (100 mg) and
succinic anhydride (23.4 mg) was dissolved in CH2Cl2, and 4-
dimethylaminopyridine was subsequently added as a catalyst. After
stirring for 48 h at 50 °C, the end product was extracted using
deionized water. CS was activated by EDC/NHS catalyst systems, and
then ethanediamine was added dropwise and stirred for 12 h. The
mixture of acetylated PTX was added into the reaction solution. After
12 h reaction, CPs were dialyzed against deionized water and then
lyophilized. The chemical structure of CP was characterized by FTIR
and 1H NMR. CPN were prepared through a sonication method.
To prepare PTX and Su-loaded CBN (PTX/Su@CBN), PTX and
Su were dissolved in DMSO and then added dropwise into the CP
solution under magnetic stirring. The solution was treated with a
probe-type sonicator (NingBo Scientz, work time 2 s, rest time 5 s,
200 W) for 10 min and dialyzed against deionized water. Finally, both
free drugs and the CS hydrogel shell were dissolved in deionized
water and added dropwise into the obtained mixture solution under
magnetic stirring. After 12 h stirring, the final solution was sonicated
and dialyzed against deionized water. The morphology and hydro-
dynamic diameter of the nanoparticles were examined by TEM
(Philips FEI Tecnai G2 20s-TWIN, Netherlands) and DLS (LPSA,
Malvern Instruments, U.K.). The amounts of PTX and Su loaded in
the nanoparticles were extracted and measured by HPLC.
4.3. Disassembly of CBN Using GSH. CBN were placed in
dialysis bags and immersed in release media in the presence of various
concentrations of GSH (10 μM, 2 mM, 10 mM) at 37 °C with gentle
shaking. The size distribution and morphology change were
monitored at each time point. Cypate, a hydrophobic dye, was
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loaded into CBN to visualize the stimuli-responsive drug release
profile. After immersion in media with different concentrations of
GSH at 37 °C for 12 h, the morphology change was observed. The
drug release profile of PTX/Su@CBN was analyzed using a similar
dialysis method, with the release media containing various
concentrations of GSH. At every designated time interval, 3 mL of
dialysate was withdrawn and replaced with the same volume of fresh
release medium. The concentrations of PTX and Su in the release
media were determined by HPLC analysis.
4.4. In Vivo Release of PTX and Su in Blood. Free drugs and
PTX/Su@CBN with the same dosage were intravenously injected
into mice (∼220 g). Blood samples were collected in heparinized
tubes at each time point, followed by centrifugation to obtain plasma.
Then, the free PTX and Su were extracted from plasma, and the
concentrations of PTX and Su were determined by HPLC analysis.
4.5. Targeting of CBN to CD44-Expressing Cells. Flu was
encapsulated in CBN (Flu@CBN) to evaluate the targeting capacity
of CBN. The human cell lines L02 (normal liver cells), U87MG
(human malignant glioma), HepG2 (human liver cancer cells), MCF-
7 (human breast cancer), MDA-MB-231(human breast cancer), and
MSCs (normal primary human adipose-derived mesenchymal stem
cells) were incubated with Flu@CBN for 4 h and measured using
laser scanning confocal microscopy (LSCM, FV1000, Olympus,
Japan) and hyperspectral microscopy after washing with PBS three
times. The nucleus was labeled by Hoechst.
To further study the targeting ability of CBN, 3D cultured MDA-
MB-231 and MSCs were used. Cells were cultured in 96-well plates
coated with a layer of sterilized agarose-based DMEM (2% w/v) at a
density of 1 ×105 cells/mL. After 10 days of culture, the
mammospheres were collected for further experimental use.46 The
spheres were incubated with Flu@CBN for 8 h, washed with PBS, and
fixed in 4% paraformaldehyde. The sample was then incubated with
DOI: 10.1021/acsami.8b19798
ACS Appl. Mater. Interfaces 2019, 11, 5851−5861
ACS Applied Materials & Interfaces
3% BSA to block potential nonspecific binding, followed by
incubation with primary antibody of CD44 (Abcam, Cambridge,
U.K.). After washing three times and incubated in the dark for 1 h
with the secondary antibody (goat anti-rabbit IgG tagged with Alexa
Fluor 568 from Abcam, Cambridge, U.K.), 2D cultured cells were
incubated with CBN for 4 h and incubated with CD44 antibody as
mentioned above. The samples were then washed and examined using
LSCM.
To investigate the binding mechanism of CBN, CS was conjugated
with Flu to form CS-Flu. The experimental steps were as follows: Flu
was activated by EDC/NHS catalyst systems and then added
dropwise into CS, which was dissolved in ethylenimine. After 12 h
reaction, CS-Flu was dialyzed against deionized water then
lyophilized. To confirm the CD44 receptor specificity, the RNA
interference experiment of CD44 was also performed. The siRNAs
against CD44 and CD44s were designed and synthesized by Biomics
Biotechnology Co., Ltd. (Nantong, China). The siRNAs were
transfected into cells using Lipofectamine 2000. After incubating
with CS-Flu and CD44 antibody, the fluorescence signal was detected
using LSCM.
4.6. In Vitro Imaging. All the animal experiments were conducted
under the Animal Management Rules of the Ministry of Health of the
People’s Republic of China and the Guidelines for the Care and Use
of Laboratory Animals of China Pharmaceutical University. Tumor
models were generated by injecting tumor cells into the left axillary
fossa of nude mice. Cypate, an NIR fluorescent dye, was encapsulated
into CBN for in vivo imaging (Cy@CBN). After injection of Cy and
Cy@CBN into the tail vein separately, the tumor-bearing mice were
imaged using the NIR fluorescence imaging system at different time
points. Each treatment group (n = 10) was sacrificed up to 24 h after
injection, and the harvested tissues were cut into small pieces to
examine the fluorescence intensity using LSCM. To confirm the
specificity of CBN to tumor tissues, Flu@CBN were intravenously
injected into the MDA-MB-231-bearing mice. The mice were
sacrificed up to 24 h post-injection, the organs and tumors were
harvested and cut into small pieces. After incubating with CD44
antibody, LSCM was used to evaluate the fluorescence intensity. The
nucleus was labeled by Hoechst.
4.7. In Vitro Antitumor Efficacy and Molecular Mechanism.
The cytotoxicity of PTX/Su@CBN, PTX+Su, and CS was evaluated
by the MTT assay. MDA-MB-231, HepG2, and MCF7 cells were
grown in 96-well plates with 5 ×103 cells per well and incubated for
24 h. Then, various concentrations of drugs were added into the
media and incubated with cells for 48 h. Afterward, MTT was added
to each well and incubated for 4 h, and dimethyl sulfoxide was used to
dissolve the formazan crystals. The optical density was measured at
560 nm. According to the MTT results obtained, the IC50 values of
different groups were calculated by SPSS.
PTX-resistant cell line MDA-MB-231/Taxol was used to study the
antitumor efficacy of PTX/Su@CBN in drug-resistant tumor cells.
The annexin V and propidium iodide Kit (Invitrogen, CA, USA) was
used to determine the mode and extent of apoptosis according to the
manufacturer’s instructions. MDA-MB-231/Taxol cells were cultured
in a six-well plate and treated with PBS, CS, PTX, PTX+Su, and PTX/
Su@CBN. Subsequently, cells were incubated for 30 min in the dark
after adding annexin V-FITC and propidium iodide, and flow
cytometry was used to differentiate the stained cells. The cytotoxicity
of CS, PTX, PTX+Su, and PTX/Su@CBN was evaluated using MDA-
MB-231/Taxol cells. Cells were grown in 96-well plates with 5 ×103
cells per well and incubated for 24 h. Then, various concentrations of
drugs were added into the media and incubated with cells for 48 h.
Afterward, MTT was added to each well and incubated for 4 h, and
dimethyl sulfoxide was used to dissolve the formazan crystals. The
optical density was measured at 560 nm.
For Western blot, MDA-MB-231/Taxol cells were grown in a six-
well plate and incubated with PTX/Su@CBN, PTX+Su, and CS for
24 h; saline was used as the control. Then, cells were collected and
lysed to isolate the proteins. Proteins were separated using
electrophoresis and then transferred to nitrocellulose membranes.
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Research Article
After blocking, membranes were incubated with the antibody.
Reactive bands were observed with chemiluminescence.
To assess the pathway of apoptosis, NF-κB, COX-2, Akt, and Bcl-
XL were measured using ELISA kits. MDA-MB-231/Taxol cells were
collected and lysed after treating with different groups. The level of
proteins was monitored at 450 nm. MDA-MB-231/Taxol cells were
treated with different groups followed by incubation with COX-2 and
Bcl-XL antibodies as mentioned above. The nucleus was labeled by
Hoechst. The Flu signal was detected using LSCM.
4.8. Distribution and Therapeutic Efficacy of PTX/Su@CBN
in Tumor-Bearing Mice. PTX/Su@CBN were intravenously
injected into tumor-bearing mice. After 12 h, the mice were sacrificed,
and the tissues were removed immediately after perfusion with saline.
Plasma and tissue concentrations of drugs were determined by HPLC
analysis. Free drugs and PTX/Su@CBN with the same dosage were
intravenously injected into mice (∼220 g). Blood samples were
collected in heparinized tubes at each time point, followed by
centrifugation to obtain plasma. The mixture was treated through
sonication with high power to break the structure of the nanoparticles,
resulting in the drug release. Then, the free PTX and Su were
extracted, and the concentrations of PTX and Su were determined by
HPLC analysis. The pharmacokinetic parameters AUC and T1/2
were calculated through WinNonlin.
MDA-MB-231/Taxol tumor-bearing mice were randomly assigned
into five groups (n = 10) and treated with different drugs [saline,
PTX, CBN, PTX+Su, PTX/Su@CBN, PTX+Su (double dose)]. The
tumor volume and body weight of each mouse were monitored every
other 2 days over a period of 28 days. The median survival times of
different groups were calculated using SPSS. To further investigate the
therapeutic effects of these treatments, the mice were sacrificed, and
the tissues were excised for histological examination after treatment.
The tissue sections were stained with hematoxylin and eosin (H&E)
and observed by a BX41 bright-field microscope (Olympus, Japan).
For the immunohistochemistry assay, sample tumors were collected
after treatment for subsequent CD31 immunohistochemistry,
according to the manufacturer’s instructions (KeyGen Biotech,
Nanjing, China).
4.9. Statistical Analysis. Significant differences were determined
using Student’s t test, where differences were considered significant
when p < 0.05. All data are expressed as mean ± standard error of the
mean.
■
ASSOCIATED CONTENT
* Supporting Information
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsami.8b19798.
FTIR and 1H NMR of CP; ζ-potential of CS, CP, and
CBN; diameter changes of PTX/Su@CBN under
different temperatures; plasma concentrations of free
drugs after intravenous injection with PTX/Su@CBN or
PTX+Su; fluorescence images of CS-Flu-incubated
MDA-MB-231 and MSCs; MTT assay of MDA-MB-
231, HepG2, MCF7, and MDA-MB-231/Taxol cell
lines; Western blot analysis and apoptosis of CS
incubated cells; in vivo retention of PTX/Su@CBN
(PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: guyueqingabc@163.com.
ORCID
Yueqing Gu: 0000-0002-6189-8323
Author Contributions
∥
M.Z. and Y.M. contributed equally. The manuscript was
written through contributions of all authors. All authors have
given approval to the final version of the manuscript.
DOI: 10.1021/acsami.8b19798
ACS Appl. Mater. Interfaces 2019, 11, 5851−5861
ACS Applied Materials & Interfaces
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We acknowledge financial support from the National Natural
Science Foundation of China (NSFC 81220108012,
61335007, 81371684, 81000666, 81171395, and 81328012),
the 973 Key Project (2015CB755504), and the Priority
Academic Program Development of Jiangsu Higher Education.
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5861 DOI: 10.1021/acsami.8b19798

ACS Appl. Mater. Interfaces 2019, 11, 5851−5861