Selective uptake of chitosan polymeric micelles by circulating monocytes for enhanced tumor targeting

Carbohydrate Polymers 229 (2020) 115435

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Selective uptake of chitosan polymeric micelles by circulating monocytes for
enhanced tumor targeting

T

Xiqin Yanga, Keke Liana, Yanan Tanb, Yun Zhub, Xuan Liua, Yingping Zenga, Tong Yua,
Tingting Menga, Hong Yuana, Fuqiang Hua,⁎
a
b

College of Pharmaceutical Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, People’s Republic of China
Ocean College, Zhejiang University, 1 Zheda Road, Zhoushan 316021, People’s Republic of China

A R T I C LE I N FO

A B S T R A C T

Keywords:
Chitosan polymeric micelles
Delivery mechanism
Circulating monocytes
Tumor targeting

Micelles are one of the most investigated nanocarriers for drug delivery. In this study, polymeric micelles based

on chitosan were prepared to explore the delivery mechanism which was critical for enhancing tumor targeting
but still remain elusive. The chitosan polymer COSA was synthesized and the polymeric micelles showed good
self-assembly ability, good dispersion stability and low toxicity. After being intravenously administered, the
micelles were selectively taken up by circulating monocytes in a receptor-mediated way (almost 94% uptake in
Ly-6Chi monocytes, below 7% in all other circulating cells) and reach the tumor with the subsequent travel of
these cells. In addition, the micelles in macrophages (differentiated from circulating monocytes) can be exocytosed and subsequently taken up by cancer cells. The delivery mechanism of COSA micelles is directional for
the novel strategies to enhance tumor targeting and the micelles are promising candidates for diseases in which
monocytes are directly implicated.

1. Introduction
In the past decades, nanotechnology is a promising approach for
drug delivery in cancer therapy. Unfortunately, the limited therapeutic
efficacy is a trend found across many nanoparticle formulations
(Bertrand, Wu, Xu, Kamaly, & Farokhzad, 2014). To improve cancer
treatment, worldwide attention is captured on engineering a myriad of
nanocarriers to settle some of great problems in cancer therapy
(Bhushan, 2015; Kaounides, Yu, & Harper, 2007). One such examples is
micelles. Micelles have the advantage of a stealth shell-hydrophobic
core structure, which is capable of encapsulating a variety of waterinsoluble drugs without altering their chemical structures (Gref et al.,
1994; Houdaihed, Evans, & Allen, 2017). Therefore, micelles have been
explored as one of the main nanocarriers for cancer nanomedicine
aimed at delivering drugs to tumors (Cabral et al., 2011; Eetezadi,
Ekdawi, & Allen, 2015; Elvin, Haifa, & Mauro, 2015).
Chitosan (CO), as a kind of natural carbohydrate polysaccharides,
has attracted much attention as an excipient for the preparation of
micelles due to the desirable properties like bioavailability, non-toxicity, biodegradability, stability, and affordability. Stearic acid (SA),

which are compatible with the cellular membrane, are good for promoting cellular uptake. With these in mind, the chitosan polymeric
micelles (COSA), which combine the advantages of CO and SA, were
constructed and expected to show great potential as nanocarriers in

cancer therapy. Unexpectedly, after being intravenously administered,
a considerable proportion of COSA micelles were delivered to the center
of tumor which was frequently the hypoxic/necrotic regions and rendered inaccessible for nanoparticles delivered through the typical mechanism (blood vessels leakiness) (Choi et al., 2007; Owen et al., 2011).
In addition, COSA micelles were mainly accumulated in macrophages.
For this reason, we chose to reveal the COSA micelles delivery mechanism which was critical for overcoming the delivery obstacles but
still remain elusive.
At present, micelles are assumed to be delivered via several targeting mechanisms, particularly extravasation. It is clear that blood
cells including monocytes, macrophages and dendritic cells express
glycoprotein receptors such as mannose receptors, Dectin 1 receptors,
Toll-like receptor 2 and 4 (Liu & Zeng, 2013; Macri, Dumont, Johnston,
& Mintern, 2016). While chitosan, as a cationic polysaccharide, can
bind with the glycoprotein receptors expressed in blood cells, resulting

⁎

Corresponding author.
E-mail addresses: (X. Yang), (K. Lian), (Y. Tan), (Y. Zhu),
(X. Liu), (Y. Zeng), (T. Yu), (T. Meng), (H. Yuan),
(F. Hu).
/>Received 2 September 2019; Received in revised form 26 September 2019; Accepted 3 October 2019
Available online 04 October 2019
0144-8617/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
( />

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4.0 cm−1.
Dynamic light scattering (DLS) was used to determine the particle

size and zeta potential. The transmission electron microscopy (TEM,
JEM-1230, JEOL) was used to observe the morphology of micelles.
Pyrene was used as a probe, and fluorescence spectroscopy was used to
determine the critical micelle concentrations (CMC) of COSA. The
substitution degree of amino groups was also measured by the TNBS
method as previously described (Hu, Zhang, You, Yuan, & Du, 2012).

in the endocytic of chitosan based nanocarriers by these cells (Chen,
2015; Seferian & Martinez, 2000). Besides, following recruitment to
tissues, circulating monocytes can differentiate into macrophages
within the tissues (Frederic et al., 2010; Jakubzick, Randolph, &
Henson, 2017; Warren & Vogel, 1985). Inspired by these facts, we hypothesized that monocytes in blood took up COSA micelles and deposited them in the tumor.
In this study, the characterizations of COSA micelles were investigated. The distribution of COSA micelles in tumor was analyzed
and the amount of tumor macrophages that internalized micelles was
quantified. To reveal the delivery mechanism that directed COSA micelles accumulation in the tumor, the interaction between COSA micelles and blood cells was explored. Particularly, the process by which
COSA (accumulated in monocytes-derived macrophages) reached
cancer cells were further investigated.

2.4. Dispersion stability of COSA
The stability of polymeric micelles in serum or in different temperatures was investigated by determining the particle size of micelles
(Lu, Owen, & Shoichet, 2011; Tan et al., 2019). Briefly, COSA micelles
in deionized water at the concentration of 0.5 mg mL−1 were prepared.
To investigate the stability of COSA micelles in serum, the prepared
micelles aqueous solution was supplemented with 10% fetal bovine
serum (FBS, v/v), and then the particle size of micelles was determined
by DLS at predetermined time points. To investigate the stability of
COSA micelles in different temperatures, the prepared micelles aqueous
solution were stored at 4 °C, 25 °C and 37 °C, and then the particle size
of micelles in different temperatures was determined by DLS at predetermined time points.
In addition, to investigate the stability of drug-loaded micelles,

Doxorubicin base (DOX) was used as the model drug to test the in vitro
drug release from micelles in phosphate buffered saline (PBS, pH 7.4).
To obtain DOX-loaded micelles, 2 mg/mL of DOX/DMSO was added
dropwise into a 2 mg/mL COSA aqueous solution and stirred for 2 h.
Then, the mixture solution was dialyzed in DI water overnight and then
centrifuged at 8000 rpm for 10 min. The supernatant was collected as
the COSA/DOX micelles. To investigate the in vitro release of DOX from
COSA/DOX micelles, the COSA/DOX micelles was dialyzed against PBS
in an incubator shaker with horizontal shaking (75 rpm) at 37 °C.
COSA/DOX aqueous solution (1.0 mL) was dialyzed in 20.0 mL of PBS
(MWCO: 3.5 kDa). At predetermined time points, all of the medium
outside of the dialysis bag was acquired and replaced with fresh PBS.
The DOX concentration of all the samples was determined with a
fluorescence spectrophotometer, and the assays were repeated three
times.

2. Materials and methods
2.1. Reagents
95% deacetylated chitosan (CO, Mw = 450 kDa, Yuhuan, China)
was degraded with enzymes to acquire low molecular weight CO
(Mw = 19.9 kDa). Stearic acid (SA) and D-mannose were supplied by
Shanghai Chemical Reagent Co, Ltd. 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC) were purchased from Shanghai Medpep Co,
Ltd. Fluorescein isothiocyanate (FITC), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), β-glucan, Lipopolysaccharide
(LPS) and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were obtained
from Sigma-Aldrich Inc. 1, 1′-dioctadecyl-3, 3, 3′, 3′-tetramethyl indotricarbocyanine iodide (DiR) was obtained from Life Technologies
(Carlsbad, CA, USA). PE-labeled anti-Gr-1, PE/Cy7-labeled anti-F4/80,
APC-labeled anti-Ly6C, anti-αvβ3 and Percp/Cy5.5-labeled anti-CD11b
were purchased from Biolegend (San Diego, CA). Other chemicals used
were of chromatographic grade or analytical grade.
2.2. Cell culture and animals

4T1 and RAW264.7 cells were purchased from the Cell Bank of
Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy
of Sciences (Shanghai, China) and cultured in DMEM supplemented
with 10% fetal bovine serum (FBS, v/v), 10000 U mL−1 streptomycin
and 10000 U mL−1 penicillin at 37 C in a humidified incubator with 5%
CO2. 6–8 week-old female BALB/c mice were purchased from the
Shanghai Silaike Laboratory Animal Limited Liability Company. All
animal experiments were carried out in compliance with the Zhejiang
University Animal Study Committee’s requirements for the care and use
of laboratory animals in research.

2.5. The distribution of COSA in tumor
To prepare the tumor-bearing mice models, 1 × 105 4T1 cells was
implanted to the right mammary gland of 6–8 week-old female BALB/c
mice. To detect the in vivo distribution of COSA micelles, near infrared
dye DiR was encapsulated in COSA micelles according to the preceding
protocol. DiR loaded COSA micelles were intravenously injected into
the tail vein of tumor bearing BALB/c mice. The mice were imaged at
predetermined time points by a Maestro in vivo Imaging System (CRI
Inc., Woburn, MA). After 24 h, the mice were sacrificed, followed by
collection of heart, liver, spleen, lung, kidney and tumor. The fluorescence images of these tissues were obtained by using a Maestro in vivo
Imaging System (CRI Inc., Woburn, MA).
To investigate the distribution of COSA in tumor, FITC labeled
COSA was prepared as previously described (Zhu et al., 2018). Briefly,
2.0 mg/mL FITC (C21H11NO5S) ethanol solution was added dropwise
into 1.0 mg/mL COSA aqueous solution (COSA: FITC = 1:1, mol: mol),
then kept stirring overnight and dialyzed (MWCO = 7 000 Da) against
pure water. After 24 h of injection of FITC-COSA micelles, the tumors
were collected. Then the tumors were sectioned and stained with antibodies to examine by confocal laser scanning microscopy.

2.3. COSA polymer synthesis and characterization
The COSA polymer was synthesized in the presence of EDC. Briefly,
20 mL of ethanol was used to dissolve stearic acid (SA) and EDC, and
the mixed solution was stirred for 1 h at 60 °C. Then, 20 mL of deionized
water (DI water) was used to dissolve 0.3 g CO, and the solution was
incubated at 60 °C for 20 min. Then, the mixed solution was added into
the CO solution. After stirring for another 14 h, the reaction solution
was collected and dialyzed against DI water for 2 days. Finally, the
products were collected by lyophilization after three purifications with
ethanol.
1
H NMR spectroscopy was used to elucidate the structure of COSA.
Briefly, 5 mg COSA was dissolved in 0.5 mL D2O. The samples were
measured by 1H NMR spectrometer (AC-80, Bruker Biospin, Germany).
In addition, fourier-transform infrared spectroscopy (FTIR) was used to
confirm the structure of COSA. The samples were sliced by KBr tableting and examined using a Bruker Tensor 27 model infrared spectrometer with a scan range of 400–4000 cm–1 and a resolution of

2.6. Quantification of macrophages that internalized COSA micelles
To quantify the accumulation of FITC-COSA in the tumor cell subsets, tumors were isolated at 4 h, 18 h and 24 h after intravenous
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injection of FITC-COSA. The samples were grinded and filtered using a
cell strainer (Qin et al., 2018). The obtained single cell suspension was
centrifuged at 350 g for 10 min at 4 °C. The cell pellets were washed
using PBS buffer. Before antibody labeling, all the cells were pre-incubated with anti-CD16/CD32 mAb. Then cells were labelled with the

antibodies (anti-αvβ, PE/Cy7-labeled anti-F4/80 and Percp/Cy5.5-labeled anti-CD11b) and analyzed by flow cytometry.

COSA/Fe2O3.
Cells were exposed to COSA/Fe2O3 for different duration and collected, washed twice with PBS and centrifuged at 350 × g for 10 min.
The cells were pre-fixed with formaldehyde overnight and then dehydrated with increasing concentrations of ethanol (50, 60, 70, 80, 90,
and 100%) for 15 min each, and stained with 2% uranyl acetate in 70%
ethanol overnight, then embedded in Epon. Ultrathin sections of macrophages were cut using a sliding ultramicrotome and the thin sections
were supported by copper grids and observed using a TEM system.

2.7. The interaction between COSA micelles and blood cells
Mouse whole blood from eyeball was drawn into tubes containing
heparin sodium at 4 h, 18 h and 24 h after intravenous injection of
FITC-COSA. Red blood cell lysis buffer (Biolegend, San Diego, CA) was
added to tubes and vortexed for several seconds. After incubated 15 min
at room temperature, blood samples were centrifuged at 350×g for
10 min. Before antibody labeling, the remaining immune cells were preincubated with anti-CD16/CD32 mAb and then stained with specific
antibodies (PE-labeled anti-Gr-1, APC-labeled anti-Ly6C and Percp/
Cy5.5-labeled anti-CD11b) for flow cytometry analysis.

2.11. Cancer cells uptake of COSA excreted by macrophages
Three-dimensional (3D) models were used to investigate whether
the excreted COSA would indeed be taken up by cancer cells. To construct 3D models, 4T1 cells, RAW264.7 cells and 3T3 cells were mixed
at the same number. 2 × 105 mixed cells were seeded in 96-well plates
pretreated with 2% agarose to construct the cell spheres as previously
described (Yang et al., 2018).
After 3days, the cell spheres were divided into two groups. One
group was added with FITC-COSA and incubated for different duration.
Another group was treated with FITC-COSA for 24 h and washed with
PBS and then incubated with fresh culture medium (without FITCCOSA) for different duration. The group without culture medium replacement was used as control. All the cell spheres were collected at
determined time points and digested with enzymes to obtain single cell

suspension. Then the cells were pre-incubated with anti–CD16/CD32
mAb and labelled with antibodies (anti-αvβ, PE/Cy7-labeled anti-F4/
80 and Percp/Cy5.5-labeled anti-CD11b) for flow cytometry analysis.

2.8. Cellular uptake mechanisms
To reveal the cellular uptake mechanisms of COSA, monocytes from
peripheral blood were isolated according to the previous protocol
(Macparland et al., 2017). Whole blood was collected into tubes containing heparin sodium. Peripheral blood mononuclear cells (PBMC)
were isolated from blood by gradient centrifugation in Ficoll-paque Plus
(GE Healthcare). To purify monocytes from PBMC, the negative-selection-based monocyte isolation kit II (Miltenyi Biotec, Auburn, CA) was
used, and then the purified monocytes were suspended in RPMI-1640
medium supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin/streptomycin.
The collected monocytes were respectively pre-incubated with
specific ligands (mannose, β-glucosan and lipopolysaccharide) of glycoprotein receptors (mannose receptors, Dectin 1 receptors and Tolllike receptor 2 and 4). After removal of inhibitor solutions, cells were
washed twice with PBS and then incubated with 20 μg/mL of FITCCOSA for another 10 min. The cells without inhibitor treatment were
used as control. The cellular uptake of FITC-COSA was measured by
flow cytometry.

2.12. Statistical analysis
All the data were reported as mean ± SD. Differences between
groups were tested using the two-tailed student’s t-test. The differences
with p < 0.05 were considered statistically significant.
3. Results and discussion
3.1. The synthesis and characteristics of COSA
The polymer COSA was synthesized by reactions between chitosan
(CO) amine groups and stearic acid (SA) carboxyl groups in the presence of EDC (Fig. 1A). The COSA structure was confirmed based on the
1
H NMR and FTIR spectroscopy. As shown in Fig. 1B, new peaks at
approximately 1.00 ppm indicated the synthesis of COSA. In addition,

the absorption peaks in FTIR spectra at about 3091 cm−1, 1645 cm−1,
1521 cm−1 and 1288 cm−1 which resulted from amide bonds
(eCONHe) indicated the amidation reaction between CO and SA
(Fig. 1C).
The particle size and zeta potential of COSA were investigated
(Fig. 2 and Table 1). The average size of COSA was determined as
85.93 ± 0.68 nm and COSA micelles showed positive zeta potential
(23.17 ± 0.90 mv) which was good for cellular uptake. In addition, the
spherical morphology of micelles was presented in the TEM images
(Fig. 2A). The degree of amino substitution (SD%) of COSA was measured as 7.03% (Table 1) and the polymer COSA could self-assemble
into nano-scaled micelles. Fig. 2B showed the variation of the I1/I3 ratio
against the logarithmic concentration (Log C) of COSA. The inflection
point corresponded to the critical micelle concentration (CMC) value
which was 52.02 μg/mL. The relatively low CMC values indicated the
good self-assembly ability and structural stability of COSA micelles.
The stability of polymeric micelles in serum is critical for in vivo
applications (Cao et al., 2013). Therefore, the particle size of COSA
micelles in solution with 10% serum (v/v) was investigated. The adsorption of blood proteins onto the micelles surface can contribute some
changes in particle size of micelles and the protein-particle interactions
can increase the particle size by 3–35 nm (Dobrovolskaia et al., 2009;

2.9. Cytotoxicity evaluation
The cytotoxicity of COSA was evaluated by MTT assay (Cheng et al.,
2017). Briefly, 6 × 103 monocytes were seeded in 96-well plates. After
12 h incubation, a series of concentrations of COSA were added to the
cells and co-cultured for another 48 h. 15 μL MTT solution (5 mg/mL)
was added to each well and incubated for another 4 h. After removing
the medium, the cells were incubated with 200 μL Dimethyl sulfoxide
(DMSO) in an automated shaker. Finally, the absorbance of each well at
570 nm was read by an automatic reader (BioRad, Model 680, USA).

2.10. Cellular uptake and exocytosis of COSA
To determine in vitro cellular uptake, 1 × 105 RAW264.7 cells were
seeded in 6-well plates and incubated in 5% CO2 for 8 h. Different
concentration of FITC-COSA micelles were added to each well and incubated with cells. Then the cells were rinsed with PBS, collected and
analyzed by flow cytometry.
Transmission electron microscope (TEM) was used to investigate the
cellular uptake and exocytosis of COSA. Firstly, Fe2O3 loaded micelles
were prepared as previous description (Tan et al., 2017). Briefly, Fe2O3
nanoparticles solution (5 nm, 5 mg/mL in oleic acid) was added to
COSA micelles under being sonicated with a probe type ultrasonicator
(JY92-II, Ningbo scientz biotechnology Co., Ltd., China) at 100 W for
30 min. The solution was centrifuged at 1500×g for 20 min to obtain
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Fig. 1. Synthesis of COSA. (A) The synthetic scheme of COSA. (B) The 1H NMR spectra of COSA. (C)The FTIR spectra of COSA.

obvious fluorescent signals, which indicated the ability of COSA micelles to accumulate at the tumor site.
To effectively kill cancer cells, the drug delivery system should be
targeted to cancer cells. In order to investigate whether the COSA micelles were exactly delivered to cancer cells, the tumors were collected
for immunofluorescent analysis at 24 h after intravenous administration
of COSA. As shown in Fig. 4A, a considerable proportion of micelles
were delivered to the center of tumor which is frequently the hypoxic/
necrotic regions and rendered inaccessible for nanoparticles delivered
through the typical mechanism (blood vessels leakiness). In addition,
macrophages (in red) showed much more overlaps with micelles (in

green) compared with cancer cells. As shown in Fig. 4B, up to 68.10%
of tumor macrophages internalized COSA at 24 h. While as the targeted
cells, cancer cells did not actively interact with micelles and the proportion was less than 3% which can be negligible when compared with
macrophages. The result indicated that macrophages were the key cells
in the sequestration of intravenously administrated micelles.

Hak Soo et al., 2007; Monopoli et al., 2011). As shown in Fig. 3, the
particle size of COSA micelles in solution with serum was larger than
that without serum, which resulted from the interactions between micelles and blood proteins. As expected, with or without serum, there
was no obvious change in COSA micelle diameters at different time
points. Besides, the stability of COSA in different temperatures was also
tested. As shown in Fig.S1, no obvious change of the micelle particle
size was observed in different temperatures, which also indicated the
stability of COSA micelles. In addition, to investigate the stability of
drug-loaded micelles, DOX was chosen as the model drug to test the in
vitro drug release from COSA/DOX micelles in PBS (pH 7.4). The in
vitro drug release curves in Fig. S2 showed that less than 25% of DOX
was released from COSA/DOX micelles after 72 h, illustrating that the
drug-loaded micelles were very stable under physiological conditions.
All these results suggested the good dispersion stability of COSA which
was desirable for in vivo applications as nanocarriers.

3.2. Distribution of COSA in tumor
3.3. Selective uptake of COSA by circulating monocytes
To effectively inhibit tumor growth, drug delivery system should be
targeted to tumors. The in vivo distribution of COSA micelles was
macroscopically investigated. As shown in Fig. S3, the tumor showed

The hypoxic and necrotic regions of tumor are accessible for
monocytes (Anselmo et al., 2015; Murdoch, Giannoudis, & Lewis, 2004)

Fig. 2. Characterizations of COSA. (A)The size distribution and transmission electron microscopy (TEM) image of COSA. The image represented one of three
experiments with similar results. (B)The critical micelle concentration (CMC) of COSA.
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Table 1
Characterizations of COSA.
Micelles

Diameter (nm)

PDI

Zeta potential (mv)

CMC (μg/mL)

SD%

COSA

85.93 ± 0.68

0.13 ± 0.04

23.17 ± 0.90

52.02 ± 5.67

7.03 ± 0.84

Data represent the mean ± standard deviation (n = 3).

Jakubzick et al., 2017; Warren & Vogel, 1985). It was found that a
considerable proportion of COSA micelles were delivered to the center
of tumor and the micelles were mainly accumulated in macrophages.
Inspired by these facts, we hypothesized that circulating monocytes
took up COSA micelles and deposited them in tumor.
Intrigued by the hypothesis, we used highdimensional 14-parameter
(12-colour) FACS analysis to identify the subset(s) of immune cells that
took up COSA in blood. Interestingly, of all the myeloid cells, only a
single monocyte subset—Ly-6Chi monocytes—displayed substantial
COSA uptake. As shown in Fig. 5, up to 94.30% of CD11b—Ly-6Chi
monocytes took up COSA micelles at 18 h after intravenous injection. In
contrast, neutrophils, which expressed higher levels of surface CD11b
and Gr-1, took up negligible amounts of COSA (less than 2% of neutrophils took up the micelles at 18 h). Similarly, the cellular uptake of
COSA by other circulating white blood cells was also negligible in
comparison with Ly-6Chi monocytes (Fig. S4). The result demonstrated
that COSA micelles can be internalized by circulating cells in blood and
showed high selectivity for Ly-6Chi monocytes.
It was confirmed that COSA micelles were selectively taken up by
circulating monocytes, which would raise the question: what was the
mechanism of cellular uptake by circulating monocytes? To answer the
question, we investigated the mechanism of cellular uptake and found

Fig. 3. Particle size of COSA micelles with/without serum at different time
points. Data was expressed as mean ± standard deviation (n = 3).

but inaccessible for nanoparticles delivered through blood vessels leakiness (Choi et al., 2007; Owen et al., 2011). Tumors can generate
molecular gradients that attract circulating monocytes which can differentiate into macrophages within the tissues (Frederic et al., 2010;

Fig. 4. Macrophages internalized COSA micelles. (A) Immunofluorescent staining of tumor. COSA was labeled by FITC (green), cell nucleus were labeled by DAPI
(blue), macrophages were labeled by anti-F4/80 antibodies (red) and cancer cells were labeled by anti-αvβ3 antibodies (red). The images represented one of three
experiments with similar results. (B) Flow cytometry plots showing FITC-COSA selective accumulation in tumor macrophages. Flow cytometry plot data were
representative of n = 3 mice per group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. Selective uptake of FITC-COSA by circulating monocytes. Flow cytometry plots showing selective uptake of FITC-COSA into Ly-6Chi monocytes. Blood was
harvested at 4 h, 18 h and 24 h after intravenous injection of FITC-COSA and stained with specific antibodies for flow cytometry analysis. Flow cytometry plot data
were representative of n = 3 mice per group.
Fig. 6. The cellular uptake of COSA by monocytes. (A) The cellular uptake of COSA by
monocytes. “F” represents FITC-COSA, “M” represents mannose, “L” represents lipopolysaccharide (LPS) and “β” represents β-glucan.
Data was expressed as mean ± standard deviation (n = 3). (*p < 0.05, **p < 0.01). (B)
In vitro cytotoxicity against monocytes after
treatment with COSA for 48 h. Data was expressed as mean ± standard deviation
(n = 6).

selectively taken up by circulating monocytes and reach the tumor with
the subsequent travel of these cells. The delivery mechanism was independent of the blood vessels leakiness, and could afford new strategies to improve tumor targeting by increasing monocyte homing to
tumors. In addition, monocytes can easily enter and travel throughout

tumors. Thus, the hypoxic and necrotic regions of tumor, which were
rendered inaccessible for nanoparticles delivered through blood vessels
leakiness, can now be reached with this delivery mechanism.
Moreover, we quantified the monocytes in tumor and found that
monocytes continued to home to tumor over time (Fig. 7B). To investigate whether this mechanism (targeting tumors via monocyte) has
a substantial effect on the total amount of COSA accumulating in tumor,
we assessed the effect by computing the proportion of COSA in tumor
contained within monocytes and comparing this with the total amount
of COSA in the tumor (which may arrive as a consequence of other
targeting mechanisms such as extravasation (Smith et al., 2013)). As
shown in Fig. 7C, ∼41% of COSA in tumor on day 1 were due to
monocyte delivery and the proportion was increased to nearly 50% on
day 2, which suggested that this delivery mechanism can account for a
considerable proportion of COSA delivered to tumors.

that the cellular uptake of COSA was obviously inhibited by mannose,
especially in the group pretreated with mannose and β-glucan simultaneously (Fig. 6A). The result indicated that COSA micelles can be
internalized mainly by mannose receptor-mediated mechanism and
secondly by Dectin 1 receptor-mediated mechanism. Beside, we investigated the cytotoxicity of COSA against monocytes with MTT assay.
The result revealed that COSA micelles showed low toxicity to monocytes (Fig. 6B), which can ensure the intrinsic homing property of
monocytes.
3.4. The delivery of COSA to tumor by circulating monocytes
After confirming that COSA can be selectively took up by circulating
monocytes in a receptor-mediated way, we then investigated whether
monocyte would indeed deposited COSA in tumor. When COSA micelles were intravenously injected, APC-labeled anti-mouse Ly-6C Abs
(a specific marker of monocytes) was subcutaneously injected around
the tumor to stain monocytes homing to tumor from the blood (Chu,
Dong, Zhao, Gu, & Wang, 2017). As shown in Fig. 7A, monocytes (in
red) showed lots of overlaps with micelles (in green), which indicated
that monocytes can home to tumor after taking up COSA in blood. It can

be concluded that the intravenously administered micelles were
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Fig. 7. COSA-laden monocytes enter the tumor. (A) Immunofluorescent staining of tumors. COSA was labeled by FITC (green), cell nucleus were labeled by DAPI
(blue) and monocytes were labeled by anti-Ly6C antibodies (red). (B) Monocytes accumulating in tumors. (C) Relative amounts of COSA that were ferried in the
tumor via monocytes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

we then used 3D cell models to further investigate whether cancer cells
would took up COSA exocytosed by macrophages. One group of cell
spheres was pretreated with COSA for 24 h and then the exposure solution was replaced with fresh culture medium (without COSA). The
group of cell spheres, without culture medium replacement, was used as
control.
As shown in Fig. 8C, the total amount of macrophages taking up
COSA in control group (the first row) was increased within 24 h and
after that it was decreased. While in another group (the second row),
the amount of macrophages taking up COSA was decreased over time,
which resulted from the continued exocytosis. In contrast, the situation
was different for cancer cells (Fig. 8D). In control group (the first row),
the total amount of cancer cells that took up COSA showed continued
increase during 48 h, although much lower than that of macrophages.
Similarly, the amount in another group (the second row) also increased
over time. The uptake of COSA by cancer cells in control group resulted
from remaining COSA in culture medium or the exocytosed COSA by
macrophages. Once replacing the exposure solution with fresh culture
medium without COSA, the increased uptake by cancer cells was only

due to the exocytosed COSA. It can be concluded that macrophages can
exocytose the internalized COSA and the excreted COSA would be taken
up by cancer cells. This interaction between macrophages and cancer
cells showed great significance and could demonstrate novel ways to
influence cancer cells for cancer therapy.

3.5. The interaction between macrophages and cancer cells
COSA micelles were selectively taken up by circulating monocytes.
Subsequently, the COSA-loaded monocytes were recruited to tumors
and became the source of tumor-infiltrating macrophages. We then
asked how COSA micelles which located in macrophages were delivered to cancer cells to realize therapeutic efficacy. Based on previous
study, macrophages were able to exocytose the internalized nanoparticles (Jiang et al., 2017; Oh & Park, 2014). Accordingly, we hypothesized that the internalized COSA could be excreted by macrophages.
First, flow cytometry was used to investigate the COSA endocytosis
of macrophages exposed to 5, 10, 20 and 40 μg/mL COSA. As shown in
Fig. S5, the cellular uptake became saturated at the COSA concentration
of 20 μg/mL. Then, we further determined the cellular uptake of COSA
over time in 20 μg/mL COSA exposure. It was surprising that the
amount of cells taking up COSA obviously decreased at 36 h and 48 h in
comparison with that at 24 h (Fig. S6A). These data, although limited,
partly suggested the occurrence of COSA exocytosis. In addition,
transmission electron microscope (TEM) images revealed the endocytosis process of COSA, which began with the interaction between
COSA and cell membrane (Fig. 8A). However, when we replaced the
exposure solution with fresh culture medium (after 4 h exposure), the
amount of cells that took up COSA significantly decreased with prolonged incubation time (Fig. S6B) and the fluorescence intensity of the
culture medium increased over time (Fig. S7), which suggested the
occurrence of COSA exocytosis by macrophages. Moreover, the TEM
images also showed that the cells exocytosed internalized contents via
vesicle-related secretion (Fig. 8B).
Having shown that macrophages would excrete internalized COSA,

4. Conclusions
In summary, the chitosan polymer COSA was synthesized and the
polymeric micelles showed good self-assembly ability, good dispersion
stability and low toxicity. After being intravenously administrated, the
7


Carbohydrate Polymers 229 (2020) 115435

X. Yang, et al.

Fig. 8. Cancer cells uptake of COSA secreted by macrophages. (A) Typical TEM images showing endocytosis. (B) Typical TEM images showing cellular exocytosis.
The images represented one of three experiments with similar results. (C) Cellular uptake of COSA by macrophages at different time points. (D) Cellular uptake of
COSA by cancer cells at different time points. Gating strategy: cells taking up COSA were identified first from all the cells based on FITC staining. Macrophages were
then identified from FITC+ cells by F4/80+ gating and cancer cells were identified from FITC+ cells by αvβ3+ gating. Flow cytometry plot data represented one of
three experiments with similar results.

to tumors. In addition, the internalized COSA can be exocytosed by
macrophages and then taken up by cancer cells. This interaction between macrophages and cancer cells would demonstrate novel ways to
influence cancer cells for cancer therapy. Overall, the delivery mechanism identified in this work is directional for enhancing tumor

COSA micelles were selectively taken up by nearly 94% of circulating
monocytes (Ly-6Chi monocytes) in a receptor-mediated way. The subsequent travel of these cells resulted in a considerable proportion of
COSA accumulation in tumor. This delivery mechanism can afford new
strategies to improve tumor targeting by increasing monocytes homing
8


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X. Yang, et al.

targeting and the COSA micelles exhibited great potential in cancer
therapy, particularly in the treatment of diseases in which monocytes
are directly implicated.

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Declaration of Competing Interest
The authors declare no conflicts of interest.
Acknowledgement
This work was National Nature Science Foundation of China (Grant
Nos. 81773648).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
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