Carbohydrate Polymers 222 (2019) 115004

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

Review

Advances in chitosan-based nanoparticles for oncotherapy T

a,b a,b,⁎ a,b a,b a,b a,b,⁎
Enhui Zhang , Ronge Xing , Song Liu , Yukun Qin , Kecheng Li , Pengcheng Li
a
Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
b
Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, No. 1 Wenhai Road, Qingdao 266237, China

A R T I C LE I N FO A B S T R A C T

Keywords: Chitosan has attracted considerable attention as an anti-tumor drug carrier material in recent years, which is due
Chitosan to its biocompatibility and biodegradability, as well as the simple and mild preparing techniques of drug-loaded
Nanoparticle nanoparticles. Chitosan-based nanoparticles can deliver various anti-tumor agents to specific tumor tissues by
Anti-tumor passive and active targeting mechanisms, including traditional chemotherapeutic agents, DNA or siRNA, pro-
Drug delivery
teins, photosensitizers and so on. In this review, we summarized the factors affecting the anti-tumor efficacy of
chitosan-based nanoparticles, to aid exploring the function-structure relationship. The recent studies on chit-
osan-based nanoparticles for oncotherapy were highlighted, including their structures, properties and phar-
macological effects. Finally, we offered our perspectives on the challenges and future development of this area.

1. Introduction
Chitosan, a unique alkaline polysaccharide in nature, is derived
from chitin by a process of deacetylation, comprising of β-(1→4)-linked
D-glucosamine and N-acetyl-D-glucosamine units. It was firstly prepared
by Charles Rouget in 1859, who boiled chitin in the presence of con-
centrated potassium hydroxide to obtain chitosan (Rouget, 1859). The
modified chitin was named “chitosan” after 35 years later by Felix
Hoppe Seyler (Hoppe-Seyler, 1894) and the structure of chitosan was
finally resolved in 1950 (Baldrick, 2010). In the wake of development
of drug delivery systems in 1970s, chitosan continuously attracted the
attention as a promising candidate for the drug carrier material due to
its favorable properties such as biocompatibility, biodegradability, non-
toxicity and low immunogenicity (Garcia-Fuentes & Alonso, 2012).
The safety is always the priority concern for a qualified pharma-
ceutical excipient, and chitosan is commonly considered to be a safe
and biocompatible material. The oral administration of chitosan with
dose of 6.75 g for 8 weeks induced no observable effects on serum vi-
tamin, carotenes levels or other safety parameters in human volunteers
(Tapola, Lyyra, Kolehmainen, Sarkkinen, & Schauss, 2008). Further-
more, intravenous injection of chitosan with 7.1–8.6 mg/kg/day in
rabbits had no obvious effects in 65 days (Kean & Thanou, 2010). In a
phase II clinical trial, percutaneous injection of holmium-166/chitosan
complex for the treatment of hepatocellular carcinoma on patients in-
duced very little discomfort and disruption of daily activities for pa-
tients (Kim et al., 2006). From most studies, chitosan exhibited non- or
minimal toxicity, which made it widely regarded as a safe material in
drug delivery (Smith, Perelman, & Hinchcliffe, 2014).
One of the most essential properties of chitosan is the cationic
nature, which exerts advantages as a desirable drug carrier due to the
enhanced adhesion to the negatively charged mucosal surface by elec-
trostatic interaction, resulting in the improvement on drug inter-
nalization into targeted cells (He, Davis, & Illum, 1998; Lee et al., 2014;
Narayanan, Jayakumar, & Chennazhi, 2014). On the other hand, the
cationic property of chitosan also makes it potential to complex abun-
dant anionic nucleic acid, protecting from degradation by nuclease in
serum, achieving efficient gene therapy (Rudzinski & Aminabhavi,
2010).
However, chitosan is only soluble in an aqueous acidic medium but
not at neutral pH, which is a major hurdle for its application. The
abundant amino and hydroxyl groups on chitosan backbone represent
target moieties for chemical modifications to improve the aqueous

Abbreviations: Mw, molecular weight; DD, degree of deacetylation; PEG, poly(ethylene glycol); FITC, fluorescein isothiocyanate; EPR, enhanced permeability and
retention; APBA, N-3-acrylamidophenylboronic acid; MRP, multidrug resistance-associated protein; TPP, tripolyphosphate; mPEG, methoxy PEG; EGCG, epigallo-
catechin-3-gallate; Ce6, chlorin e6; CT, X-ray computed tomography; MR, magnetic resonance; PAMAM, polyamidoamine; ZWC, zwitterionic chitosan; VEGF,
vascular endothelial growth factor; MDR, multi drug resistance; P-gp, P-glycoprotein; mTERT, mouse telomerase reverse transcriptase; PEI, poly(ethylene imine);
PLR, poly-L-arginine; PLL, poly-L-lysine; PAH-Cit, poly(allylamine hydrochloride)-citraconic anhydride; γ-PGA, poly(γ-glutamic acid); PpIX, protophorphyrin IX;
DEAP, 3-diethylaminopropyl isothiocyanate; CpG, cytosine-guanine; IL-12, interleukin-12
⁎
Corresponding authors at: Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences,
Qingdao 266071, China.
E-mail addresses: xingronge@qdio.ac.cn (R. Xing), pcli@qdio.ac.cn (P. Li).

https://doi.org/10.1016/j.carbpol.2019.115004
Received 21 February 2019; Received in revised form 27 May 2019; Accepted 17 June 2019
Available online 19 June 2019
0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
E. Zhang, et al.
Fig. 1. Structural features of chitosan and their influences on chitosan-based nanoparticles for oncotherapy.
solubility, as well as endow chitosan with new functionalities, such as
targeted drug delivery and environment-sensitive drug release, enhan-
cing the therapeutic effects and reducing side effects. The most common
chitosan derivatives used for drug carriers include thiolated chitosan,
trimethyl chitosan, carboxymethyl chitosan, glycol chitosan and so on
(Sarmento & Bruno, 2012).
In this review, we first described the influence of chitosan structural
features on anti-tumor efficacy of nanoparticles and the characteriza-
tion of medical grade chitosan. Furthermore, we highlighted recent
research advances in anti-tumor chitosan nanoparticles, categorized by
the types of delivered drugs. Finally, we made a brief comment on the
future development and challenges of chitosan nanoparticles for on-
cotherapy (Tables 1–3).
2. Factors affecting the efficacy of chitosan-based nanoparticles
The structural features of chitosan and its derivatives, such as mo-
lecular weight (Mw), degree of deacetylation (DD), degree and site of
substitutions, have great impact on physiochemical properties as well
as the anti-tumor efficacy of chitosan-based nanoparticles (Fig. 1).
Choosing appropriate chitosan is a priority issue for rational designed
nanoparticles. The Mw of commercial chitosan ranges from 10 kDa to 2
000 kDa and the DD is between 60% and 100%. In 2003, Huang et al.
firstly conducted a comprehensive assessment of effects of Mw and DD
on cellular uptake and cytotoxicity of chitosan nanoparticles. The Mw
of chitosan in this study ranged from 10 kDa to 213 kDa and DD of 46%
to 88%. The cellular uptake results showed positive correlation with
zeta potential of nanoparticles, and the uptake ratios reduced by 26%
and 41% with the decrease of Mw and DD, respectively. The decrease of
DD also attenuated the cytotoxicity of nanoparticles against A549 cells,
but the impact of Mw was smaller (Huang, Khor, & Lim, 2004). Fur-
thermore, Park et al. evaluated the effect of Mw (20 kDa, 100 kDa,
250 kDa) on the physiochemical properties and in vivo activities of
glycol chitosan nanoparticles. The size and zeta potential did not differ
significantly, but nanoparticles with Mw 250 kDa exhibited longer cir-
culation time and better tumor selectivity (Park et al., 2007). On the
contrary, nanoparticles fabricated by amphiphilic chitosan derivatives
with low Mw (5 kDa, 10 kDa, 20 kDa) also showed desirable in vitro
cytotoxicity against HepG2 cells. However, no sufficient in vivo tests
were reported to confirm the efficacy of nanoparticles based on chit-
osan with low Mw (Zhang, Huo, Zhou, Yu, & Wu, 2009). Based on
above studies, Mw is not a key factor that influences the physiochemical
characteristics of chitosan nanoparticles, but relatively high Mw en-
sures the longer circulation time of nanoparticles in bloodstream, which
subsequently guarantees the high tumor selectivity. High DD produces
high surface charge density of nanoparticles, resulting in enhanced
cellular uptake and anti-tumor efficacy.
As for DNA, the binding ability of chitosan with DNA and trans-
fection efficiencies changes along with chitosan Mw. However, di-
vergent results were observed in exploring the influence of Mw on
transfection efficiency of chitosan/DNA complexes. In some studies,
2
Carbohydrate Polymers 222 (2019) 115004
higher Mw (> 200 kDa) was better (Huang, Fong, Khor, & Lim, 2005;
Weecharangsan et al., 2008) but lower Mw (< 10 kDa) had success in
other cases (Köping-Höggård et al., 2004; Sato, Ishii, & Okahata, 2001).
There is a subtle balance between stability and dissociation inside cells
influenced by chitosan Mw. Higher Mw increases the stability of chit-
osan/DNA complexes but delayed their dissociation and subsequent
gene expression in cells, while lower Mw has the opposite effects. The
balance also exists in chitosan/siRNA complexes, but there are few
reports on successful delivery of siRNA based on chitosans with low Mw
(< 10 kDa). A study reported that chitosan/siRNA nanoparticles with
low Mw (below 10 kDa) had almost no gene silencing efficacy, while
those with higher Mw (114 and 170 KDa) exhibited effective silencing
efficacy, which was comparable to commercial reagents (Liu et al.,
2007). Furthermore, complete binding of siRNA into chitosan nano-
particles (weight ratio of chitosan and siRNA, 50:1) could be observed
with chitosans Mw of 25 kDa at least but not those with Mw below
10 kDa (Fernandes et al., 2012).
The DD of chitosan determines the positive charge density and the
binding capacity with DNA/siRNA. Higher DD is conducive to escape
from the endo-lysosomal compartment for transfection (Huang et al.,
2005; Liu et al., 2007). DD of more than 46% is a basic requirement for
obtaining stable chitosan-based DNA complexes (Mao, Sun, & Kissel,
2010). Kiang et al. reported the disparity of in vitro and in vivo trans-
fection efficiency of chitosan/DNA complexes with large Mw chitosan
(390 kDa) and DD of 90%, 70% and 62%. The decrease of DD induced
the decrease of in vitro gene expression due to the instability of com-
plexes, but this instability, in turn, increased the in vivo gene expression
(Kiang, Wen, Lim, & Leong, 2004). As for siRNA, Malmo et al. proved
that fully N-acetylated chitosan had superior silencing efficacy com-
pared to partially N-acetylated chitosan (Malmo, Sørgård, Vårum, &
Strand, 2012). Most cases suggested that DD of more than 80% is
mandatory for efficient delivery of DNA/siRNA and transfection effi-
ciency (Alameh et al., 2018; Buschmann et al., 2013; Liu et al., 2007;
Ragelle, Vandermeulen, & Préat, 2013).
In addition, the degree and site of substitutions grafted onto chit-
osan are also important factors in synthesis of chitosan nanoparticles. Li
and co-workers synthesized a series of N-deoxycholic acid-N,O-glycol
chitosans with different degree of substitution of glycol and deoxycholic
acid group. The increase of deoxycholic acid, the hydrophobic group,
resulted in the increase of particle size, entrapment efficiency and drug
loading content of paclitaxel significantly (Li et al., 2010). Furthermore,
it was found that the content of hydrophobic groups could influence the
stability and deformability of nanoparticles based on N-5β-cholanic
acid-glycol chitosan (CNPs). Despite that the prepared nanoparticles
were over 300 nm in size, they could deform and pass through the
0.2 μm pore filter, as shown in Fig. 2. CNPs with smaller wt.% of 5β-
cholanic acid exhibited excellent deformability but unsatisfactory sta-
bility in serum. Thus, the balanced stability and deformability of na-
noparticles ensured the superior in vivo tumor targeting efficiency.
Amongst, nanoparticles with 23 wt.% of 5β-cholanic acid exhibited the
highest tumor accumulation (Na et al., 2012). Moreover, the site of
E. Zhang, et al.
carboxymethylation on chitosan is also closely related to the anti-tumor
activity and cellular uptake of carboxymethyl chitosan-based nano-
particles. O-carboxymethylation showed superior activity to N-carbox-
ymethylation and N,O-carboxymethylation, which might be attributed
to the reservation of amino groups that facilitate cellular uptake
(Zhang, Xing et al., 2017). Besides, as for nanoparticles prepared by
chitosan and poly(ethylene glycol) (PEG) derivatives, the addition of
excessive PEG could decrease the zeta potential due to the shielding of
nanoparticle surface by PEG (Huo et al., 2012).
The characteristics of chitosan and its derivatives, such as Mw, DD,
degree and site of substitutions, have remarkable impact on the efficacy
of chitosan-based nanoparticles. It is important to note that these fac-
tors are not independent but exert combined influence on the nano-
particles. Therefore, choosing proper chitosan as nanocarriers needs
comprehensive consideration on the relationship of above factors with
the properties of payloads, preparing method of nanoparticles, target
disease and so on.
3. Characterization of medical grade chitosans
Chitosan is a common medical material and pharmaceutical ex-
cipient extracted from natural sources, so a standard for medical grade
chitosan is necessary. Establishing the standard will ensure the batch-
to-batch consistency of production and therapeutic efficacy as well as
safety of chitosan materials. Different pharmacopeias and societies
provided standard guides for chitosan or its salts. United States
Pharmacopoeia (USP) regards the DD of chitosan between 70.0% and
95.0% as acceptance criteria and the residue on ignition should be not
more than 1.0%. There are also requirements on the limit of lead,
mercury, chromium, nickel, cadmium, arsenic as well as iron.
Furthermore, the level of bacterial endotoxins should meet the re-
quirements of related dosage form monographs using chitosan (United
States Pharmacopoeia Convention, 2016). Recent studies showed that
the endotoxin content played an important role in immune responses,
which was related to Mw, DD of chitosan, and endotoxin structure
(Gjoseva et al., 2018; Ravindranathan, Koppolu, Smith, & Zaharoff,
2016). Besides, American Society for Testing and Materials (ASTM) also
provide a standard guide for chitosan salt as starting materials used for
biomedical and tissue-engineered medical product applications. The
guide lists a series of detection methods for DD, Mw, viscosity, dry
matter content, ash content, insolubles. Similar to USP, the content of
endotoxin is also listed as one of the major impurities of chitosan
products. Other impurities include protein content, heavy metal content
and microorganisms (American Society for Testing & Materials, 2011).
3
Carbohydrate Polymers 222 (2019) 115004
Fig. 2. The deformability test of Cy5.5-labeled
CNPs with different wt.% of 5β-cholanic acid to
glycol chitosan by measuring the fluorescence
intensity before and after filtration at different
pore sizes (A). The fluorescence intensities be-
fore and after filtration (B). The rigid and non-
deformable polystyrene nanoparticles (PSNPs)
were employed as control. Reproduced with
permission from (Na et al., 2012). Copyright
2012, Elsevier. Abbreviation: GC, glycol chit-
osan.
4. Applications in drug delivery
4.1. Chitosan-based systems as carriers of chemotherapeutic agents
Currently, chemotherapy is one of the most essential tools for on-
cotherapy in clinic, which is usually used to shrink the tumor size be-
fore or after surgery. Common chemotherapeutic agents, including
doxorubicin, paclitaxel, docetaxel, cisplatin, camptothecin and so on,
have exhibited stronger cytotoxicity against tumor cells than normal
cells and made enormous contributions in oncotherapy for the past
decades. However, these drugs still suffer severe drawbacks, such as
dose-dependence and occasional lethal cardiomyopathy of doxorubicin,
various severe side effects caused by usage of organic solvent in taxane
injections. Numerous studies have been conducted to solve these pro-
blems by incorporating chemotherapeutic agents with various chitosan-
based nanoparticles. According to the difference in preparing method,
the nanoparticles can be divided into three categories: self-assembled
nanoparticle, ionic cross-linked nanoparticle and polyelectrolyte com-
plex. Their structures are depicted in Fig. 3.
4.1.1. Self-assembled nanoparticles
Self-assembled nanoparticles have a micelle-like structure with a
hydrophobic inner core and a hydrophilic out shell, which are fabri-
cated by amphiphilic chitosan derivatives (Fig. 3). In aqueous phase,
the hydrophobic chains spontaneously aggregated together to form a
reservoir for both soluble and sparingly soluble drugs, and hydrophilic
chains served as a shell surrounded the core being exposed to the
aqueous phase.
Hydrophobically modified glycol chitosan is one of the most com-
monly used derivatives to fabricate self-assembled nanoparticles. Kwon
and co-workers synthesized a series of hydrophobic moieties con-
jugated glycol chitosans as nanocarriers, including 5β-cholanic acid,
fluorescein isothiocyanate (FITC), deoxycholic acid, palmitoyl group
and so on. The encapsulated drugs included both soluble and insoluble
molecules, such as cisplatin, camptothecin, paclitaxel, docetaxel and so
on (Bei et al., 2014; Hwang, Kim, Kwon, & Kim, 2008; Kim et al., 2005,
2008; Min et al., 2008). A recent study demonstrated that glycol chit-
osan-5β-cholanic acid-based nanoparticles have longer circulation time
and stronger liver tumor targeting ability than liposomes, polystyrene
nanoparticles with similar diameters (Na et al., 2016). Furthermore,
conjugating 2-(4-(vinylbenzyloxy)-N,N-diethylnicotinamide) oligomers
with glycol chitosan could significantly increase the drug loading
content up to 24.2 wt.% of paclitaxel, and the in vivo anti-tumor activity
was significantly stronger than the free drug and Abraxane® (Koo et al.,
2013). The conjugation of octylamine could increase the drug loading
content over 30%, and the tumor inhibition rate was 90.3% compared
E. Zhang, et al. Carbohydrate Polymers 222 (2019) 115004

with paclitaxel of 58.7% in MDA-MB-231 tumor bearing mice (Huo

(Wang, Zhen et al., 2013)

(Lee, Termsarasab et al.,

(Hsiao, Mu et al., 2015)
et al., 2016). For further improvement of selective delivery of drugs, a

(Huang et al., 2015)

variety of ligands were often introduced in glycol chitosan nano-

(Chen et al., 2018)

(Yang et al., 2017)

(Huo et al., 2012)

(Huo et al., 2016)

(Liu & Yeo, 2013)

(Tsai et al., 2018)
(Min et al., 2008)

(Zhu et al., 2018)

(He & Yin, 2017)

(Hu et al., 2015)

(Jia et al., 2014)

particles, such as somatostatin (Huo et al., 2012), peptides (Key et al.,
2016; Zhang et al., 2019), folic acid (Yu et al., 2013) and so on. Besides,
Reference

carboxymethyl chitosan is another soluble chitosan derivative and

2017)
widely studied as drug carriers. As with glycol chitosan, carboxymethyl
chitosan can self-assemble into nanoparticles after hydrophobically
modified, such as quercetin (Wang et al., 2014), linoleic acid (Tan &
in vitro and in vivo
in vitro and in vivo
in vitro and in vivo
in vitro and in vivo

in vitro and in vivo

in vitro and in vivo

in vitro and in vivo

in vitro and in vivo

in vitro and in vivo
in vitro and in vivo

in vitro and in vivo

in vitro and in vivo
Biological status

Liu, 2009), 4-mercaptobenzoic acid (Gao, Liu et al., 2014) or hydro-

phobic drug, methotrexate (Wang et al., 2011). Unlike glycol chitosan,
the surfaces of carboxymethyl chitosan nanoparticles were negative
in vitro

in vitro

in vitro
charged, which also showed high cellular uptake and strong anti-tumor
efficacy. Huang et al. synthesized hexanoyl-grafted carboxymethyl
chitosan as drug carriers coated with an anti-EGFR antibody layer for
both oral and intravenous delivery of paclitaxel

chemo-photodynamic-gene therapy, bioimaging

high redox sensitivity of drug release in tumor

delivery of demethoxycurcumin. The prepared nanoparticles exhibited

overcome microenvironment mediated drug
high hydrophobic drug loading, targeting

8-fold reduction of tumor volume in A549 xenograft lung tumor mice

surface charge reversible nanoparticles

targeted chemo-photodynamic therapy

compared with the control group (Huang et al., 2015).

enhance the penetration into tumor

co-delivery of doxorubicin and its

Amphiphilic chitosan-grafted copolymers have also been in-

enhance passive targeting ability
enhance active targeting ability
enhance active targeting ability

overcome multidrug resistance

synergistical anti-tumor effect

vestigated to fabricate self-assembled nanoparticles. Hsiao et al. syn-

delivery to glioblastoma cells
redox-sensitive drug release
high drug loading content,

thesized an amphiphilic hexanoyl-chitosan-PEG copolymer, which was

used to coat paclitaxel-loaded and chlorotoxin-grafted iron oxide na-
Anti-tumor strategy

noparticles for targeted delivery of paclitaxel to glioblastoma cells.

chemosensitizer

Chlorotoxin enhanced cellular uptake of nanoparticles in U-118 MG

cells and therefore improved the anti-tumor activity (Hsiao, Mu,
resistance

Stephen, Fang, & Zhang, 2015). Furthermore, Jiang et al. designed a

delicate nanoparticle by polymerization of N-3-acrylamidophe-
nylboronic acid (APBA) in the presence of chitosan, then forming am-
cancer associated fibroblasts,

NIH/3T3 cells, SKOV-3 cells

phiphilic copolymers by the coordination between boronic acid of poly-

SH-SY5Y cells, H-22 cells

MCF-7 cells, EMT-6 cells

APBA and amino groups of chitosan. The nanoparticles were then

SKOV-3 cells, L-02 cells

MKN45 cells, GES cells

Caco-2 cells, H22 cells

modified by iRGD and encapsulated doxorubicin. iRGD enhanced the

MDA-MB231 cells

MDA-MB231 cells

tumor penetration and inhibition of prepared nanoparticles both in 3-D

U-118 MG cells

multicellular spheroids and tumor-bearing mice compared with non-

MCF-7 cells

MCF-7 cells
Target cell

A549 cells

A549 cells

HeLa cells

conjugated ones. The in vivo tumor inhibition rate of nanoparticles was

4T1 cells

81%, while that of free drug was 24% (Wang, Zhen et al., 2013).
Chitosan oligosaccharide, depolymerized from chitosan, has low
viscosity and better aqueous solubility. Therefore, amphiphiles can be
Representative studies on chitosan-based nanoparticles for delivery of chemotherapeutic agents.

Ce6, doxorubicin, P-gp shRNA

synthesized via one-step reaction of hydrophobic modification on

methotrexate, mitomycin C

chitosan oligosaccharide. Xu et al. grafted ferrocene onto chitosan oli-

gosaccharide to fabricate stimuli-responsive nanoparticles. The nano-
demethoxycurcumin

particles could rapidly release the payloads under the synergistic effect
of low pH and oxidative stimulation (Xu, Wang, Li, & Wang, 2014).
camptothecin

gemcitabine
doxorubicin

doxorubicin
doxorubicin

doxorubicin

Furthermore, Zhu and co-workers constructed a doxorubicin-loaded

paclitaxel

paclitaxel

paclitaxel
paclitaxel

curcumin
Payload

nanoparticle based on telmisartan and stearic acid-conjugated chitosan

oligosaccharide. Telmisartan introduced nanoparticles to sequentially
target angiotensin II type I receptor (AT1R) overexpressed cancer as-
hexanoyl-chitosan-PEG, chlorotoxin-conjugated iron oxide nanoparticles

magnetic mesoporous silica nanoparticles coated with alginate/chitosan

chitosan-poly(N-3-acrylamidophenylboronic acid) modified with iRGD

sociated fibroblasts (CAFs) and tumor cells. After the elimination and
glycol chitosan-N-deoxycholic acid, octreotide-PEG-deoxycholic acid

apoptosis of CAFs, the nanoparticles acquired deeper permeability in

telmisartan and stearic acid-conjugated chitosan oligosaccharide

tumor masses and the tumor cells became more vulnerable to che-
motherapeutic agents (Zhu et al., 2018). Besides, Wang et al. in-
carboxymethylhexanoyl chitosan modified with anti-EGFR

vestigated the molecular dynamics of paclitaxel-loaded salicylic acid-

chitosan oligosaccharide nanoparticles. They found that the en-
capsulation process was majorly driven by van der Waals and hydro-
trimethyl chitosan-CSKSSDYQC (CSK) peptide

phobic interactions. Moreover, the self-assembled nanoparticles loading

PAMAM dendrimers, zwitterionic chitosan
chitosan oligosaccharide-SS-stearylamine

hydrophobic drugs were proved to be highly water-soluble, and the

chitosan oligosaccharide-indomethacin
folic acid-trimethyl chitosan-paclitaxel

optimal drug loading content was 10% (w/w) (Wang, Zhang, Wei, &
Wang, 2013).
glycol chitosan-5β-cholanic acid

polyelectrolyte multilayers
glycol chitosan-SS-octylamine

Except for being encapsulated, drugs can also be covalently con-

jugated to the hydrophilic polymers, self-assembling to form nano-
EGF-conjugated chitosan

particles, which is known as polymer-drug conjugate. The idea was

PEGylated chitosan

applied in anti-tumor drugs by Ringsdorf in 1975 (Duncan, 2006). The

conjugates usually have smaller size, which facilities deeper penetra-
tion (Wicki, Witzigmann, Balasubramanian, & Huwyler, 2014). Kwon
Material

et al. conjugated doxorubicin to glycol chitosan backbone with a cis-

Table 1

aconityl spacer, which acted as a pH-sensitive linkage. The conjugates

accumulated in tumors via EPR effect and showed long retention time in

4
E. Zhang, et al.
Fig. 3. The structures of self-assembled nanoparticle, ionic cross-linked nanoparticle and polyelectrolyte complex.
blood circulation (Hyung Park et al., 2006; Son et al., 2003). Similarly,
redox-responsive drug delivery was achieved by conjugating doxor-
ubicin to chitosan oligosaccharide via disulfide linkage (Su et al., 2015).
Furthermore, He et al. grafted paclitaxel and folic acid onto trimethyl
chitosan backbone for both oral and intravenous delivery of paclitaxel.
The conjugates could self-assemble into nanoparticles with 18.8 folds
higher intestinal transport of paclitaxel compared to the free drug. The
enhanced anti-tumor activity of these nanoparticles compared with the
free drug was also shown in H22 tumor bearing mice (He & Yin, 2017).
In addition, the nanoparticles can be functionalized by conjugation
of specific ingredients to polymers. Indomethacin was a chemosensi-
tizer for doxorubicin in cells expressing multidrug resistance-associated
protein (MRP). A doxorubicin-loading nanoparticle based on chitosan
oligosaccharide-indomethacin conjugate significantly enhanced the
anti-tumor efficacy for doxorubicin, the chemosensitizing mechanism
of which was shown in Fig. 4. After intravenously injected, the nano-
particles accumulated in tumor tissue by EPR effect, and internalized
into tumor cells via endocytic uptake. Furthermore, intravenous injec-
tion in rats exhibited much lower clearance of doxorubicin from na-
noparticles than the free drug (Lee, Termsarasab et al., 2017).
4.1.2. Ionic cross-linked nanoparticles
By exploiting the cationic property of chitosan, ionic cross-linked
chitosan nanoparticles are fabricated through electrostatic interaction,
in which the amino groups on the backbone interact with polyanionic
cross-linking agents such as tripolyphosphate (TPP), CaCl2, Na2SO4 and
so on (Fig. 3). This method was firstly reported by Bodmeier et al. in
1989 (Bodmeier, Oh, & Pramar, 1989), and has been widely utilized in
preparation of drug delivery systems encapsulating proteins, peptides,
gene materials and low molecular drugs (Katas & Alpar, 2006; Park,
Fig. 4. The chemosensitizing mechanism of doxorubicin-loaded chitosan oli-
gosaccharide-indomethacin nanoparticles for reversing multidrug resistant
cancer. Reproduced with permission from (Lee, Termsarasab et al., 2017).
Copyright 2017, Elsevier. Abbreviations: DOX, doxorubicin; IDM, in-
domethacin; CI, chitosan oligosaccharide-indomethacin conjugate.
5
Carbohydrate Polymers 222 (2019) 115004
Saravanakumar, Kim, & Kwon, 2010; Ragelle et al., 2014; Tsai, Yu,
Huang, & Lee, 2018; Yang, Fu, Wang, & He, 2009). Unlike the chemical
cross-linking, the process of ionically cross-linking offers simple and
mild preparation conditions without the usage of toxic reagents. Fur-
thermore, the physiochemical properties of nanoparticles, such as size
and surface charge, can be readily customized by ionic cross-linking
with the adjustment of processing parameters, which subsequently in-
fluences drug encapsulation efficiency and release profile (Gan & Wang,
2007; Gan, Wang, Cochrane, & McCarron, 2005). However, the ionic
cross-linked chitosan nanoparticles are weak in controlling the high
burst release of encapsulated drugs (Boonsongrit, Mitrevej, & Mueller,
2006; Gan & Wang, 2007).
Chen and colleagues synthesized chitosan nanoparticles using TPP
as the cross-linker in acid solution and then chemically conjugated
methoxy PEG (mPEG) and methotrexate to the surface of nanoparticles.
The in vitro and in vivo results showed the prepared nanoparticles en-
hanced the targeting ability to HeLa cells overexpressing folate re-
ceptors, approximately twice higher than folic acid-modified mPEG-
chitosan nanoparticles (Chen et al., 2014). Similarly, Zhang et al.
conjugated both mitomycin C and methotrexate to mPEG-chitosan na-
noparticles. Methotrexate not only enhanced the cellular uptake of
nanoparticles by folic acid receptor-mediated endocytosis, but also
exerted synergistic anti-tumor efficacies together with mitomycin C
against H22 tumors. More importantly, the nanoparticles induced no
body weight loss of mice compared with an obvious weight loss caused
by treatment of mitomycin C and methotrexate (Jia et al., 2014). Fur-
thermore, other ligands, such as folic acid, herceptin antibody and
glycyrrhetinic acid, were also reported to improve tumor targeting of
ionic cross-linked chitosan nanoparticles (Arya, Vandana, Acharya, &
Sahoo, 2011; Tian et al., 2010; Yang, Gao, & Kjems, 2014).
Additionally, Mukhtar et al. used water soluble chitosan and TPP to
fabricate nanoparticles encapsulating epigallocatechin-3-gallate
(EGCG), a polyphenol from green tea, as an oral drug delivery system.
The release of EGCG from nanoparticles was slow in stimulated gastric
juice but fast in intestinal fluid, which ensured the stability of nano-
particles in stomach. Furthermore, EGCG-loading nanoparticles ex-
hibited significant in vivo inhibiting effects against prostate and mela-
noma tumors (Khan et al., 2014; Siddiqui et al., 2014).
4.1.3. Polyelectrolyte complexes
Chitosan-based polyelectrolyte complex nanoparticles are also
formed via electrostatic interactions. After mixing chitosan with nega-
tive charged polyelectrolytes in solution, the polymeric chains interact
with each other to form strong but reversible electrostatic networks
without using cross-linkers (Fig. 3). The whole process is also facile,
simple and avoids using toxic reagents. Various polyanions have been
reported to form polyelectrolyte complex nanoparticles with chitosan,
including alginate, carrageenan, pectin, poly(acrylic acid) and so on
(Hamman, 2010). Stokke et al. investigated the factors that influence
the physicochemical properties of polyelectrolyte complexes formed by
chitosan and alginate. The net charge ratio and Mw of polymers were
the most essential parameters that affected size, zeta potential and pH
of prepared complexes. Additionally, the speed and diameter of the
dispersing element of the homogenizer, and the mixing order might also
E. Zhang, et al. Carbohydrate Polymers 222 (2019) 115004

cause size change of complexes (Sæther, Holme, Maurstad, Smidsrød, &

(Jadidi-Niaragh et al.,
(Lee, Ku et al., 2017)

(Zhang et al., 2015)

(Zhang et al., 2016)

Stokke, 2008).

(Noh et al., 2011)

(Han et al., 2015)

(Wei et al., 2013)
(Lee et al., 2015)
A recent study reported the preparation of magnetic mesoporous

(Li et al., 2015)

silica nanoparticles coated with alginate/chitosan polyelectrolyte mul-

Reference
tilayers for co-delivery of P-gp shRNA, doxorubicin and photosensitizer

2017)
chlorin e6 (Ce6). The polyelectrolyte multilayers endowed this multi-
functional system with pH-sensitive property, enhanced shRNA binding
affinity and better biocompatibility. The in vivo animal studies showed

in vitro and in vivo

in vitro and in vivo

in vitro and in vivo

in vitro and in vivo

in vitro and in vivo

in vitro and in vivo

in vitro and in vivo

Biological status
that the nanoparticles exerted high therapeutic efficacy by combined
chemotherapy, photodynamic and gene therapy. Meanwhile, the Fe3O4-
Au core of the nanoparticle enabled dual-modal MR and CT imaging of

in vitro
in vivo
tumors (Yang et al., 2017). Furthermore, Yeo and co-workers mixed
polyamidoamine (PAMAM) dendrimers with zwitterionic chitosan
(ZWC) and obtained stable electrostatic complexes at pH 7.4. Interest-

combined therapy by siRNA and chemotherapeutics

suppress the expression of CD73 and potentiate the

optimize the binding affinity of siRNA and carriers

ingly, the coating of ZWC on the surface of PAMAM dendrimers reduced
its cytotoxicity against normal cells caused by high surface charge, but
the complex disintegrated at acid pH due to the charge change of ZWC.

activate mitochondria apoptosis pathway

This pH-dependent charge conversion made it potential as nanocarrier

surface charge reversible nanoparticles

delivering drugs to tumors in acid environment (Liu & Yeo, 2013).
Moreover, another study demonstrated that the coating of chitosan on

silence the expression of Mcl-1

overcome multidrug resistance

effect of dendritic cell vaccine
hyaluronic acid-paclitaxel nanoparticles could protect the conjugates

anti-angiogenesis, apoptosis
from degradation in acid environment of stomach and intestine, but
fairly fast release was shown at pH 7.4, which enhanced the bioavail-

Anti-tumor strategy
ability of orally administrated paclitaxel (Li et al., 2013).

anti-angiogenesis
4.2. Chitosan-based systems as carriers of therapeutic genes

Gene-based therapy has become an important part in the develop-

ment of oncotherapy, which is accomplished by delivering exogenous
nucleic acids (DNA, siRNA, etc.) capable of modulating gene expression

QGY-7703 cells, HeLa cells

Caco-2 cells, Lewis lung
into tumor cells. As the therapeutic nucleic acids can be easily degraded
by endonucleases in serum and the extracellular matrix, development of

HepG2/ADM cells
effective delivery vectors is the key issue for successful gene therapy
(Yin et al., 2014). Chitosan has gained large attention for delivery gene carcinoma cells
Target cell

HeLa cells

HeLa cells
PC-3 cells

PC-3 cells

products due to its non-toxicity, biodegradability, low immunogenicity

4T1 cells
KB cells

as well as the cationic property. The negatively charged DNA and siRNA
can bind to protonated amines on chitosan backbone via electrostatic
Representative studies on chitosan-based nanoparticles for delivery of chemotherapeutic genes.

interaction, which protect them from degradation by nucleases

paclitaxel, TERT siRNA

(Jayakumar et al., 2010; Ragelle et al., 2013). However, the transfec-

VEGF siRNA, Bcl-2

RAPSI nanoflower

tion efficiency of native chitosan is relatively low due to in vivo in-

stability and insufficient cellular release. Therefore, chemical mod-
Bcl-2 siRNA,
Mcl-1 siRNA

VEGF siRNA

VEGF siRNA
CD73 siRNA

P-gp siRNA,
doxorubicin
lonidamine

ifications or incorporation with functional materials are in need for

Payload

(siRNA)

developing powerful delivery systems (Ragelle et al., 2013). In recent

siRNA

years, numerous studies have reported various chitosan-based delivery

systems for delivering different DNA and siRNA with excellent anti-
urocanic acid-modified galactosylated trimethyl chitosan, poly(allylamine

tumor efficacies preclinically.

chitosan-PEI-lonidamine/triphenylphosphine, poly(acrylic acid)-PEG-folic

4.2.1. Nanoparticles based on chitosan derivatives

N-((2-hydroxy-3-trimethylammonium) propyl) chitosan chloride

Kwon et al. synthesized self-crosslinked nanoparticles based on

thiolated glycol chitosan and poly-siRNA, which was polymerized via
reducible disulfide linkages. Chitosan and poly-siRNA formed stable
complexes through both electrostatic interaction and disulfide cross-
linking. This system achieved successful in vivo delivery of Bcl-2 and
N-succinyl chitosan-poly-L-lysine-palmitic acid

vascular endothelial growth factor (VEGF) siRNA with desirable tumor

specificity and transfection efficiency (Lee et al., 2012, 2015; Yoon
hydrochloride)-citraconic anhydride
chitosan-polymer conjugates/complexes

et al., 2014). Furthermore, Kwon and colleagues prepared a polymeric

siRNA nanoflower by a novel designed rolling circle transcription (RCT)
method, which was then complexed with thiolated glycol chitosan and
α-tocopherol oligochitosan

verified for its efficacy in PC-3 xenograft tumors. The complexes

thiolated glycol chitosan

thiolated glycol chitosan

showed desirable particle stability when confronted with polyanion

Chitosan derivatives

competition or nuclease and also excellent anti-tumor activity with

chitosan-agmatine
chitosan-lactate

tumor inhibition rate of 75% at dose of 1.5 mg/kg. (Lee, Ku et al.,

2017).
acid
Material

Trimethylated chitosan, a water soluble chitosan derivative, has

Table 2

enhanced complexing ability between chitosan and anionic nucleic acid

(Gao, Wang et al., 2014). Yin et al. prepared a series of galactosylated

6
E. Zhang, et al.
Fig. 5. The structure of chitosan-PEI nanoparticle co-delivering lonidamine and Bcl-2 siRNA (A). The process of hierarchically targeting and mechanism of mi-
tochondrial apoptosis pathway induced by Bcl-2 siRNA and lonidamine (B). Reproduced with permission from (Zhang et al., 2015). Copyright 2015, Elsevier.
Abbreviations: PAA, poly(acrylic acid); PPF, poly(acrylic acid)-polyethylene glycol-folic acid; LND, lonidamine; mPTP, mitochondrial permeability transition pore;
RISC, RNA-induced silencing complex.
trimethyl chitosan-cysteine/siRNA complexes to study the effect of
siRNA binding affinity on anti-tumor efficacy. The strong binding
strength provided excellent stability of complexes in serum but in-
efficient release of siRNA inside the cells, while the weak binding
strength came to the opposite results. The authors suggested that tai-
loring the binding strength between carriers and siRNA was important
for improving transfection efficiency and anti-tumor activity (Han,
Tang, & Yin, 2013).
Furthermore, Ren et al. prepared folic acid-modified hydroxypropyl
chitosan nanoparticles delivering antisense oligodeoxynucleotides for
overcoming tumor drug resistance. The nanoparticles selectively accu-
mulated in folic acid receptor-high expressing tumor cells and inhibited
multi drug resistance (MDR) 1 gene and P-glycoprotein (P-gp) levels
(Wang, Tao, Zhang, Wei, & Ren, 2010). Moreover, N-(2-hydroxy-3-tri-
methylammonium)propyl chitosan chloride, a partially quaternized
chitosan, was utilized for oral delivery of mouse telomerase reverse
transcriptase (mTERT) siRNA and paclitaxel simultaneously. This
chitosan derivative enhanced intestinal absorption and cellular uptake,
which resulted in synergistic anti-tumor effect by siRNA and paclitaxel
(Wei et al., 2013).
In addition, Li and co-workers prepared pH-sensitive charge-con-
versional nanocomplexes based on dimethylmaleic anhydride modified
chitosan-agmatine conjugates, which could condense VEGF siRNA into
nanoparticles. The zeta potential of nanoparticles transformed from
negative charge into positive charge at acid pH, facilitating tumor
cellular uptake and subsequent VEGF mRNA knockdown (approxi-
mately 79.7%) and cell apoptosis (Li et al., 2015).
7
Carbohydrate Polymers 222 (2019) 115004
4.2.2. Nanoparticles based on chitosan-polymer conjugates/complexes
The conjugation or addition of functional polymers in chitosan
formulations can cover the shortage of chitosan and achieve more ef-
ficient transfection. Poly(ethylene imine) (PEI) is one of the most stu-
died amine-rich polymeric materials for nucleic acid delivery. Due to
the abundant amino groups on its chains, PEI has high charge intensity
at sponge effect. Nevertheless, PEI is also known to be toxic against
normal cells due to the large positive charge. The incorporation of PEI
with chitosan nanoparticles is often carried out via either chemical
modification or addition as an excipient. PEI improved the endosomal
escape capacity of chitosan nanoparticles and the subsequent trans-
fection efficiency, and also, the cytotoxicity is reduced compared with
native PEI nanoparticles (Buschmann et al., 2013; Huh et al., 2010).
Jiang et al. designed a lonidamine-conjugated chitosan-g-PEI nano-
particle for Bcl-2 siRNA delivery, hierarchically targeting to tumor cells
and mitochondria. The structure and targeting mechanism were dis-
played in Fig. 5. After folic acid-mediated internalization into target
cells, lonidamine was delivering into mitochondria by triphenylpho-
sphine (TPP) and induced apoptosis. Meanwhile, siRNA was released
into cytoplasm and suppressed anti-apoptotic Bcl-2 expression, re-
sulting in significant anti-tumor activity via mitochondrial signaling
pathway synergistically with lonidamine. The proliferation rate of HeLa
cells treated by the nanoparticles after 72 h was only 11.9%, which was
significantly lower than groups treated by lonidamine or siRNA (Zhang
et al., 2015). Similarly, cationic polymers like poly-L-arginine (PLR)
(Noh et al., 2010), poly-L-lysine (PLL) (Zhang et al., 2016) and poly-
guluronate (Lee et al., 2009) were used to increase gene stability and
E. Zhang, et al. Carbohydrate Polymers 222 (2019) 115004

transfection efficiency of chitosan nanoparticles.

(Hsiao, Chuang et al.,

(Wang et al., 2015)
(Ding et al., 2018)
Except for cationic polymers, some anionic polymers were reported

(Gao et al., 2016)

(Zhang, Lv et al.,

(Liu et al., 2018)

(Shi et al., 2017)

(Xu et al., 2012)
(Li et al., 2017)
to improve the in vivo performance of chitosan/siRNA systems. Yin et al.
incorporated anionic poly(allylamine hydrochloride)-citraconic anhy-
Reference

2017) dride (PAH-Cit) with urocanic acid-conjugated galactosylated trimethyl

2015)
chitosan for delivery of VEGF siRNA. PAH-Cit could enhanced the en-
dosomal escape capability due to its property of hydrolyzing into ca-

in vitro anti-tumor activity and in vivo

tionic poly(allylamine) in endosomes (Han, Tang, & Yin, 2015). Fur-
thermore, Babu et al. coated negatively charged polylactic acid
nanoparticles with cationic chitosan shells, co-delivering P62siRNA, β5
plasmid DNA and cisplatin for reversing cancer drug resistance. After
the release of therapeutic agents into cisplatin-resistant 2008/C13
in vitro and in vivo

in vitro and in vivo

in vitro and in vivo

in vitro and in vivo

in vitro and in vivo

ovarian cancer cells, the treatment with these nanoparticles caused P62
biocompatibility
Biological status

inhibition and restoration of β5 expression, which significantly en-

hanced the anti-tumor activity of cisplatin (Babu et al., 2014). More-
in vitro
in vitro

over, the effectiveness of chitosan/siRNA nanoparticles was also im-

in vivo

proved when incorporated with anionic poly(γ-glutamic acid) (γ-PGA).

The incorporation of γ-PGA improved the cellular uptake, intracellular
chemo-photothermal therapy, two-

unpackage, transfection efficiency compared with chitosan/siRNA na-

therapy for deep-

noparticles. In particular, chitosan/γ-PGA based siRNA nanoparticles

exhibited a knockdown efficiency of 72% after 24 h post transfection
imaging,

and 55% after 72 h, compared with chitosan/siRNA nanoparticles with

photodynamic therapy,
therapy

therapy

therapy

photothermal therapy

photothermal therapy
Anti-tumor strategy

50% after 24 h and only 30% after 72 h (Liao et al., 2010).

photon bioimaging

immunotherapy
immunotherapy
photodynamic

photodynamic
photodynamic

photodynamic
tumor-specific

seated tumors

4.3. Chitosan-based systems as carriers of other therapeutic agents

Photodynamic therapy is a rapidly growing treatment for oncology

due to its minimal toxicity compared with chemotherapy and radio-
therapy. Selective delivery of water-insoluble photosensitizers into
target tissues is one of the greatest challenges for successful therapeutic
B16 cells, bone marrow
HepG2 cells, H22 cells

derived dendritic cells

A549 cells, L02 cells

outcome (Agostinis et al., 2011; Lucky, Soo, & Zhang, 2015). Lee at al.
prepared an intelligent polymer-drug conjugate based on glycol chit-
osan, on which a pH-sensitive block, 3-diethylaminopropyl iso-
DU145 cells

Hep3B cells
MCF-7 cells
Target cell

CT26 cells
HeLa cells

PC-3 cells

thiocyanate (DEAP) group, photosensitizer Ce6 and PEG was grafted.

The conformation of conjugates could switch from self-assembled na-
Representative studies on chitosan-based nanoparticles for delivery of other chemotherapeutic agents.

noparticles at neutral pH into uncoiled molecules when exposed in acid

environment. Based on this property, the conjugates exhibited pH-in-
dependent singlet-oxygen generation, which was crucial for photo-
toxicity against tumor cells (Park et al., 2011). Furthermore, Ding et al.
prepared an amphiphilic polymer by conjugating Ce6 onto glycol
whole tumor cell lysate
heptamethine cyanine

mono-hydroxylphenyl

chitosan backbone, which subsequently self-assembled into nano-

Zinc phthalocyanine

triphenylporphyrin
indocyanine green

particles. The cytotoxicity of Ce6 was markedly attenuated but the

doxorubicin, CD

therapeutic efficacy was improved (Ding et al., 2018). Moreover, in

polyaniline

order to improve efficiency of photodynamic therapy against deep-se-

Payload

ated tumors, Gao and co-workers synthesized a novel nanosystems

IL-12
Ce6

consisting of Zinc phthalocyanine-loaded nanoparticles coated with c

(RGDyK)-conjugated N-succinyl-N-octyl chitosan. A significant im-
Zinc phthalocyanine nanoparticle coated with c(RGDyK)-N-succinyl-

DMA-chitosan oligosaccharide-PEG as the shell, mesoporous silica

provement of therapeutic efficacy by this nanosystem was shown

mono-hydroxylphenyl triphenylporphyrin-chitosan carbon dots

compared with traditional photodynamic therapy. The authors also

introduced a sequential treatment of nanosystem followed by Doxil,
which could simultaneously enhance the efficacy with a tumor inhibi-
coated gold nanorod modified with peptide RLA

tion rate of 79% compared with 56% of Doxil alone, and also reduced
folic acid/heptamethine cyanine-modified chitosan

the cardiotoxicity of Doxil (Gao et al., 2016).

As an extension of photodynamic therapy, photothermal therapy
has also attracted large attention for precise cancer treatment. Liu et al.
utilized chitosan oligosaccharide-PEG-2,3-dimethylmaleic anhydride
PEG-chitosan coated CD nanogels

(DMA) copolymer as a shell protecting mesoporous silica coated gold

nanorods for delivery of indocyanine green. The composition and in
mannose- alginate, chitosan

vivo working process of this nanosystem was shown in Fig. 6. The

N- polyaniline-chitosan

chitosan-based outer shell could prevent the nanoparticles from serum

N-octyl chitosan
glycol chitosan-Ce6

chitosan glutamate

absorption and prolonged the blood circulation time. The amide bonds
between chitosan and DMA broke down in acidic microenvironment,
resulting in the decomposition of the shell. This composite nanosystem
Material

achieved successful photodynamic and photothermal therapy for breast

Table 3

cancer at the same time with minimal side effects (Liu et al., 2018).
Furthermore, Wang and colleagues prepared PEG-chitosan coated

8
E. Zhang, et al. Carbohydrate Polymers 222 (2019) 115004

Fig. 6. The preparation and in vivo working process of a multi-

functional nanosystem for simultaneous photodynamic and pho-
tothermal therapy against breast cancer. Reproduced with per-
mission from (Liu et al., 2018). Copyright 2018, Elsevier.
Abbreviations: AuNR, gold nanorods; MSN, ; APTES, 3-amino-
propyltriethoxysilane; ICG, indocyanine green; β-CD, β-cyclodex-
trin; PTT, photothermal therapy; PDT, photodynamic therapy (For
interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article).

carbon dots (CD) nanogels loading doxorubicin for pH and NIR light- taken up by dendritic cells and increase serum IFN-γ and IL-4. Fur-
sensitive drug release, bioimaging, and chemo-photothermal therapy. thermore, this novel vaccine significantly delayed the tumor growth
The hydroxyl and amine groups of chitosan help form hydrogen bonds partially due to cytotoxic T lymphocytes response (Shi et al., 2017).
with hydroxyl and carboxyl groups of CDs as well as ether oxygens of
PEG. Besides, the abundant amine groups on chitosan could modulate
5. Challenges of translation from laboratory to clinic
the drug release by pH-sensitive swelling/deswelling effect. This novel
nanosystem provided high anti-tumor activity against DU145 prostate
Till now, there were no chitosan-based nanoparticles approved for
cancer cells due to the synergistic effect of chemo-photothermal
treatment of cancers in clinic or being tested clinical trials. The clinical
therapy (Wang et al., 2015). Moreover, Lu et al. designed a chitosan-
translation of anti-tumor chitosan nanoparticles is confronted with
coated CuS nanoparticles incorporated with immunoadjuvants, oligo-
many challenges. First of all, there are only a few studies testing the in
deoxynucleotides containing cytosine-guanine (CpG) motifs, which
vivo safety of chitosan, especially chitosan administrated by intravenous
exerted anti-tumor activity through both photothermal and im-
injection, because most preclinical anti-tumor chitosan-based nano-
munotherapy. The nanoparticles were disintegrated after laser excita-
particles as described in this review were designed as injections.
tion, allowing the system reassembled into chitosan-CpG complexes,
Additionally, the chemical versatility of chitosan makes it more difficult
subsequently activated host antitumor immunity. The combined
to carried out, because each modification produced a new chemical
therapy showed more effective than immunotherapy or photothermal
entity which should be assessed individually. However, some dis-
therapy alone, resulting in synergistic effects against both primary-
advantages of chitosan, such as the poor water solubility and inefficient
treated and distant-untreated tumors (Guo et al., 2014).
endosomal escaping ability, compel it to make structural modification.
Chitosan and its derivatives also attract large attention as drug
Furthermore, chitosan-based nanoparticles shared some common
carriers for cancer immunotherapy. Interleukin-12 (IL-12) is a cytokine
problems with other nano-drug delivery systems. For example, the
with immunomodulatory and angiogenesis activities, and it has been
targeting mechanisms which are considered as the rationale of design
widely investigated as either an anti-tumor agent or a vaccine adjuvant.
and development of anti-tumor nanomedicine, like EPR effect and ac-
However, the systematic toxicity of IL-12 seriously hindered its clinical
tive targeting, are often being questioned (Danhier, 2016; Mcneil, 2016;
applications (‘Mac’Cheever, 2008; Del Vecchio et al., 2007). Wang et al.
Wilhelm, Tavares, & Chan, 2016, 2016b). It is still uncertain whether
incorporated IL-12 with chitosan glutamate nanoparticles using TPP as
the targeting mechanisms of nanoparticles displayed in rodents works
cross-linker. The intravenously administrated nanoparticles sig-
in humans due to the heterogeneity of clinical tumors. Therefore, taking
nificantly inhibited the growth of liver metastasis tumors of colorectal
a deep insight into the correlation of patient tumors and nanomedicine
cancer and reduced the toxicity caused by IL-12. Furthermore, in-
behavior is necessary for further clinical translation.
filtration of microphages and T cells was induced during the im-
In addition, the large-scale production is another problem for clin-
munotherapy, which might be related to the tumor inhibition (Xu et al.,
ical translation of chitosan-based nanoparticles. Chitosan, composed of
2012). Besides, in order to enhance the immunotherapeutic efficacy of
a broad range of poly-N-acetyl-glucosamine, varies in Mw and DD,
tumor cell lysates (TCL), Shi et al. encapsulated TCL into nanoparticles
which depends on the sources, extraction and preparation processes,
based on mannose-modified alginate and chitosan, targeting specific
and its derivatives change from degrees of substitutions. Improved
dendritic cells. The prepared nanoparticles vaccine could be readily
processing and characterization in production are in need.

9
E. Zhang, et al.
Furthermore, compared to traditional chemotherapeutic agents, the
reproducibility and quality control of nanoparticles are far more com-
plicated. How to retain the batch-to-batch consistency is a key problem
for large-scale production. Besides, the high cost of large-scale pro-
duction discouraged pharmaceutical companies from clinical transla-
tion of nanomedicine.
6. Conclusion and perspectives
Chitosan-based nanoparticles present many advantages for anti-
tumor drug delivery. Various anti-tumor agents, such as hydrophilic or
hydrophobic chemotherapeutic drugs, nucleic acids, photosensitizers
and cytokines, can be incorporated into chitosan-based nanoparticles
and delivered to specific tumor cells via EPR effect and ligand-receptor
interaction. Furthermore, the cationic property of chitosan facilitates
endosomal escape of nanoparticles and the efficient release of nucleic
acids into cytoplasm. Numerous studies demonstrated that chitosan-
based nanoparticles not only caused obvious side effects on experi-
mental animals, but also alleviated the toxicity of chemotherapeutic
agents or other ingredients like PEI. In a number of cases, chitosan-
based nanoparticles exhibited exciting in vivo anti-tumor efficacy and
favorable targeting ability, which indicated there is tremendous scope
in clinical application.
Overall, numerous studies presented exciting anti-tumor efficacy of
chitosan-based nanoparticles in recent years. Chitosan and its deriva-
tives are capable of targeting delivery of various anti-tumor agents,
which can be applied to a variety of treatment for oncotherapy. The
future studies should be focus on not only development of more effi-
cient and simple chitosan-based nanoparticles for oncotherapy, but also
their safety after intravenously administrated as well as targeting me-
chanisms by using more clinical-relevant tumor models.
Acknowledgements
This work is supported by National Key R&D Program of China
(2018YFC0311305); Special projects of Foshan science and technology
innovation team (2017IT100054).
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