Review

Journal of
Biomedical Nanotechnology
Copyright © 2016 American Scientific Publishers
All rights reserved Vol. 12, 1585–1603, 2016
Printed in the United States of America www.aspbs.com/jbn

Functional Chitosan Nanoparticles in Cancer Treatment

Shaozhi Fu1 ∗ , Jiyi Xia2 , and Jingbo Wu1
1
Department of Oncology, The Affiliated Hospital of Luzhou Medical College, Luzhou Medical College, Luzhou 646000, P. R. China
2
Department of Science and Technology, Luzhou Medical College, Luzhou 646000, P. R. China

With exponential growth in nanotechnology and material chemistry, the application of a variety of nanomaterials in biomed-
ical field has attracted increasing interest, especially in cancer diagnosis and treatment. Nanoparticles have greatly
revolutionized the therapy pattern of cancer owing to their specific targeting ability, prolonged circulation time, enhanced
therapeutic efficacy, and decreased systemic side effects. Chitosan, a modified natural polysaccharide with excellent bio-
compatibility, has been intensively studied for use as carriers to deliver various drugs and genes in cancer treatment. This
review provides an insight into the advances in chitosan-based nanoparticles with a focus on its therapeutic potential in
cancer treatment.
KEYWORDS: Chitosan Nanoparticles, Anticancer Drug, Drug Carrier, Gene Delivery, Biodegradability, Biocompatibility, Toxicity,
Structure Modification, Positive Targeting, Active Targeting, Blood Circulation Time, Multi-Drug Resistance, Blood Brain Barrier, Cancer
Diagnosis, In Vivo Imaging, Photodynamic Therapy, Thermotherapy, Individualized Cancer Therapy.

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CONTENTS Directly Publishers
Copyright: American Scientific Conjugated Chemotherapeutic Drugs with
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1585 Chitosan Backbone . . . . . . . . . . . . ........ . . . . . . . . 1597
Source, Structure, and Physicochemical Encapsulation of Metal Compounds . . ........ . . . . . . . . 1598
Properties of Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1586 Encapsulation of Protein . . . . . . . . . ........ . . . . . . . . 1598
Source and Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . 1586 Problems and Outlook . . . . . . . . . . . . ........ . . . . . . . . 1599
Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . 1587 Acknowledgements . . . . . . . . . . . . ........ . . . . . . . . 1599
Modification of Chitosan Nanoparticles . . . . . . . . . . . . . . . . . 1588 References . . . . . . . . . . . . . . . . . . ........ . . . . . . . . 1599
Chitosan Nanoparticles Modified by
Carbon-Based Materials . . . . . . . . . . . . . . . . . . . . . . . . . 1589
Chitosan Nanoparticles Modified by Natural Polysaccharide . . 1589 INTRODUCTION
Chitosan Nanoparticles Modified by Chemically It is well established that cancer has become one of the
Synthesized Copolymer . . . . . . . . . . . . . . . . . . . . . . . . . 1590 most serious threat to human health. It is estimated that
Chitosan Nanoparticles Modified by Low Molecular
there will be 12 million cancer deaths worldwide in 2030.1
Weight Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1590
Chitosan Nanoparticles Modified by Peptide, Protein . . . . . . . 1591 Especially in China, as a developing country with a large
Functions of Chitosan Nanoparticles . . . . . . . . . . . . . . . . . . . 1592 population, cancer incidence and death rates keep rising
Enhancing Anti-Tumor Efficacy . . . . . . . . . . . . . . . . . . . . 1592 year by year due to environmental pollution. According
Improving Special Targeting Tumor Tissue . . . . . . . . . . . . . 1592 to an estimate, six persons are diagnosed with cancer
Prolonging Blood Circulation Time . . . . . . . . . . . . . . . . . . 1592
every passing minute. This serious situation has brought
Reversal of Multi-Drug Resistance . . . . . . . . . . . . . . . . . . 1593
Crossing Blood-Brain Barrier . . . . . . . . . . . . . . . . . . . . . . 1593 a heavy burden to the society and patients. Among exist-
Diagnosis, Detection and Imaging . . . . . . . . . . . . . . . . . . . 1594 ing cancer treatments, chemotherapy is by far the most
Photodynamic Therapy and Imaging . . . . . . . . . . . . . . . . . 1594 employed form of intervention, however, patients face
Thermotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1595 many severe problems caused by chemotherapeutic agents,
Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1596
such as detrimental side effects, drug resistance and high
Chitosan Nanoparticles in Drug Delivery . . . . . . . . . . . . . . . . 1597
Encapsulation of Chemotherapeutic Agents Into Chitosan . . . . 1597
cost.
In the process of exploring novel cancer therapy,
∗
thanks to the progress in material science and process
Author to whom correspondence should be addressed.
Email: shaozhifu513@163.com technology, nanodrug development has emerged as a
Received: 17 May 2015 promising approach to overcome the shortcomings of con-
Revised/Accepted: 7 September 2015 ventional chemotherapeutic agents. Researchers worldwide

J. Biomed. Nanotechnol. 2016, Vol. 12, No. 8 1550-7033/2016/12/1585/019 doi:10.1166/jbn.2016.2228 1585

Chitosan Nanoparticles in Cancer Treatment Fu et al.

are now combining nanotechnology with drug delivery
system (DDS) to achieve interesting and useful results.
So far many nanoformulations of anticancer drugs have
been developed and studied in clinical trails. For instance,
Abraxane® and Genexol-PM, two nanoformulations of
paclitaxel (PTX) have been approved for clinical use by
Food Drug Administration (FDA) in the past decade.2
Therapeutic agents and their carriers are the two basic
components of nanoformulations. The antitumor effect is
achieved by carriers delivering drug selectively to the
tumor cells. Therefore, the role of carriers in DDS is vital.
The carriers for loading anticancer drugs are commonly
composed of amphiphilic polymers or hydrophilic biopoly-
mers. Based on the source of the composition matrix, drug
carriers can be classified into two types: chemosynthetic
and natural. Among chemically synthesized polymers,
polyesters, poly(ether-esters), polyurethanes, and poly-
carbonates, have received considerable attention. Specif-
ically, poly(lactic-co-glycolic acid) (PLGA), poly(lactic
acid) (PLA), poly(-caprolactone) (PCL) or their polyethy-
lene glycol (PEG)-copolymers are well studied and widely
used as drug carriers.3 Natural biopolymers such as chi-
tosan, collagen, cellulose, and fibrin, are also investi-
gated intensively in pharmaceutical field owing to their
unique characteristics.4 5 In particular, chitosan nanopar-
ticles (CSNPs) have drawn considerable attention as
anticancer drug delivery carriers because of their easy
accessibility, excellent stability, low toxicity, and easy
modification.6 Herein, we aim to review various aspects
of chitosan-based nanoparticles for drug delivery in cancer
treatment.
SOURCE, STRUCTURE, AND
PHYSICOCHEMICAL PROPERTIES
OF CHITOSAN
Source and Structure
As a natural polysaccharide, chitosan is manufactured on
a large scale by alkaline N -deacetylation of chitin in

Shaozhi Fu received his Ph.D. from the State Key Lab of Biotherapy, Sichuan Univer-
sity, China in Chemical Biology in 2012. After finishing his graduate work, he became
a research assistant professor in Luzhou Medical College. He has served as a core
member of the Radiobiological Center in the Department of Oncology at the Affiliated
Delivered
Hospital ofby Ingenta
Luzhou to: Main
Medical CID since
College is 80004805
2012. His(JPP)
research interests include targeted
IP: 146.185.205.29 On: Wed, 07 Dec 2016
drug delivery system, biomedical nanotechnology04:41:09
and degradable biomaterials.
Copyright: American Scientific Publishers

Jiyi Xia received his M.D. from Luzhou Medical College, China in Clinical Medicine
in 2011. Then he became an experimentalist in Medical Experimental Center of the
Affiliated Hospital of Luzhou Medical College. He has served as a core member of
the Research Center of Drug and Functional Food in Luzhou Medical College since
2012. Currently, He is a member of the Department of Science and Technology, Luzhou
Medical College. His research interests include molecular biology, especially in cancer-
specific protein.

Jingbo Wu is a professor at the department of Oncology at the Affiliated Hospital of

Luzhou Medical College, China. He received his bachelor’s degree from the Department
of clinical medicine in the Chongqing Medical University in 1984. And then, he served
as a clinician in the Affiliated Hospital of Luzhou Medical College. In 1995, he got his
M.D. in medical oncology from Sun Yat-Sen Medical University, China. He is currently
the director of the Department of Oncology at the Affiliated Hospital of Luzhou Medical
College. His main interests include multimodality treatment of cancer, nanodrugs and
natural anti-caner products.

1586 J. Biomed. Nanotechnol. 12, 1585–1603, 2016

Fu et al.
commercial production. Chitin is an abundant biopolymer
isolated from the exoskeleton of crustaceans, such as crabs
and shrimps.7 Deacetylation of chitin and protonation
of chitosan is shown in the Figure 1. The proportion
of the two repeating units (glucosamine and N -acetyl-
glucosamine units) determines the degree of deacetylation
of the polymer.
Physicochemical Properties
Water Solubility
The aqueous solubility of chitosan has a great effect on
its processing conditions and application range. Chitosan
is insoluble in aqueous medium at neutral pH but is sol-
uble in slightly acidic environment owing to the amine
groups on its backbone. However, the solubility of chi-
tosan in neutral and basic pH media can be improved
by quaternization to form trimethylammonium chitosan
derivatives. Moreover, the molecular weight of chitosan
also greatly influences its solubility and degradability. Chi-
tosans and its derivatives having lower molecular weights
and lower degrees of deacetylation exhibit greater solu-
8 in the DD.
bility and faster degradation. Owing to the presence of
protonatable amine groups, chitosan is a positively charged
polymer at acidic pH (Fig. 1). This unique characteris-
tic endows it the ability to interact electrostatically with
polyanions when chitosan is used as carriers for deliv-
ery of drugs or genes. It has been confirmed
Delivered bythat chitosan
Ingenta to: Mainmissing
with positive charge binds to cellIP: 146.185.205.29
membranes resultingOn:
in a Wed,can
decrease in the transepithelial electricalCopyright:
resistance American
(TEER) Scientific
Figure 1. Chemical structure of chitin, chitosan, and proto-
nated chitosan.
J. Biomed. Nanotechnol. 12, 1585–1603, 2016
Chitosan Nanoparticles in Cancer Treatment
of cell monolayers as well as an increase in the paracellu-
lar permeability.8 9
Biodegradability
Biodegradability is a key property of polymers which are
used as carriers in DDS and as scaffolds in tissue engi-
neering. It not only determines the application potential
and range of biomaterials used in clinic, but also con-
trols the metabolic fate of these polymers in the body.
Chitosan, as one of the biodegradable biopolymers, not
only contains abundant amino groups, but also possesses
hydrolysable glycosidic bonds in its backbone. Therefore,
chitosan can be degraded to nontoxic oligosaccharides of
variable length by proteases, mainly lysozyme, in vivo.
Subsequently, the produced oligosaccharides can be incor-
porated in metabolic pathways or excreted out.10 Its degra-
dation rate is related to its molecular weight, the degree
of deacetylation (DD), material shape, size, and adminis-
tration route. Generally, higher molecular weight chitosan
has a lower degradation rate. Similar trend is observed
with DD, the degradation rate decreases with an increase
Chitosan is often used as a scaffold or carrier of active
biomolecules in tissue engineering. For this purpose, it
is necessary for chitosan to possess enough persistence
time to allow the growth and extension of newborn tis-
sue. For various applications as a relevant candidate for
CID isor80004805
damaged (JPP)
tissue and organ, chitosan scaffolds
07 Dec 2016 04:41:09
be easily processed into different shapes by method-
Publishers
ologies, such as hydrogels, foams or sponges, and fibrous
membranes. Based on the adopted processing methods and
inherent properties of chitosan matrix, these biomaterials
with various morphologies may undergo in vivo degrada-
tion in days to weeks. For example, a chitosan derivative
(N ,O-carboxymethyl chitosan, with molecular weight of
200 kDa and 92% of DD) hydrogel persisted for ∼5 days
when it was used as a wound healing dressing.11 In a
yet another case, a chitosan-gelatin tridimensional scaffold
was observed even after 5 weeks of implantation into tooth
sockets of rats.12
Degradation of chitosan is also greatly influenced by
the administration route. After oral administration, chi-
tosan is degraded chemically as well as enzymatically via
hydrolysis of glucosamine–glucosamine, glucosamine-N -
acetyl-glucosamine, and N -acetyl-glucosamine-N -acetyl-
glucosamine linkages of chitosan.10 It has been confirmed
that chitosan and its derivatives are excellent nasal absorp-
tion enhancers. It widely used in formulations such as
solutions, gels, powders, and nanoparticles to deliver a
large amount of drugs owing to their unique efficacy in
improving the nasal bioavailability. When the chitosan-
based drugs are administrated by nasal route, they may
undergo the enzymatic degradation in the nasal cavity
by many enzymes including cytochrome-P450 dependent
monooxygenase, aldehyde dehydrogenase, peptidases and
proteases, etc.13
1587
Chitosan Nanoparticles in Cancer Treatment Fu et al.

Toxicity
Among natural polymers, chitosan is widely regarded
as non-toxic, biocompatible, and biodegradable polysac-
charide. Several products based on chitosan have been
proved by the FDA for use in wound dressing. How-
ever, when chitosan and its derivatives are used to deliver
drugs and genes, the toxicity of nanosized chitosan parti-
cles should not be ignored because the nanoparticles may
enter systemic circulation from the gastrointestinal tract,
nasal cavity, or alveolar sacs, thereby causing various lev-
els of toxicity to the human body.10 14 Also, in order to
achieve a specific targeting therapy, structural modification
of chitosan is carried out. This may lead to some toxicity
depending on the toxicity of the reactant(s) used.
Some studies have reported the toxicity evaluation of
CSNPs. The toxicity level of CSNPs may be related to sev-
eral factors including the properties of the parent material
which was used to prepare the nanoparticles, particle-size,
and the interacting cell type.15 Huang et al. reported that
the binding affinity and cell uptake capacity of CSNPs
reduced with the decrease of molecular weight (Mw) and
DD of the parent polymer. Cell uptake fell by 26% when
Mw was decreased from 213,000 to 10,000 and by 41%
when DD was lowered from 88% to 46%. Additionally,
the uptake data was also influenced by the zeta potential
of the nanoparticles. Chitosan molecules and nanoparticles
showed comparable cytotoxicityDelivered
profiles against the A549
by Ingenta to: Main CID is 80004805 (JPP)
cells and exhibited similar IC50 IP: IC20 values.16 On: Wed, 07 Dec 2016 04:41:09
and146.185.205.29
Since liver is the main detoxifying Copyright:
organ in theAmerican
human Scientific Publishers
body, hepatic toxicity of CSNPs should be taken into
consideration. CSNPs are known to show certain neg-
ative effects on hepatic cells. A study carried out by
Qi et al. reported that the incubation of human hepato-
cellular carcinoma-derived cell line (BEL7402 cells) with Figure 2. Investigation of the mechanism of toxicity caused
by chitosan nanoparticles. (A) cellular apoptosis was
CSNPs at 40 nm for 24 and 48 h displayed cytotoxicity determined using acridine orange staining of chitosan
with IC50 values of 15.01 and 6.19 g/mL, respectively.17 nanoparticle-exposed embryos at 96 post-fertilization.
Loh et al. also evaluated the hepatotoxicity of CSNPs.15 (B) Flow cytometric analysis of intracellular reactive oxygen
The cell toxicity analysis results revealed that human liver species in the control group (left panel) and 5 mg/L chitosan
nanoparticle-treated group (right panel). (C) Western blot
cells could tolerate 0.5% w/v of CSNPs (18 ± 1 nm,
analysis of HSP70 in zebrafish embryos. Reproduced with
7.5 ± 1.0 mV in culture medium) for up to 4 h. When permission from [14], Y. L. Hu, et al., Toxicity evaluation
the concentrations of chitosan nanoparticle were above of biodegradable chitosan nanoparticles using a zebrafish
0.5% w/v, cell membrane integrity was compromised as embryo model. Int. J. Nanomed. 6, 3351 (2011). © 2011,
evidenced by leakage of alanine transaminase into the DovePress.
extracellular milieu. Additionally, CYP3A4, an enzyme
Therefore, chitosan toxicity must be taken into account
found predominantly in the liver and intestinal enterocytes,
when CSNPs are used in systemic drug delivery.
showed a dose-dependent increase in its activity. CYP3A4
is responsible for approximately 60% of CYP-mediated
drug metabolism in human. Hu et al. used a zebrafish MODIFICATION OF CHITOSAN
embryo model to investigate the toxicity of CSNPs.14 Their NANOPARTICLES
results indicated that with an increase in the concentration Molecular backbone of chitosan contains reactive amino
of CSNPs, embryos showed a decreased hatching rate and and hydroxyl groups that can be readily decorated with
increased mortality. The physiological stress in zebrafish various ligands, functional groups and moieties.18 After the
caused by the nanoparticles is also evident by increased appropriate modification, beneficial properties can be
cytotoxicity, high expression of reactive oxygen species, as obtained including enhanced solubility, biocompatibility,
well as overexpression of heat shock protein 70 (Fig. 2).14 active targeting, etc. (Table I).

1588 J. Biomed. Nanotechnol. 12, 1585–1603, 2016

Fu et al. Chitosan Nanoparticles in Cancer Treatment

Table I. Modified chitosan derivatives used in anti-cancer drug delivery.

Modifier(s) Loading drug/gene(s) Cancer/cells Functions Ref.

Hyaluronic acid MiR-34a/Doxorubicin Breast cancer Combination therapy [26]

Heparin CdtB Gastric cancer Enhancing therapy efficacy [24]
PLGA Survivin siRNA Bladder cancer Gene therapy [29]
PNIPAM 5-FU – Thermo- and pH-sensitive [30]
PAMAM Lysozyme L929 fibroblast cells Gene therapy [31]
PEG ATRA U87MG cells Inhibiting tumor cell migration [32]
PEG-PCL Paclitaxel/Rutin – Prolonging drug release [33]
PLA Anthraquinone HepG2 cells Enhancing antitumor activity [34]
Folic acid (FA) pEGFP-N1 HeLa, MCF-7 Gene therapy [21]
CuAA HeLa, MCF-7, Hep-G2 Targeted delivery [35]
5-ALA HT29 cells Photodynamic detection [86]
NaYF4: Yb/Er H460 lung cancer cells Molecular diagnosis [96]
Oleic acid Cy5.5/ION U87MG cells Optical/MR imaging [36]
D,L-Lactic acid BSA – Prolonged drug delivery [39]
Glycol Paclitaxel Breast cancer Tumor-targeting therapy [54]
siRNA MCF-7/ADR Overcoming multi-drug resistance [72]
AuNPs CT-26 colon cancer cells Tumor-targeting CT imaging [90]
PpIX HT29 cells Photodynamic imaging and therapy [94]
Paclitaxel B16F10 melanoma tumor Enhanced therapeutic efficacy [123]
Cisplatin SCC7 cell-derived tumor Enhanced anti-tumor efficacy [134]
RGD peptide B16F10 melanoma tumor Antiangiogenic therapy [150]
Glycyrrhetinic acid DOX HepG2 Liver-cancer targeting therapy [41]
5-FU SMMC-7721 cells Liver-cancer targeting therapy [42]
Galactose (GC) 5-FU SMMC-7721 cells Liver-cancer targeting therapy [44]
Herceptin Gemcitabine Pancreatic cancer Enhanced antiproliferative activity [49]
Silk fibroin Proteins HepG2/CCL-13 cells Liver-cancer targeting therapy [50]
RGD peptide NaYF4:Yb/Er U87-MG/MCF-7 cells Photodynamic therapy [51]

Notes: Abbreviation: Hyaluronic acid (HA); Polyamidoamine (PAMAM); Poly(N-isopropylacrylamide) (PNIPAM); Poly(lactic-co-glycolic acid) (PLGA);
Delivered
Poly(ethylene glycol) (PEG), Methoxy poly(ethylene by Ingenta to:
glycol)–poly(lactic acid)Main CID isPoly(ethylene
(mPEG-PLA); 80004805glycol)–poly(-caprolactone)
(JPP) (PEG-PCL); All-trans
IP: 146.185.205.29
retinoic acid (ATRA); Green fluorescent protein On: Wed, 07
(pEGFP-N1); Copper acetylacetonate Dec 5-aminolevulinic
(CuAA); 2016 04:41:09 acid (5-ALA); Iron oxide nanoparticles (ION);
Bovine serum albumin (BSA); Gold nanoparticles Copyright: American
(AuNPs); Protoporphyrin Scientific
IX (PpIX); Publishers
Doxorubicin (DOX).

Chitosan Nanoparticles Modified by
Carbon-Based Materials
Carbon nanotubes (CNT) are carbon cylinders composed
of benzene rings. Although their insolubility in water
and organic solvents may cause toxicity, they are being
applied in biomedical fields as carriers to deliver pro-
tein, drugs, and sensors for DNA detection, inter alia.19
For the purpose of improving water-solubility and multi-
functionalization, they can be chemically modified by
conjugation with entities such as proteins, peptides, and
bipolymers. However, the use of organic solvents may
limit polymer functionalization. To address the problem,
Li et al. developed multi-walled carbon nanotube chitosan
nanoparticle (MWCNT CSNP) hybrids by an ionotropic
gelation process. The in situ synthesis technique was
conducted at extremely mild room temperature condi-
tions without the use of any toxic solvents. The pre-
pared MWCNT-CSNP hybrids improved the model protein
immobilization efficiency 0.8 times and simultaneously
decreased the cellular toxicity by ∼50% compared with
carboxylated MWCNT.20 MWCNTs with varying length
were also conjugated with chitosan-folic acid nanoparticles
(CS-FA NPs) by ionotropic gelation process. The surface
functionalization improves the transfection efficiency and
decreases the cytotoxicity of MWCNTs.21 Therefore, the
MWCNT-CS-FA NPs can be used as gene delivery vectors
for cancer treatment.
Chitosan Nanoparticles Modified by
Natural Polysaccharide
Heparin is a biodegradable, biocompatible, and negatively
charged polysaccharide with many carboxylic groups in its
molecular structure. It is relatively stable in vitro but it
can be degraded by hydrolysis and in vivo enzymolysis.22
Thus, it can be used to stabilize functional nanoparti-
cles via the ionic interaction between cationic chitosan
and anionic heparin. A study reported by Yuk et al.
revealed that gold-deposited iron oxide NPs immobilized
into the glycol chitosan/heparin network showed enhanced
tumor-specific targeting (Fig. 3).23 Lai et al. used heparin-
modified chitosan NPs to load cytolethal distending toxin
(CdtB) for the treatment of gastric cancer. Like CdtB
itself, the CdtB-encapsulated nanoparticles could enhanc
cell-cycle arrest at G2/M leading to apoptosis. The mech-
anism for CdtB-encapsulated nanoparticles-induced cell
death was mediated by ATM-dependent DNA damage
checkpoint responses.24
Hyaluronic acid (HA) is a biodegradable and lin-
ear polysaccharide with high binding capacity to HA
receptors (trans-membrane glycoprotein CD44). Therefore,

J. Biomed. Nanotechnol. 12, 1585–1603, 2016 1589

Chitosan Nanoparticles in Cancer Treatment Fu et al.

Figure 3. In vivo noninvasive NIR images and quantification analysis of the Cy5.5/composite NPs with or without heparin.
Reproduced with permission from [23], S. H. Yuk, et al., Glycol chitosan/heparin immobilized iron oxide nanoparticles with a
tumor-targeting characteristic for magnetic resonance imaging. Biomacromolecules 12, 2335 (2011). © 2011, American Chemical
Society.

HA modified DDS can specifically improve drug accu- and/or to the tumor site, thereby increasing the therapeutic
mulation in cancer cells over-expressing CD-44. For response.29
example, polyelectrolyte complex nanoparticles based on Poly(N -isopropylacrylamide) (PNIPAA) is a widely
hyaluronic acid/chitosan (HA/CS) showed potent cytotox- studied polymer with excellent thermosensitivity. The
icity and higher uptake efficiency in C6 cells after load- PNIPAA-modified CSNPs has the potential to exhibit
ing a water-insoluble curcuminoid.25 Deng et al. evidenced unique thermo-responsibility. Zhang et al. prepared self-
simultaneous co-delivery of chemotherapeutic agent dox- assembled chitosan-graft-poly(N -isopropylacrylamide)/
orubicin (DOX) and tumor suppressive
DeliveredmiRNA-34a
by Ingenta into
to: Maincarboxymethyl
CID is 80004805 cellulose
(JPP) nanoparticles to load
IP: 146.185.205.29
triple negative breast cancer cells by HA-CSNPs. ItOn: notWed, 07 Dec 2016
5-fluorouracil 04:41:09
(5-FU). The obtained polyelectrolyte com-
only improved the antitumor activity Copyright:
of DOX, butAmerican
it also Scientific Publishers were thermosensitive and had an
plex nanoparticles
suppressed tumor cells migration by targeting Notch-1 average diameter of about 200 nm.30
26
signaling. Besides, HA-CSNPs also have a great poten- Polyamidoamine dendrimers are a new type of synthetic
tial in ocular gene delivery.27 polymer characterized by a branched spherical shape and a
Apart from the above mentioned polysaccharides, other high density surface charge. Zhang et al. grafted carboxyl
polysaccharides such as alginate, starch, pectin, and car- group-poly(amidoamine) onto carboxymethyl chitosan for
boxymethyl cellulose are also used to fabricate polyelec- preparing core–shell nanoparticles.31 The self-assembled
28
trolyte complexes with chitosan for drug delivery. dendrimer nanoparticles did not display significant cyto-
toxicity in the range of concentrations below 3.16 mg/ml
Chitosan Nanoparticles Modified by Chemically and showed excellent properties as highly potent and non-
Synthesized Copolymer toxic intracellular carriers for protein delivery.
Many chemically synthesized copolymers like polyesters In addition to these, other polymers, such as polyethy-
and polyamides have been used directly as drug carriers lene glycol, methoxy poly(ethylene glycol)–poly(lactic
owing to their excellent biocompatibility and controllable acid) (mPEG-PLA), poly(ethylene glycol)–poly(-
degradability. They can also be used to improve the bio- caprolactone) (PEG-PCL), have also been grafted onto
logical and chemical properties of chitosan by chemical chitosan for fabricating various carrier nanoparticles.32–34
conjugation. For example, Chen et al. used paclitaxel and rutin as
Poly(lactic-co-glycolic acid) (PLGA) is a biocompati- model drugs for the evaluation of the controlled release
ble and degradable polymer that has been widely used to capacity of the chitosan-PEG-PCL nanoparticles.33 Wang
encapsulate and deliver various chemotherapeutic drugs, et al. prepared cationic biodegradable mPEG-PLA-CSNPs
siRNAs, DNAs, peptides, and proteins. Chitosan-modified as a small interfering ribonucleic acid (siRNA) delivery
34
PLGA nanoparticles can protect biologic agents from system for hepatitis B.
degradation during systemic circulation, thereby enhanc-
ing therapeutic efficacy. Martin et al. reported that Chitosan Nanoparticles Modified by Low
PLGA-chitosan (chitosan with a low molecular weight Molecular Weight Compounds
of 2.5 kDa) nanoparticles have the capacity to transport The compounds with low molecular weight offer
large amounts of surviving siRNA across the urothelium unique characteristics in modification of nanoparticles.

1590 J. Biomed. Nanotechnol. 12, 1585–1603, 2016

Fu et al.
The modified NPs may exhibit targeting efficacy, lower
toxicity, longer circulation time, etc. Folate, a low molec-
ular weight compound, is often used as a modifier owing
to high-expression of folate receptor in many cancer
cells. Folic acid can be covalently conjugated to chi-
tosan molecules via its gamma-carboxyl moiety. The
folic acid-tagged hydrophobic-modified chitosan NPs has
been shown to load curcumin and successfully deliver
the drug to folate receptor over-expressed cancer cells.35
Oleic acid is often used to prepare magnetic iron oxide
nanoparticles (Fe3 O4 NPs), and the oleic acid-decorated
Fe3 O4 NPs are usually encapsulated in various carriers
for use as imaging probes to detect tumors. However,
the interface compatibility between Fe3 O4 NPs and carri-
ers can greatly influence the loading efficacy and imag-
ing quality. Oleic acid is used to modify the polymeric
carriers to improve the interface compatibility. After graft-
ing with oleic acid, oleoyl-chitosan self-assembles into
core–shell structures in aqueous solution and provides
36
the effective core compartment for loading Fe3 O4 NPs.
A high-performance nanoparticle is prepared by physi-
cally complexing succinate-modified chitosan with folic
37
acid-modified chitosan. The resulting nanoparticles have
an average diameter and zeta potential of 110.0 nm and
18.6 mV, respectively. They are very stable in aqueous
suspension and are readily engulfed by oral cancer cells
via folate-receptor-mediated endocytosis. Therefore, this
kind of chitosan nanoparticle is Delivered by Ingenta
an excellent vector to:
for Main CID isfunctions.
IP: 146.185.205.29 On:
oral-specific delivery of 5-aminolevulinic acid for fluores-
Copyright: American Scientific
cent endoscopic detection, and it can be used for pho-
todynamic detection of oral cancer. Jin et al. prepared
N -octyl-O-sulfated chitosan (NOSC) with a viscosity aver-
age molecular weight of 65–70 kDa for the prepara-
tion of PTX-loaded micelles.38 The PTX micelles showed
superior blood persistence, tumor accumulation, and ther-
apeutic efficacy after intravenous injection into the tumor-
bearing mice.
D,L-Lactic acid has also been used as a modifier to dec-
orate chitosan by grafting lactic acid onto amino groups
in chitosan. After incorporating the lactyl segment into
the chitosan backbone, the resulting nanoparticle showed
a high protein encapsulation (96%) and a prolonged drug
release rate (15%) over 4 weeks.39 Glycyrrhetinic acid
(GA) is one of the main bioactive compounds of licorice
and is used widely as medicine for the treatment of
many diseases. It is also used as a ligand to target liver
because of the abundance of receptors for GA on the hep-
atocyte membrane.40 Tian et al. used GA-modified sul-
fated chitosan (GA-SCS) to prepare doxorubicin-loaded
41
micelles (DOX/SA-SCS micelles). The IC50 of the pre-
pared micelles against liver cancer cells (HepG2 cells) was
54.7 ng/mL, remarkably lower than that of the no-GA-
modified micelles. Moreover, the DOX/SA-SCS micelles
showed higher affinity for the liver cancer cells (HepG2
cells) than for the normal liver cells (Chang liver cells).
Another study reported that 5-FU-conjugated GA-modified
J. Biomed. Nanotechnol. 12, 1585–1603, 2016
Chitosan Nanoparticles in Cancer Treatment
chitosan nanoparticles can target the liver, and have sig-
nificantly inhibited tumor growth in an orthotropic liver
cancer mouse model.42
In order to enhance liver-targeted drug delivery, galac-
tosylated nanocarrier is another ideal option. Asialo-
glycoprotein receptors are expressed predominantly on
hepatocytes for clearance of galactose-terminated gly-
coproteins, and have been identified on a continuous
human hepatoma cell line, HepG2.43 Therefore, the ther-
apeutic activity can be improved by the interactions
between the ligand and liver cancer cell receptors. Cheng
et al. used galactosylated chitosan (GC) to prepare 5-FU
loaded nanoparticles (GC/5-FU). Analysis of apoptosis
pathways indicated that GC/5-FU upregulates p53 expres-
sion at both protein and mRNA levels. In vivo antitumor
evaluation showed that the sustained release of GC/5-FU
nanoparticles was more effective at targeting hepatic can-
cer cells than 5-FU monotherapy in the mouse orthotropic
liver cancer mouse model.44 Owing to its excellent active
targeting ability, galactosylated chitosan has been widely
used as nanocarriers for delivering various therapeutic
drugs and genes in cancer treatment.45–48
Chitosan Nanoparticles Modified by
Peptide, Protein
The bioactive molecules including peptides and proteins
are also used to prepare functionalized chitosan NPs with
unique 80004805 (JPP)
Wed, 07 Dec 2016 04:41:09
In order
to enhance selective delivery of anti-tumor
Publishers
drugs to tumor sites, herceptin was conjugated with
gemcitabine-loaded CSNPs (HER2-Gem-CSNPs).49 The
targeted NPs displayed marked cytotoxicity along with an
enhanced S-phase arrest, leading to apoptosis in compari-
son with free gemcitabine and unconjugated gemcitabine-
loaded nanoparticles due to higher cellular binding with
eventual uptake and prolonged intracellular retention.
Thus, HER2-Gem-CSNPs are able to provide an efficient
and targeted delivery of gemcitabine for pancreatic cancer
treatment.
Silk fibroin is an insoluble protein with low immune or
inflammatory response and favorable biological response
characteristics. Therefore, silk fibroin-modified CSNPs are
very suitable for chemotherapeutic delivery in cancer treat-
ment because of their better stability, low toxicity, simple
and mild preparation methods.50 Zhou et al. developed
a kind of upconversion nanoparticle (UCNP-Ppa-RGD)
by using a photosensitizer pyropheophorbide a (Ppa) and
RGD peptide c (RGDyK) to co-modify chitosan and
wrap NaYF4:Yb/Er nanocrystals.51 The UCNP-Ppa-RGD
nanoparticles exhibited low dark toxicity and high photo-
toxicity against cancer cells upon 980 nm laser irradiation
at an appropriate dose indicating that they may be used
for near-infrared photodynamic therapy with an improved
tumor targeting specificity.
Besides the above mentioned peptides and proteins,
other proteins have also been applied to modify chitosan
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Chitosan Nanoparticles in Cancer Treatment
for preparing the nanoparticles with unique physicochem-
ical properties. For example, lectin conjugated chitosan
nanoparticles showed fluorescence characteristics with a
potential to be used for disease detection and drug
delivery.52
FUNCTIONS OF CHITOSAN
NANOPARTICLES
Even though chitosan nanoformulations are seldom used
in cancer treatment up till now, multi-functional CSNPs
are showing promise in personalized therapy. The nanopar-
ticles may show improved anti-tumor efficacy and spe-
cific targeting ability due to modification of chitosan
nanocarriers or encapsulation of multiple therapeutic
agents.
Enhancing Anti-Tumor Efficacy
In clinical treatment, many anticancer drugs with tra-
ditional formulation are being despised due to certain
disadvantages such as poor water solubility, short cir-
culation time in vivo, poor targeting and high side
effects.53 Nanomedicine holds a great potential in resolv-
ing these problems. Nanoparticles have been considered
to be effective carriers because they can stay unrec-
ognized during blood circulation, reduce the adverse
reactions and increase the therapeutic efficacy.3 As one
of drug carriers, CSNPs mayDelivered by Ingenta
show enhanced to: Mainticles
antitu-
mor efficacy in cancer treatment IP:owing
146.185.205.29 On: Wed, 07 Dec 2016 04:41:09
to many unique
characteristics.5 Water soluble chitosanCopyright: American
naonoparticles can Scientific
improve water solubility of hydrophobic drugs.54 55 The
biodegradability of chitosan matrix may ensure that encap-
sulated drugs are released in a controlled fashion.4 18 The
small-sized CSNPs can pass through biological barriers
in vivo and deliver drugs to the tumor site owing to
the enhanced permeation and retention or active target-
ing ability.47 The specific targeting of modified CSNPs
can improve bioavailability of therapeutic agents and
reduce systemic toxicity. Therefore, antitumor efficacy
can be enhanced by multi-functional CSNPs or mul-
tiple drug loading strategy. For example, co-delivery
of paclitaxel and survivin shRNA-expressing plasmid
(iSur-pDNA) by folate-modified amphiphilic linoleic acid
and poly(-malic acid) double grafted CSNPs exhib-
ited enhanced antitumor efficacy and prolonged survival
period as compared with single delivery of PTX or
iSur-pDNA. 56
Improving Special Targeting Tumor Tissue
According to the targeting pattern, therapeutic nanopar-
ticles are commonly divided into passive and active
targeting nanoparticles. As mentioned above, passive tar-
geting nanoparticles accumulation in tumor tissues mainly
depends on the enhanced permeation and retention. Active
targeting based on ligand-receptor interactions is an impor-
tant way of increasing specific targeting ability of drug
1592
Fu et al.
delivery system. It has been intensively studied in various
drug delivery systems. The nanoparticles based on galac-
tosylated chitosan (GC) and 5-FU can be more effective at
targeting hepatic cancer cells than 5-FU monotherapy in
the orthotropic liver cancer mouse model because Asialo-
glycoprotein receptor (ASGPR) found on membranes of
hepatocytes shows specificity for glycoproteins.44 Gly-
cyrrhetinic acid-modified sulfated chitosan (GA-SCTS)
micelles specifically target the liver cancer cells (HepG2
cells) and display rapid and significant ability to target
the liver in vivo.41 42 An aptamer conjugated hyaluro-
nan/CSNPs were prepared and used as carriers for targeted
delivery of 5-FU by Ghasemi et al. The prepared NPs
showed significantly higher targeting ability in (MUC1+
human colorectal adenocarcinoma as compared with the
free drug.57
Prolonging Blood Circulation Time
During the development of effective drug delivery
nanoparticles, one major obstacle is the rapid clearance
from blood. The ability of drug-loaded nanoparticles
to circulate in the bloodstream for a prolonged period
of time is often a prerequisite for successful targeted
delivery.58 59 They should evade the phagocytic uptake by
reducing opsonization by blood proteins, hence increasing
the bioavailability of the drug. The in vivo fate of nanopar-
CID isis 80004805 (JPP) on the chemical and physi-
mainly dependent
cal properties of the NPs, including size, surface charge
and
Publishers
surface chemistry. 60 61
In order to overcome these
obstacles, some shielding groups including PEG, polyvinyl
alcohol, and polysaccharides are adsorbed or grafted on
the surface of NPs for masking the NPs because these
groups or polymers can hinder the hydrophobic and elec-
trostatic interactions that help plasma proteins bind to par-
ticle surface.62–64
For the purpose of decorating NPs, PEG seems to
be an ideal candidate and has been extensively used
to coat the surface of NPs, as it successfully weakens
the uptake by cells of mononuclear phagocytic system
(MPS) and leads to prolonged blood circulation time.65
Some CSNPs also display prolonged blood circulation
time after PEG modification. Parveen et al. reported that
PLGA-CS-PEG nanoparticles showed dramatic prolonga-
tion in blood circulation, as well as reduced macrophage
uptake, with only a small amount of the nanoparticles
sequestered in the liver.58 Long blood circulation of drug
also has great advantage in the treatment of blood malig-
nancies. Termsarasab et al. developed doxorubicin (DOX)-
loaded nanoparticles based on PEG-conjugated chitosan
oligosaccharide-arachidic acid (CSOAA-PEG) for poten-
tial application to leukemia therapy.64 In vivo clearance
rate of DOX from the CSOAA-PEG nanoparticle group
was slower than other groups, thereby extending the cir-
culation period.
J. Biomed. Nanotechnol. 12, 1585–1603, 2016
Fu et al. Chitosan Nanoparticles in Cancer Treatment

Reversal of Multi-Drug Resistance MCF-7/ADR tumors and downregulate P-gp expression to

Multi-drug resistance (MDR) is a major barrier for lim-
iting the therapeutic effects of chemotherapeutic agents
in cancer treatment. Once a patient suffers from MDR,
the therapy efficiency is reduced and leads to the fail-
ure of the treatment. P-glycoprotein-mediated resistance
is the most extensively studied MDR pathway.66 It has
been confirmed that nanoparticles may be able to reverse
the drug resistance because it may avoid recognition by
the P-glycoprotein (P-gp) efflux pump by means of being
enveloped in an endosome when entering the cell, leading
to high intracellular drug concentrations. 67–69
To overcome MDR, Jin et al. used amphiphilic
N -octyl-O-sulfate chitosan (NOSC) copolymer to pre-
pare paclitaxel-encapsulated micelles (PTX-M). Owing to
the combination of the inhibiting P-gp effect of NOSC
and the bypassing P-gp action of the intact PTX-M, the
micelles had superior blood persistence, tumor accumu-
lation, and therapeutic efficacy after intravenous injection
into the tumor-bearing mice (Fig. 4).38 Besides load-
ing of the chemotherapeutic agents, CSNPs are also
used to deliver genes for the purpose of MDR reversal.
Zhang et al. used magnetic-Fe3O4 chitosan nanoparticle-
encapsulated MDR1 siRNA to investigate the effects of
MDR1 gene on the reversal of MDR in the glioblastoma
cell line BT325. Their results revealed that the expres-
sion of MDR1 at both the mRNA Delivered
and P-gp by protein
Ingentalevel
to: Maina CID
IP: 146.185.205.29
decreased, which increased sensitivity On: Wed,
to chemotherapy
in vitro.70 Yhee et al. also confirmedCopyright: American Scientific Publishers
that the thiolated
glycol chitosan could deliver Pgp-targeted poly-siRNA
and form stable nanoparticles. The resulting nanoparticles
could not only protect siRNA molecules from enzymatic
degradation, but also accumulate in adriamycin-resistant
sensitize cancer cells after intravenous administration.71
Crossing Blood-Brain Barrier
As the biological interface between the brain and the
blood, the blood-brain barrier (BBB) takes responsibil-
ity for transporting essential nutrients and protecting the
brain.72 The BBB constitutes an efficient organization of
tight junctions between endothelial cells in the brain tis-
sues. The barrier can prevent harmful substances including
chemotherapeutic agents entering into the brain intersti-
tium and protect the cells of nervous system.73 74 However,
the existence of BBB brings tremendous negative impact
on therapeutic effects of brain tumors when chemother-
apeutic drugs are used. Therefore, various nanoparti-
cles are being the optional strategies for overcoming the
problem.75–77 Especially, it has been demonstrated that
cationic nanoparticles showed prominent efficacy in per-
meating the BBB due to their cationic charge.72 78
As a cationic polymer, CSNPs receive much attention
in preparation of nanoformulations or nanoprobes for the
treatment and diagnosis of brain cancers. Veiseh et al.
used chitosan as a linker and stabilizer to develop a tar-
geting nanoprobe (NPCP-CTX-Cy5.5) for the purpose of
selective accumulation in brain tumors across the BBB.72
The nanoprobe is comprised of an iron oxide nanoparti-
cle coated with a PEGylated chitosan-branched copolymer,
is 80004805
targeting (JPP)
ligand, chlorotoxin (CTX), and a near-IR flu-
07 Dec 2016 04:41:09
orophore, Cy.5.5. The magnetofluorescent nanoprobe was
able to traverse the BBB and remain in brain tumors
for 70 hours. Stephen et al. developed redox-responsive
magnetic nanoparticles by using chitosan-PEG copolymer
to coat magnetic iron oxide nanoparticles for targeted
convection-enhanced delivery (CED) of O6 -benzylguanine

Figure 4. Scheme of collaborative mechanism-induced improvement on the PTX-resistant cellular uptake and therapeutic effi-
cacy of PTX by PTX-M: passive targeting of the intact PTX-M to the tumor tissues by the EPR effect (A) and the mechanism of
enhanced accumulation of PTX inside tumor cells by PTX-M via a combination mechanism of the inhibiting P-gp effect of NOSC
and the bypassing P-gp action of the intact PTX-M (B). Reproduced with permission from [38], X. Jin, et al., Paclitaxel-loaded
N-octyl-O-sulfate chitosan micelles for superior cancer therapeutic efficacy and overcoming drug resistance. Mol. Pharm. 11, 145
(2014). © 2014, American Chemical Society.

J. Biomed. Nanotechnol. 12, 1585–1603, 2016 1593

Chitosan Nanoparticles in Cancer Treatment
to brain tumors.79 The NPs remained within the tumor
boundary at 48 hours post CED. The in vivo efficacy of the
NPs in combination with temozolomide showed a 3-fold
increase in survival as compared to untreated animals.
Although the specific mechanisms for BBB passage are
yet to be elucidated, it has been proven that P-glycoprotein
plays a vital role in the BBB. P-gp is an ATP-dependent
drug transport protein that is expressed in cells at the
anatomical and physiological barriers in mammalian tis-
sues and also in malignant cells.80 81 Much effort has
focused on finding ways to overcome drug efflux from
P-gp expressing cells. Therefore, the strategies to avoid
P-gp mediated drug efflux at the BBB such as the use of
specific inhibitors, altering the gene regulation and injec-
tion of siRNA, are being used to reduce the expression of
P-gp in brain endothelial cells.72 Mallmo et al. developed
siRNA-CSNPs to silence P-gp in a brain BBB model.82
The nanoparticles led to a significant reduction in P-gp
substrate efflux and improved the delivery and efficacy of
doxorubicin.
Diagnosis, Detection and Imaging
With the process of nanomedicine and molecular imag-
ing, a variety of tumor-targeting nanoparticles have been
designed and extensively studied for cancer theranostics.83
The functional NPs can chemically interact with biomark-
ers to alter the signals for imaging and provide biological
Delivered by Ingenta
information on pathological lesions. Therefore, they areto: Main CID is 80004805
IP: 146.185.205.29 On: Wed,
expected to achieve early diagnosis and personalized ther-
Copyright: American Scientific Publishers
apy in cancer treatment in the near future.84
Owing to excellent biocompatibility and available func-
tional groups on chitosan, it is often used to encapsu-
late or decorate other nanoparticles for preparing various
multifunctional nanoparticles. For the purpose of disease
diagnosis and detection, Yang et al. developed a high-
performance nanoparticle by using alginate to physically
complex with folic acid-modified chitosan for fluorescent
endoscopic detection of colorectal cancer.85 Zhu et al.
developed a probe using nanoparticles of miR-155 MB
self-assembled with chitosan (CS-miR-155 MB) to image
86
the expression of miR-155 in lung cancer cells. The
CSNPs showed higher fluorescence intensity and trans-
fection efficiency, thus can be used for detecting miRNA
expression in living cells. Hajdu et al. prepared gadolin-
ium (Gd) ions loaded nanoparticles using poly-gamma-
87
glutamic acid (-PGA) and chitosan biopolymers. MRI
measurements and MRI/PET fusion images revealed that
the targeting NPs could accumulate in tumors and provide
a suitable means for the early diagnosis of tumors based
on their overexpression of folate receptors.
Chitosan is commonly used to coat on surfaces of
the magnetic iron oxide nanoparticles for the preparation
of various magnetic resonance imaging contrast agents
for molecular imaging. Zhao et al. developed a host-
metal-guest coordination-bonding system to co-deliver pH-
responsive drug DOX and Fe3 O4 . They prepared the
1594
Fu et al.
mesostructured Fe3 O4 /CSNPs by a facile solvothermal
approach, and then used it to load DOX because chi-
tosan can act as a host molecule to form the NH2 Zn(II)
DOX coordination architecture.88 To obtain dual imaging
of near-infrared (NIR) and magnetic resonance imaging
(MRI), Lee et al. encapsulated oleic acid-decorated iron
oxide nanoparticles (ION) in oleic acid-conjugated chi-
tosan (oleoyl-chitosan) (Fig. 5).36 During in vivo evalua-
tion, both NIR and MR imaging showed the detectable
signal intensity and enhancement in tumor tissues via
enhanced permeability and retention effect after intra-
venous injection of ION-loaded Cy5.5-conjugated oleoyl-
chitosan nanoparticles. Ex vivo fluorescence images and
Prussian blue staining images also confirmed that the
nanoparticles accumulated in tumor tissues. Sun et al.
modified the surface of Au nanoparticles with biocompat-
ible glycol chitosan for tumor-targeting imaging. Several
tumor sites of metastatic liver cancer were successfully
located in the CT images when glycol chitosan-AuNPs
(0.1 ml, 50 mg Au/ml) were injected intravenously into
the mice.89
Chitosan can also be transformed into a nanoprobe
for use in fluorescence imaging. Ryu et al. developed
a CB-sensitive nanoprobe (CB-CSNP) consisting of self-
quenched CB-sensitive fluorogenic peptide probes conju-
gated onto the surface of tumor-targeting glycol chitosan
nanoparticles.90 The prepared CB-CSNP had a spherical
nanoparticle structure(JPP)
with a diameter of 280 nm and its
07 Dec 2016 04:41:09
fluorescence intensity was strongly quenched in physiolog-
ical condition. Evaluation of CB-CSNP in three metastatic
mouse models demonstrated the potential to discriminate
metastases in vivo through non-invasive CB molecular
imaging.
Photodynamic Therapy and Imaging
Photodynamic therapy (PDT) is becoming a promising
and non-invasive method for cancer treatment owing to its
unique therapeutic characteristics (Fig. 6).91 Under light
irradiation at certain wavelength, photosensitizer (PS) pro-
duces cytotoxic singlet oxygen (1 O2  to kill tumor cells
through apoptosis or necrosis.51 92 Moreover, the selec-
tive accumulation of photosensitizers in tumor tissues also
can be employed in photodynamic imaging (PDI) because
the photosensitizers can provide an intense fluorescence
signal.93 94 However, the photosensitizers are greatly lim-
ited in clinical use owing to their poor water solubility,
non-specific skin phototoxicity, inadequate tumor-targeting
selectivity, etc. One way for overcoming these shortcom-
ings is the use of nanocarriers to deliver photosensitizers
for enhancing their tumor specificity by the so-called EPR
effect.95 Therefore, chitosan is often used as coating poly-
mer to modify photosensitizers because of its nontoxicity,
excellent biocompatibility, good water-solubility and avail-
ability for further modification of functional groups.51 95
As a key component of PDT, the most used
photosensitizers include protoporphyrin IX (PpIX),
J. Biomed. Nanotechnol. 12, 1585–1603, 2016
Fu et al. Chitosan Nanoparticles in Cancer Treatment

Figure 5. In vivo NIRF images of U87MG-bearing mice. The images were obtained (a) before injection and at (b) 1 h, (c) 3 h,
and (d) 5 h after injection of Ion-Cy5.5-oleyl-chitosan nanoparticles. (e) Ex vivo NIRF images of major tissues excised from the
mice at 5 h postinjection. (f) A quantification of the ex vivo tissues was recorded as average radiance (p/s/cm2/sr). All data are
represented as mean ± SD (n = 3). Reproduced with permission from [36], C. M. Lee, et al., Oleyl-chitosan nanoparticles based
on a dual probe for optical/MR imaging in vivo. Bioconjug. Chem. 22, 186 (2011). © 2011, American Chemical Society.
Delivered by Ingenta to: Main CID is 80004805 (JPP)
IP: 146.185.205.29 On: Wed, 07 Dec 2016 04:41:09
pyropheophorbide a (Ppa), and other porphyrin derivatives.
Copyright: nanoparticles
American Scientific (UCNP-Ppa-RGD).51 The prepared nano-
Publishers
They all show excellent tumor-targeting specificity and structure exhibited low dark toxicity and high phototoxi-
photodynamic imaging ability in many tumor models. Lee city against U87-MG cells upon 980 nm laser irradiation
et al. conjugated the photosensitizer PpIX with glycol chi- at an appropriate dosage. The study of UCNPs has the
tosan polymer to prepare the tumor-targeting nanoparticles potential for cancer theranostics, individualized therapeu-
(PpIX-GC-NPs) for photodynamic imaging and therapy.93 tics, multimodality medicine, etc.
The PpIX-GC-NPs showed the self-quenching effect that
is in the ‘off’ state with no fluorescence signal and photo- Thermotherapy
toxicity with light exposure. After in vivo administration, Because of the thermosensitivity of tumor cells, ther-
the total photon counts of PpIX-GC-NPs in tumor tissue motherapy has been an important support to other
are 1.41 and 2.1 folds higher than that of PpIX-CNP and forms of cancer treatments in clinics. Taking the advan-
free PpIX-treated mice, respectively. tage of passive/active targeting ability of nanoparticles,
However, in order to overcome the limited therapeutic chemotherapeutic agents and functional nanoparticles can
depth of current PDT technologies, upconversion nanopar- be encapsulated simultaneously into CSNPs for combina-
ticles have gained increasing attention in recent years tion therapy. As a kind of thermotherapy, magnetic fluid
due to their unique upconversion luminescent properties.96 hyperthermia (MFH) is a new approach based on nan-
They enable the conversion of low-energy photons (NIR otechnology to deposit heat power in deep tissues by over-
photons) into high-energy photons (visible to UV photons) coming limitations of conventional heat treatments. After
via the multi-photon processes.97 98 Therefore, the specific infiltration into the target tissues with nanosized mag-
characteristics of upconversion nanoparticles including low netic particles, the power of an alternating magnetic field
toxicity, weak background fluorescence, high detection is transformed into heat.101 For the purpose of combi-
sensitivity, and deep light-penetration in tissues endow nation hyperthermia with chemotherapy for the treatment
them with great potential for use in in vivo bioimaging, of liver cancer, Li et al. developed carboplatin-Fe@C-
NIR-triggered drug/gene delivery applications and pho- loaded CSNPs by reverse microemulsion method using the
todynamic therapy.99 100 Zhou et al. used pyropheophor- nanocages Fe@C as magnetic cores and chitosan as the
bide a (Ppa) and RGD peptide c (RGDyK) to modify matrix.102 In vivo experiments showed that tumor tempera-
chitosan for preparation of NIR-to-visible upconversion ture reached 42.6 ± 0.2  C within 10 min in the alternating

J. Biomed. Nanotechnol. 12, 1585–1603, 2016 1595

Chitosan Nanoparticles in Cancer Treatment Fu et al.

Choi et al. developed a very effective hyperthermia system

based on Pluronic F68-conjugated chitosan and GNRs for
photothermal cancer therapy. The prepared nanoplatform
has a reasonable tumor-targeting efficiency and is highly
effective and safe for transducing NIR light into localized
heat.104

Figure 6. Schematic diagram of enhanced PDT by utiliz-
ing the surface plasmonic effect of the AuNR to simul-
taneously increase the absorption coefficient and reduce
photo1degradation of the photodynamic dye ICG. Meso-
porous silica provided the second photoprotection for ICG by
facilitating the formation of ICG aggregates. The double pho-
toprotection will significantly enhance the stability of loaded-
ICG in comparison with the single photoprotection of silica
shell. Reprinted with permission from [91], Y. Li, et al., Local-
ized electric field of plasmonic nanoplatform enhanced pho-
todynamic tumor therapy. ACS Nano 5, 11529 (2014). © 2014,
American Chemical Society.
Delivered by Ingenta to: Maininterfering
IP: 146.185.205.29 On: Wed, 07 Dec 2016 04:41:09
magnetic field after injection; and theCopyright:
temperatures American
in the Scientific Publishers
right hepatic lobes and the rectum were significantly lower
than in the tumor and the constant temperature could last
up to 30 min. The carboplatin-Fe@C-loaded CSNPs can
specifically target liver cancer tissue by static magnetic
field and with the application of alternating magnetic field,
effectively raise tumor tissue temperature and facilitate
tumor apoptosis. Therefore, the combination of chemother-
apy and MFH is likely to be a new safer and efficacious
therapy model in cancer treatment.
Photothermal therapy is an attractive technique for treat-
ing solid tumors in aminimally invasive manner by using
NIR laser light-generated heat to destroy tumor cells
(Fig. 7).103–105 Light-absorbing materials e.g., photothermal
conducting agents, as the source of photothermal effect,
play a key role in photoactivated cancer therapy. Among
reported NIR absorbents, gold nanocrystals, including gold
nanorods, silica-cored nanoshells, and gold nanocages,
were most extensively investigated due to their excellent
biocompatibility and tunable surface plasmon resonance
property to convert NIR light into local heat. Duan et al.
used DOX-conjugated chitosan derivatives to cover gold
nanorods (GNRs) for preparation of functional nanocar-
riers (DOX-CS-GNR).106 Because of the combination of
chemical and photothermal effects, the prepared nanocar-
riers showed good optical properties and enhanced antitu-
mor activity compared with photothermal therapy alone.
Gene Therapy
Gene therapy holds a promising alternative for can-
cer treatment. However, the major shortcoming of gene
therapy is the gene transfection rate.107 Currently, there
are two main types of vectors that are used in gene ther-
apy, viral or non-viral gene delivery systems. Although
the viral gene delivery system shows a high transfec-
tion yield, it still has many disadvantages, such as onco-
genic effects and immunogenicity.108–110 Therefore, as the
alternate, nonviral gene delivery system gains importance.
Especially, numerous synthetic chemicals, natural poly-
mers, or lipids have been used as nonviral vectors for
enhancing the efficiency of gene delivery.111 Among them,
as a natural nontoxic polysaccharide, chitosan has the
potential for gene delivery applications. Its biodegrad-
ability, biocompatibility, and the ability to protect DNA
against DNase degradation are added advantages.107
RNAi-based therapy is a highly specific method for
gene silencing which holds a dominant position in cancer
gene therapy. However, the delivery efficiency of free short
CID is 80004805
RNA (siRNA)(JPP) is quite low and most of the
free siRNA is rapidly degraded following i.v. injection.112
Therefore, in order to improve the delivery efficiency
and prolong the existence time, chitosan-based nanopar-
ticles are often used to load siRNA and form a vari-
ety of nanocomplexes because its positive charge allows
transport across cellular membranes and subsequent endo-
cytosis. Han et al. developed Arg-Gly-Asp (RGD) peptide-
labeled chitosan nanoparticles (RGD-CSNP) as a novel
tumor targeted delivery system for siRNA.113 In vivo tumor
vascular targeting was achieved by using RGD-CSP to
deliver growth-promoting gene PLXDC1-targeted iRNA
into the  3 integrin-positive tumor endothelial cells in
the A2780 tumor-bearing mice.
Some chitosan derivatives also show good DNA-
binding capacity and gene therapy efficacy after func-
tional modification. For example, Hu et al. prepared
a non-viral vesicle vector which was composed of
low-molecular weight polyethylenimine-conjugated stearic
acid-g-chitosan oligosaccharide (CSOSA-g-PEI) micelles.
The micelles had much lower cytotoxicity and corre-
sponding transfection efficiency in comparison with Lipo-
fectamine 2,000 in both human cancer cells (Hela and
MCF-7). Moreover, after intravenous injection in tumor-
bearing mice, CSOSA-g-PEI/plasmid pigment epithelium-
derived factor formulation could effectively localize in the
tumor tissue and suppress the tumor growth (above 60%
tumor inhibition) without systematic toxicity.114 In addi-
tion to those mentioned above, other chitosan-based

1596 J. Biomed. Nanotechnol. 12, 1585–1603, 2016

Fu et al. Chitosan Nanoparticles in Cancer Treatment

Figure 7. (A) Infrared thermal imaging of P-GNRs-DOX (1.0 mg mL−1 , contained 28 g Au mL−1 ) under 2.5 W cm−2 irradiation by
808 nm laser at different time and (B) infrared thermal imaging of tumor under the photothermal heating by 808 nm laser irradiation
in normal saline and P-GNRs-DOX injected mice under 2.5 W cm−2 irradiation. Reprinted with permission from [105], J. F. Liao,
et al., Combined cancer photothermal-chemotherapy based on doxorubicin/gold nanorod-loaded polymersomes. Theranostics
5, 345 (2015). © 2015, Theranostics Publishing Team.

nanoparticles have being widely studied to deliver siRNA an important role in cancer treatment. For example,
for gene silencing and these nanocomplexes have shown lactosyl-norcantharidin TMC nanoparticles (Lac-NCTD-
great potential in gene therapy.115–120 TMC-NPs) were prepared using an ionic cross-linkage
In addition, the use of chitosan vectors to deliver process.130 The Lac-NCTD-TMC-NPs had an average
short hairpin RNA (shRNAs) to knock-down genes particle size of about 120.6 nm and it showed the
has attracted considerable attention in cancer treat- strongest antitumor activity on the murine hepatocar-
ments. Chen et al. designed a novel nanoparticle cinoma 22 subcutaneous models. Hu et al. prepared
(Au-PEI/CS-Aco/PEI/shRNA) for delivering shRNA by chondroitin sulfate-chitosan (ChS-CS) nanoparticles and
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using a three-layered polyethyleneimine (PEI)-coated gold
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07 Dec 2016their 04:41:09
cellular uptake, cytotoxicity, and transep-
nanocomplex interlaid with a pH-responsive charge- Scientific
Copyright: American ithelial transport in human epithelial colorectal adenocar-
Publishers
reversible chitosan-aconitic anhydride (CS-Aco) as a car- cinoma (Caco-2) fibroblasts.131
rier. The prepared nanocomplexes displayed higher shRNA In addition to natural extracts, many first-line
delivery efficiency than 25 kDa PEI, and efficiently deliv- chemotherapeutic agents are also encapsulated in CSNPs
ered shABCG2 to tumors and markedly silenced the for cancer treatment owing to good biodegradability and
expression of ABCG2.121 biocompatibility of chitosan. Among them, 5-FU42 44 57 132
and cisplatin133 134 are well studied. For example, Xu et al.
CHITOSAN NANOPARTICLES IN developed an effective pH and oxidation dual-responsive
DRUG DELIVERY carrier by using ferrocene-modified chitosan oligosac-
Encapsulation of Chemotherapeutic charide nanoparticles for 5-FU delivery.132 Kim et al.
Agents Into Chitosan used hydrophobic cholanic acid-modified glycol chitosan
Chemotherapeutic agents play a central role in can- (HGCs) to encapsulate cisplatin (CDDP) and they believed
cer intervention. Because of their significant antitumor that HGC nanoparticles are a promising carrier for the
effect, various chemotherapeutic drugs, including natural anticancer drug CDDP.133 Arya et al. prepared herceptin-
or synthetic agents, were intensively investigated. As a conjugated gemcitabine-loaded CSNPs to target the deliv-
typical example of chemotherapeutic agent of plant ori- ery of gemcitabine for the treatment of pancreatic cancer.49
gin, paclitaxel was encapsulated into CSNPs by many
researchers. These systems showed a good controlled Directly Conjugated Chemotherapeutic
release behavior and enhanced antitumor efficacy in many Drugs with Chitosan Backbone
tumor cells such as MDA-MB231 human breast cancer Even though chitosan is extensively used as a drug car-
cells, B16F10, A2780 cells, Caco-2 cells, HepG2 cells, rier for delivery of various chemotherapeutic drugs, it only
etc.54 122–125 Additionally, other plant-derived anticancer acts as a vehicle to load drugs and not play a role in can-
agents and their synthetic derivatives, such as baicalein, cer treatment. Therefore, a prodrug strategy was proposed
camptothecine, curcumin, and resveratrol, were loaded on owing to rich reactive groups on chitosan backbone. Some
chitosan nanocarriers for tumor therapy.126–129 the hydrophobic anticancer drug can be directly conju-
Anticancer compounds of animal origin have also gated onto hydrophilic chitosan chains resulting in better
shown excellent antitumor activity and could play hydrophilicity and bioavailability of drugs. For example,

J. Biomed. Nanotechnol. 12, 1585–1603, 2016 1597

Chitosan Nanoparticles in Cancer Treatment
DOX was simply conjugated with chitosan via an acid-
cleavable hydrazone bond.135 The prepared prodrugs could
directly disperse in an aqueous solution without the use
of any organic solvent. The resulting Chitosan-hz-DOX
nanoparticles with size lower than 200 nm had an excel-
lent pH-sensitive drug release property as well as antitu-
mor activity. The Chitosan-hz-DOX particles were stable
at a neutral pH while disassembled at acidic pHs, result-
ing in a fast release of DOX. This was desired for the
internalization process of prodrug nanoparticles into can-
cer cells. Lu et al. developed a series of melphalan-O-
carboxymethyl chitosan conjugates. These conjugates had
a remarkably improved water solubility (160–217 times
that of melphalan solubility) and 52–70% of cytotoxicity
against RPMI8226 cells in vitro as compared with free
melphalan.136
Encapsulation of Metal Compounds
Several metals and their compounds find special appli-
cations in cancer diagnosis and treatment owing to their
unique properties. For example, platinum compounds have
significant antitumor efficacy and broad spectrum anti-
cancer activity attributable to their remarkable cytotoxic-
ity against tumor cells. Gold nanoparticles are often used
in NIR photothermal therapy due to their tunable sur-
face plasmon resonance property to convert NIR light into
local heat.106 Magnetic iron oxide nanoparticles
Delivered canto:
by Ingenta beMaintoCID
used as a magnetic resonance imaging agent and tumor-
IP: 146.185.205.29 On: Wed,
Copyright:
targeting drug carriers because they have American
excellent mag- Scientific
netic response.23 70
In addition to above mentioned metallic compounds,
several other metal nanoparticles and compounds are also
encapsulated in chitosan for cancer therapy. Several cop-
per compounds have demonstrated anticancer activity.
For example, the complex containing 1,3-diketone group
of copper ion and acetylacetone (CuAA) was delivered
by folic acid-tagged hydrophobic-modified CSNPs.35 The
NPs could induce apoptosis and reduce mitochondrial
membrane potential of cancer cells. It could also be suc-
cessfully delivered to folate receptor over-expressed cancer
cells. Sanpui et al. used chitosan nanocarriers (CS NCs)
to deliver silver nanoparticles (Ag NPs) for induction of
apoptosis in human colon cancer cells (HT 29) (Fig. 8).137
At a much lower concentration (0.33 g/mL) of Ag NPs,
the viability of HT 29 cells was reduced to 50%. The
oxidative stress and mitochondrial dysfunction were found
to play an important role in the apoptosis induced by the
Ag-CS NCs.
Encapsulation of Protein
Therapeutic peptides/proteins and protein-based antigens
are being extensively utilized for the management and cure
of many diseases, particularly oncologic and metabolic
diseases.138 Although they are frequently used in clinic and
almost exclusively administered by parenteral injections,
1598
Fu et al.
Figure 8. Schematic illustration of induced apoptosis in HT
29 cells by chitosan nanocarriers loading silver nanoparti-
cles. Reprinted with permission from [137], P. Sanpui, et al.,
Induction of apoptosis in cancer cells at low silver nanopar-
ticle concentrations using chitosan nanocarrier. ACS Appl.
Mater. Interf. 3, 218 (2011). © 2011, American Chemical
Society.
the therapeutic efficacy and biocompatibility are greatly
weakened due to their inherent instability, poor pharma-
cokinetics, and potential toxicity.139–141 Therapeutic pro-
teins have high molecular weight (MW > 1,000 Da) and
good hydrophilicity which significantly affect their ability
cross
is cell membranes.
80004805
142
(JPP) Moreover, proteins are easily
and
07rapidly
Dec 2016 removed from the bloodstream after under-
04:41:09
Publishers
going proteolytic cleavage in vivo.143 Additionally, the
conditions of production and storage of protein drug for-
mulations may cause adsorption, aggregation, and denatu-
ration. This reduces their effectiveness considerably under
in vivo conditions.144 145
To circumvent these issues and further improve the
bioavailability of proteins, a large number of investigations
have been carried out by employing numerous methods,
including addition of nonionic surfactants, metal chelators,
enzyme inhibitors, etc.142 Furthermore, many chitosan-
based nanoparticles have been designed and extensively
tailored to deliver various proteins according to the types
of proteins and their practical purposes.146 147 In Caco-2
cell and rat model, N -trimethyl chitosan chloride (TMC)
was confirmed as a potent mucosal permeation enhancer
for the peptide drug buserelin at neutral pH. Coadminis-
tration of buserelin with TMCs resulted in mean bioavail-
ability values between 6 and 13% which was greatly
higher than that of intraduodenally applied buserelin with
0.8% absolute bioavailability.148 Self-assembled glycol
chitosan nanoparticles also show high efficacy for sus-
tained and prolonged delivery of peptide drugs in cancer
therapy.149–151 Kime et al. developed hydrophobically-
modified glycol chitosan (HGC) nanoparticles conjugated
with interleukin-4 receptor (IL-4R) binding peptides (I4R),
and then tested them in mice bearing IL-4R-positive
tumors. Their study showed that HGC-I4R nanoparticles
J. Biomed. Nanotechnol. 12, 1585–1603, 2016
Fu et al.
exhibited enhanced IL-4R-dependent cellular uptake in
tumors compared to nonconjugated nanoparticles, con-
sequently leading to better therapeutic and imaging
efficacy.151
PROBLEMS AND OUTLOOK
Even though chitosan has many advantages in drug deliv-
ery, there still are several problems which can not be
ignored. One problem is the potential toxicity caused by
modification of chitosan. Chitosan has poor solubility and
unmodified CSNPs can encapsulate only some hydrophilic
drugs. In order to make chitosan encapsulate hydrophobic
drugs easily, structure modification is necessary. There-
REFERENCES
fore, various modifiers, catalysts or crosslinking agents
18
are widely used to decorate CSNPs. Moreover, some
toxic organic solvents are also used during the process of
modification. The use of these chemicals may bring obvi-
ous side-effects due to their intrinsic toxicity. Therefore,
after each chemical reaction, the unreacted reagents and
reactants must be removed completely to prevent confus-
ing results.10 Also, the further investigation is required on
the biocompatibility of modified chitosan and its deriva-
tives. Another problem is retaining the encapsulation effi-
ciency through mass production of drug loading systems.
Commonly, high drug loading efficacy and encapsulation
efficiency can be achieved at a small scale in lab. How-
Delivered by Ingenta to: Main CID
ever, it is very difficult to maintain the same high load-
IP: 146.185.205.29 On: Wed, 07Riahi,
ing efficacy and stability during a large-scale production.
Copyright: American Scientific Publishers and endoxifen by chitosan nanoparticles. PLoS
Additionally, although nanosized particles have a wide
range of potential biomedical and pharmaceutical applica-
tions, the unique physicochemical properties of nanopar-
ticles may cause potential risks to human body and the
environment. Some studies have demonstrated that CSNPs
may also exhibit different cytotoxicity profiles depending
on the properties of the parent material used to synthe-
size the nanoparticles, the size of the nanoparticles and
the interacting cell types.10 15 Therefore, potential toxicity
of chitosan-based nanoformulations should be taken into
consideration including their in vivo distribution in normal
tissues and organs.
Even though a large-scale application of CSNPs in
clinic may take some time, the advances in nanotech-
nology and polymer chemistry is poised to unravel the
full potential of the chitosan-based nanodrug delivery
systems have the great potential for the development
of multifunctional “smart” nanoparticles that may facil-
itate the realization of individualized cancer therapy.18
So far, a variety of multifunctional CSNPs have been
evaluated for application to cancers detection, diagnosis,
simultaneous treatment and in vivo imaging. Although
some barriers still exist in the development of chitosan-
based nanoformulations, we still believe that chitosan
nanocarriers have broad application prospects in cancer
therapy.
J. Biomed. Nanotechnol. 12, 1585–1603, 2016
Chitosan Nanoparticles in Cancer Treatment
Declaration of Interest Statement
The authors declare no conflicts of interest. The authors
alone are responsible for the content and writing of the
paper.
Acknowledgments: This work was financially sup-
ported by the Union Project of Luzhou City and Luzhou
Medical College (14JC0144, 2013LZLY-J40), Young
Scholars Fund of Luzhou Medical College (2012QN-14),
Science and Technology Project of Luzhou City (2013-
134), Youth Talent Fund of the Affiliated Hospital of
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KANSER TEDAVİSİNDE FONKSİYONEL KİTOSAN NANOPARTİKÜLLERİ
Shaozhi Fu, Jiyi Xia ve Jingbo Wu tarafından yazılan bu makalede kitosanın kanser
tedavilerinde nasıl ve hangi amaçla kullanıldığına dair bilgiler verilmiştir.
Kanserin insan sağlığını nasıl tehdit ettiğini bilmeyen yoktur.Öyle ki 2030 yılına kadar dünya
çapında 12 milyon insanın kanser hastalığı yüzünden öleceği ön görülüyor. Özellikle Çin gibi
gelişmiş ve büyük ülkelerde çevre kirliliği yüzünden yıldan yıla kanser hastalığı ve ölümü
artmakta.Kanser tedavilerinde en çok kemoterapi gibi tedavi yöntemleri kullansa da bu
tedavi yöntemlerinin çok ciddi yan etkileri bulunur.Yeni kanser tedavisi keşfetme sürecinde
malzeme bilimi sayesinde önemli gelişmeler yaşanmıştır.Kitosan da bu tedavi
yöntemlerinden biridir.Doğal bir polisakkarit olan kitosan büyük ölçekte kitinin alkali N-
deasetilasyonu ile üretilir.Kitin bol bir polimerdir ve kitin kabuklu hayvanların kabuğunda
bulunur.Kitosan çeşitli kemoterapötik ilaçların dağıtımı için ilaç taşıyıcısı olarak yaygın
kullanılsa da sadece ilaç yükleme aracı görevi görür ve kanser tedavisinde rol oynamaz.Bu
nedenle başka bir şey önerildi.Kitosan omurgasında ki zengin reaktif gruplar sayesinde bazı
hidrofobik antikanser ilacı hidrofilik kitosan zincirlerine doğrudan konjuge edilebilir ve bu da
iyi bir sonuç verir.