Application_of_chitosan_and_chitosan_derivatives_as_biomaterials1

Review
Application
of
chitosan
and
chitosan
derivatives
as
biomaterials
Changyong
Choi
JoungPyo
Nam
JaeWoon
Nah *
Department
of
Polymer
Science
and
Engineering
Sunchon
National
University
255
Jungangro
Suncheon
Jeollanamdo
Republic
of
Korea
Contents
Introduction







































































































1
Genetic
materials
delivery
by
chitosan

















































































2
ChitosanDNA
polyplexes























































































3
ChitosanRNA
polyplexes























































































3
Genetic
materials
delivery
by
chitosan
derivatives








































































3
Hydrophilic
material
modified
chitosan











































































4
Hydrophobic
material
modified
chitosan










































































4
Cationic
material
modified
chitosan














































































5
Targeting
ligand
modified
chitosan















































































6
Thiol
group
modified
chitosan



















































































7
Amino
acid
and
peptide
modified
chitosan









































































7
Genetic
materials
delivery
by
anionic
materialchitosan
complexes


























































7
Anion
polymer
and
chitosan
complexes











































































8
Anionic
biopolymer
and
chitosan
complexes







































































8
Polypeptide
and
chitosan
complexes














































































8
Conclusion








































































































9
Acknowledgements

































































































9
References








































































































9
Introduction
Gene
therapy
uses
genetic
materials
(eg
deoxyribonucleic
acids
(DNA)
or
ribonucleic
acid
(RNA))
as
a
pharmaceutical
agent
to
treat
various
diseases
Gene
therapy
has
the
following
three
main
mechanisms
(1)
delivering
missing
genes
(2)
replacing
defective
genes
and
(3)
gene
silencing
undesired
gene
expression
[1]
Through
these
mechanisms
gene
therapy
treats
a
wide
range
of
diseases
Consequently
the
interest
in
gene
therapy
is
increasing
Despite
these
advantages
use
of
genetic
materials
in
gene
therapy
is
limited
due
to
rapid
degradation
by
nuclease
large
size
poor
cellular
uptake
high
anionic
charge
density
and
Journal
of
Industrial
and
Engineering
Chemistry
33
(2016)
1–10
A
R
T
I
C
L
E
I
N
F
O
Article
history
Received
24
August
2015
Received
in
revised
form
13
October
2015
Accepted
20
October
2015
Available
online
24
October
2015
Keywords
Chitosan
Chitosan
derivatives
Biomaterials
Delivery
system
A
B
S
T
R
A
C
T
Chitosan
is
a
linear
polysaccharide
composed
of
randomly
distributed
b(14)linked
Dglucosamine
and
NacetylDglucosamine
It
is
one
of
the
major
cationic
polymers
and
the
second
most
abundant
polysaccharides
in
nature
It
is
extensively
used
to
the
biomedical
and
the
industrial
fields
Specially
chitosan
has
been
studied
much
in
the
field
of
gene
therapy
during
the
last
decade
for
its
biocompatibility
and
noncytotoxicity
However
it
has
several
problems
such
as
solubility
low
transfection
efficiency
and
low
specialty
on
targeted
disease
To
solve
these
problems
various
strategies
have
been
reported
to
enhance
them
This
review
briefly
introduces
various
strategies
of
chitosan
carrier
ß
2015
The
Korean
Society
of
Industrial
and
Engineering
Chemistry
Published
by
Elsevier
BV
All
rights
reserved
* Corresponding
author
Tel
+82
61
750
3566
fax
+82
61
750
5423
Email
address
jwnah@sunchonackr
(JW
Nah)
Contents
lists
available
at
ScienceDirect
Journal
of
Industrial
and
Engineering
Chemistry
jou
r
n
al
h
o
mep
ag
e
w
ww
elsevier
co
m
loc
atejiec
httpdxdoiorg101016jjiec201510028
1226086Xß
2015
The
Korean
Society
of
Industrial
and
Engineering
Chemistry
Published
by
Elsevier
BV
All
rights
reservednonspecificity
[1–4]
To
overcome
these
problems
vectors
are
used
for
safe
delivery
of
genetic
materials
in
gene
therapy
Generally
vectors
can
be
classified
into
two
types
One
of
them
is
a
viral
vector
which
is
commonly
used
to
deliver
genetic
material
into
the
cells
The
viral
vectors
such
as
retroviruses
lentiviruses
adenoviruses
and
adenoassociated
viruses
are
very
effective
in
achieving
high
transfection
efficiency
however
their
availability
for
therapeutic
use
in
the
human
body
is
limited
because
of
immune
responses
safety
problems
high
cost
and
low
transgenic
size
[5–
9]
The
other
type
is
nonviral
vectors
which
are
preferred
as
safer
alternatives
to
viral
vectors
for
gene
therapy
The
viral
vectors
such
as
liposome
protein
and
cationic
polymer
have
many
advantages
including
stability
safety
low
immune
response
and
cell
targeting
properties
[510]
Thus
in
recent
years
the
interest
in
nonviral
vector
is
increasing
and
active
research
has
been
reported
Cationic
polymers
are
widely
used
as
carriers
for
nonviral
genetic
materials
delivery
[11–13]
They
can
condense
with
genetic
materials
through
electrostatic
interaction
to
form
polyplexes
and
facilitate
the
cellular
uptake
by
cells
[1213]
In
addition
the
amine
group
of
polyplexes
is
quick
on
the
uptake
of
the
cell
absorbing
protons
facilitating
the
escape
of
the
polyplexes
from
endosome
or
lysosome
through
a
triggered
osmotic
swelling
effect
[121415]
Chitosan
is
a
linear
polysaccharide
composed
of
randomly
distributed
b(14)linked
Dglucosamine
(deacetylated
unit)
and
NacetylDglucosamine
(acetylate
unit)
–
a
structure
very
similar
to
that
of
cellulose
As
such
chitosan
is
one
of
the
major
cationic
polymers
[1617]
It
is
obtained
by
the
alkaline
deacetylation
of
chitin
which
is
the
second
most
abundant
polysaccharides
in
nature
after
cellulose
Chitosan
forms
inter
and
intramolecular
hydrogen
bonding
owing
to
amine
and
hydroxyl
groups
therefore
it
has
a
rigid
crystalline
structure
[18]
Chitosan
has
a
various
bioactivities
due
to
the
abundant
primary
amino
groups
in
the
chitosan
main
chain
For
this
reasons
the
chitosan
is
extensively
used
to
the
biomedical
fields
such
as
drug
andor
gene
delivery
and
the
industrial
fields
such
as
water
treatment
(eg
harmful
algae
control)
heavy
metal
flocculants
and
functional
foods
[19]
Chitosan
is
soluble
in
an
acid
solution
but
insoluble
at
natural
and
alkaline
pH
values
because
of
the
pKa
value
of
chitosan
of
about
65
[20]
The
solubility
of
chitosan
is
significantly
dependent
on
the
degree
of
deacetylation
(DDA)
When
DDA
of
chitosan
is
40
chitosan
is
soluble
up
to
a
pH
of
9
Whereas
DDA
of
chitosan
is
80
it
is
soluble
only
up
to
a
pH
of
65
[18]
Moreover
the
molecular
weight
(MW)
of
chitosan
and
the
ionic
strength
of
the
solution
influence
the
solubility
of
chitosan
Reporting
on
the
chemical
properties
of
chitosan
including
cationic
properties
Sanford
pointed
out
that
the
high
charge
density
at
pH
<
65
forms
gels
with
polyanions
adheres
to
negatively
charged
surfaces
chelates
certain
transitional
metals
and
is
readily
susceptible
to
chemical
modification
[21]
During
the
last
decade
chitosan
has
been
extensively
used
as
a
gene
carrier
for
gene
therapy
by
applying
the
chemical
properties
described
above
It
has
also
been
extensively
studied
as
nonviral
derived
cationic
natural
polymers
for
a
number
of
pharmaceutical
and
biomedical
applications
due
to
its
biocompatibility
biodegradability
to
normal
body
constituents
nontoxic
hemostatic
bacteriostatic
fungistatic
spermicidal
anticancerogen
anticholesteremic
properties
easily
susceptible
to
chemical
modification
[21–24]
In
addition
chitosan
is
tightly
condensed
with
negatively
charged
genetic
materials
protecting
genetic
materials
against
nuclease
degradation
due
to
cationic
property
as
a
positive
charge
[2526]
ChitosanDNA
polyplexes
have
been
reported
to
transfect
into
various
cell
types
(eg
human
embryonic
kidney
cells
(HEK293)
[13]
cervical
cancer
cells
(HeLa
cell)
[13]
primary
chondrocytes
[27]
Chinese
hamster
ovary
cells
(CHOK1)
[28]
fibroblast
cells
(NIH
3T3)
[29]
and
epithelioma
papulosum
cyprinid
cells
(EPC)
[30])
This
review
briefly
introduces
the
strategies
of
chitosan
and
chitosan
derivatives
that
have
been
reported
as
genetic
materials
delivery
carrier
in
various
gene
therapies
Genetic
materials
delivery
by
chitosan
The
amine
groups
of
chitosan
are
positively
charged
in
acidic
mediums
and
provide
a
strong
electrostatic
interaction
with
negatively
charged
mucosal
surfaces
or
other
macromolecules
such
as
genetic
materials
[18]
Therefore
chitosan
has
been
used
as
a
delivery
carrier
for
gene
delivery
in
order
to
enhance
transfection
efficiency
and
protect
genetic
materials
against
nuclease
Howev
er
to
effectively
transfer
genetic
material
by
using
chitosan
the
MW
and
DDA
of
chitosan
must
be
considered
Over
the
years
many
studies
have
reported
on
the
effect
of
various
MW
and
DDA
of
chitosan
to
transfer
genetic
material
into
the
cells
effectively
The
MW
and
DDA
of
chitosan
have
an
influence
on
its
biological
and
physicochemical
properties
The
DDA
affects
biodegradability
and
results
in
very
low
rates
of
enzymatic
degradation
of
acetyl
groups
[3132]
The
transfection
efficiency
and
binding
affinity
of
chitosanDNA
system
were
found
to
increase
with
the
increase
of
MW
and
DDA
[3133]
Kiang
et
al
have
demonstrated
that
destabilization
of
particles
cause
the
decreased
DDA
in
a
decrease
in
luciferase
expression
levels
in
HEK293
HeLa
and
cervical
carcinoma
(SW756)
cells
in
vitro
[13]
The
high
MW
(100
kDa)
of
chitosan
was
found
to
have
several
advantages
in
genetic
materials
delivery
forming
extremely
stable
polyplexes
with
genetic
materials
delaying
the
release
of
the
genetic
materials
and
forming
the
physical
shape
of
the
polyplexes
[2534]
However
high
MW
of
chitosan
has
pharmaceutical
drawbacks
such
as
low
solubility
at
physiological
pH
slow
dissociation
and
release
of
genetic
materials
and
high
viscosity
at
concentrations
used
for
in
vivo
delivery
[183435]
These
factors
of
chitosan
with
high
MW
led
to
slow
onset
of
action
[34]
To
overcome
these
problems
of
chitosan
with
high
Mw
numerous
studies
have
been
conducted
by
using
low
MW
chitosan
Jang
et
al
have
reported
that
low
molecular
weight
watersoluble
chitosan
(LMWSC)
is
prepared
using
a
novel
salt
removedmethod
to
enhance
the
solubility
of
chitosan
under
physiological
pH
[36]
They
characterized
the
structure
of
LMWSC
having
free
amine
group
by
using
fourier
transform
infrared
(FTIR)
and
nuclear
magnetic
resonance
(NMR)
and
investigated
DDA
and
MW
of
LMWSC
from
which
salt
is
removed
by
using
NMR
and
viscometer
The
results
demonstrated
that
the
LMWSC
is
successfully
prepared
and
it
can
be
used
in
pharmaceutical
and
food
industry
[36]
Bloomfield
[37]
and
Strand
et
al
[38]
demonstrated
that
the
low
MW
chitosan
provides
stable
polyplexes
without
aggregation
soluble
at
neutral
pH
reduces
viscosity
and
is
more
easily
dissociated
polyplexes
than
using
high
MW
chitosan
in
gene
therapy
The
pDNA
loaded
chitosan
microspheres
are
prepared
by
using
a
precipitation
technique
for
in
vitro
tranfection
in
rat
prostate
adenocarcinoma
cell
line
(MATLyLu)
[39]
The
MW
of
chitosan
and
the
amount
of
plasmid
influenced
the
in
vitro
transfection
in
the
cells
The
pDNA
loaded
chitosan
microspheres
prepared
with
low
MW
chitosan
expressed
slightly
higher
transfection
than
the
high
molecular
weight
chitosan
[39]
Ko¨pingHo¨gga˚ rd
et
al
reported
on
the
properties
and
efficiency
of
low
MW
(<5
kDa)
and
they
found
that
24mer
(approximately
3
kDa–4
kDa)
of
chitosan
forms
stable
complexes
and
gives
a
high
level
of
gene
expression
comparable
to
the
high
MW
of
chitosan
[40]
Furthermore
it
was
found
that
the
stability
and
transfection
efficiency
of
chitosanused
polyplexes
has
been
dependent
not
only
on
the
MW
and
DDA
of
chitosan
but
also
on
the
pH
of
transfection
medium
[30313440–43]
serum
concentration
[1343–45]
and
concentrations
of
genetic
materials
[30313846]
The
pH
of
transfection
medium
was
affected
to
C
Choi
et
al

Journal
of
Industrial
and
Engineering
Chemistry
33
(2016)
1–102form
the
shape
in
coils
(physiological
pH)
or
globules
in
acid
pH
condition
The
amount
of
globules
of
polyplexes
formed
with
chitosan
increased
as
the
pH
decreased
ranging
from
65
to
35
and
led
to
high
level
transfection
efficiency
In
addition
amine
group
of
chitosan
of
pKa
value
(about
65)
was
activated
in
low
pH
environment
and
formed
with
globules
by
charge
inversion
[40]
Guliyeva
et
al
reported
that
pDNA
is
released
from
chitosan
microparticles
with
pH
21
mediums
in
shorter
periods
than
pH
647
mediums
and
the
released
amounts
are
higher
than
other
pH
conditions
in
pH
45
and
pH
647
because
of
different
solubilities
of
chitosan
in
acidic
and
basic
pHs
of
mediums
[42]
Lavertu
et
al
[31]
Nydert
et
al
[41]
and
Nimesh
et
al
[43]
demonstrated
that
transfection
efficiency
is
more
effective
at
acidic
pH
than
natural
pH
in
HEK293
cell
Through
the
results
of
transfection
efficiency
in
vitro
Lavertu
et
al
demonstrated
that
transfection
efficiency
is
highly
sensitive
to
DDA
MW
concentrations
of
genetic
materials
and
medium
pH
[31]
For
efficient
gene
delivery
system
that
uses
chitosan
the
optimal
conditions
have
yet
to
be
established
including
MW
DDA
medium
pH
concentrations
of
genetic
materials
and
serum
stability
ChitosanDNA
polyplexes
Chellat
et
al
experimented
on
the
metalloproteinase
and
cytokine
production
by
the
human
monocytic
leukemia
cell
line
(THP1)
macrophages
using
chitosanDNA
(ChDNA)
nanoparticle
and
their
results
indicated
that
the
ChDNA
nanoparticles
do
not
induce
the
release
of
proinflammatory
cytokines
whereas
MMP9
was
significantly
increased
[47]
ChitosanDNA
nanoparticles
were
prepared
for
nonviral
gene
transfer
in
animal
models
of
fetal
gene
therapy
to
characterize
the
chitosanDNA
nanoparticle
stability
within
amniotic
fluid
in
vitro
[48]
Chitosan
protected
pDNA
from
enzymatic
degradation
despite
the
chitosan
aggregate
in
amniotic
fluid
The
transfection
efficiency
of
chitosanDNA
nanoparticles
shows
high
levels
in
vivo
than
in
vitro
transfection
due
to
bioavailable
property
of
chitosan
in
vivo
and
the
long
exposure
time
of
chitosanDNA
nanoparticles
in
vivo
[48]
Niu
et
al
investigated
human
insulin
band
by
using
the
gel
electrophoresis
system
in
harvested
tissues
such
as
stomachs
intestines
and
rectums
after
human
insulin
gene
transfection
using
gene
wrapped
chitosan
nanoparticles
and
they
detected
that
the
human
insulin
bands
are
found
only
in
the
harvested
intestines
of
diabetic
rats
[29]
Yang
et
al
evaluated
the
potential
of
chitosan
in
DNA
vaccine
delivery
via
mucosa
and
their
results
show
that
low
MW
of
chitosan
has
lower
binding
affinity
to
DNA
but
higher
transfection
efficiency
than
the
high
MW
of
chitosan
and
the
intranasal
vaccination
of
chitosan
(low
MW)DNA
polyplexes
elicits
signifi
cant
systemic
immune
responses
[49]
Roy
et
al
investigated
the
effect
of
chitosanDNA
nanoparticles
on
food
allergy
therapy
and
demonstrated
that
chitosanDNA
nanoparticles
are
effective
in
controlling
murine
anaphylactic
responses
[50]
This
result
indicates
that
the
chitosan
has
potential
utility
in
treating
food
allergy
ChitosanDNA
nanospheres
were
prepared
to
investigate
the
potential
of
chitosanDNA
nanospheres
against
acute
respira
tory
syncytial
virus
(RSV)
infection
and
they
were
found
to
have
a
greater
potential
against
acute
RSV
infection
than
the
controls
owing
to
the
results
of
induction
of
RSVspecific
immunoglobulin
G
(IgG)
antibodies
nasal
immunoglobulin
A
(IgA)
antibodies
interferong
and
cytotoxic
T
lymphocytes
production
in
lung
and
splenocytes
[51]
ChitosanRNA
polyplexes
Recently
small
interfering
ribonucleic
acid
(siRNA)
has
been
studied
as
a
new
therapeutic
tool
for
gene
expressionimplicated
disease
[5253]
To
silence
target
genes
siRNA
offers
a
potentially
new
therapeutic
strategy
with
high
specificity
by
reducing
undesirable
gene
expression
[54–56]
However
using
siRNA
should
consider
the
limitations
such
as
rapid
degradation
short
halflife
and
low
internalization
in
the
therapeutic
[5457]
Therefore
in
gene
therapy
that
uses
siRNA
a
carrier
system
is
required
for
the
protection
against
nuclease
degrada
tion
and
delivery
of
siRNA
into
target
cell
Kenneth
et
al
reported
that
the
chitosansiRNA
nanoparticles
enhance
green
fluorescent
protein
(EGFP)
gene
knockdown
in
both
human
lung
carcinoma
cells
(H1299)
and
murine
peritoneal
macrophages
also
showing
similar
results
in
transgenic
EGFP
mice
after
nasal
administration
[58]
In
another
research
EGFP
gene
knockdown
was
enhanced
by
increasing
nitrogenphosphorus
(NP)
ratios
ranging
from
50
to
150
and
high
MW
of
chitosan
[59]
Malmo
et
al
reported
that
the
results
of
flow
cytometry
and
the
knockdown
efficiency
assay
in
H1299
cells
indicated
the
most
efficient
gene
silencing
is
achieved
by
using
the
fully
deNacetylated
chitosan
with
intermediate
chin
lengths
(degrees
of
polymerization
(DPn)
100–300)
[60]
Aerosolised
chitosansiRNA
nanoparticles
were
prepared
to
detect
pulmonary
gene
silencing
in
transgenic
EGFP
mice
[61]
This
research
demonstrated
that
in
vivo
study
results
showed
significant
EGFP
gene
silencing
above
the
68
reduction
of
fluorescence
ratio
compared
to
the
mismatch
group
in
transgenic
EGFP
mice
dosed
with
the
aerosolized
chitosansiRNA
nanoparticle
and
the
exact
siRNA
dosage
improved
the
effect
of
gene
silencing
more
than
the
intranasal
administration
did
[61]
Ji
et
al
showed
that
chitosansiRNA
nanoparticle
which
is
four
and
a
half
LIM
domains
protein
2
(FHL2)
siRNA
formulated
with
chitosan
could
knock
down
about
696
FHL2
gene
expression
This
result
is
very
similar
to
the
reduced
FHL2
gene
expression
transfected
by
the
commercial
transfection
agent
called
Lipo
fectamine
[52]
Alameh
et
al
prepared
chitosansiRNA
nanocom
plexes
to
enhance
gene
silencing
with
dipeptidyl
peptidase
IV
(DPPIV)
siRNA
This
research
revealed
an
80
silencing
of
the
DPPIV
gene
compared
to
nontransfected
cells
[62]
To
induce
mucosal
secretory
IgA
(SIgA)
secretion
the
VPIencoded
chitosan
DNA
(pcDNA3VP1)
which
is
the
major
structural
protein
of
coxsackievirus
B3
(CVB3)
was
prepared
by
vortexing
DNA
with
chitosan
[63]
ChitosanDNA
(pcDNA3VP1)
successfully
induced
mucosal
SIgA
secretion
and
significantly
reduced
the
viral
load
after
acute
CVB3
infection
in
mice
Thus
this
research
indicates
that
chitosan
may
be
a
promising
vaccine
candidate
for
protection
against
infection
Genetic
materials
delivery
by
chitosan
derivatives
Despite
the
excellent
properties
of
biodegradability
biocom
patibility
and
nontoxicity
chitosan
has
been
mainly
limited
in
biomedical
field
because
of
several
disadvantages
One
of
them
is
low
solubility
at
natural
pH
and
alkaline
pH
[2064]
Another
disadvantage
is
the
low
transfection
efficiency
due
to
the
relatively
low
cationic
density
in
chitosan
that
causes
less
compact
of
chitosangenetic
material
complexes
[64–66]
Other
disadvan
tages
are
lack
of
cell
specificity
and
low
escape
property
from
endosomes
into
the
cytoplasm
[6467]
To
overcome
these
disadvantages
many
research
efforts
have
been
reported
in
recent
years
to
enhance
solubility
transfection
efficiency
release
and
cell
specificity
of
chitosan
derivatives
The
following
sections
discuss
various
derivatives
including
hydrophilic
moiety
hydrophobic
moiety
cationic
moiety
anion
moiety
and
target
moiety
modified
chitosan
which
aim
at
enhancing
the
property
of
chitosan
for
effective
genetic
materials
delivery
Chitosan
derivatives
have
been
prepared
using
a
variety
of
methods
and
materials
to
enhance
application
of
chitosan
The
classification
of
various
strategies
to
prepare
chitosan
derivatives
is
shown
in
Table
1
C
Choi
et
al

Journal
of
Industrial
and
Engineering
Chemistry
33
(2016)
1–10
3Hydrophilic
material
modified
chitosan
Hydrophilic
modification
to
chitosan
backbone
was
introduced
to
improve
transfection
efficiency
for
increased
water
solubility
at
physiological
pH
and
improved
intracellular
DNA
release
[6869]
Polyethylene
glycolation
(PEGylation)
and
trimethylation
are
the
strategies
used
most
to
improve
the
solubility
of
chitosan
PEG
has
a
high
solubility
in
water
low
cytotoxicity
and
high
cell
permeability
properties
therefore
it
is
a
good
candidate
to
induce
hydrophilic
part
to
chitosan
backbone
In
addition
PEG
can
increase
plasma
halflives
and
shield
them
from
inactivation
by
the
immune
system
[7071]
Trimethylation
of
chitosan
backbone
enhances
water
solubility
of
chitosan
at
broader
pH
range
which
leads
to
compact
interaction
with
pDNA
[72–75]
Trimethylation
chitosan
(TMC)
is
capable
of
opening
the
tight
junctions
of
cells
at
physiological
pH
which
led
to
enhance
paracellular
permeability
[727476]
Zhang
et
al
prepared
PEGconjugated
chitosanDNA
(CSDNA
PEG)
nanoparticles
and
demonstrated
that
the
transfection
efficiency
of
the
PEGylated
nanopartecles
improved
more
than
PEG
unconjugated
chitosanDNA
nanoparticles
both
in
vitro
and
in
vivo
experiment
[70]
Kean
et
al
investigated
the
transfection
efficiency
of
the
trimethylated
oligomeric
chitosan
(TMO)
and
trimethylated
polymeric
chitosan
(TMC)
prepared
with
low
molecular
weight
[75]
The
transfection
efficiency
of
TMO
and
TMC
with
various
degrees
of
quaternization
showed
a
greater
efficiency
than
high
molecular
weight
(25
kDa)
polyethyleneimine
(PEI)
in
epithelial
breast
cancer
(MCF7)
cells
Moreover
TMO
and
TMC
also
showed
appreciable
transfection
in
monkey
kidney
fibroblasts
(COS7)
cells
[75]
Unfortunately
chitosan
which
has
at
higher
degrees
of
quaternization
led
to
increased
cytotoxicity
[75–
78]
PEGylated
TMC
was
conjugated
with
different
grafting
ratios
and
PEG
chain
lengths
to
increase
solubility
and
decrease
cytotoxicity
[77]
The
half
maximal
inhibitory
concentration
(IC50)
value
of
PEGylated
TMC
was
421
mgmL
while
PEG
unmodified
TMC
showed
higher
toxicity
with
an
IC50 value
of
96
mgmL
in
NIH
3T3
cells
Hydrophobic
material
modified
chitosan
Hydrophobic
moiety
modified
chitosan
polyplexes
formed
with
DNA
have
several
advantages
such
as
alleviation
of
serum
inhibition
facilitated
intracellular
DNA
dissociation
efficient
protection
from
enzymatic
degradation
and
improved
cell
membrane
permeation
over
the
unmodified
chitosan
polyplexes
[79–81]
Hydrophobic
moiety
modified
chitosan
forms
amphi
philic
polymer
by
chemically
attaching
hydrophobic
functional
moieties
to
chitosan
The
modified
hydrophobic
group
can
be
a
charge
neutralization
or
even
a
charge
inversion
of
chitosanDNA
polyplexes
[82]
The
strategy
of
introducing
hydrophobic
group
to
chitosan
has
been
tried
with
various
hydrophobic
moiety
including
alkyl
group
(as
steric
acid
alkyl
bromide
and
caproic
acid)
[64657983–85]
bile
acid
(as
a
deoxycholic
acid)
[8687]
and
5b
cholanic
acid
[66]
Stearic
acid
grafted
chitosan
oligosaccharide
(CSOSA)
is
synthesized
by
coupling
reaction
with
a
1ethyl3(3dimethyla
minopropyl)
carbodiimde
(EDC)
[65]
CSOSA
formed
micelle
by
selfaggregation
in
aqueous
solution
The
critical
micelle
concen
tration
(CMC)
of
CSOSA
is
about
0035
mgmL
with
154
amino
substituted
degree
of
CSO
The
transfection
efficiency
of
CSOSA
micelles
with
pDNA
(pEGFPC1)
is
about
15
higher
than
that
of
chitosan
oligosaccharide
(CSO)pDNA
particles
(about
2)
In
addition
CSOSApDNA
micelles
are
not
interfered
in
the
presence
of
10
serum
Zhu
et
al
prepared
dodecylated
chitosanpDNA
nanoparticles
(DCDNPs)
with
a
mean
diameter
of
approximately
90–180
nm
for
local
gene
delivery
via
endovascular
stents
coated
[85]
The
DCDNPs
containing
pDNA
(EGFPC1)coated
stents
showed
high
level
of
green
fluorescent
protein
(GFP)
expression
in
cells
which
grew
on
the
stent
surface
and
along
the
adjacent
area
Table
1
The
classification
of
various
strategies
to
provide
the
advantage
in
chitosan
Modification
Materials
Advantage
Ref
Hydrophilic
material
modification
Trimethyl
group
High
solubility
low
cytotoxicity
high
transfection
efficiency
[71–77]
PEG
[697076]
Hydrophobic
material
modification
Alkyl
group
High
transfection
efficiency
alleviate
serum
inhibition
enhanced
protection
efficiency
improved
cell
membrane
permeation
[63647882–84]
Bile
acid
[8586]
5bcholanic
acid
[65]
Cationic
material
modification
PEI
Improve
cationic
density
enhance
condensation
capability
high
transfection
efficiency
effectively
escape
from
endosome
[87–92]
Urocanic
acid
[6693]
Imidazole
[9495]
Diethyleneamine
[96]
Spermine
[4]
Targeting
ligand
modification
Saccharide
Hepatocyte
targeting
(Galactose)
To
achieve
cellspecificity
High
cellular
uptake
High
transfection
efficiency
[101–106]
Macrophages
targeting
Dendritic
cell
targeting
(Mannose)
[107–110]
Hepatocyte
targeting
(Lactose)
[111]
Folic
acid
Folate
receptor
on
present
various
tumor
(ovarian
lung
breast
colon
etc)
[112–114]
Transferrin
Transferrin
receptor
on
present
on
malignant
cells
[115]
RGD
peptide
Integrin
anb3
targeting
[116]
Thiol
group
modification
Thioglycolic
acid
Increase
extracellular
stability
high
cellular
uptake
improved
intracellular
release
high
transfection
efficiency
[138–140]
Cystamine
[132]
2Iminothiolane
[141]
Amino
acid
and
peptide
modification
TAT
peptide
Enhance
cell
penetrating
High
cellular
uptake
improved
intracellular
release
high
transfection
efficiency
[144145]
Cystein
Induced
thiol
group
[134147]
Arginine
Induced
hydrophilicity
[146]
Nonpolar
amino
acids
(alanine
valine
leucine
isoleucine)
Induced
hydrophobicity
[1]
C
Choi
et
al

Journal
of
Industrial
and
Engineering
Chemistry
33
(2016)
1–104Caproic
acid
grafted
chitosan
(CGC)
was
prepared
to
investigate
the
effect
of
the
degree
of
substitutions
of
hydrophobic
group
on
binding
affinity
with
pDNA
cellular
uptake
transfection
efficiency
and
biocompatibility
[79]
The
transfection
efficiency
of
CGCpDNA
(gWizGFP
or
gWizbGal)
nanoparticles
exhibited
a
higher
gene
expression
compared
to
chitosanpDNA
nanoparticles
but
did
not
depend
on
the
degree
of
substitution
of
caproic
acid
group
ranging
from
5
to
25
The
optimal
formulation
is
CGC15
because
substitution
of
a
large
percentage
of
hydrophobic
groups
induced
unstable
polyplexes
and
led
to
low
levels
of
transfection
efficiency
[79]
Liu
et
al
investigated
the
gene
transfection
of
Nalkylated
chitosan
prepared
with
alkyl
bromide
(various
alkyl
chain
lengths
as
butyl
octyl
dodecyl
and
hexadecyl
bromide)
[83]
Various
N
alkylated
chitosanplasmid
encoding
chloramphenicol
acetyl
transferase
(pcDNA
31CAT)
complexes
exhibited
a
greater
transfection
efficiency
than
naked
DNA
and
unmodified
chito
sanpcDNA
31CAT
complexes
and
increased
the
transfection
efficiency
by
increasing
alkyl
chin
ranging
from
4
to
16
in
mouse
skeletal
muscle
cell
lines
(C2C12)
Jang
et
al
had
reported
previously
that
deoxycholic
acid
conjugated
chitosan
oligosaccharide
nanoparticles
(COSDs)
pre
pared
by
coupling
reaction
between
primary
amine
groups
in
COS
and
Nhydroxysuccinimide
(NHS)activated
deoxycholic
acid
(DOCA)
[86]
COSDspDNA
nanoparticles
whose
optimal
formula
tion
is
COS3D25
(with
1–3
kDa
and
5
degree
of
substitution
of
DOCA)
showed
a
great
potential
for
gene
carrier
with
high
level
of
gene
transfection
efficiencies
even
in
the
presence
of
serum
compared
to
the
deoxycholic
acid
unmodified
chitosanpDNA
nanoparticles
and
polyLlysine
(PLL)pDNA
nanoparticles
Yoo
et
al
reported
that
hydrophobically
modified
glycol
chitosan
(HGC)
prepared
by
coupling
reaction
with
hydrophobic
moiety
(5bcholanic
acid)
spontaneously
formed
nanoparticles
by
a
hydrophobic
interaction
between
HGC
and
hydrophobized
DNA
showing
a
higher
transfection
efficiency
than
naked
DNA
and
a
commercialized
transfection
agent
such
as
superfect
in
vivo
[66]
Fig
1
shows
the
structures
of
chitosan
derivatives
which
are
modified
with
hydrophilic
group
or
hydrophobic
group
Cationic
material
modified
chitosan
Since
the
major
disadvantage
of
chitosan
to
delivery
of
genetic
materials
is
the
low
transfection
efficiency
due
to
the
relatively
low
cationic
density
in
chitosan
[64–66]
many
studies
reported
the
cationic
densityenhanced
chitosan
through
various
cationic
groups
including
PEI
[88–93]
Urocanic
acid
[6794]
imidazole
[9596]
diethylethylamine
[97]
and
spermine
[4]
modification
The
enhanced
cationic
density
of
chitosan
can
play
a
crucial
role
in
endosomal
rupture
through
proton
sponge
mechanism
which
can
lead
to
high
transfection
efficiency
Although
PEI
is
the
most
effective
nonviral
vector
on
cationic
polymers
because
of
its
high
pH
buffering
capacity
[98]
it
can
be
highly
toxic
depending
on
the
dose
and
molecular
weight
and
its
nonbiodegradable
property
is
also
the
main
concern
when
PEI
is
used
for
a
longterm
[92]
Therefore
to
enhance
the
transfection
efficiency
and
decrease
toxicity
the
method
of
inducing
PEI
to
chitosan
backbone
has
been
studied
Wong
et
al
demonstrated
that
PEIgchitosan
synthesized
by
performing
cationic
polymeri
zation
of
aziridine
to
watersoluble
oligochitosan
(MW
34
kDa)
showed
good
DNA
condensation
capability
low
cytotoxicity
and
high
gene
transfection
efficiency
both
in
vitro
and
in
vivo
[88]
Lu
et
al
prepared
LM
PEI
grafted
chitosan
(Nmaleated
form)
for
gene
delivery
[89]
The
DNA
condensation
capability
of
PEI
grafted
chitosan
(NMCgPEI)
was
increased
by
increasing
chitosan
molecular
weight
ranging
from
5
to
50
kDa
under
the
same
condition
as
condensation
ratios
(ww)
NMCgPEI
improved
more
buffering
capacity
than
ungrafted
chitosan
but
it
is
still
lower
than
that
of
PEI
In
addition
the
buffering
capacity
of
NMCgPEI
was
decreased
by
increasing
the
molecular
weight
of
chitosan
Therefore
the
transfection
efficiency
of
NMC5kgPEI
and
NMC10k
gPEI
copolymers
has
a
relatively
higher
gene
transfection
ability
Fig
1
Chitosan
derivatives
with
hydrophilic
group
(A)
or
hydrophobic
group
(B)
A
(a)
trimethylated
chitosan
[74]
(b)
pegylated
chitosan
[69]
B
(a)
deoxycolic
acid
modified
chitosan
[85]
(b)
5bcholanic
acid
modified
chitosan
[65]
C
Choi
et
al

Journal
of
Industrial
and
Engineering
Chemistry
33
(2016)
1–10
5than
NMC50kgPEI
in
293
T
cell
and
HeLa
cell
Lu
et
al
prepared
N
succinyl
chitosan
(NSC)
to
graft
with
LM
PEI
(800
Da)
[91]
NSC
graftedPEI
(NSCgPEI)
was
synthesized
by
coupling
reaction
using
different
amounts
(02
and
04mmol)
of
coupling
agent
as
EDC
The
grafted
degree
(GD)
of
PEI
molecules
grafted
to
the
saccharide
unit
of
NSCgPEI
was
measured
to
be
0167
(NSCg
PEI1
used
02mmol
of
EDC)
and
0266
(NSCgPEI1
used
04
mmol
of
EDC)
respectively
The
transfection
efficiency
of
NSCg
PEIDNA
complexes
was
higher
than
PEI
(25
kDa)DNA
complexes
and
it
was
increased
by
increasing
GD
in
HeLa
and
Chinese
hamster
ovary
(CHO)
cells
Wang
et
al
demonstrated
that
urocanic
acid
(UAC)modified
chitosanmediated
efficient
p53
gene
transfer
which
is
hepato
cellular
carcinoma
(HCC)
growth
inhibiting
genes
[99–101]
could
induce
apoptosis
significantly
inhibiting
the
growth
of
human
hepatoblastoma
(HepG2)
cells
in
vitro
[67]
Kim
et
al
also
demonstrated
the
transfection
efficiency
of
urocanic
acidmodified
chitosan
(UAC)
in
293
T
cells
[94]
UACDNA
complex
showed
good
DNA
binding
ability
high
protection
of
DNA
from
nuclease
attack
cytotoxicity
lower
than
PEI
and
high
transfection
efficiency
with
increased
UA
contents
in
the
UAC
ranging
from
20
to
70
The
imidazole
ring
of
urocanic
acid
may
be
playing
a
role
in
endosomal
rupture
through
proton
sponge
mechanism
[94]
In
addition
Moreira
et
al
[95]
and
Ghosn
et
al
[96]
used
imidazole
moiety
to
enhance
transfection
efficiency
through
proton
sponge
mechanism
of
imidazole
Moreira
et
al
investigated
the
transfection
efficiency
of
imidazole
moiety
modified
chitosan
(CHimi)
in
the
presence
of
bafiomycin
A1
the
vacuolar
type
H+ATPase
inhibitor
[96]
To
improve
cationic
density
Jiang
et
al
prepared
spermine
induced
chitosan
(CHIgSPE)
through
imine
reaction
between
periodateoxidized
chitosan
and
spermine
as
a
gene
carrier
in
vitro
and
in
vivo
[4]
CHIgSPEDNA
complexes
showed
a
good
transfection
efficiency
while
CHIgSPEGFP
showed
a
GFP
expression
higher
than
that
of
the
chitosanGFP
complexes
without
toxicity
Fig
2
shows
the
cationic
groupmodified
chitosan
structure
Targeting
ligand
modified
chitosan
Targeting
ligand
provides
cellspecificity
to
chitosan
whereby
the
target
ligand
modified
chitosan
has
high
cellular
uptake
and
high
transfection
efficiency
Various
targeting
ligands
such
as
galactose
[102–107]
mannose
[108–111]
lactose
[112]
folate
[113–115]
transferrin
[116]
arginineglycineaspartic
acid
(RGD)
[117]
have
been
reported
to
provide
cellspecificity
and
high
transfection
efficiency
to
chitosan
Hepatocyte
possesses
asialoglycoprotein
receptor
(ASGR)
that
is
known
to
be
present
in
hepatocytes
at
a
high
density
of
500000
receptors
per
cell
and
internalize
galactoseterminal
(asialo)
glycoproteins
also
retained
on
several
human
hepatoma
cell
lines
[118–120]
Kim
et
al
demonstrated
that
galactosylated
water
soluble
chitosan
(GC)
coupled
between
lactobionic
acid
and
chitosan
showed
a
very
high
luciferase
activity
with
DNA
(pCI
Luc)
in
HepG2
cells
wellknown
model
cells
of
parenchymal
cells
in
the
liver
which
have
rich
ASGPR
receptors
on
the
surface
of
cells
[103]
Furthermore
the
result
of
a
competition
assay
clearly
demonstrated
that
GC
transferred
the
gene
through
receptor
mediated
transfection
system
Gao
et
al
also
prepared
galactosy
lated
low
molecular
weight
chitosan
(galLMWC)
through
a
method
similar
to
Kim
et
al
[103]
and
demonstrated
the
transfection
efficiency
of
galLMWCDNA
complexes
in
various
cell
lines
[106]
The
bGalactosidase
activity
of
galLMWCDNA
complexes
has
shown
a
high
transfection
efficiency
in
ASGPR
over
expressed
cell
as
a
HepG2
while
the
bGalactosidase
activity
of
galLMWCDNA
complexes
showed
a
very
low
transfection
efficiency
on
HeLa
cells
because
HeLa
cells
have
no
ASGRR
To
enhance
the
transfection
efficiency
Lu
et
al
[104]
and
Kim
et
al
[105]
prepared
the
PEI
and
galactose
induced
to
chitosan
The
cellular
uptake
of
Nsuccinylchitosangraftpolyethylenimine
lactobionic
acid
(NSCgPEILA)YoYo1
labeled
DNA
(pGL3)
complexes
was
found
to
be
located
mainly
in
the
cytoplasm
of
most
cells
(HepG2)
and
a
few
green
dots
were
found
in
the
nuclei
using
a
confocal
microscope
[104]
In
addition
the
cellular
uptake
of
(NSCgPEILA)YoYo1
labeled
DNA
complexes
increased
the
degree
of
LA
substitution
Macrophages
express
a
mannose
receptor
that
is
used
for
mannosemediated
endocytosis
or
phagocytosis
[121]
of
various
antigens
and
drug
delivery
systems
[122]
to
target
macrophages
Hashimoto
et
al
reported
that
DNAmannosylated
chitosan
(DNA
manchitosan)
complexes
were
found
through
mannose
receptor
mediated
gene
transfer
whereby
the
transfection
efficiency
is
enhanced
[109]
pDNAmanchitosan
or
pDNAchitosan
complexes
were
observed
in
different
intracellular
transport
in
macrophages
using
the
confocal
laser
microscope
pDNAmanchitosan
com
plexes
are
delivered
inside
the
cells
while
they
are
localized
in
early
phogosomes
near
the
plasma
membrane
In
addition
the
transfection
efficiency
of
pDNAmanchitosan
(5
substitution
degree
of
mannose)
was
observed
to
be
higher
than
chitosan
and
pDNAmanchitosan
(10)
Therefore
the
low
substitution
degree
of
mannose
at
5
in
chitosan
is
sufficient
for
gene
delivery
to
macrophages
Jiang
et
al
prepared
the
mannose
modified
chitosangPEI
(ManChigPEI)
as
a
gene
carrier
for
murine
Fig
2
Cationic
group
modification
chitosan
A
PEI
modified
chitosan
[90]
B
urocanic
acid
modified
chitosan
[93]
C
imidazole
modified
chitosan
[94]
D
diethyleneamine
modified
chitosan
[96]
and
E
spermine
modified
chitosan
[4]
C
Choi
et
al

Journal
of
Industrial
and
Engineering
Chemistry
33
(2016)
1–106macrophage
cells
(Raw
2647)
[110]
ManChigPEI
was
observed
to
have
high
transfection
efficiency
in
Raw
2647
cell
and
the
mannose
receptor
expressed
cell
(antigen
presenting
cells
(APCs))
in
contrast
to
ManChigPEI
that
was
observed
to
have
a
very
low
transfection
efficiency
in
HeLa
cell
the
untargeted
cell
This
result
of
transfection
efficiency
with
ManChigPEI
through
mannose
receptormediate
gene
transfer
is
similar
to
the
results
of
the
competition
assay
in
the
presence
50
mM
of
mannose
Kim
et
al
prepared
the
mannosylated
chitosan
(MC)
which
exhibited
much
enhanced
interleukin12
(IL12)
gene
transfer
efficiency
to
dendritic
cells
that
reside
within
the
tumor
through
mannose
receptormediated
endocytosis
between
MC
and
mannose
recep
tor
of
dendritic
by
directly
injection
into
tumor
[111]
The
delivered
pmIL12
genes
induced
high
expression
levels
of
cytokines
such
as
IL12
p70
and
interferong
(IFNg)
As
a
result
the
growth
and
angiogenesis
of
the
tumor
were
clearly
suppressed
Folate
receptors
(FR)
are
known
to
be
overexpressed
in
various
cancer
cells
surface
such
as
ovarian
lung
breast
colon
and
kidney
cells
and
rarely
found
on
normal
cell
surface
[115123]
The
folate
conjugated
polymer
including
cationic
liposomes
[124]
PLL
[125]
and
PEI
[126]
have
been
reported
to
be
cellular
uptake
into
the
FR
present
on
tumor
by
receptormediated
endocytosis
Mansouri
et
al
[113]
and
Chan
et
al
[114]
reported
that
folatechitosanDNA
nanoparticles
and
chitosangPEGfolateDNA
complex
are
char
acterized
for
gene
therapy
Viola
et
al
reported
on
the
enhanced
transfection
efficiency
of
histidinetrimethylated
chitosanfolate
PEG
(HTFP)
polymers
as
a
gene
delivery
vector
[115]
HTFPDNA
(pGL3luc)
polyplexes
showed
more
enhanced
transfection
efficiency
than
histidinetrimethylated
chitosan
(HTMC)
and
HTFP
with
excess
folate
Transferrin
(TF)
has
been
widely
applied
with
many
advantages
as
a
targeting
ligand
to
achieve
targeting
of
anticancer
agent
proteins
and
therapeutic
genes
to
primary
proliferating
malignant
cells
that
overexpressed
transferrin
receptors
(TfR)
[127–
129]
Kadiyala
et
al
prepared
transferrinconjugated
chitosan
DNA
nanoparticle
(NP
ratio
of
3)
to
clarify
the
influence
of
the
intrinsic
properties
including
polymer
chain
length
charge
ratio
nanoparticle
size
surface
charge
and
ligand
conjugation
[116]
The
presence
of
TF
in
chitosanDNA
nanoparticle
enhances
the
transport
of
nanoparticles
Han
et
al
prepared
RGDlabeled
chitosan
(RGDCH)
to
enhance
targeted
gene
silencing
[117117]
RGD
peptide
binds
with
anb3
integrin
which
is
overexpressed
in
a
wide
range
of
tumors
and
rarely
found
on
normal
cell
[130–132]
siRNARGDCH
nanopar
ticles
showed
the
targeted
silencing
of
multiple
growhpromoting
genes
(eg
POSTN
FAK
and
PLXDC1)
in
the
human
epithelial
ovarian
cancer
models
such
as
SKOV3ip1
HeyA8
and
A2780
In
addition
the
PLXDC1
siRNARGDCH
nanoparticles
delivered
into
the
anb3
integrinpositive
tumor
endothelial
cells
in
the
A2780
tumorbearing
mice
were
observed
to
have
a
higher
gene
silencing
efficiency
than
control
(siRNARGDCH
nanoparticles)
and
PLXDC1
siRNACH
nanoparticles
[117]
Thiol
group
modified
chitosan
The
thiol
group
introduced
into
several
carrier
systems
increases
the
extracellular
stability
and
improves
the
intracellular
release
properties
owing
to
its
enhanced
properties
such
as
the
formation
of
reducible
disulfide
bonds
between
introduced
thiol
groups
in
carrier
[133–136]
In
addition
the
thiol
group
introduced
into
chitosan
as
a
carrier
can
promote
its
mucoadhesive
potential
because
of
the
formation
of
disulfide
bonds
between
the
thiol
group
of
polymer
and
mucin
glycoproteins
on
the
cell
membrane
whereby
thiol
groupmodified
polymer
enhances
the
cellular
uptake
[137138]
Various
materials
such
as
cystamine
[133]
thiglycolic
acid
(TGA)
[139–141]
and
2iminothiolane
[142]
have
been
used
to
introduce
thiol
group
into
chitosan
for
efficient
gene
transfer
Varkouhi
et
al
reported
that
the
introduction
of
thiol
groups
to
trimethylated
chitosan
(TMCSH)
by
cystamine
modification
enhances
the
extracellular
stability
of
the
complexes
(siRNA
TMCSH)
and
promotes
the
intracellular
release
of
siRNA
[133]
siRNATMCSH
polyplexes
were
observed
to
have
a
high
gene
silencing
efficiency
and
good
stability
against
competing
anionic
macromolecules
as
a
hyaluronic
acid
while
siRNATMC
polyplexes
hardly
show
any
silencing
activity
in
the
same
condition
Thiolated
chitosan
(CSH)
nanocomplexes
TGAmodified
to
chitosan
and
DNA
are
prepared
to
enhance
transfection
efficiency
[139]
CSH360
(thiol
groups
360
mmol
of
chitosan)DNA
nano
complexes
induced
a
significantly
higher
GFP
expression
than
the
thiol
group
unmodified
chitosanDNA
nanocomplexes
in
various
cell
lines
including
HEK293
madindarby
canine
kidney
(MDCK)
and
human
larynx
carcinoma
cell
(Hep2)
In
addition
disulphide
crosslinked
CSH360DNA
nanocomplexes
are
observed
to
show
a
sustained
DNA
release
and
continuous
expression
in
cultured
cells
for
over
60
h
after
transfection
Martien
et
al
reported
that
thiolated
chitosan
when
synthesized
by
introducing
TGA
to
chitosan
via
amide
bond
formation
mediated
by
a
carbodiimide
showed
a
higher
transfection
efficiency
than
the
thiol
group
unmodified
chitosan
in
human
colorectal
carcinoma
cell
lines
(Caco2)
[140]
and
Caco2
differentiated
cell
culture
system
[141]
Amino
acid
and
peptide
modified
chitosan
Recently
chitosanpeptide
derivatives
have
received
increasing
interest
in
the
drug
and
gene
delivery
system
due
to
their
beneficial
property
such
as
enhanced
cell
adsorption
and
excellent
safety
profile
[1143144]
Malhotra
et
al
[145]
and
Katas
et
al
[146]
demonstrated
that
TAT
peptide
(cellpenetrating
peptide
(RKKRRQRRR))
chitosan
can
be
used
to
enhance
gene
therapeutic
as
a
gene
carrier
To
introduce
the
function
of
cellpenetrating
hydrophilicity
hydrophobicity
and
thiol
group
to
chitosan
amino
acids
are
used
in
several
studies
[1136147148]
Gao
et
al
prepared
arginine
chitosan
(ArgCs)DNA
selfassemble
nanoparticles
(ACSNs)
to
overcome
its
poor
water
solubility
and
low
transfection
efficiency
[147]
Arginine
of
ArgCs
can
increase
the
pKa
value
of
chitosan
which
leads
to
high
solubility
The
transfection
efficiency
of
ACSNs
is
found
not
only
to
be
much
higher
than
that
of
chitosanDNA
nanoparticles
(CSNs)
but
also
similar
level
as
Lipofectamine
Loretz
et
al
[136]
and
Zhao
et
al
[148]
reported
that
thiolated
chitosan
improves
the
transfection
efficiency
through
cysteine
modification
because
of
the
sulfide
group
introduced
to
chitosan
Layek
et
al
reported
on
the
hydrophobic
amino
acid
grafted
chitosan
(AGC)
with
Lalanine
Lvaline
Lleucine
and
Lisoleucine
to
enhance
membrane
permeability
[1]
The
cellular
uptakes
of
AGCA
AGCV
AGCL
and
AGCI
polylexes
with
pDNA
are
624
846
978
and
983
respectively
The
GFP
expression
efficiency
of
AGCamino
sampDNA
polyplexes
is
similar
to
the
results
of
cellular
uptake
at
AGCI

AGCL
>
AGCV
>
AGCA
Fig
3
shows
the
structure
of
chitosan
derivatives
which
are
modified
with
targeting
ligand
thiol
group
or
amino
acid
Genetic
materials
delivery
by
anionic
materialchitosan
complexes
Several
anionic
materials
have
been
used
to
improve
the
properties
(eg
low
transfection
efficiency
low
solubility
and
serum
stability)
of
chitosan
as
a
genetic
materials
delivery
vector
Anionic
materials
formed
the
complexes
with
chitosan
through
gelation
or
electrostatic
interaction
C
Choi
et
al

Journal
of
Industrial
and
Engineering
Chemistry
33
(2016)
1–10
7Anion
polymer
and
chitosan
complexes
Tripolyphosphate
(TPP)
a
poly
anion
can
form
nanoparticles
with
chitosan
by
using
the
ionotropic
gelation
between
positively
charged
chitosan
and
negatively
charged
TPP
[149150]
In
addition
TPP
was
used
as
crosslinker
like
a
polyanionic
linker
because
of
its
unique
properties
of
nontoxicity
and
instant
gelling
ability
[151]
Gaspar
et
al
reported
that
the
particle
size
of
chitosanTPPpDNA
nanocapsules
influences
the
parameters
like
the
chitosan
degree
of
deacetylation
and
formulation
ratio
(chitosan
to
TPP)
[152]
The
particle
size
of
chitosanTPPpDNA
nanocapsules
was
increased
when
the
chitosan
degree
of
deacetylation
increased
in
the
presence
of
pDNA
Katas
et
al
reported
on
the
chitosanTPP
nanoparticles
prepared
through
ionic
gelation
with
two
different
types
of
chitosan
(hydrochloride
form
and
glutamate
form)
and
each
type
with
two
different
MW
[153]
siRNA
(targeting
against
pGL3
luciferase
gene)
associated
with
chitosanTPP
nanoparticles
that
used
hydrochloride
form
of
chitosan
showed
a
high
gene
silencing
efficiency
In
addition
the
gene
silencing
efficiency
of
siRNA
associated
with
chitosanTPP
nanoparticles
increased
with
the
increasing
MW
(270–470
kDa)
Wang
et
al
demonstrated
the
potential
of
chitosanTPP
nanopar
ticle
to
deliver
short
hairpin
RNA
(shRNA)
[154]
ChitosanTPP
mediator
successfully
delivered
GFP
expression
vector
pSUPER
into
the
human
rhabdomyosarcoma
cell
(RD)
and
showed
a
higher
GFP
expression
than
the
chitosan
mediator
In
addition
transform
ing
growth
factor
(TGFB1)specific
shRNA
was
effectively
delivered
using
chitosanTPP
mediator
into
the
RD
cells
that
reduce
the
TGFB1
level
When
delivered
into
the
mice
through
chitosanTPP
mediator
with
four
type
shRNA
–
shRNAun
(negative
control)
shRNAa
shRNAb
and
shRNAc
–
after
RD
cell
inoculation
in
nude
mice
for
2
weeks
the
tumor
volume
in
the
four
groups
was
decreased
to
939
767
455
and
625
of
the
control
tumor
size
respectively
Anionic
biopolymer
and
chitosan
complexes
Hyaluronic
acid
(HA)
is
another
anionic
biopolymer
widely
used
in
biomedical
application
for
its
excellent
cytocompatibility
cell
adhesion
morphogenesis
inflammation
regulation
and
biocompatibility
[155–158]
In
addition
HA
can
also
interact
with
the
CD44
receptor
which
is
expressed
in
the
human
cornea
and
conjunctiva
[159160]
that
led
to
regeneration
of
corneal
and
conjunctiva
epithelial
cells
[161]
Fuente
et
al
demonstrated
that
optimal
formulation
of
HAchitosan
nanoparticles
(HACS
NP)
was
found
to
be
12
(HAchitosan
oligomer
(CS)
of
various
HACS
NP
formulations
including
HACS
(11
12
and
21)
hyaluronic
acid
oligomer
(HAO)CS
(11
12
and
21)
HACSO
(12)
and
HAOCSO
(11
12))
[160]
Ravin˜a
et
al
reported
that
the
cell
toxicity
of
HA
CSgrafted
PEG
(HACSgPEG
21
of
formulation
ratio)
showed
a
decrease
with
high
amount
of
nanoparticles
(HACSgPEGDNA)
at
10181
mgcm2 due
to
HA’s
high
biocompatibility
compared
to
CSgPEG
or
HACSgPEG
12
or
HACSgPEG
12
formulations
[162]
In
addition
HACSgPEGDNA
(pEGFP)
was
found
to
be
effective
gene
transfection
efficiency
in
all
formulations
(11
12
and
21)
and
HACSgPEGsiRNA
(against
the
EGFP)
significantly
silenced
the
EGFP
expression
compared
to
nontreated
cells
Lin
et
al
demonstrated
that
the
prepared
HAchitosanDNA
complex
multilayer
not
only
has
good
cytocompatibility
but
also
possesses
the
in
vitro
gene
transfection
ability
[163]
Lu
et
al
investigated
the
pDNA
delivered
by
HACS
vectors
to
enhance
transfection
optimal
environment
[164]
Various
factors
including
transfection
medium
pH
NP
ratio
pDNA
concentrations
and
MW
of
chitosan
were
investigated
The
transfection
efficiency
of
HACSpDNA
nanopar
ticles
was
significantly
higher
than
that
of
CSDNA
nanoparticles
at
the
following
condition
medium
pH
68
5
of
NP
ratio
4
mgmL
of
pDNA
concentration
and
50
kDa
of
chitosan
MW
respectively
Polypeptide
and
chitosan
complexes
Poly(gglutamic
acid)
(PGA)
a
naturally
occurring
peptide
is
biodegradable
watersoluble
and
nontoxic
[54165]
Therefore
PGA
and
its
derivatives
have
been
employed
as
a
carrier
in
biomedical
field
such
as
oral
delivery
of
insulin
[166167]
and
protein
vaccines
delivery
[168]
Liao
et
al
reported
that
chitosan
siRNAPGA
complexes
significantly
expedite
the
onset
of
gene
knockdown
and
also
enhance
their
inhibition
efficiency
and
prolong
the
duration
of
gene
silencing
[54]
In
addition
the
cellular
uptake
of
chitosansiRNAPGA
is
increased
when
the
PGA
ratio
increases
from
0
to
50
Generally
anionic
complexes
are
not
taken
up
well
by
cells
owing
to
the
electrostatic
repulsion
between
anionic
complexes
and
negatively
charged
cell
membranes
[54]
However
PGAcoated
cationic
polymers
significantly
enhance
their
cellular
uptake
suggesting
that
there
might
be
a
PGAspecific
receptormediated
pathway
[54165169]
In
addi
tion
Peng
groups
reported
on
the
cellular
uptake
and
transfection
Fig
3
Chitosan
derivatives
with
targeting
ligand
(A)
thiol
group
(B)
or
amino
acid
(C)
A
(a)
galactosylated
chitosan
[106]
(b)
mannosylated
chitosan
[109]
and
(c)
lactosylated
chitosan
[111]
B
(a)
thioglycolic
acid
conjugated
chitosan
[140]
and
(b)
cystamine
modified
trimethylated
chitosan
[132]
C
amino
acid
modified
chitosan
[1134146]
C
Choi
et
al

Journal
of
Industrial
and
Engineering
Chemistry
33
(2016)
1–108efficiency
[165]
and
the
mechanisms
of
cellular
uptake
and
intracellular
trafficking
[170]
of
chitosanDNAPGA
(CSDNAPGA)
complexes
as
a
gene
delivery
vector
Transfection
and
intracellular
uptake
of
CSDNAPGA
which
is
prepared
at
different
chitosan
DNAPGA
(NPC)
ratios
increased
via
a
specific
proteinmediated
endocytosis
when
NPC
ratios
increased
ranging
from
1010
to
1014
[165]
The
mechanisms
of
cellular
uptake
and
intracellular
trafficking
of
CSDNAPGA
were
investigated
with
various
inhibi
tors
on
the
uptake
such
as
chlorpromazine
(clathrinmediated
uptake
inhibitor)
wortamannin
(phosphatidyl
inositol3phos
phate
inhibitor)
cytochalasin
D
(actin
polymerization
and
membrane
ruffling
or
macropinocytosis
inhibitor)
filipin
(caveo
laemediated
endocytosis
inhibitor)
or
genistein
(caveolaemedi
ated
endocytosis
inhibitor)
respectively
[170]
As
a
result
of
the
uptake
test
with
endocytosis
inhibitor
the
CSDNAPGA
complexes
are
internalized
via
macropinocytosis
and
caveolaemediated
pathway
Conclusion
Chitosan
chitosan
derivatives
and
chitosananionic
materials
complexes
can
be
designed
by
enhancing
the
physicochemical
properties
for
genetic
materials
delivery
for
gene
therapy
The
MW
DDA
genetic
materials
concentration
and
serum
stability
of
chitosan
and
the
various
modifications
(hydrophilic
group
hydrophobic
group
cationic
group
targeting
ligand
thiol
group
and
amino
acid)
are
very
important
factors
in
preparing
chitosan
derivatives
to
enhance
the
efficiency
of
gene
therapy
In
addition
anionic
materials
such
as
TPP
HA
and
PGA
also
enhance
the
cellular
uptake
and
transfection
efficiency
of
chitosangenetic
materials
This
review
focused
on
the
chitosan
derivatives
chitosananionic
materials
preparing
techniques
and
sitespecific
targeting
to
improve
the
chitosan
properties
for
genetic
materials
delivery
These
factors
modifications
and
complexation
with
anionic
materials
effectively
improved
the
chitosan
properties
including
solubility
in
aqueous
solution
toxicity
in
HM
chitosan
buffering
capacity
escapes
in
endosome
cellular
uptake
genetic
material
release
from
chitosan
based
polyplexes
transfection
efficiency
and
silencing
efficiency
However
most
of
these
results
were
obtained
from
experiments
in
vitro
Therefore
further
research
is
needed
on
chitosan
for
gene
therapy
in
vivo
Research
on
chitosan
chitosan
derivatives
and
chitosananionic
materials
complexes
still
needs
to
understand
the
effects
of
the
character
istics
of
the
gene
carriers
on
cellular
entry
and
intracellular
trafficking
processes
Moreover
most
of
the
researches
used
HM
chitosan
Research
on
in
vivo
and
LM
chitosan
is
called
for
the
development
of
genetic
materials
delivery
Acknowledgements
This
work
was
supported
by
National
Research
Foundation
of
Korea
(NRF)
grant
funded
by
the
Ministry
of
Science
ICT
&
Future
Planning
(No
NRF2014R1A2A1A10053027)
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Journal
of
Industrial
and
Engineering
Chemistry
33
(2016)
1–1010

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