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Highly
efficient
gold
nanorods
assisted
laser
phototherapy
for
rapid
treatment
on
mice
wound
infected
by
pathogenic
bacteria
§
M.
Shahnawaz
Khan
d
,
Mukesh
L.
Bhaisare
a
,
Judy
Gopal
a,e
,
Hui-Fen
Wu
a,b,c,d,
*
a
Department
of
Chemistry
and
Center
for
Nanoscience
and
Nanotechnology,
National
Sun
Yat-Sen
University,
Kaohsiung,
70,
Lien-Hai
Road,
Kaohsiung
80424,
Taiwan
b
School
of
Pharmacy,
College
of
Pharmacy,
Kaohsiung
Medical
University,
Kaohsiung
807,
Taiwan
c
Institute
of
Medical
Science
and
Technology,
National
Sun
Yat-Sen
University,
Kaohsiung
80424,
Taiwan
d
Doctoral
Degree
Program
in
Marine
Biotechnology,
National
Sun
Yat-Sen
University
and
Academia
Sinica,
Kaohsiung
80424,
Taiwan
e
Department
of
Molecular
Biotechnology,
Konkuk
University,
Seoul
143-701,
Korea
Introduction
Wound
infection
and
its
treatment
require
high
level
of
advanced
research,
due
to
frequent
occurrence
of
pathogenic
infections.
Wound
healing
is
a
regenerative
process,
which
needs
the
coordinated
regulation
of
various
cell
regeneration
machiner-
ies
[1].
The
major
steps
in
wound
healing
are
inflammation,
proliferation
(tissue
formation)
and
tissue
remodeling
[2].
Healing
of
wound
is
a
sequential
process
in
which
biological
and
cellular
factors
are
released
resulting
in
fibroblast
proliferation,
synthesis
and
deposition
of
collagen,
revascularization,
and
wound
contrac-
tion
[3,4].
Pseudomonas
aeruginosa
is
one
of
the
major
causes
of
wound
infection,
which
can
lead
to
life-threatening
problems
to
immu-
nologically
challenged
patients
and
for
those
who
suffer
from
cystic
fibrosis
[5–7].
It
has
a
high
mutation
rate
which
will
lead
to
frequent
resistance
to
antibiotic
treatment.
Therefore,
an
effective
treatment
strategy
is
needed
which
can
be
used
independently
or
combined
with
antibiotics.
Lasers
have
been
used
in
medical
treatment
such
as
healing
of
wound,
treating
tumors
and
dental
diseases,
and
in
pain
reduction
[3].
Recent
progress
in
nanos-
ciences
applies
metallic
nanoparticles
including
Ag
[8],
Au
[9,10]
to
treat
pathogenic
infections.
Ag
NPs
have
been
used
to
kill
the
bacteria,
virus,
and
cancer
cells
[11,12].
Gold
nanorods
(Au
NRs)
are
excellent
in
photo
stability,
biocompatibility,
and
easy
to
be
modified
with
antibodies
[13]
than
the
Ag
NPs
[14].
Au
NRs
can
strongly
absorb
near-infrared
radiation
(NIR)
by
manipulating
their
shapes
and
size
[15,16]
and
thereby
facilitate
effective
energy
transfer
to
the
surrounding
environment
[17,18]
resulting
in
effective
killing
to
the
pathogenic
bacteria.
NIR
with
low
intensity
can
be
used
to
treat
human
tissues.
Thus,
Au-NRs
were
employed
to
focus
heat
exposure
for
effective
killing
of
pathogens.
The
cell
death
could
be
due
to
shock
waves,
bubble
formation
and
thermal
disintegration
[7].
Furthermore,
as
pulse
wave
lasers
have
‘‘quench
periods’’
which
is
the
time
period
when
laser
pulse
is
shut
off
resulting
in
less
tissue
heating
and
damage
[19,20].
Hashmi
et
al.
compared
the
difference
in
continuous
laser
and
pulsed
lasers
(more
effective)
for
treatment
[21].
Conventional
treatment
of
Journal
of
Industrial
and
Engineering
Chemistry
xxx
(2016)
xxx–xxx
A
R
T
I
C
L
E
I
N
F
O
Article
history:
Received
10
August
2015
Received
in
revised
form
11
November
2015
Accepted
5
December
2015
Available
online
xxx
Keywords:
MALDI-MS
Mice
wound
healing
Pseudomonas
aeruginosa
Nd-YAG
laser
A
B
S
T
R
A
C
T
Treatment
of
wound
infection
is
one
of
the
most
challenging
problems
to
be
addressed
in
infectiously
microbiological
world.
This
is
mainly
due
to
the
pathogen’s
ability
for
fast
mutation
and
generating
severely
antibiotic
resistance
to
antimicrobial
treatment.
Pathogens
causing
wound
infection
treated
by
photothermal
methods
can
avoid
drug
resistance
problem.
Beside,
rapid
and
highly
effective
wound
treatment
method
in
modern
medical
technology
is
extremely
required.
Therefore,
we
have
proposed
a
novel
method
by
using
gold
nanorods
(Au
NRs)
to
assist
the
Nd-YAG
laser
(1064
nm)
for
photothermal
killing
pathogenic
bacteria
(Pseudomonas
aeruginosa)
for
directly
healing
the
wound
infection
on
the
(albino)
mice.
In
all
experiments,
the
bacteria
numbers
were
calculated
by
the
plate
count
method
and
we
also
used
MALDI-MS
to
evaluate
the
effectiveness
of
the
treatment
and
the
physiological
condition
of
wound
infection.
The
current
approach
can
be
used
to
control
severe
skin
infections
from
antibiotic
resistant
pathogens
in
wounds.
ß
2015
The
Korean
Society
of
Industrial
and
Engineering
Chemistry.
Published
by
Elsevier
B.V.
All
rights
reserved.
§
Contract/grant
sponsor:
Ministry
of
science
and
technology
(MOST),
Taiwan.
*Corresponding
author
at:
National
Sun
Yat-Sen
University,
Department
of
Chemistry
and
Center
for
Nanoscience
and
Nanotechnology,
70,
Lien-Hai
Road,
Kaohsiung
80424,
Taiwan.
Tel.:
+886
7
5252000
3955;
fax:
+886
7
5253909.
E-mail
address:
hwu@faculty.nsysu.edu.tw
(H.-F.
Wu).
G
Model
JIEC-2750;
No.
of
Pages
10
Please
cite
this
article
in
press
as:
M.S.
Khan,
et
al.,
J.
Ind.
Eng.
Chem.
(2016),
http://dx.doi.org/10.1016/j.jiec.2015.12.011
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
ate/jiec
http://dx.doi.org/10.1016/j.jiec.2015.12.011
1226-086X/ß
2015
The
Korean
Society
of
Industrial
and
Engineering
Chemistry.
Published
by
Elsevier
B.V.
All
rights
reserved.
wound
infection
typically
employs
high
doses
of
antibiotics
or
incision
drainage
wound
packing
procedures.
However,
these
treatments
have
drawbacks
like
antibiotic
resistance
and
drainage
of
wound
packing
procedures
are
severely
afflicting
procedures.
To
solve
these
problems,
we
employed
Au
NRs
assisted
laser
for
photothermal
treatment
directly
on
the
wound
infection
of
mice.
The
Au
NRs
exposed
to
lasers
can
act
as
light-activated
antimicrobial
agents,
thereby
efficiently
killing
the
bacterial
colonies
[22].
Due
to
laser
exposure,
the
Au
NRs
could
generate
high
heat
for
effectively
killing
pathogens
at
the
wound
infection
sites
without
killing
the
mice
tissues
[23].
MALDI-MS
(Matrix-
assisted
laser
desorption/ionization
Mass
Spectrometry),
is
a
valuable
instrument
for
fast
detection
of
biological
samples
and
biomolecules
based
on
sample’s
molecular
weight
and
their
fragmentation
patterns
[24,25].
In
biology,
it
was
also
used
to
monitor
the
infection
kinetics
of
Staphylococcus
aureus
at
the
wound
site
and
the
immune
response
of
mice
[42].
Thus,
we
also
used
it
in
our
study
to
understand
the
physiological
condition
of
wound
infection,
and
the
efficiency
of
Au
NR
to
assist
the
photothermal
killing
of
bacteria
in
the
laser
treatment.
Materials
and
methods
All
chemicals
used
in
this
study
were
more
than
99%
purity.
Hydrogen
tetrachloroaurate
was
obtained
from
Alfa
Aesar.
Acetonitrile,
hydrochloric
acid
and
trifluoroacetic
acid
(TFA)
were
purchased
from
Wako
Pure
Chemicals
(Osaka,
Japan).
Sodium
borohydride
was
bought
from
Kochlight
laboratories
Ltd
(coli
brooks;
England).
Cetyl
trimethylammonium
bromide
(CTAB)
and
ascorbic
acid
were
obtained
from
Sigma
Aldrich.
Silver
nitrate
was
taken
from
Mallinckrodt
Company.
The
water
used
for
all
experiments
including
the
cleaning
of
glasswares
was
purified
by
using
a
Milli-Q
water
purification
system
(Millipore,
Bedford,
MA,
USA).
Instruments
Transmission
electron
microscopy
(TEM)
images
were
gener-
ated
using
a
Philip
CM200
(Switzerland)
at
accelerating
voltage
of
100
kV.
The
UV–vis
spectra
of
Au-NRs
were
recorded
by
a
double
beam
UV–vis
spectrophotometer
(U3501,
Hitachi,
Japan).
A
vortex
agitator
(VM,
2000,
Digi
system
Laboratory,
Taipei,
Taiwan)
was
used
for
proper
mixing
of
the
sample
solution
with
the
matrix
solution.
A
MALDI-TOF
mass
spectrometer
(Microflex,
Bruker
Daltonics,
(Germany)
was
used
for
the
analysis
of
bacterial
(protein)
peaks
and
mice
(blood)
peaks.
All
data
were
recorded
in
the
Linear
and
positive-ion
mode.
The
MS
system
was
operated
under
accelerating
voltage
of
20
kV
and
using
a
pulsed
nitrogen
laser
(337
nm,
4-ns
pulses
at
10.0
Hz,
60.0
m
J).
96
wells
of
the
MALDI-MS
target
plate
was
used
and
the
spectra
were
taken
by
applying
300
laser
pulses.
Microtomy
of
mice
organs
were
performed
by
Hestion
ERM
3000
semi-automatic
Microtome
(Australia).
Pseudomonas
aeruginosa
culture
and
sample
collection
from
wound
site
for
MALDI-MS
and
TVC
(total
viable
counts)
Single
colony
of
pure
culture
of
P.
aeruginosa
was
grown
in
agar
plates
(Luria
Bertania)
in
LB
broth
by
incubating
at
37
8C
for
12
h.
After
the
incubation,
the
bacterial
culture
suspension
was
prepared
to
infect
on
the
wound
sites
of
mice.
15
colonies
of
P.
aeruginosa
were
suspended
in
1
mL
of
double
distilled
sterile
water
in
eppendorf
tubes.
Twenty
eppendorf
tubes
were
centrifuged
(Mikro
22R,
Andreas
Hettich
GmbH
&
Co.
KG,
Germany)
at
3000
rpm
for
4
min.
After
the
centrifugation,
the
supernatant
was
discarded
and
the
bacterial
pellets
were
collected
in
one
tube.
By
using
a
sterile
pipette,
the
bacterial
suspension
(5
10
7
cells/100
m
L)
was
dropped
at
the
fresh
wounds
of
each
mouse
(100
m
L).
After
the
spreading
the
infection
by
the
P.
aeruginosa
at
the
wound
sites,
the
mice
were
put
back
to
their
cages
bedded
with
sterile
tissue
papers.
From
the
infected
wound
site,
a
swab
was
collected
by
sterile
cotton
swab
in
eppendorf
tubes
which
contains
10
m
L
of
sterile
double
distilled
water,
exudate
from
the
wound
infection
around
10
m
L
were
taken
out
by
using
1
mL
insulin
syringe
of
26
ga.
All
samples
collected
from
mice
at
each
time
point
were
suspended
in
100
m
L
of
dd
water,
then
vortexed
for
5
min
using
a
vortex
machine
(VM
2000,
Digi
System
Calculation,
and
Taipei,
Taiwan).
Then,
they
were
centrifuged
at
3000
rpm
for
4
min.
Finally,
the
bacterial
pellet
was
collected
and
spotted
(5
m
L
in
triplicates)
onto
the
MALDI-MS
target
plate
for
analysis
[42].
100
m
L
of
the
samples
were
serially
diluted
in
double
distilled
water
and
dropped
on
LB
agar
for
estimating
the
Total
Viable
Counts
(TVC)
of
bacteria
by
the
microbiological
standard
procedures.
Nd-YAG
laser
for
wound
infection
treatment
Nd-YAG
Laser
with
1064
nm
in
wavelength,
1
mm
spot
size.
The
Pulse
repetition
rate
for
laser
is
10
Hz,
power
155
mW/10
laser
shot
and
energy
100
mJ,
was
applied
for
the
treatment
of
wound
infection
in
all
mice
experiments.
Power
was
measured
by
ophir
(Nova
display-ROHS)
56SN:
612989,
www.ophiropt.com/
laser-measurement.
Sensor
Model:
30(150)
A-HE-17ROHS
(Made
in
Israel)
Synthesis
of
gold
nanorods
(Au-NRs)
The
Au
NRs
were
synthesized
via
seeded-mediated
growth
method
[26].
For
making
the
seed
solution,
the
following
molar
concentrations
of
solutions
were
prepared:
7.5
mL
of
0.2
M
CTAB
solution
was
added
into
0.25
mL
of
0.01
M
HAuCl
4
in
a
flask.
0.6
mL
of
NaBH
4
(0.01
M,
ice
cooled)
was
added
drop
wisely
until
the
solution
turned
to
light
brown.
After
vigorous
stirring
for
2
min,
the
solution
was
kept
aside
undisturbed
at
room
temp.
10
mL
of
0.01
HAuCl
4
was
mixed
with
237.5
mL
of
0.1
M
CTAB
in
a
beaker,
which
turns
the
solution
to
orange.
1.6
mL
of
0.1
M
of
ascorbic
acid
was
added
and
slowly
inverts
solution
3–4
times
until
it
becomes
colorless.
Finally,
2
mL
of
seed
solution
was
added
dropwisely
into
the
growing
solution.
The
maximum
absorption
of
wavelength
was
optimized
by
adding
nitric
acid
in
the
growth
solution
[27].
The
solution
was
undisturbed
about
12
h,
till
it
turned
to
reddish
brown.
Centrifuged
the
Au
NRs
solution
at
5000
rpm
till
removed
the
froth
of
CTAB,
which
is
responsible
for
toxicity.
To
solve
the
problem
of
toxicity,
the
Au
NRs
were
further
modified
with
Chitosan.
The
gold
nanorods
were
confirmed
by
using
TEM
and
UV
spectrophotometer.
The
methodology
of
the
experimental
proce-
dures
for
the
use
of
laser
on
mice’s
wound
treatment
is
shown
in
the
Scheme
1.
Optimization
of
laser
exposure
time
and
Au
NR’s
concentration
to
against
P.
aeruginosa
To
determine
the
minimum
exposure
of
Nd-YAG
laser
against
P.
aeruginosa.
100
m
L
of
bacterial
suspension
placed
in
a
microtiter
plate
and
treated
with
Nd-YAG
laser
with
respect
to
different
time
intervals
from
0
to
300
s.
After
treatment,
the
viable
bacterial
colonies
were
counted
by
the
traditional
plate
count
method.
P.
aeruginosa
(BCRC
10303)
standard
cultures
were
brought
from
the
Bioresource
Collection
and
Research
Center
(BCRC,
Hsin-Chu,
Taiwan).
The
bacterial
viability
after
laser
treatment
revealed
that
the
Nd-YAG
laser
treatment
resulted
in
effectively
killing
of
bacteria.
In
control,
the
bacterial
counts
were
about
1.5
10
9
cfu/mL;
laser
M.S.
Khan
et
al.
/
Journal
of
Industrial
and
Engineering
Chemistry
xxx
(2016)
xxx–xxx
2
G
Model
JIEC-2750;
No.
of
Pages
10
Please
cite
this
article
in
press
as:
M.S.
Khan,
et
al.,
J.
Ind.
Eng.
Chem.
(2016),
http://dx.doi.org/10.1016/j.jiec.2015.12.011
treatment
showed
significant
decreased
in
the
total
bacterial
counts.
Further
exposure
of
bacterial
sample
in
time
intervals
from
0,
120,
180,
240
and
300
s
led
to
reduction
in
the
bacterial
population.
The
last
two
results
(240
and
300
s)
for
laser
treatment
revealed
that
the
cell
viability
would
not
be
influenced
and
is
approximately
the
same
number
on
increasing
the
exposure
time.
According
to
the
efficiency
of
Nd-YAG
laser
on
decreasing
bacterial
viability,
laser
exposure
of
240
s
was
selected
as
the
optimal
parameter.
The
results
for
exposure
treatment
with
Nd-YAG
laser
are
tabulated
in
Table
1a.
The
investigated
concentrations
of
Au
NRs
were
varied
from
2.25
m
g/mL
to
11.25
m
g/mL.
Constant
time
interval
of
240
s
of
laser’s
exposure
was
used
for
finding
the
optimal
concentration
of
Au
NRs.
As
increasing
the
concentration
of
Au
NRs,
the
bacterial
counts
were
recorded
in
Table
1b.
The
optimum
concentration
of
the
Au
NRs
was
found
to
be
9.0
m
g/mL;
beyond
this
concentration
of
Au
NRs,
the
viability
of
bacterial
counts
was
similar
so
this
concentration
was
used
in
all
experiments.
All
experiments
were
performed
in
triplets.
Animal
experiments
to
confirm
the
wound
healing
treatment
by
laser
Fifteen
healthy
male
albino
mice
(eight
weeks)
with
a
mean
weight
20
5
g
were
obtained
from
the
Bio
Lasco
Co.
Ltd,
Taipei,
Taiwan.
They
were
kept
at
room
temp
(25
8C)
with
a
12
h
light/dark
cycle
and
were
provided
with
food
and
water.
The
ethics
committee
of
National
Sun
Yat-Sen
University,
Taiwan
has
approved
the
experimental
protocols
for
our
animal
experiments.
These
mice
were
anaesthetized
by
using
ketamine
hydrochloride
(50
mg/kg/
bodyweight)
injected
via
the
intraperitoneal
route
[28].
The
fur
of
mice
was
removed
from
dorsal
side
using
an
electronic
clipper
and
the
skin
was
cleaned
by
alcohol
and
povidine
iodine.
Three
circular
wounds
were
made
on
thoracic
and
lumbar
regions
of
area
0.5
0.5
cm
2
using
a
No.
15
blade
and
scalpel.
The
Nd-YAG
Laser
was
irradiated
on
two
regions
of
wound
at
45
˚and
60
˚for
deeper
penetration
at
a
distance
of
10
cm
for
each
wound
for
240
s
of
time
period.
For
histological
studies,
mice
were
euthanized
on
3rd
and
12th
day
and
their
treated
tissue
was
sectioned
by
Microtomy
instrument
which
were
stained
by
Haemotoxolin
and
Eosin
dye.
Three
wounds
were
made
on
each
mouse
on
its
dorsal
side
and
infected
by
P.
aeruginosa,
which
can
be
named
as
1st,
2nd
and
3rd
wound
(W1,
W2
and
W3),
respectively.
Individual
treatment
is
named
as:
first
treatment
is
the
control
experiments
(W1);
the
second
treatment
(W2)
is
treated
with
Nd-YAG
laser
only
and
third
Scheme
1.
(a)
Schematic
diagram
showing
the
methodology
of
whole
experiment.
Table
1a
The
optimization
of
exposure
time
by
counting
cfu
of
bacterial
suspension
by
Nd-
YAG
laser.
S.
no.
Time
(s)
Cfu/mL
1.
00
1.5
10
9
2.
120
3.9
10
8
3.
180
2.2
10
7
4.
240
4.8
10
6
5.
300
3.6
10
6
Table
1b
The
optimization
of
Au
NRs
concentration
by
exposing
the
Nd-YAG
laser
on
bacterial
suspension
and
counting
colony
forming
units
by
plate
count
method.
S.
no.
Time
(s)
Vol.
of
bacterial
solution
(
m
L)
Conc.
of
Au
NRs
450
m
g/mL
(stock
solution)
(
m
g/mL)
cfu/mL
1.
00
100
2.25
2.7
10
9
2.
240
100
4.5
3.1
10
8
3.
240
100
6.75
3.5
10
7
4.
240
100
9.0
2.6
10
6
5.
240
100
11.25
1.2
10
6
M.S.
Khan
et
al.
/
Journal
of
Industrial
and
Engineering
Chemistry
xxx
(2016)
xxx–xxx
3
G
Model
JIEC-2750;
No.
of
Pages
10
Please
cite
this
article
in
press
as:
M.S.
Khan,
et
al.,
J.
Ind.
Eng.
Chem.
(2016),
http://dx.doi.org/10.1016/j.jiec.2015.12.011
wound
treatment
(W3)
was
treated
by
the
Nd-YAG
laser
with
the
assistant
of
Au-NRs
for
photothermal
killing
bacteria
and
healing
of
wound.
Nd-YAG
laser
(1064
nm)
was
applied
on
each
wound
site
for
240
s
and
immediately
the
mice
were
put
inside
the
laminar
flow
to
collect
exudate
from
the
wound
surface
using
a
sterile
swab
in
500
m
L
of
PBS
solution.
10
m
L
from
this
bacterial
sample
was
taken
in
a
Petri
plate
for
the
bacterial
count
after
pouring
the
nutrient
agar
medium
on
the
plate.
The
laser
treatment
was
started
after
the
bacterial
infection
was
spread
on
the
wound
surface
of
mice
after
3
days.
Au
NRs
in
liquid
form
around
100
m
L
were
pouring
carefully
on
the
wound
surface
just
before
giving
the
laser
therapy
till
the
last
day
of
treatment.
The
total
Bacterial
colony
count
was
calculated
after
laser
exposure
for
each
set
of
animal
models.
Differentiate
between
bacterial
proteins
and
mouse
wound
proteins
To
differentiate
between
the
MALDI-MS
peaks
originated
from
the
infected
bacteria
or
the
wound
site
proteins
of
mice,
we
took
the
samples
directly
from
the
mice
wound
site
and
spotted
onto
the
stainless
steel
plates
with
Sinapinic
acid
(SA)
as
a
matrix
for
MALDI-MS
detection.
For
understanding
the
concentration/popu-
lation
of
P.
aeruginosa,
a
single
colony
plated
on
LB
agar
and
inoculated
for
24
h
at
37
8C
was
examined.
By
using
a
sterile
loop
P.
aeruginosa
was
scrapped
and
dispersed
into
400
m
L
of
sterile
PBS
and
vortexed
for
10
min
and
centrifuged
at
5000
rpm
for
5
min.
The
obtained
pellet
was
spotted
on
the
MALDI-MS
stainless
steel
plate
along
with
SA
matrix
for
identification
of
bacterial
peaks.
MALDI-MS
spectra
of
P.
aeruginosa
were
confirmed
from
the
infected
sites
for
those
peaks
observed
at
m/z,
3680,
4489,
5038,
5798,
6435,
6749,
7288,
7690,
8196,
8881,
9418,
10,370,
11,656,
12,515,
14,146
which
were
reported
previously
[28–30].
The
10,108
m/z
peak
signifies
the
mice-interaction
triggered
protein
peak
released
at
the
wound
site
[42].
The
15
kDa
peak
is
assigned
as
the
blood
peak
of
mice
whereas
7288
m/z
belong
to
b
defensin
and
10,370
m/z
is
the
a
defensin
[31–34,42].
These
b
defensin
proteins
are
related
to
cysteine-rich
(broad
spectrum)
antimicro-
bial
peptide
family;
a
-defensin
are
present
in
the
mammalian
leukocytes
as
major
granule
constituents
at
the
infection
sites.
Wound
size
determination
For
measuring
the
size
of
wounds
during
the
treatment,
a
graph
paper
was
used
and
marked
the
size
of
wound
on
the
paper
everyday
[35].
During
the
whole
experiment,
the
size
was
monitored
and
the
values
were
recorded
after
the
treatment
with
Nd-YAG
laser
and
Au
NRs.
At
the
end
of
experiment,
the
graph
was
plotted
according
to
the
percentage
of
area
of
wounds.
Result
and
discussion
Characterization
of
the
Au
NRs
After
synthesis
of
the
Au
NRs,
TEM
and
UV
were
performed
to
confirm
the
formation
of
Au
NRs.
Fig.
1(a)
shows
the
UV–vis
absorbance
spectra
of
Au
NRs.
It
clearly
distinguishes
the
Au
NR
formation
from
the
gold
nanoparticles
as
it
has
two
absorption
peaks.
Absorption
at
525
nm
and
1060
nm
were
due
to
the
surface
Plasmon
oscillations
of
shorter
transverse
(TSPR)
direction
and
higher
absorption
longitudinal
oscillations
(LSPR),
respectively.
The
TEM
image
of
Au
NRs
in
Fig.
1(b)
reveals
the
aspect
ratio
of
about
3.8
(length:
27.2
nm,
width:
9.3
nm).
Fig.
1(c)
shows
the
enhancement
of
temperature
by
Au
NRs
which
was
observed
by
heating
the
solution
(0
m
g/mL
to
45
m
g/mL)
and
the
temperature
reached
to
57.4
8C
which
is
sufficient
to
kill
the
bacteria.
Fig.
1(d)
demonstrates
increased
temperature
by
using
Au
NRs
and
Nd-YAG
laser
and
was
recorded
by
using
a
thermal
imaging
camera
(FLIR
E8,
Sweden)
which
shows
the
maximum
enhancement
of
temperature
can
be
reached
to
56.7
8C.
Fig.
2(a)
shows
the
experimental
photo
of
Nd-YAG
Laser
exposure
on
the
bacterial
broth
for
the
calculation
of
the
colony
forming
units
of
bacteria.
In
the
next
photograph
of
Fig.
2(b)
shows
the
zoom
image
of
the
exposure
of
Nd-YAG
Laser
on
the
microtiter
plate
with
different
concentrations
of
Au
NRs.
Fig.
2(c)
shows
the
exposure
of
Nd-YAG
laser
on
mice’s
wound,
where
as
in
Fig.
2(d)
the
IR
images
were
taken
during
the
irradiation
of
Nd-YAG
laser
with
Au
NRs
showing
temperature
elevation
reached
to
55.1
8C
on
the
surface
of
wound.
Fig.
3(a)
shows
the
TEM
images
of
the
control
P.
aeruginosa
without
any
treatment.
In
the
next
figure
(b)
P.
aeruginosa
was
treated
by
only
Nd-YAG
laser
for
240
s,
the
morphology
remain
same
up
to
this
exposure
time,
as
it
has
a
less
impact
of
laser
for
this
duration.
In
Fig.
3(c)
when
it
was
exposed
with
Au
NRs
and
Nd-YAG
laser,
we
can
observed
the
lethal
effect
of
killing
P.
aeruginosa;
its
total
morphology
changed
including
disruption
in
plasma
membrane.
It
was
observed
that
most
of
the
bacterial
cells
were
killed
by
this
new
treatment.
These
results
confirmed
the
feasibility
of
this
technique
in
curing
the
skin
wound
infections
which
can
avoid
the
resistance
of
mutation
of
bacteria
if
treated
by
antibiotics.
Nd-YAG
laser
used
in
triple
wounds
on
mice
skin
Three
different
locations
wounds
were
created
on
mice’s
back
namely
as
Wound
(W1)
near
the
lumbar
or
neck
region,
Wound
2
(W2)
in
middle
of
body
region
and
Wound
3
(W3)
near
tail
region.
We
observed
that
the
Nd-YAG
laser
was
effective
to
infected
W2
which
showed
better
recovery
than
the
control
set
of
infected
W1.
Also,
the
duration
of
treatment
can
be
greatly
reduced
when
heat-
absorbing
Au
NRs
were
used
in
combination
with
the
Nd-YAG
laser.
Additionally,
infected
W3
exhibited
signs
of
hair-follicle
regeneration,
which
is
a
hallmark
of
wound
healing.
After
treatment,
CFU
was
calculated
by
using
1
mL
of
insulin
syringe.
Mice’s
wound
exudate
was
collected
in
a
laminar
flow
and
subjected
to
serial
dilution
for
counting
the
bacterial
colonies
[42].
After
applying
the
treatment,
the
total
viable
counts
revealed
that
by
applying
this
treatment,
the
bacterial
counts
can
be
decreased.
The
Laser
treatment
with
photothermal
agents
was
performed
and
continued
for
12
days
for
these
three
different
groups
of
mice
on
their
infected
wounds.
Michael
R.
Hamblin
proposed
the
parameters
that
can
affect
the
healing
of
wound
[36].
There
are
many
transcriptional
factors
regulated
by
the
changes
in
cellular
redox
state.
For
instance,
Redox
factor-1
(Red
f-1),
dependent
activator
protein-1
(AP-1),
nuclear
factor
(NF-W3),
activation
transcription
factor
c-AMP
response
element-binding
protein
(ATF/CREB),
hypoxia-inducible
factor
(HIF)-1
alpha.
A
research
based
on
He-Ne
laser
(632
nm)
exposure
at
wounds
on
nude
mice
showed
improvement
in
the
tensile
strength
in
about
1
to
2
weeks.
After
2
months,
the
collagen
amount
was
increased
as
compared
to
the
control
wound
[37].
Laser
therapy
stimulates
the
expression
of
cytokines
and
other
growth
factors
for
wound
healing
[38].
Additionally,
it
also
results
in
enhanced
fibroblast
which
proliferates
the
transportation
of
proteins
like
basic
fibroblast
factor
and
hepatocyte
growth
factor
[37–39].
The
important
proteins
like
vascular
endothelial
growth
factor,
which
is
responsible
for
the
neovascularization
also
expressed
abun-
dantly
[40].
Other
report
showed
that
the
transforming
growth
factor
beta
expression
can
also
be
regulated
by
the
application
of
laser
as
it
is
important
in
collagen
synthesis
from
fibroblast
[41].
It
also
facilitates
the
formation
of
smooth
muscle
alpha-actin
and
desmin
[41,42].
In
our
study,
laser
along
with
Au
NRs
were
applied
on
the
wounds
every
day
for
240
s,
their
CFU
and
MALDI-MS
spectra
was
recorded
on
the
3rd,
6th
and
12th
days.
M.S.
Khan
et
al.
/
Journal
of
Industrial
and
Engineering
Chemistry
xxx
(2016)
xxx–xxx
4
G
Model
JIEC-2750;
No.
of
Pages
10
Please
cite
this
article
in
press
as:
M.S.
Khan,
et
al.,
J.
Ind.
Eng.
Chem.
(2016),
http://dx.doi.org/10.1016/j.jiec.2015.12.011
Table
2
represents
the
wounds
(W1,
W2,
and
W3)
Colony
forming
units
of
bacteria
on
third
day
after
treatment
with
Nd-YAG
laser
and
with
Au
NRs.
The
bacterial
counts
for
W1,
W2
and
W3
were
2.8
10
9
cfu/mL,
2.7
10
9
cfu/mL
and
3.1
10
9
cfu/mL
on
the
3rd
day,
respectively.
Every
day
the
mice
were
subjected
to
the
same
treatment,
parameters
and
conditions.
In
Fig.
4(B)
W1
which
was
regarded
as
a
control,
not
being
subjected
to
any
treatment
after
infection
had
almost
the
same
CFU
of
2.9
10
8
cfu/mL
on
Fig.
1.
(a)
UV–vis
spectra
of
Au
NRs
showing
both
the
absorption
of
longitudinal
surface
Plasmon
resonance
and
transverse
Plasmon
resonance.
(b)
TEM
images
of
Au
NRs.
(c)
Temp
enhancement
curve
of
Au
NRs
upon
heating
by
Nd-YAG
laser.
(d)
Thermal
image
was
taken
using
thermal
imaging
camera
(FLIR)
upon
irradiation
of
Nd-YAG
laser
(1064
nm).
Fig.
2.
The
optimization
of
laser
employed
in
the
experiment.
(a)
Setup
picture
of
working
Nd-YAG
laser
on
bacterial
suspension.
(b)
The
microtiter
plate
utilized
for
bacterial
suspension
for
optimized
condition.
(c)
The
Nd-YAG
laser
exposed
on
the
mice
wound.
(d)
IR
images
taken
during
the
irradiation
of
Nd-YAG
laser
and
Au
NRs.
M.S.
Khan
et
al.
/
Journal
of
Industrial
and
Engineering
Chemistry
xxx
(2016)
xxx–xxx
5
G
Model
JIEC-2750;
No.
of
Pages
10
Please
cite
this
article
in
press
as:
M.S.
Khan,
et
al.,
J.
Ind.
Eng.
Chem.
(2016),
http://dx.doi.org/10.1016/j.jiec.2015.12.011
6th
day.
We
found
the
difference
in
W2
as
treated
with
laser;
its
CFU
was
1.2
10
8
cfu/mL.
W3
which
was
treated
by
Au
NRs
and
Nd-YAG
laser
exhibited
some
signs
of
healing
as
evident
by
decreased
in
the
bacterial
counts
to
3.1
10
7
cfu/mL.
On
12th
day,
W1
showed
decrease
in
CFU
i.e.
5.6
10
7
cfu/mL,
due
to
self-
immunity
of
the
mice
immune
system.
In
the
case
of
W2,
the
result
was
relatively
promising
as
CFU
ceased
to
1.3
10
7
cfu/mL;
thereby
the
size
of
wound
was
also
decreased.
In
W3
the
condition
was
remarkably
good
as
the
wound
size
was
decreased
and
hairs
were
also
re-grow
at
the
W3
nearby
regions.
Its
CFU
count
was
decreased
to
1.8
10
6
cfu/mL.
According
to
the
above
results,
when
both
the
Nd-YAG
laser
and
Au
NRs
were
used
simultaneous-
ly,
it
augments
the
killing
efficacy
of
the
pathogenic
species.
The
vast
reduction
in
the
infected
bacterial
colony
proved
that
laser
combined
with
heat
absorbing
nanomaterial
is
more
effective
than
the
single
use
of
laser.
MALDI-MS
studies
of
wound
condition
in
mice
Fig.
5(a)
depicts
the
MALDI-MS
spectra
in
which
(a)
shows
the
background
peaks
of
P.
aeruginosa
proteins
for
reference.
The
second
spectra
of
W1
(b)
was
taken
from
the
infected
wound
of
mice
which
also
shows
the
15
kDa
peak,
which
is
a
well-known
mass
peak
of
the
blood
[43].
The
bacterial
peaks
did
not
appear
more
in
numbers
and
the
blood
protein
peaks
suppressed
the
bacterial
peaks.
The
spectrum
showed
in
(c)
was
taken
from
W2
in
which
infection
was
spread
and
treated
by
Nd-YAG
laser
alone.
It
can
be
seen
clearly
in
the
spectra
that
the
number
of
bacterial
peaks
also
appears
along
with
the
blood
peaks.
Similarly,
when
the
W3
was
treated
with
Au
NRs
and
Nd-YAG
laser,
the
peak
of
11,736
kDa
was
disappeared
and
the
blood
peaks
became
broader,
which
may
be
a
result
of
the
blood
evaporation
from
the
wound
surface
(d).
The
removal
of
the
bacterial
peaks
signified
the
killing
and
starts
the
beginning
of
the
healing
process.
On
the
6th
day,
the
MALDI-MS
spectra
were
taken
from
the
samples
obtained
for
mice’s
wounds.
As
explained
earlier,
in
the
previous
spectrum,
the
first
spectrum
was
taken
as
a
control
for
the
bacterial
peaks
in
Fig.
5(b)
(a)
whereas
the
second
spectra
were
taken
from
the
W1
(b)
which
was
not
treating
by
any
external
source.
In
this
spectrum,
it
is
observed
that
the
blood
peaks,
which
are
marked
by
star,
are
more
prominent
as
compared
to
the
spectra
from
(c)
W2
and
(d)
W3
of
same
figure.
Its
peak
intensity
decreased
as
the
physiological
conditions
showed
slight
improvement
like
dryness
at
the
periphery
of
wound,
skin
growth,
and
follicular
regeneration.
On
12th
day
of
post-infection
in
Fig.
5c,
the
first
spectrum
(a)
is
from
P.
aeruginosa
which
was
used
as
a
control
background
for
the
comparison
of
blood
peaks
from
the
bacterial
peaks.
In
the
second
spectra
(b)
of
W1
showed
a
mixture
of
peaks
of
P.
aeruginosa
and
blood
taken
directly
from
the
wound
surface
of
mice,
which
showed
decrement
in
the
intensity
of
all
MALDI-MS
peaks.
It
showed
that
in
case
of
laser
treatment,
infection
along
with
healing
was
slowly
treated.
In
the
third
spectra
(c)
of
W2,
most
of
the
bacterial
peaks
are
suppressed
and
the
blood
peak
of
15
kDa
was
also
suppressed.
In
the
last
spectra
of
W3
(d)
showed
a
remarkable
decreased
in
bacterial
peaks
which
correlate
well
with
low
CFU
results.
This
spectrum
showed
that
the
bacterial
and
blood
protein
peaks
were
no
more
prominently
dominant
in
MALDI-MS
spectra
[43,44].
All
the
bacterial
peaks
were
disappeared
and
very
low
intensity
of
15
kDa
peak
was
observed,
which
may
due
to
little
pus
or
blood
remains
at
the
center
of
the
wound
sites.
When
Au
NRs-mediated
laser
exposure
was
performed,
the
healing
efficien-
cy
was
remarkably
improved.
On
12th
day,
the
improved
wound
healing
is
evident
by
the
decreased
in
CFU
counts,
along
with
no
observation
of
15
kDa
peak
in
MS
spectra.
The
above
results
of
CFU
and
MALDI-MS
would
be
more
meaningful
if
it
can
be
shown
in
the
photographs
as
well.
Thus,
the
photographs
were
taken
just
after
these
three
wounds
were
mounted
on
the
dorsal
surface
of
mouse
in
Fig.
6(a).
The
second
photograph
in
Fig.
6(b)
was
taken
after
3
days,
when
the
P.
aeruginosa
infection
started
on
the
W1,
W2
and
W3
wounds.
The
yellow
color
of
wounds
surround
periphery
and
in
middle
indicates
the
beginning
of
infection.
Final
results
can
be
seen
in
the
last
photographs
in
Fig.
6(c),
which
was
taken
on
the
12th
day
of
the
wound
treatment.
In
this
photograph,
the
first
wound
W1
starting
from
lumbar
region
showing
dryness
and
hardness
on
the
surface
but
little
yellow
pus
at
the
periphery
of
the
wound.
The
W2
Fig.
3.
(a)
TEM
images
of
control
P.
aeruginosa.
(b)
Treated
by
only
Nd-YAG
laser
for
240
s,
upto
this
exposure
time,
the
morphology
remain
same.
(c)
Exposure
of
P.
aeruginosa
with
Au
NRs
and
Nd-YAG
laser,
morphology
changed
including
disruption
in
plasma
membrane.
Table
2
Bacterial
counts
of
the
wounds
after
the
Nd-YAG
laser
treatment
for
(a)
third
day
treatment
(b)
sixth
day
treatment
(c)
twelfth-day
treatment.
S.
no.
Wound
1
(cfu/mL)
Wound
2
(cfu/mL)
Wound
3
(cfu/mL)
3rd
Day
2.8
10
9
2.7
10
9
3.1
10
9
6th
Day
2.9
10
8
1.2.
10
8
3.7
10
7
12th
Day
5.6
10
7
1.3
10
7
1.8
10
6
M.S.
Khan
et
al.
/
Journal
of
Industrial
and
Engineering
Chemistry
xxx
(2016)
xxx–xxx
6
G
Model
JIEC-2750;
No.
of
Pages
10
Please
cite
this
article
in
press
as:
M.S.
Khan,
et
al.,
J.
Ind.
Eng.
Chem.
(2016),
http://dx.doi.org/10.1016/j.jiec.2015.12.011
Fig.
4.
(A)
Third
day
(B)
sixth
day
(C)
twelfth
day-
of
MALDI-MS
analysis
on
(a)
control
cells
of
P.
aeruginosa
(b)
pure
blood
sample
for
reference
of
blood
peak
(c)
Wound
treated
with
Nd-YAG
(1064
nm)
laser
only
(d)
wounds
treated
with
Nd-YAG
(1064
nm)
laser
and
Au
NRs.
M.S.
Khan
et
al.
/
Journal
of
Industrial
and
Engineering
Chemistry
xxx
(2016)
xxx–xxx
7
G
Model
JIEC-2750;
No.
of
Pages
10
Please
cite
this
article
in
press
as:
M.S.
Khan,
et
al.,
J.
Ind.
Eng.
Chem.
(2016),
http://dx.doi.org/10.1016/j.jiec.2015.12.011
shows
decreased
in
size
and
better
healing
result.
The
best
result
can
be
seen
in
W3
where
wound
size
almost
finished
and
speed-up
the
hair
growth
and
follicular
regeneration.
We
changed
the
location
of
wounds
on
mice
dorsal
surface
and
gave
the
same
treatment
in
similar
conditions
to
verify
whether
changes
in
position
of
wounds
have
different
result.
We
found
our
laser
treatment
with
Au
NRs
therapy
has
same
effect
on
changing
the
location
of
wound
as
shown
in
Fig.
6(d,
e
and
f).
Wounds
which
Fig.
5.
(a)
Three
fresh
wounds
namely
as
W1,
W2,
and
W3
on
mice
model.
(b)
P.
aeruginosa
infection
on
W1,
W2
and
W3
wounds.
(c)
After
12
days
of
the
treatment
on
W1,
W2
and
W3.
(d),
(e)
and
(f)
show
changed
in
the
treatment
and
location
of
wounds
on
the
dorsal
surface
of
animal
model
by
using
Au
NRs
and
Nd-YAG
laser.
Fig.
6.
Photographs
of
control
and
treated
wound
tissue
section
stained
by
Haemotoxolin
and
Eosin
taken
after
3rd
and
12th
day
exposure
by
Au
NRs
and
Nd-YAG
laser
(10
objective,
scale
bar
=
100
m
m).
(For
interpretation
of
the
references
to
color
in
this
figure
legend,
the
reader
is
referred
to
the
web
version
of
this
article.)
M.S.
Khan
et
al.
/
Journal
of
Industrial
and
Engineering
Chemistry
xxx
(2016)
xxx–xxx
8
G
Model
JIEC-2750;
No.
of
Pages
10
Please
cite
this
article
in
press
as:
M.S.
Khan,
et
al.,
J.
Ind.
Eng.
Chem.
(2016),
http://dx.doi.org/10.1016/j.jiec.2015.12.011
were
treated
by
Nd-YAG
laser
heal
first
as
compared
to
other
wounds.
Histological
studies
Histological
examination
provides
profound
studies
in
under-
standing
the
effect
of
this
treatment
on
the
surface
of
wound.
Microtomy
of
upper
part
of
tissue
by
this
treatment
on
the
three
wounds
was
performed
very
carefully.
In
Fig.
7
the
upper
panel
shows
photos
of
wound
taken
on
starting
the
infection
after
3rd
day.
Photos
of
lower
panel
was
taken
on
12th
day
after
the
treatment,
the
yellow
arrows
show
the
damaged
tissues
caused
by
the
pathogen
on
control
wound.
Adjacent
photo
is
belonging
to
wound
which
was
treated
by
only
Nd-YAG
laser,
the
black
part
of
the
tissue
(inside
yellow
rectangle)
pointed
out
the
destruction
caused
by
laser.
The
results
of
this
treatment
is
shown
in
the
last
tissue
section
which
retained
its
architecture
after
treatment
by
Au
NRs-mediated
Nd-YAG
laser
in
which
no
disorder
can
be
observed
clearly.
According
to
Lyon
et
al,
study
was
conducted
on
effect
of
laser
on
wounds
found
that
the
tensile
strength
of
tissues
increased
and
collagen
content
also
enhanced
[45].
Similar
positive
results
were
also
found
by
Redy
et
al.
in
which
they
found
that
by
using
the
laser
on
wound
surface
augmented
the
soluble
neutral
salt
and
insoluble
collagen
as
compared
to
control,
which
helps
in
fast
healing
[46].
Another
study
was
performed
by
Dadpay
et
al.
found
increased
in
concentration
of
fibroblast,
blood
vessel
endothelium
and
decreased
macrophages
concentration
which
are
the
signs
of
fast
healing
[19].
Fig.
8
shows
the
final
three
wound’s
size-area
percentage
taken
during
the
whole
treatment.
For
measuring
the
size
of
wounds
the
graph
paper
was
used
and
marked
on
the
paper
the
size
of
W1,
W2
and
W3
every
day.
For
W1,
the
light
black
lines
are
depicting
the
size
of
wound,
which
was
reduced
to
80%
of
its
original
size
in
19
days.
W2
which
is
represented
by
the
dotted
lines
was
treated
by
the
Nd-YAG
laser;
it
showed
80%
healing
in
14
days
than
the
W1.
The
dark
lines
represent
the
W3,
which
was
treated
by
Au
NRs-mediated
Nd-YAG
laser
treatment
and
showed
tremendous
improvement
in
wound
healing,
80%
of
wound
healed
in
just
9
days.
It
showed
about
twice
faster
healing
rate
than
the
W1
control
without
any
laser
treatment.
The
stages
of
the
wound
healing
as
homeostasis,
inflammation,
proliferation
and
remodeling
stages
are
depicted
and
tabulated
in
Fig.
9.
The
present
study
can
significantly
help
in
tracking
the
wound
healing
process,
determining
the
bacterial
response
to
antibiotics,
comparing
healing
rate
among
different
patients.
Thereby,
adjusting
the
antibiotic
doses
and
facilitating
the
wound
healing
process
by
Au
NRs-mediated
laser
therapy.
This
study
also
helps
in
curtailing
the
opportunistic
infections
on
skin
wounds
by
an
accident
from
cut
or
burn.
Fig.
7.
The
correlation
of
wound
size
area
by
treatment
of
laser
therapy.
(a)
W1-
Control
wound
(b)
W2-Nd-YAG
laser
treated
(c)
W3-Treated
by
Au
NR’s
and
Nd-
YAG
laser.
Fig.
8.
Schematic
representation
of
the
wound
healing
process.
M.S.
Khan
et
al.
/
Journal
of
Industrial
and
Engineering
Chemistry
xxx
(2016)
xxx–xxx
9
G
Model
JIEC-2750;
No.
of
Pages
10
Please
cite
this
article
in
press
as:
M.S.
Khan,
et
al.,
J.
Ind.
Eng.
Chem.
(2016),
http://dx.doi.org/10.1016/j.jiec.2015.12.011
Conclusions
An
efficient
cure
strategy
for
medical
application
is
develop
by
using
Au
NRs
based
Nd-YAG
laser
therapy
to
facilitate
the
rapid
wound
healing
and
reducing
the
chance
for
drug
resistance
from
pathogenic
infections
present
on
dorsal
surface.
After
treatment
by
Au
NRs
and
Nd-YAG
laser,
the
bacterial
counts
were
calculated
by
colony
forming
units
to
evaluate
the
efficiency
of
this
new
treatment
method.
The
disappearance
of
the
pathogenic
bacterial
peaks
in
the
MALDI-MS
can
be
the
evidence
of
healing
of
the
wound.
Our
results
have
proved
that
the
wound
which
were
treated
by
Au
NRs
with
Nd-
YAG
laser
show
significant
results
in
fast
healing,
increased
follicular
regeneration
and
hair
growth
than
the
wound
which
was
not
treated
by
Nd-YAG
laser
alone
or
Au
NRs
assisted
Nd-YAG
laser
treatment.
This
therapy
method
is
a
new
treatment
platform
which
can
be
widely
applied
in
many
fields
such
as
burn
skin
infections,
tumor
targeting,
deep
infection
in
organs,
infection
inside
bones,
control-
ling
drug
resistant
bacteria
and
virus
etc
which
can
be
treated
rapidly
without
screening
of
pathogens
tests.
Acknowledgement
All
authors
thank
the
only
source
of
financial
support
from
the
ministry
of
science
and
technology
(MOST)
of
Taiwan
to
carry
out
this
project
by
the
grants
of
NSC
101-2113-M-110-001-MY3
and
104-2113-M-110-003-MY3.
Judy
Gopal
thanks
the
KU
research
professor
program,
Konkuk
University.
References
[1]
R.F.
Diegelmann,
M.C.
Evans,
Front.
Biosci.
9
(2004)
283.
[2]
M.C.
Robson,
T.A.
Mustoe,
T.K.
Hunt,
Am.
J.
Surg.
176
(1998)
80.
[3]
A.J.
Makowski,
E.D.
Jansen,
J.M.
Davidson,
A.M.
Jansen,
Lasers
Surg.
Med.
44
(2012)
233.
[4]
A.R.
Medrado,
L.S.
Pugliese,
S.R.
Reis,
Z.A.
Andrade,
Lasers
Surg.
Med.
32
(2003)
239.
[5]
F.
Festini,
R.
Buzzetti,
C.
Bassi,
C.
Braggio,
D.
Salvatore,
G.
Taccetti,
G.
Mastella,
J.
Hosp.
Infect.
64
(2006)
1.
[6]
J.B.
Lyczak,
C.L.
Cannon,
G.B.
Pier,
Microbes
Infect.
2
(2006)
1051.
[7]
R.S.
Norman,
J.W.
Stone,
A.
Gole,
C.J.
Murphy,
T.L.
Sabo-Attwood,
Nano
Lett.
8
(2008)
302.
[8]
S.
Galdiero,
A.
Falanga,
M.
Vitiello,
M.
Cantisani,
V.
Marra,
M.
Galdiero,
Molecules
16
(2011)
8894.
[9]
F.
Ratto,
P.
Matteini,
N.
Tiwari,
F.
Rossi,
S.K.
Kulkarni,
L.
Menabuoni,
R.
Pini,
Nanomed.:
Nanotechnol.
Biol.
Med.
5
(2009)
143.
[10]
E.
Yasun,
B.
Gulbakan,
I.
Ocsoy,
Q.
Yuan,
M.I.
Shukoor,
C.
Li,
W.
Tan,
Am.
Chem.
Soc.
14
(2012)
6008.
[11]
N.K.
Palanisamy,
N.
Ferina,
A.N.
Amirulhusni,
Z.M.
Zain,
L.J.
Ping,
R.
Durairaj,
J.
Hussaini,
J.
Nanobiotechnol.
12
(2014)
2.
[12]
A.M.
Gobin,
L.L.
West,
O.N.
Patrick,
D.M.
Watkins,
N.J.
Halas,
R.A.
Drezek,
Lasers
Surg.
Med.
37
(2005)
123.
[13]
P.P.
Joshi,
Y.S.J.
Joon,
K.V.
Sokolov,
W.G.
Hardin,
S.
Emelianov,
Bioconjug.
Chem.
24
(2013)
878.
[14]
Q.
Zhang,
V.M.
Hitchins,
A.M.
Schrand,
S.M.
Hussain,
P.L.
Goering,
Nanotoxicology
55
(2011)
284.
[15]
C.
Gui,
D.X.
Cui,
Cancer
Biol.
Med.
9
(2012)
221.
[16]
X.
Huang,
H.E.
El-Sayed,
W.
Qian,
M.A.E.
Sayed,
J.
Am.
Chem.
Soc.
128
(2006)
2115.
[17]
C.
Loo,
A.
Lowery,
N.
Halas,
J.
West,
R.
Drezek,
NanoLett.
5
(2005)
709.
[18]
G.V.
Maltzahn,
J.H.
Park,
K.Y.
Lin,
N.
Singh,
C.S.R.
Mesters,
W.E.
Berdel,
E.
Ruoslahti,
M.J.
Sailor,
S.N.
Bhatia,
Nat.
Mater.
109
(2011)
545.
[19]
M.
Dadpay,
Z.
Sharifian,
B.
Bayat,
M.
Bayat,
A.
Dabbagh,
J.
Photochem.
Photobiol.,
B:
Biol.
111
(2012)
1.
[20]
F.A.
Al-Watban,
X.Y.
Zhang,
J
Clin.
Laser
Med.
Surg.
22
(2004)
15.
[21]
J.T.
Hashmi,
B.
Kurup,
Y.Y.
Huang,
S.K.
Sharma,
D.L.D.
Taboada,
J.D.
Carroll,
M.R.
Hamblin,
Lasers
Surg.
Med.
42
(2010)
450.
[22]
P.S.
Zolfaghari,
S.
Packer,
M.
Singer,
S.P.
Nair,
J.
Bennett,
C.
Street,
M.
Wilson,
BMC
Microbiol.
9
(2009)
27.
[23]
N.
Kojic,
E.M.
Pritchard,
H.
Tao,
M.A.
Brenckle,
J.P.
Mondia,
P.
Panilaitis,
F.
Omenetto,
D.L.
Kaplan,
Adv.
Funct.
Mater.
12
(2012)
3793.
[24]
M.
Karas,
F.
Hillenkamp,
Anal.
Chem.
60
(1988)
2299.
[25]
T.
Krishnamurthy,
U.
Rajamani,
P.L.
Ross,
Rapid
Commun.
Mass
Spectrom.
10
(1996)
883.
[26]
M.A.
El-Sayed,
Acc.
Chem.
Res.
34
(2001)
257.
[27]
M.
Fournelle,
W.
Bost,
I.H.
Tarner,
T.
Lehmberg,
E.
Weiß,
R.
Lemor,
R.
Dinser,
Nanomed.:
Nanotechnol.
Biol.
Med.
8
(2012)
346.
[28]
M.L.
Bhaisare,
H.N.
Abdelhamid,
B.S.
Wua,
H.F.
Wu,
J.
Mater.
Chem.
B
2
(2014)
4671.
[29]
B.S.
Wu,
H.N.
Abdelhamid,
H.F.
Wu,
RSC
Adv.
4
(2014)
3722.
[30]
J.C.
Liu,
W.J.
Chen,
C.W.
Li,
K.K.
Mong,
P.J.
Tsai,
T.L.
Tsai,
Y.C.
Lee,
Y.C.
Chen,
Analyst
134
(2009)
2087.
[31]
G.
Judy,
N.
Hasan,
M.
Manikandan,
H.F.
Wu,
Sci.
Rep.
3
(2013)
1260.
[32]
G.
Judy,
N.
Hasan,
H.F.
Wu,
Biosens.
Bioelectron.
39
(2013)
57.
[33]
K.R.
Dilip,
N.G.
Brien,
L.
Britta,
A.
Gunvor,
J.G.
William,
J.
Mass
Spectrom.
39
(2004)
289.
[34]
T.S.
Michael,
T.
Hiroki,
J.O.
Andre
´,
Infect.
Immun.
79
(2011)
459.
[35]
R.W.
Bohannon,
Phys.
Ther.
63
(1983)
1622.
[36]
M.R.
Hamblin,
Mechanism
of
low
level
light
therapy.
hwww.photobiology.info.
hamblin.html
www.mgh.harvard.edu/wellman/people/mhamblin.aspi.
[37]
R.
Duan,
T.C.Y.
Li,
H.
Guo,
L.B.
Yao,
Lasers
Surg.
Med.
29
(2001)
174.
[38]
V.K.
Poon,
L.
Huang,
A.
Burd,
J.
Photochem.
Photobiol.,
B:
Biol.
81
(2005)
1.
[39]
J.T.
Hashmi,
Y.Y.
Huang,
S.K.
Shama,
K.
Taboada,
J.D.
Carrol,
M.R.
Hamblin,
Lasers
Surg.
Med.
42
(2010)
450.
[40]
Z.Q.
Lin,
T.
Kondo,
Y.
Ishida,
T.
Takayasu,
N.
Mukaida,
J.
Leukocyte
Biol.
73
(2003)
713.
[41]
N.
Kipshidze,
V.
Nikolaychik,
M.H.
Keelan,
L.R.
Shankar,
A.
Khanna,
R.
Kornowski,
M.
Leon,
J.
Moses,
Lasers
Surg.
Med.
28
(2001)
355.
[42]
A.
Khanna,
L.R.
Shankar,
M.H.
Keelan,
R.
Kornowski,
M.
Leon,
K.J.N.
Moses,
Cardiovasc.
Radiat.
Med.
1
(1999)
265.
[43]
L.L.
Narayana,
J.
Gopal,
H.F.
Wu,
Analyst
137
(2012)
3372.
[44]
M.S.
Khan,
G.
Gedda,
H.F.
Wu,
J.
Gopal,
Anal.
Methods
6
(2014)
5304.
[45]
R.F.
Lyons,
R.P.
Abergel,
R.A.
White,
R.M.
Dwyer,
J.C.
Costel,
Uitto,
J.
Ann.
Plast.
Surg.
(1987)
1847.
[46]
R.G.
Kesava,
Lasers
Surg.
Med.
33
(2003)
344.
M.S.
Khan
et
al.
/
Journal
of
Industrial
and
Engineering
Chemistry
xxx
(2016)
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Please
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