Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect...
-
Upload
astri-ggamjong-xiao-lu -
Category
Documents
-
view
222 -
download
0
Transcript of Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect...
-
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
1/29
International Journal of Nanomedicine Dovepressopen access to scientific and medical research
Open Access Full Text Article O r i g i n a l R e s e a r c h
Synthesis of PLGA nanoparticles of
tea polyphenols and their strong invivo protective eect againstchemically induced !A damage
This article "as pu#lished in the follo"ing ove Press $ournal%
&nternational 'ournal of
!anomedicine () April *+()
!um#er of times this article has #een vie"ed
Amit ,umar Srivastava(
Priyan-a .hatnagar*
/adhuli-a Singh(
San$ay /ishra(
Pradeep ,umar*
0ogesh"er Shu-la(
,ailash 1hand Gupta(2*
(Proteomics La#oratory2 &ndian&nstitute of Toxicology Research
31S&R42 Luc-no"2 &ndia5 *!ucleic AcidResearch La#oratory2 &nstitute ofGenomics and &ntegrative .iology31S&R42 elhi 6niversity 1ampus2 &ndia
Abstract: In
spite of proficient
results of severalphytochemicals in
preclinical
settings, the
conversion rate
from bench to
bedside is not very
encouraging.
Many reasons are
attributed to this
limited success,
including
inefficient
systemic delivery
and bioavailability
under in vivo
conditions. To
achieve improved
efficacy,
polyphenolic
constituents of
black (theaflavin
[TF] and green
(epigallocatechin!
"!gallate [#$%$]
tea in poly(lactide!
co!glycolide
nanoparticles
(&'$!)&s *ere
entrapped *ith
entrapment efficacy of 7+- and /-,
respectively. Further, their preventive potential
against 0,+!dimethylben1anthracene (2M3!induced 2) damage in mouse skin using
2) alkaline un*inding assay *as evaluated.
&retreatment (topically of mouse skin *ith
either TF or #$%$ (+44 g5mouse doses
e6hibits protection of 78."7- and ."-,
respectively, against 2M3!induced 2)
damage. 9o*ever, pretreatment *ith TF!loaded
&'$!)&s protects against 2) damage
/7.7+- by +54th dose of bulk, 0+.0:- by
+5+4th dose of bulk, and 0.7/- by +58th dose
of bulk. ;imilarly, 8+.- (+54th of bulk,
80./"- (+5+4th of bulk, and /".+7- (+58th of
bulk prevention *as noted using #$%$!loaded
&'$!)& doses. These results sho*ed that tea
polyphenol!loaded &'$!)&s have 7"4!fold
dose!advantage than bulk TF or #$%$ doses.
dditionally, TF! or #$%$!loaded &'$!)&s
sho*ed significant potential for induction of
2) repair genes (XRCC1, XRCC3, and
ERCC3 and suppression of 2) damage
responsive genes (p53, p21, MDM2,
GADD45, and COX-2 as compared *ith
respective bulk TF or #$%$ doses. Taken
together, TF! or #$%$!loaded &'$!)&s
sho*ed a superior ability to prevent 2M3!
induced 2) damage at much lo*er
concentrations, thus opening a ne* dimension
in chemoprevention research.
Keywords: 2) alkaline un*indingassay, mouse skin, 2) repair
1orrespon
dence%
0ogesh"er
Shu-laProteomic
s
La#oratory2 &ndian &nstitute
of Toxicology Research
31S&R42
PO .ox 8+2 /G/arg2 Luc-no"6P **9++(2 &ndia
Tel +:( ;**
*:9)8*84 has gained
importance for the encapsulation of a *ide variety of drugs as it
is biodegradable, biocompatible, capable of controlling the
release of the incorporated entity, and less to6ic.+/,+0
The choice
of &'$ *as also supported by the fact that it can be used to
form stable nanoparticles ()&s, and has already been approved
for use in humans by the B; Food and 2rug dministration.+
Thus, it has been chosen as the polymer scaffold to fabricate
the )&s used in the present study.
Mammalian skin forms a
hurdle bet*een the host and
surrounding
environments.+:,4
9o*ever,
it is also a ma=or route of
entry for several to6ic agents
and a potential target of
environmental ha1ards, *hich
can cause several skin!related
abnormalities including skin
cancer.+
#pidemiological
stud!ies suggest that almost
one!third of all ne* cancers
diagnosed annually
*orld*ide originate from
skin.
;everal studies sho*
an association bet*een
different human cancers and
lifestyle5dietary habitsA
additionally, detailed studiesof muta!tional events in
human cancers have provided
evidence for a direct
involvement of
environmental
mutagens5carcinogens in the
progression of different types
of cancer.",7
In the present study, a
&'$!based drug
development strategy *asfollo*ed to encapsulate TF
and #$%$. The authors
envisioned that )&s!
mediated delivery of
bioactive catechins from
black tea (TF and green tea
(#$%$ could be useful in
enhancing their protective
potential against 0,+!
dimethylben1[a]anthracene
(2M3!induced 2)damage in mouse skin
tissue.
&ateria's andmethods1hemicals&'$ 84>84 (molecular *eight
[MC] 74D08 k2a, polyvinyl
alcohol (MC "4 k2a, #$%$,
TF, and 2M3 *ere purchased
from ;igma!ldrich (;t 'ouis,
M?, B;. 9ydro6ylapatite
and ),)!dimethylformamide
*ere purchased from ;isco
Eesearch 'aboratory (Mumbai,
India. ll other chemicals
used *ere of analytical grade
purity and procured locally.
Formulationof TFE or=G1GEloadedPLGAE!PsTF! or #$%$!loaded &'$!
)&s *ere prepared using the
standard solvent evaporation
method,8
*ith minor
modifications. 3riefly, a 4.8m' a
-
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
5/29
1452submit your manuscript @ """?dovepress?com
&nternational 'ournal of!anomedicine *+()%8
Dovepress
http://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.com -
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
6/29
Dovepress Protection against !A damage #y nanotied tea polyphenols
by *ashing *ith *ater, and finally, the )&s *ere free1e!dried
*ith a Bnivapo +849 Bnicryo free1e dryer (Bnie
-
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
7/29
The mean particle si1e and distribution, as *ell as the
=valuation of TF or =G1Grelease
1eta potential of the TF! or #$%$!loaded &'$!)&s *as from !Psdetermined by a dynamic light scattering techni
-
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
8/29
-
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
9/29
1454submit your manuscript @ """?dovepress?com
&nternational 'ournal of!anomedicine *+()%8
Dovepress
http://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.com -
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
10/29
Dovepress Protection against !A damage #y nanotied tea polyphenols
,ab'e 1 1haracteristics of TFJ=G1G loadedE!Ps
"anopartic'es - ie'd #i)e nm eta potentia' rug 3oading ncapsu'ation
+I m - 6ciency -
=G1GEPLGA :9?:+ (*
-
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
11/29
9igure 1 Particle sie distri#ution of epigallocatechinE)Egallate 3A4 andtheaMavin4nanoparticles? 3
"ote: Nalues are represented as meanstandard deviation?
&nternational 'ournal of !anomedicine *+()%8 submit your manuscript @ """?dovepress?com 1455
http://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.com -
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
12/29
Srivastava et al Dovepress
having all the properties re
-
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
13/29
145$submit your manuscript @ """?dovepress?com
&nternational 'ournal of!anomedicine *+()%8
Dove press
http://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.com -
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
14/29
Dovepress Protection against !A damage #y nanotied tea polyphenols
A 100 EGCG PLGA NPs90
80
release 70
60
50
%d
rug
40
30
20
100
0 1 2 3 4 5 6 7 8 9 10
Numer !" days
B #0 $% PLGA NPs80
70
release
60
&0
%d
rug
40
30
20
'0
0
0 1 2 3 4 5 6 7 8 9 10
Numer !" days
9igure % lnEvitro releaseA4and TFprole34PLGAE!Ps?of =G1G 3
"ote: Nalues are represented as meanstandard deviation?
Abbre7iations: =G1G2epigallocatechinE)Egallate5 PLGAE!P2 poly3lactideEcoEglycolide4 nan
time interval of 7 hours follo*ing the doses of TF or #$%$. ;ignificant
protection (78."7- *as observed at the dose of +44 g5mouse of bulk TF
in 7 hours (Figure 8, *hile TF!loaded &'$!)&s at doses 8, +4, and 4g5mouse provided protection of /7.74-, 0+.+:-, and 0.7/-,
respectively, *hich clearly sho*s about a "4!fold dose!advantage over
the bulk counterpart. ;imilarly,
*ith bulk #$%$ (+44 g5
mouse, .- protection *as
observed, *hile #$%$!loaded
&'$!)&s at 8, +4, and 4
g5mouse dose provided
8+.-, 80./"-, an
protection, res
sho*ing about a "!f
advantage over bulk
9o*ever, &'$!lo
($roup KI and #$%$
25
rea(s
20
s
trand
15
!"
10
Numer
5
0
II
9igure 4 Reduction in /.AEinduced strand #rea-s#y #ul- and poly3lactideEcoEglycolide4Eloaded teapolyphenols?
"otes: ata sho"n is the meanstandard deviationof all the animals5
indicatesP,+?+;5Qindicatessignifigniccantrof nanotied teapolyphenol over its#ul-2 P,+?+;?
Abbre7iation:/.A2
-
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
15/29
&nternational 'ournal of !anomedicine *+()%8submit your manuscript @
"""?dovepress?com 145
Dove press
http://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.com -
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
16/29
Srivastava et al Dovepress
%
re*enti!n
80
60
4
0
20
0
III IV V VI VII
VIII IX X
Gr!us
9igure 5 Percentage 34 prevention in
-
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
17/29
I
!ld+,ange
2
1
.
1
.
0.
0
.
0
MDM2
-
#*
I II
III
IV
V
VI
VII
VIII
!
ld+,ange
2
.
1
.
1
0
.
0
p21
-
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
18/29
* * #*#*
#*
-
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
19/29
I II III IV V VIVIIVIII
!ld+,ange
2
1
.
COX-2
#* #* #*
#*
I II III IV V VI VIIVIII
9igure $ =ect of
#ul- and PLGAE
loaded tea
polyphenols on the
transcription of !A
d"as uantied #y
densitometryIE
actinand? expressedas a fold dierence
against
"otes: atasho"nis the result of threedierentexperimentsP,+?+;incomparisonto"ithGroup&&5Qsimindilcartesresultsignic?Teect ofnanotied teapolyphenol over its#ul-2 P,+?+;? Lanes%&2 1ontrol5 &&2 /.A3;* gJmouse45 &&&2TF 3(++ gJmouse4+/.A5 &N2 TFE
loaded PLGAE!Ps 3;gJmouse4
+
/.A5N2TFEloadedPLGAE!Ps
3*
+
Ab
-
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
20/29
145;submit your manuscript @ """?dovepress?com
&nternational 'ournal of!anomedicine *+()%8
http://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.com -
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
21/29
Dovepress Protection against !A damage #y nanotied tea polyphenols
!ld+,ange
'.
'.2
0./
0
.
0I
!ld+,ange
0./
0
.
0I
II III
IV V
VI VII
VIII
XR
!ld+,ange
'. XRCC3
1-1
-1 -1'.2
1
0./
0.
0
I II III IV V VI VII VIII
XRCC3
a historical perspective. Can'e (ett.+::0A++7(+D>"+8D"+0.
5.McJay 2', 3lumberg 3. The role of tea in human health> an update. )
Am C#ll *!t. 44A+(+>+D+".
6.'eone M, hai 2, ;areth ;, Jitada ;, Eeed %, &ellecchia M. %ancer pre!
vention by tea polyphenols is linked to their direct Inhibition of antiapop!totic 3cl!!family proteins. Can'e Res. 44"A/"(">++D++.
7.hmad ), Mukhtar 9. $reen tea polyphenols and cancer> biological
mechanisms and practical implications.*!t Re%. +:::A80(">0D".
8.Hang %;, Maliaka &, Meng K. Inhibition of carcinogenesis by tea. Ann!Re% P+ama'#l T#i'#l. 44A7(+>8D87.
9.%houdhury ;E, 3alasubramanian ;, %he* H%, 9an 3, Mar8D"+.
+. &rasad ;, Jaur , Eoy &, Jalra ), ;hukla H. Theaflavins induce $5M
arrest by modulating e6pression of p+*af+5cip+, cdc8% and cyclin 3
in human prostate carcinoma &%!" cells. (ie 0'i. 440A+(+0D
+>+""D+""+.
+". ;iddi nanocarriers shape up for long life. *at
*an#te'+n#l. 440A(7>4"D47.
+8. 2emming . 3ig challenges and nanosolutions. *an#te'+n#l#g$.
4++A(:>:44+.
+/. 9u ;, hang H. #ndostar!loaded $!&'$ nanoparticles> in vitro and
in vivo evaluation..nt ) *an#me&i'ine. 4+4A7>8>+4":D+47.
+0. Hallapu MM, $upta 3J, aggi M, %hauhan ;%. Fabrication of curcumin
encapsulated &'$ nanoparticles for improved therapeutic effects in
metastatic cancer cells.) C#ll#i& .ntea'e 0'i. 4+4A"8+(+>+:D:.
+. ain E. The manufacturing techni"0"D"8.
+. Eockley &F, Trieff ), Cagner r EF, Tyring ;J. )onsunlight risk factors
for malignant melanoma. &art I> chemical agents, physical conditions,
and occupation..nt ) Demat#l. +::7A""(/>":D74/.
. emal , ;iegel E, Card #, et al. %ancer statistics 44. CA Can'e)
Clin. 44A8(>0+D:/.
". Murray %', 'ope1 2. T+e Gl#al "!&en # Disease a C#mpe-+ensi%e Assessment # M#talit$ an& Disailit$ #m Diseases, .n!ies
an& Ris a't#s in 1667 an& P#e'te& t# 2727. %ambridge> 9arvard
Bniversity &ressA +::/.
7. 'yle 3, Mares!&erlman , Jlein 3#, Jlein E, $reger '. ;upplement
users differ from nonusers in demographic, lifestyle, dietary and health
characteristics.) *!t. +::A+(+>"88D"/.
8. #smaeili F, 9osseini!)asr M,
Ead!Malekshahi M, ;amadi ),
tyabi F, 2inarvand E.
&reparation and antibacterial
activity of rifampicin!loaded poly
lactide!co!glycolide
nanoparticles. *an#me&i'ine.
440A"(>+/+D+/0.
/. ;*ami , ggar*al , &athak ,
&atnaik ;, Jumar &, $upta J%.
Imida1olyl!I modified
nanoparticles for enhanced genedelivery. .nt ) P+am.
440A""8(+D>+4D+:.
0. Jhan T9, &rasad ', nuradha,
;ultana ;. ;oy isoflavones
inhibits the genoto6icity of
ben1o(apyrene in ;*iss albino
mice. 8!m Ep T#i'#l.
448A7(">+7:D+88.
. )igam ), &rasad ;, ;hukla H.
&reventive effects of lupeol on
2M3 induced 2) alkylation
damage in mouse skin. ##&
C+em T#i'#l. 440A78(++>""+D
""8.
:. rora , ;iddi+78:D+7//.
"4. $opferich . Mechanisms of
polymer degradation and erosion.
"i#mateials. +::/A+0(>+4"D
++7.
"+. ert M, 'i ;, $arreau 9. More
about degradation of '5$
derived matrices in a+8D/.
". llNmann #, $urny E, 2oelker #.2rug!loaded nanoparticles D
preparation methods and drug
targeting issues. ) P+am
"i#p+am. +::"A":(8>+0"D+:+.
"". 3arichello M, Morishita M,
Takayama J, )agai T.
#ncapsulation of hydrophilic and
lipophilic drugs in &'$
nanoparticles by the nano!
precipitation method. D!g De%
.n& P+am. +:::A8(7>70+D70/.
"7. $ovender T, ;tolnik ;, $arnett M%,
Illum ', 2avis ;;. &'$ nanopar!
ticles prepared by nanoprecipitation>
drug loading and release studies of a
*ater soluble drug. ) C#nt#l
Release. +:::A80(>+0+D+8.
"8. Feng ;, 9uang $. #ffects of
emulsifiers on the controlled
release of paclita6el (Ta6ol from
nanospheres of biodegradable
polymers. ) C#nt#l Release.
44+A0+(+>8"D/:.
"/. ;iddi proof of principle *ith
green tea polyphenol
epigallocatechin!"!gallate.
Can'e Res. 44:A/:(8>+0+D
+0+/.
"0. 9rel=ac I, a=c I, 'ah T, Filipi M.
#ffects of model
organophosphorous pesticideson 2) damage and
proliferation of 9ep$ cells.
En%i#n M#l M!tagen.
44A7:(8>"/4D"/0.
". ogelstein 3, 'ane 2, 'evine
. ;urfing the p8" net*ork.
*at!e. 444A74(/+4>"40D
"+4.
":. Cang ;, #l!2eiry C;. p0" or
p8" regulates human p8" tran!
scription to maintain cell cycle
checkpoints. Can'e Res.
44/A//(+7>/:D/::.
74. #l!2eiry C;, Tokino T,
elculescu #, et al. CF+ a
potential media!tor of p8"
tumor suppression. Cell.
+::"A08(7>+0D8.
7+. 9ollander M%, lamo I,
ackman , Cang M$, Mc3ride
?C, Fornace r. nalysis of
the mammalian gadd78 gene
and its response to 2)
damage. ) "i#l C+em.
+::"A/(">7"8D7":".
7. 'iu 9, Kiao , Hang H, et al.
%?K! e6pression is correlated
*ith #$F!%,
lymphangiogenesis and lymph
node metastasis in human
cervical cancer. Mi'#%as' Res.4++A(>+"+D+74.
7". Cang , Colf 2%, ;en 3, et al.
2imethylarsinic acid in drinking
*ater changed the morphology
of urinary bladder but not the
e6pression of 2) repair genes
of bladder transit ional
epithelium in F"77 rats. T#i'#l
Pat+#l. 44:A"0(7>78D7"0.
77. ;rivastava ;, erma M, 9enson
2#. 3iomarkers for early
detection of colon cancer.
Clini'al Can'e Res.
44+A0(8>+++D++/.
http://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.com -
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
27/29
&nternational 'ournal of !anomedicine *+()%8submit your manuscript @
"""?dovepress?com 14$1
Dove press
http://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.com -
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
28/29
-
8/10/2019 Synthesis of PLGA Nanoparticles of Tea Polyphenols and Their Strong in Vivo Protective Effect Against Chemically I
29/29
14$2submit your manuscript @ """?dovepress?com
&nternational 'ournal of!anomedicine *+()%8
Dovepress
http://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.comhttp://var/www/apps/conversion/tmp/scratch_4/www.dovepress.com