De oxi ying An i umo al D ugs ia Nanoconjuga ion: The
Case o Gold Nanopa icles and Cispla in
Joan Comenge
1,2.
, Ca men So elo
3.
, F ancisco Rome o
4
, Osca Gallego
5
, Agus ı
´Ba nadas
5
, Toma
´s
Ga cı
´a-Caballe o Pa ada
6
, Fe nando Domı
´nguez
3
*,Vı
´c o F. Pun es
1,7
*
1Ca alan Ins i u e o Nano echnology (ICN), Uni e si a Au o
`noma de Ba celona (UAB), Bella e a, Ba celona, Spain, 2In e na ional Ibe ian Nano echnology Labo a o y
(INL), B aga, Po ugal, 3Depa men o Physiology, Facul y o Medicine, San iago de Compos ela Uni e si y, San iago de Compos ela, Spain, 4Molecula Science Ins i u e,
Uni e si y o Valencia, Pa e na, Spain, 5Oncology Depa men , San Pau Hospi al, Ba celona, Spain, 6Depa men o Mo phological Sciences, School o Medicine-
Uni e si y, Clinical Hospi al, San iago de Compos ela Uni e si y, San iago de Compos ela, Spain, 7Ins i ucio
´Ca alana de Rece ca i Es udis A anc¸a s (ICREA), Ba celona,
Spain
Abs ac
Nanopa icles (NPs) ha e eme ged as a po en ial ool o imp o e cance ea men . Among he p oposed uses in imaging
and he apy, hei use as a d ug deli e y sca old has been ex ensi ely highligh ed. Howe e , he e a e s ill some
con o e sial poin s which need a deepe unde s anding be o e clinical applica ion can occu . He e he use o gold
nanopa icles (AuNPs) o de oxi y he an i umo al agen cispla in, linked o a nanopa icle ia a pH-sensi i e coo dina ion
bond o endosomal elease, is p esen ed. The NP conjuga e design has impo an e ec s on pha macokine ics, conjuga e
e olu ion and biodis ibu ion and esul s in an absence o obse ed oxici y. Besides, AuNPs p esen unique oppo uni ies
as d ug deli e y sca olds due o hei size and su ace unabili y. He e we show ha cispla in-induced oxici y is clea ly
educed wi hou a ec ing he he apeu ic bene i s in mice models. The NPs no only ac as ca ie s, bu also p o ec he
d ug om deac i a ion by plasma p o eins un il conjuga es a e in e nalized in cells and cispla in is eleased. Addi ionally,
he possibili y o ack he d ug (P ) and ehicle (Au) sepa a ely as a unc ion o o gan and ime enables a be e
unde s anding o how nanoca ie s a e p ocessed by he o ganism.
Ci a ion: Comenge J, So elo C, Rome o F, Gallego O, Ba nadas A, e al. (2012) De oxi ying An i umo al D ugs ia Nanoconjuga ion: The Case o Gold
Nanopa icles and Cispla in. PLoS ONE 7(10): e47562. doi:10.1371/jou nal.pone.0047562
Edi o : Elena A. Rozhko a, A gonne Na ional Labo a o y, Uni ed S a es o Ame ica
Recei ed July 3, 2012; Accep ed Sep embe 18, 2012; Published Oc obe 17, 2012
Copy igh : ß2012 Comenge e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s
un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
Funding: The au ho s acknowledge inancial suppo om he g an s ‘‘Plan Nacional’’ (MAT2009-14734-C02-01 and MAT2009-14734-C02-02) and NANOBIOMED-
CONSOLIDER (CSD2006-00012) om he Spanish Go e nmen . Also g an s VALTEC09-2-0085, VALTEC09-2-0089, and 2009-SGR-776 om he Ca alan Go e nmen .
The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip .
Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis .
* E-mail: e nando.domingue[email p o ec ed] (FD); [email p o ec ed] (VP)
.These au ho s con ibu ed equally o his wo k.
In oduc ion
1. NanoOncology
The global dea h a e om cance has declined only ma ginally
o e he pas se e al decades, in con as o d ama ic e e sals in
dea h a es om hea disease, s oke, and in ec ious disease o e
he same ime pe iod [1]. In his con ex , nano echnology eme ges
as a ‘‘dis up i e echnology’’ wi h a g ea po en ial o con ibu e o
imp o e cance ea men by gene a ing new diagnos ic and
he apeu ic p oduc s [2–5]. Thus, nano echnology has been
p oposed o enable esea che s o combine a se ies o ad ances;
c ea ing nanosized pa icles ha may con ain d ugs designed o
kill umo s oge he wi h a ge ing compounds designed o home
in on malignancies [6–8], and imaging agen s designed o ligh up
e en he ea lies s age o cance o moni o i s ea men [9]. Thus,
unc ionalized nanopa icles could deli e mul iple he apeu ic
agen s o umo si es in o de o simul aneously a ack mul iple
poin s in he pa hways in ol ed in cance . Howe e , despi e he
ple ho a o nanopa icles (NP), o ganic o ino ganic, and
conjuga ed chemo he apeu ic agen s which ha e shown p omising
esul s in i o, he p ecise beha io o hese conjuga es in i o is s ill
a he unknown, wi h con o e sy abou dispa i ies be ween he in
i o and in i o esul s o esul s om di e en labo a o ies. The e
a e indica ions ha small modi ica ions o he na u e o he
conjuga e ha e a s ong in luence on conjuga e in e ac ions [10],
p o ein co ona o ma ion [11,12], agg ega ion [13], deg ada ion
[14], and consequen ly biological beha io du ing he ull li e cycle
o he conjuga e inside he body [15]. By a aching he d ug o he
NP, i s physicochemical a e is modi ied. Thus, nanoca ie s can
s ongly con ibu e o modi ica ions in pha macokine ics (Table 1)
and biodis ibu ion, by leading he d ug h ough di e en
pa hways depending on he physicochemical p ope ies o he
nanoca ie (e.g., size and su ace cha ge), which is especially
appealing in he case o e y oxic d ugs [16,17]. Inside he body,
po es smalle han 1 nm ha e been epo ed in he igh junc ions
on ce ain con inuous capilla ies (including he cen al ne ous
sys em, i.e., blood-b ain ba ie , placen a and es is ba ie ) while
con inuous capilla ies (muscle, lung, skin) ha e po es o 6 nm [18].
Fenes a ed capilla ies (kidney, in es ine, some endoc ine and
exoc ine glands) ha e po es up o 50–60 nm, usually closed by a
diaph agm [18]. Finally, discon inuous capilla ies (li e , spleen,
bone ma ow) ha e po es be ween 100–1000 nm, which allow he
passage o mac omolecules be ween plasma and in e s i ium [18].
Thus, small molecules (below 6 nm, he majo i y o d ugs) leak in
PLOS ONE | www.plosone.o g 1 Oc obe 2012 | Volume 7 | Issue 10 | e47562
and ou om he blood essels and a e apidly (in minu es) clea ed
om blood ia he kidneys [19] while he passi e anspo o
mac omolecules h ough hese po ous is negligible. Thus a NP
sized be ween 6–40 nm may ollow p o ein pa hs o inally
accumula e in o gans o he mononuclea phagocy e sys em,
especially he li e and spleen, as do p o eins and p o ein
agg ega es [20], while la ge sizes o NP a e easily ecognized by
he immune sys em and also end up in li e and spleen bu wi hin
a sho e ime [21]. I is wo h no ing he e ha blood essel
pe meabili y changes in diseases such as in lamma ion and cance
[22]. In cance , he apid g ow h o umo esul s in leaky essels.
These enes a ed essels allow mac omolecules and NPs o
pe mea e h ough he umo . In addi ion, he nanopa icles a e
e ained due o he lack o a unc ional lympha ic sys em. This
e ec (Enhanced Pe meabili y and Re en ion e ec , EPR) is widely
epo ed in he li e a u e [23,24] and has been exploi ed o
passi ely accumula e nanoca ie s in umo s [4]. O he desc ibed
pa hways a e mo e complex and include he use o mig a ing
mac ophages as anspo e s o NPs and d ugs [25]. In all cases,
su ace modi ica ions allow he modi ica ion o his size-dependen
a e, o example, by making small NPs ecognizable by he
immune sys em [26] o shielding he la ge ones by means o
chemical modi ica ion such as pegyla ion [27].
Beyond oncology, small ino ganic NPs he size o a small
p o ein (5–30 nm) a e making hei way owa ds he clinic: AuNPs
a e used in cell imaging [28], a ge ed d ug deli e y [29,30], as
pho o he mal agen s o hype he mia [31], and in o he p oposed
diagnoses and he apies [32]. AgNPs display a biocidal e ec [33]
ha is cu en ly applied in comme cial p oduc s such as hospi al
equipmen and de ices. Magne ic NPs a e p esen in a ious
biomedical applica ions, e.g., he ea ly de ec ion o cance ,
diabe es, and a he oscle osis [34]. CeO
2
NPs a e being used in
biomedicine as an an ioxidan o ea diso de s caused by oxygen
adicals, such as e inal degene a ion [35] o ca diomyopa hy
[36]. Non-ino ganic nanoma e ials ha e also eached he clinics,
e.g., Doxil, which is a liposomal o mula ion (hund eds o
nanome e s in size, biocompa ible and biodeg adable) o doxo u-
bicin ha inc eases he solubili y o he ac i e ing edien and
modi ies he dosing by sus aining i o e ime.
2. The Case o Cispla in
Pla inum compounds (Figu e 1) a e a pa adigm in an icance
d ugs. Cispla in o cis-diamminedichlo opla inum(II),
[P Cl
2
(NH
3
)
2
], was o iginally syn hesized in 1845, bu no un il
1970 was i s an i umo ac i i y es ablished [37]. Today cispla in is
used o ea a ious ypes o cance s (i.e., non-small-cell lung
cance , o a ian cance , ge m cell umo s, os eosa comas, e c.),
wi h a cu e a e as high as 90% in es icula cance [38]. The
pla inum complex eac s in i o o o m adduc s wi h DNA, which
ul ima ely igge apop osis [39]. I has been p o en ha , a e
bo h passi e and ac i e cellula up ake, cispla in may eac wi h
he N
7
a om o pu ine bases in DNA [38]. Howe e , ch onic
cispla in usage esul s in esis ance ia se e al possible mechanisms
including inc eased in e ac ions wi h me allo hioneins and glu a-
hione, which deac i a e he d ug, as well as inc eased DNA epai
and/o cispla in e lux [40]. To coun e ac esis ance, which
lowe s he e iciency o cispla in signi ican ly, e y high sys emic
doses o cispla in should be adminis e ed. Un o una ely, such high
doses o cispla in esul in se e e sys emic oxici y and poo pa ien
compliance, including nausea/ omi ing, enal oxici y, gas oin-
es inal oxici y, pe iphe al neu opa hy, as henia, and o o oxici y,
which hus limi i s clinical use [40,41]. O all he oxici ies
induced by cispla in, neph o oxici y is conside ed o be he dose-
limi ing ac o [39]. Such side e ec s make i impossible o achie e
he ull bene i o he ea men in a la ge numbe o pa ien s [42].
In humans, cispla in ea men gene ally in ol es se ies o
in a enous injec ions adminis e ed e e y 3–4 weeks a a dose o
50–120 mg/m
2
(1.2–2.7 mg kg
21
). In addi ion o he undesi ed
side e ec s, he e is also a loss o d ug ac i i y in he body
associa ed wi h poo ci cula ion and poo deli e y o he umo , as
well as deac i a ion mechanisms ha i e e sibly al e he
chemis y o hese molecules be o e eaching he umo cells
[41]. Since i s disco e y, many a emp s o ind de i a i es o
cispla in ha e looked o bo h educed side e ec s and modi ied
body dis ibu ion (in o de o a ge di e en o gans), a he han
imp o ing cispla in e icacy [40,43]. He e, second-gene a ion
pla inum d ugs such as ca bopla in and oxalipla in ep esen an
imp o emen in some cance ea men s, o lung and colo ec al
espec i ely, al hough he limi a ions obse ed o cispla in ha e
no been en i ely o e come [39,40].
3. Ca ying Cispla in
Recen e o s ha e been ocused on a ge ing he umo by
using d ug deli e y sys ems o a oid he o gans o which cispla in
is oxic. As he kidney is esponsible o il a ion and emo al om
he blood o molecules smalle han 50 KDa, which co esponds
o molecula diame e s o a ound 6 nm, any la ge deli e y ehicle
will di e he d ug away om he kidney [19]. Addi ionally, NPs
accumula e in he umo due o he EPR e ec [23,24]; which is
known o be s ongly size-dependen [44,45]. The e o e, when he
a ge is a solid umo , nanome e -sized ca ie s a e expec ed o be
passi ely accumula ed on i . This case also applies when cispla in is
bound o albumin. Up o 90% o he adminis a ed cispla in is
known o bind i e e sibly o albumin [46] and hen each he
umo by EPR; howe e , his o m o cispla in is inac i e and has
no biological e ec [47]. Thus, a p ope ly designed nanoca ie
will no only anspo he cispla in o he umo , bu also p o ec i
agains plasma deac i a ion.
In his con ex , app oaches based on he encapsula ion and
anspo o cispla in ha e eme ged. S e ically s abilized polyme ic
nanopa icles, which ha e excellen s abili y in plasma, a much
longe ci cula ion ime, be e e icacy, and lowe oxici y han ee
cispla in ha e been epo ed [6,7,48,49]. Such ehicles include
lipid capsules [50] o polyme s as in P olindacH, which has a
22 kDa hyd oxyp opylme hac ilamide copolyme as a backbone
and hen a pH sensi i e glycine chela o linke [51]. O he
Table 1. Key wo ds in pha macology.
E icacy The abili y o a d ug o p oduce he desi ed he apeu ic e ec .
E iciency The abili y o a d ug o p oduce ew o no side e ec s while s ill pe o ming i s wo k.
Pha macokine ics How he body a ec s a speci ic d ug a e adminis a ion. Wha he body does o he d ug.
Pha macodynamics The s udy o he biochemical and physiological e ec s o d ugs on he body. Wha he d ug does o he body.
doi:10.1371/jou nal.pone.0047562. 001
Nanopa icles o De oxi ying An i umo al D ugs
PLOS ONE | www.plosone.o g 2 Oc obe 2012 | Volume 7 | Issue 10 | e47562
examples include soluble CNTs [52], ca bon nanoho ns [53], and
Fe
3
O
4
NPs [54]. Simila ly, Cy Immune Co p. is de eloping
AuNPs as a ca ie o TNF-aand doxo ubicin.AuNPs ha e been
ecen ly p oposed as sca olds o cispla in due o hei con olled
and ep oducible syn hesis and conjuga ion o cispla in as well as
he high loading o d ug achie ed [55]. No only cispla in, bu
o he P de i a i es, such as P (IV) p od ugs, ha e also been
loaded on AuNPs wi h main enance o he an icance e ec [56].
In closely ela ed wo k, Ren e al. [57–59] epo ed he adso p ion
o comme cial cispla in o gold colloids ia ionic in e ac ions. In
he case o adso p ion ia ionic in e ac ions on he su ace o he
nanoma e ials, an uncon olled apid libe a ion o he d ug is
obse ed as soon as he conjuga es a e dispe sed in highly ionic
media such as se um [60]. In many o he epo ed sys ems,
colloidal s abili y in he wo king en i onmen was an issue.
Ma e ials and Me hods
E hics S a emen
Mice we e ob ained om he Cen al Animal House o he
San iago de Compos ela Uni e si y (USC), a egis e ed animal
acili y ha main ains he animals unde wel a e and e hical
condi ions complying wi h he Eu opean (86/609/CEE) and
Spanish (RD223/88 and OM 13/10/89) laws on labo a o y
animal ca e and handling. In o de o amelio a e su e ing, mice
we e anaes he ized wi h 2,2,2- ib omoe hanol-2-me hyl-2-bu a-
nol (A e ineH, Sigma Ald ich) be o e xeno ansplan a ion and
be o e ecei ing he di e en ea men s epo ed he e. A e he
ea men s, mice we e eu hanized by CO
2
inhala ion. The
biological wo k ela ed o he esul s epo ed in his a icle had
he app o al o he E hical Commi ee o he USC and ollowed
he Eu opean and he Spanish legisla ions. 5 mL o blood we e
ob ained wi h in o med w i en consen om a no mal con ol
(JC, coau ho o he p esen wo k) and wi h app o al om he
E hical Commi ee o he USC.
Syn hesis and Conjuga ion o AuNP-cispla in
13 nm AuNPs we e syn hesized ollowing a seeded-g ow h
app oach and loaded wi h cispla in in a wo-s ep conjuga ion as
desc ibed in he li e a u e [61] and in he Supplemen a y
In o ma ion (Tex S1). The e a e se e al easons o using his
syn hesis: Con ol o he size is be e han ha achie ed by
classical me hods, he size dis ibu ion is e y na ow and he
concen a ion o AuNPs is highe . Mo eo e , and acco ding o ou
expe ience, he ep oducibili y is a beyond ha achie able by
using classic p o ocols. Fo mo e de ails, one can ead ou ecen
wo k [61].
In i o S abili y and pH-dependen Release
17 mL o AuNP-cispla in (2.75610
14
NP ml
–1
) we e added o
500 mL o human blood and gen ly mixed o e 24 h. Colloidal
s abili y was assayed by using DLS and UV-Vis spec oscopy. The
UV-Vis spec a o all he AuNPs in he p esen wo k we e
eco ded om 300–800 nm a 0.5 nm in e als. When needed,
app op ia e dilu ions we e pe o med o o e come he sa u a ion
limi o abso bance. The elease o cispla in in physiological
condi ions was pe o med in solu ions consis ing o 50 mM bu e
species, 120 mM NaCl, and 20% Foe al Bo ine Se um (FBS).
Bu e species we e HEPES o pH 7.6, MES o pH 6.2 and 4.4,
Glycine/HCl o pH 4.4 and 3.8 and Ace a e o pH 4.4. 100 mL
o AuNP-cispla in we e added o 900 mL o he co esponding
bu e ed solu ion and mixed o e di e ing leng hs o ime (2, 8, 24,
and 144 hou s). AuNPs we e emo ed by means o wo
cen i uga ion s eps (15 minu es, 35000 c ). The amoun o P
in he supe na an was analyzed by using ICP-MS.
P Cell In e naliza ion and DNA Accumula ion
The human lung ca cinoma de i ed cell line A549 was ob ained
om Ame ican Tissue Cul u e Collec ion (ATCC) and cul u ed in
a 1:1 mix u e o Dulbeccos Modi ied Eagles Medium (DMEM,
Sigma-Ald ich) and Hams F-12 Medium (Sigma-Ald ich), supple-
men ed o 10% ( / ) oe al bo ine se um (FBS, GIBCO-
In i ogen) and 1% ( / ) o L-glu amine, penicillin and s ep o-
mycin solu ion (GPS, Sigma-Ald ich).To quan i y P cell in e nal-
iza ion, 5610
5
cells we e pla ed in 60-mm-diame e pla es (Falcon)
and 24 hou s la e , medium was changed o ea men s dilu ed in
cul u e medium: ee cispla in o cispla in conjuga ed o AuNPs
(1.67 mg cispla in mL
21
in bo h cases). A e 0.5, 1, 3, o 24 hou s
o ea men , cells we e ypsinized and cen i uged, supe na an
was emo ed and cells we e esuspended in 0.5 mL 65% HNO
3
(Me ck). The amoun o P was de e mined by using ICP-MS
(B uke 820-MS). To quan i y cispla in bound o DNA, 10
6
cells
we e ea ed as abo e and DNA ex ac ion was pe o med by
using a comme cial ki (QIAamp DNA Blood Mini Ki ,
QIAGEN). DNA was inally esuspended in 200 mL o wa e .
Figu e 1. Di e en pla inum an icance d ugs app o ed by he FDA. The ac i e pa o each d ug is d awn in black; in all cases i is
cha ac e ized by he p esence o good lea ing g oups ha will allow he P a om o bind he a ge . In ed, he amines play a ole in he modula ion
o he ac i i y and dis ibu ion o he d ug. In he case o cispla in, he equilib ium ha spon aneously occu s inside he cell (whe e he chlo ide
concen a ion d ops om 100 mM o 4 mM) is also shown.
doi:10.1371/jou nal.pone.0047562.g001
Nanopa icles o De oxi ying An i umo al D ugs
PLOS ONE | www.plosone.o g 3 Oc obe 2012 | Volume 7 | Issue 10 | e47562
This solu ion was used o de e mine bo h he amoun o DNA and
P by using UV-Vis spec oscopy and ICP-MS, espec i ely.
TEM Cell Imaging
A549 cells we e cen i uged o 15 min a 3000 pm, ixed by
imme sion o 45 min in 2.5% glu a aldehyde in sodium
cacodyla e bu e (0.15 M, pH = 7.3), pos ixed wi h 2% osmium
e oxide in he same bu e , dehyd a ed, embedded in LRWhi e
esin, Medium G ade, and sec ioned. Sec ions we e s ained wi h
u anyl ace a e and lead ci a e and examined in a Zeiss 902
elec on mic oscope (Ca l Zeiss, F.R.G.) a 80 kV accele a ing
ol age.
Biodis ibu ion in Mice
In i o biodis ibu ion was measu ed by ea ing wo g oups o
12 emale umo -bea ing SCID mice ( h ee mice pe poin ). All
mice ecei ed a single in ape i oneal (ip) dose o 4 mg cispla in
kg
–1
mice. A 0.5, 3, 6, and 24 h a e he ea men , blood, hea ,
lungs, kidney, b ain, li e , spleen, o a ies, and umo we e
collec ed and weighed. To each o gan, p e iously weighed, we e
added 3 mL o 65% HNO
3
. The o gans we e hen kep o 3 h a
oom empe a u e in closed ubes and hen o 24 h a 60uC.
A e wa ds, 2 mL H
2
O
2
we e added o he sample and hea ed o
60uC. Finally H
2
O was added up o a inal olume o 10 mL and
il e ed wi h 0.22 mm il e s. Blood se um was dilu ed 1:20 and
measu ed by using ICP-MS. Blood cells we e esuspended in
6 mL o 65% HNO
3
and 2 mL o H
2
O
2
and hea ed in a
mic owa e o en (Miles one E hos 1) as ollows: 2 minu es a 85uC,
3.5 minu es a 135uC, and 15 minu es a 230uC. A e cooling
down, H
2
O was added up o 10 mL inal olume. The esul ing
solu ions o he abo e p ocedu es we e di ec ly measu ed using
ICP-MS o he de e mina ion o P and Au (see Supplemen a y
In o ma ion o ins umen ope a ing de ails).
Kidney His ology
P oximal ubula degene a ion was induced in mice by h ee
consecu i e ip injec ions (days 0, 3, 6) o 5 mg cispla in kg
–1
mice.
The same dosage was applied in he case o AuNP-cispla in, while
con ol mice ecei ed no ea men . Animals we e sac i iced h ee
days a e he las injec ion. Mouse kidneys and spleen we e
imme sion- ixed in 10% neu al bu e ed o malin o 24 h and
embedded in pa a in ou inely. Sec ions (4 mm hick) we e
moun ed on mic oslides and s ained wi h hema oxylin-eosin.
The specimens we e examined and pho og aphed using an
Olympus PROVIS AX70 mic oscope (Olympus, Tokyo, Japan)
equipped wi h an Olympus DP70 came a wi hou p io knowledge
o he applied expe imen al p o ocol.
The apeu ic E icacy in i o
Mice wi h se e e combined immunode iciency (SCID) be ween
8 and 14 weeks-old we e used o g ow xeno ansplan lank
umo s, one pe mouse, by subcu aneous injec ion o 20610
6
A549 umo cells. Fo moni o ing, umo s we e measu ed h ee
imes weekly and he umo olume was de e mined by he
o mula V = (A6B
2
)/2, whe e A is he la ges diame e and B is
he sho es diame e measu ed by calipe . A e ou o i e weeks,
once he umo olumes we e $100 mm
3
and mean umo size
had eached 300 mm
3
, mice we e di ided in o ou g oups o eigh
mice o ea men s, o minimize weigh and umo -size
di e ences. Tumo -bea ing mice we e ea ed by injec ion o
3 mg ee cispla in kg
–1
, 1.25 mg conjuga ed cispla in kg
–1
, and
con ols we e main ained wi hou ea men . T ea men s we e
adminis e ed in mice anes he ized by ip injec ion o 2,2,2-
ib omoe hanol-2-me hyl-2-bu anol (A e ineH, Sigma Ald ich)
wice (day 0 and 3). Mice we e moni o ed o a maximum o 10
days a e i s dose o a oid excessi e umo load, and mice weigh
and body weigh loss we e also moni o ed acco ding o good
labo a o y p ac ices o check excessi e oxici y o ea men s.
Comple e umo s o all animals we e ex ac ed and weigh ed a
endpoin o e icacy s udies.
Ano he ound o mice was s udied o a longe ea men
pe iod. Mice we e injec ed subcu aneously wi h A549-luc-C8 cells
(Calipe ); 0.5610
6
cells). Tumo s we e g own o e nine days
be o e s a ing he ea men . Tumo g ow h was moni o ed e e y
hi d day by using an in i o imaging sys em (IVISHSpec um,
Calipe ), 150 mg D-Luci e ine (kg mice)
21
was applied ip 5
minu es be o e scanning. Mice we e placed unde he CCD
came a and kep unde iso lu ane anaes hesia (1.5–2% ) du ing he
measu es. Fi e mice ecei ed h ee ip injec ions o 1.5 mg cispla in
kg
–1
, i e mice ecei ed he same amoun o cispla in conjuga ed o
AuNPs and i e mice, con ols, we e main ained wi hou
ea men .
Resul s and Discussion
AuNP Syn hesis and Func ionaliza ion
We p opose he use o cispla in a ached o 13 nm me cap-
oundecanoic acid (MUA)-capped AuNPs ia a pH-dependen
coo dina ion bond, as an e icacious an i umo d ug. The bond
be ween ca boxylic acid and P is s able unde physiologic
condi ions, bu i is b oken a acidic pH. I is well-known ha
NPs a e in e nalized ia an endocy ic pa hway [62]. Since he pH
wi hin he endosomes dec eases, he elease o cispla in a e
in e naliza ion by cells is p omo ed. The ac i e d ug (aqua ed
cispla in) is able o di use ou o he endosome and each he
nucleus. In ac , endosomal elease has been p e iously pos ula ed
as an ad anced mode o cellula deli e y [63,64]. Such p ocesses,
pH-sensi i e elease o d ugs, ha e been p oposed o en in
chemo he apy, o example, by encapsula ion o a d ug in a pH-
sensi i e polyme and subsequen elease o he d ug in he icini y
o he umo , because o he lowe pH ound he e [63]. In ou
case, he low pH a he umo (6–7) does no cause libe a ion o he
d ug. A lowe pH (<5) is needed o b eak he coo dina ion bond
be ween MUA and cispla in. Mo eo e , simul aneous moni o ing
o ehicle and d ug biodis ibu ion was possible due o he
ino ganic na u e o bo h, and his can be co ela ed o he lack o
sys emic oxici y. T acking bo h he NPs and he d ug will help in
he unde s anding how he nanoca ie s a e p ocessed by he
o ganism.
Conjuga es we e ca e ully p epa ed ega ding size, d ug
loading, su ace cha ge, and hyd ophilici y, as well as s abili y
e en a high concen a ions o AuNPs. Cispla in doses in humans
a e a ound 1–3 mg o cispla in pe kg o body weigh . Fo
ea men in mice (abou 30 g body weigh ) and aking in o
accoun ha we can load a ound 500 cispla in molecules pe NP
(which leads o he highes cispla in co e age densi y o ou
knowledge in simila NPs), we need o injec abou 750 mL o he
conjuga es solu ions wi h concen a ion o NPs as high as 0.5 mM.
The concen a ion is a se ious issue since la ge olumes canno be
injec ed in o he animals, and we can ha dly u he inc ease
conjuga e concen a ion wi hou comp omising colloidal s abili y.
Biocompa ible sodium ci a e AuNPs we e syn hesized wi h a
na ow size dis ibu ion (13.361.9 nm) and hen modi ied wi h a
MUA sel -assembled monolaye (SAM) and concen a ed by a
des abiliza ion–p ecipi a ion– esuspension p ocess (Figu e 2).
B ie ly, glycine bu e was added o dec ease he pH o 2.6 o
p o ona e MUA ca boxylic acids. A e emo ing he supe na an ,
Nanopa icles o De oxi ying An i umo al D ugs
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MUA-capped AuNPs we e esuspended in icine bu e a pH 8
concen a ing he NPs up o 2.75610
14
NP mL
–1
o a ain
easonable concen a ions o he in i o s udies. The MUA SAM
p o ided no only elec os a ic s abili y o he sys em ( -Po en ial: –
48.5 mV), bu ca boxylic g oups o u he linkage o
[P (H
2
O)
2
(NH
3
)
2
]
2+
(aqua ed cispla in). I is known ha ollowing
cispla in ([P Cl
2
(NH
3
)
2
]) adminis a ion he chlo ide ligands a e
slowly eplaced by wa e molecules, in a p ocess e med aqua ion.
The aqua ligands in he esul ing [P Cl(H
2
O)(NH
3
)
2
]
+
and
[P (H
2
O)
2
(NH
3
)
2
]
2+
a e mo e labile han Cl
–
, ac i a ing he d ug
and allowing he pla inum a om o bind o bases on DNA [39].
The [P (H
2
O)
2
(NH
3
)
2
]
2+
solu ion is ob ained by adding a solu ion
o AgNO
3
o comme cial cispla in [P (Cl)
2
(NH
3
)
2
] o p omo e he
exchange o Cl
–
o H
2
O ligands. A e pu i ica ion by ec ys al-
liza ion-washing s eps, he solid [P (H
2
O)
2
(NH
3
)
2
] (NO3)
2
was
ob ained and dissol ed in wa e be o e use. I is impo an o wo k
wi h he aqua ed o m o cispla in since H
2
O is a be e lea ing
g oup han Cl
–
and allows o ma ion o coo dina ion bonds
be ween he P molecule and he dep o ona ed MUA ca boxylic
g oups on he NP, o he wise, he comme cial cispla in molecule
will no bind co alen ly [60]. In ou con igu a ion, he eac i e
pa o he d ug is p o ec ed, which lea es he ine NH
3
moie ies
exposed o he ex e io . Thus, he d ug is p o ec ed agains plasma
deac i a ion. pH con ol is c i ical du ing he whole conjuga ion
p ocess: he pK
a
alue o a MUA SAM is epo ed o be be ween
6–8 [65], he e o e he wo king pH mus be abo e his alue o
ensu e colloidal s abili y. Howe e , a highe pH alues, aqua
ligands o he aqua ed cispla in can unde go dep o ona ion o gi e
hyd oxo complexes ha a e less eac i e (pK
a1
<5.5, pK
a2
<7.3)
[66]. Since he hyd oxyla ion eac ion is slow a pH,9, wo king a
pH 8.3 ensu es bo h colloidal s abili y and o ma ion o he
conjuga e. O he wise he conjuga e would lose elec os a ic
s abili y (a pH,pK
a
o MUA) o cispla in would be unable o
o m a coo dina ion bond (a pH.9) and i would be elec os a -
ically abso bed, and immedia ely eleased when dispe sed in high-
ionic-s eng h media such as biological luids [60].
To achie e he maximum loading o cispla in wi hou comp o-
mising colloidal s abili y, aqua ed cispla in was added in excess.
The eac ion was s opped by emo ing he excess o aqua ed
cispla in a e 25 minu es by dialysis, when su ace cha ge, as
measu ed by -po en ial, was 230.8 mV (Figu e 3d). I is
commonly accep ed ha alues abo e +/230 a e needed o
achie e colloidal s abili y [67]. This ac is in ag eemen wi h ou
obse a ions ha -po en ial alues ha a e less nega i e han –
25 mV led o des abiliza ion o he AuNPs and p ecipi a ion due
o excessi e quenching o he nega i ely cha ged ca boxylic acid
g oups by cispla in. A simila beha io was shown by C aig e al.
when using a PEG linke which has a ca boxylic e minal g oup as
well [55]. The e o e, app op ia e cispla in concen a ion and
incuba ion ime a e c ucial o main ain colloidal s abili y (Figu e 3
a, b, c). A he end, he loading o cispla in on he NP as measu ed
by induc i ely coupled plasma mass spec ome y (ICP-MS) was
42.360.8 mg P L
–1
, which ep esen s an app oxima e loading o
470 cispla in molecules pe NP. Rega ding conjuga ion, i has
been ecu en ly obse ed ha , in an excess o conjuga ing
molecule wi hou in e e ing species, homogeneous and dense
monolaye s o sel -assembled o ganic molecules can o m apidly
on he di e en NP su aces [68]; he e o e i is e en mo e
impo an o achie e homogeneous pa ial conjuga ion. I is likely
ha incuba ion o he AuNP-MUA wi h an excess o aqua ed
cispla in and hen s opping he eac ion by emo ing he excess o
cispla in by dialysis a o s he o ma ion o homogenously loaded
conjuga es.
When p epa ing NP solu ions o in i o applica ions, he
di e ence be ween NP conjuga ion solu ions and physiological
media is impo an ; he la e has highe elec oly ic concen a ion
and s ongly bu e ed pH alue. In his con ex , colloidal s abili y
in physiological media and no loss o d ug du ing i s jou ney
h ough he body should bo h be gua an eed o success o he in
i o a ge ing [16]. P io o in i o expe imen s, colloidal s abili y
was assayed in ull human blood by dynamic ligh sca e ing (DLS)
and UV-Vis spec oscopy. In DLS measu emen s, no peaks
indica ing he p esence o agg ega es in blood we e obse ed. The
12-nm shi o he AuNPs peak is due o he o ma ion o a p o ein
co ona [11]. Mo eo e , he UV-Vis spec a o AuNP–cispla in in
human blood indica ed ha AuNPs did no agg ega e in his
medium. The s eng h o he coo dina ion bond makes he link
be ween ca ie and d ug s able unde physiological condi ions.
Since his bond is pH sensi i e, inc easing he [H
+
] leads o
hyd olysis o he MUA–P bonds, eleasing he d ug in i s ac i e
o m. Only 5% o he P was eleased om he AuNPs a pH 7.6
a e 144 h, while alues as high as 40% o he P we e eached a
pH 4.4, and 67% a pH 3.8 (Figu e 4). One could expec ha he
p esence o di e en nucleophilic species, as he p esence o
p o ein, would al e he AuNP-MUA-CisP «AuNP-MUA +
CisP equilib ium. Howe e , elease seems o be independen o
he bu e ing species, as demons a ed by obse a ion o simila
beha io s in h ee di e en pH 4.4 bu e s.
P Cell In e naliza ion and DNA Accumula ion
Low cellula up ake o he d ug may limi he e icacy o a
chemo he apeu ic ea men [69], he e o e cispla in cellula
up ake and DNA binding we e employed as signs o he apeu ic
ac i i y. Adenoca cinomic human al eola basal epi helial cells
(A549) we e ea ed wi h bo h ee cispla in and cispla in
conjuga ed o AuNPs (AuNP–cispla in) a he same P concen a-
ion (1.7 mgmL
–1
). A as e up ake and highe cy oplasma ic le els
o P we e ound in he la e case (Figu e 5b). When cispla in was
conjuga ed o he AuNPs, cellula d ug con en was up o 300
imes highe a sho ime pe iods han o ee cispla in, and le els
up o 125 imes highe we e ound in he DNA a 24 h (Figu e 5b).
As can be obse ed in ansmission elec on mic oscopy (TEM)
images (Figu e 5a), AuNPs mainly eside inside esicles ha e ol e
o o m la e endosomes and endolysosomes ( esicle size and
numbe o pa icles pe esicle inc ease wi h ime). Simila ea u es
we e obse ed by con ocal mic oscopy (Figu e S2). These esicles
p o ide an acidic en i onmen ha p omo es he elease o
cispla in om he AuNPs, and i s u he escape om he
endosomes o con inue i s jou ney owa ds he DNA. No e ha
he aqua ed o m o cispla in, due o i s high labili y, is expec ed
no o a el a om he elease poin be o e eaching i s a ge o
being deac i a ed. In e es ingly, he mechanism o d ug en y in o
he cell is modi ied by conjuga ion: ee cispla in en e s he cell
mainly ia passi e di usion h ough he memb ane and by some
anspo e -media ed ou es (e.g., coppe anspo e s) [39],
whe eas cispla in a ached o AuNPs en e s ia an endocy ic
pa hway. This ac i e mechanism, oge he wi h he ca go e ec ,
allowed he apid accumula ion o cispla in. This apid accumu-
la ion o cispla in may enable us o o e come mul id ug- esis ance
mechanisms ha in ol e he o e exp ession o cispla in e lux [70]
p o eins (e.g., P-glycop o ein) o acili a e DNA- epai mecha-
nisms [40]. Addi ionally, he coa ing o he ca ie wi h se um
p o eins, mainly albumin, may also a o conjuga e up ake due o
an o e exp ession o albumin ecep o s in umo al cells [71].
Indeed, his is he claimed s a egy o enhanced up ake o
pacli axel in Ab axane [72].
Nanopa icles o De oxi ying An i umo al D ugs
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The apeu ic E iciency o AuNP–cispla in
Animal models a e pa icula ly equi ed when assaying he
he apeu ic e iciency o NPs as ehicles o d ug deli e y. Use o a
nanoca ie will lead o an inc eased blood hal -li e and a highe
accumula ion in he si e o ac ion o he d ug [73]. Thus, i is
likely ha a p ope ly designed nanoca ie should ha e a be e
esponse in i o han in i o, wi h espec o he ee d ug in e ms
o concen a ion o ac i e d ug in he o gan o in e es .
Addi ionally, issues such as umo pene abili y and oxici y migh
be modi ied as well and mus be aken in o accoun . One o he
mos common models consis s o xenog a ed umo s implan ed
subcu aneously in Se e ely Comp omised ImmunoDe icien
(SCID) mice. I umo p og ession has o be measu ed by size
(e.g., using a calipe ), ini ial umo s mus be la ge enough o be
measu able, in which case hey a e no mally poo ly i iga ed and
e en may con ain nec o ic a eas. Newe bioluminescen ech-
niques (e.g. IVIS H) a e a good al e na i e o calipe measu emen s
because hey a e mo e sensi i e and he e o e wo k p ope ly wi h
smalle umou s, bu hey depend on enzyma ic ac i i y. This
echnique has been used o ollow umo igenesis and esponse o
umo s o ea men in animal models, since he exp ession o a
bioluminescen ma ke speci ic o he implan ed umo cells is
supposed o be p opo ional o he numbe o li ing cells [74].
Howe e , en i onmen al ac o s and he apeu ic in e e ences
may cause some disc epancies be ween umo bu den and
bioluminescence in ensi y in ela ion o changes in p oli e a i e
ac i i y; his mus be aken in o accoun along wi h he di e ing
indi idual esponse o he umo and he ea men s.
In ou expe imen s, 15 umo -bea ing mice we e ea ed wi h 3
in ape i oneal (ip) injec ions o saline (con ol), ee cispla in, o
AuNP–cispla in. Doses o cispla in we e 1.5 mg cispla in kg
–1
in
bo h ea men s. No e ha di e en concen a ions a e used
h oughou he wo k. The eason o hese di e ences is he need
o explo e h ee di e en egimes: Tissue dis ibu ion, he apeu ic
window, and oxici y. In he i s case, he doses a e maximized o
ensu e de ec able le els o gold and pla inum in all issues. Fo he
he apeu ic window, we explo ed he ange o dosages which can
p e en umo g ow h e ec i ely while s aying in he sa e y ange
o he ea men (no e ha he ea men ime is much longe han
o biodis ibu ion and oxici y). O he wise, se e e cispla in-
induced oxici y would ha e led o dead animals and consequen ly
o he impossibili y o compa ing ea men s. On he o he hand,
o he oxici y assays, we ea ed he animals wi h highe
concen a ions such as a e gene ally used wi h cispla in o induce
oxici y in a sho pe iod o ime. As can be obse ed in Figu e 5d,
he conjuga ion o cispla in o he AuNPs does no a ec i s
he apeu ic e ec since bo h he sample ea ed wi h ee d ug and
ha wi h d ug conjuga ed o AuNPs showed no signi ican
Figu e 2. T ansmission Elec onic Mic oscopy (TEM) images o MUA-capped AuNPs. (a, b, c) Rep esen a i e images o AuNPs. Scale ba
ep esen s 1 mm, 200 nm, and 100 nm espec i ely. (d) A na ow size dis ibu ion (13.361.9 nm) o AuNPs is obse ed. The appea ance o he AuNPs
as syn hesized and a e concen a ion p ocess is shown in he inse in (a).
doi:10.1371/jou nal.pone.0047562.g002
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di e ences in umo g ow h, as measu ed by bioluminescence, and
bo h ea men s showed dec eased umo g ow h wi h espec o
he con ol. In a simila expe imen , bu s a ing he ea men
when umo s we e la ge enough o be measu able by calipe , he
same endencies we e main ained (Figu e S3). A he end o hese
ea men s, umo s we e ex ac ed and weighed. The a e age
weigh o umo s in bo h ea men s was 0.5960.16 g o ee
cispla in and 0.7660.20 g when conjuga ed. These alues a e
signi ican ly lowe han hose o he con ol (1.8160.54 g), wi h a
S uden ’s -dis ibu ion be ween he con ol and he ea ed mice
wi h he conjuga es o smalle han 0.05 (0.039).
Figu e 3. AuNP unc ionaliza ion. (a) UV-Vis spec oscopy o conjuga es wi h inc easing aqua ed cispla in concen a ions shows ha his
concen a ion should no exceed 0.39 mM o gua an ee colloidal s abili y. (b) When an excess o d ug was in oduced, he MUA cha ge became
p og essi ely quenched, which lead o agg ega ion o AuNPs, as deno ed by he ed-shi and u he in ensi y dec ease o SPR in ensi y in less han
one hou . (c) Red-shi o he SPR peak a he wo king condi ions: he ini ial peak o ci a e-capped AuNPs shi ed om 515 nm o 521.5 nm a e
MUA conjuga ion and o 523 nm a e cispla in conjuga ion. (d) Time e olu ion o -po en ial a e addi ion o [P (H
2
O)
2
(NH
3
)
2
]
2+
indica es he
quenching o nega i e cha ge on he MUA by he o ma ion o a coo dina ion bond be ween he ca boxylic acid g oup and aqua ed cispla in.
Conjuga es emained s able a -po en ial alues mo e nega i e han –25 mV. (e) Scheme showing he unc ionaliza ion s eps ollowed o ob ain he
cispla in deli e y sys em.
doi:10.1371/jou nal.pone.0047562.g003
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Tumo pene a ion is known o be a limi ing ac o o cance
d ug deli e y. In ac , his has been ecen ly iden i ied as he majo
limi a ion in solid umo ea men [75–77]. Despi e his ac ,
conjuga e sizes o a ound 15 nm ha e been epo ed o achie e
good pene a ion and accumula ion in he umo [24,44].
Ob iously, cispla in is smalle han he NPs and he e o e i s
pene abili y, in p inciple, could be highe . Howe e he g ea e
accumula ion o ac i e d ug when a ached o AuNPs ( he d ug is
p o ec ed agains deac i a ion by plasma p o eins) and he
possibili y o successi e ea men s (i cispla in oxici y is clea ly
educed) may o e come he lowe pene abili y o he ehicles
[75], allowing he p og essi e e osion o he umo . Ce ainly,
hese dosing s a egies could also be use ul in he case o umo
esis ance o cispla in ea men . Finally, i is also wo h no ing ha
he me allic co e o he AuNPs opens up he possibili y o
inc easing umo damage by using a combined he apy since hey
can e icien ly ac as pho o he mal he apeu ic agen s [2,78] o as
adiosensi ize s [79,80] which, oge he wi h su ge y, a e he
common s a egies used o ea cance in he clinic.
Biodis ibu ion in Mice: Co ela ion o Biodis ibu ion and
Lack o Toxici y
Inside he body, he po ous and di use on ie s o he di e en
o gans, which a e all connec ed o he blood and lympha ic
sys ems, ha e g ea ly di e ing anspo abili ies owa ds small
molecules and nu ien s, la ge p o eins, o cells. Thus, he majo i y
o d ugs a e no mally anspo ed h ough he small po es (6–
8 nm) ound in he con inuous capilla ies which a e he mos
widely dis ibu ed h oughou he o ganism [18], which causes
unwan ed side e ec s. Fo e ec i e he apy, i is necessa y o
deli e he apeu ic agen s selec i ely o hei a ge si es, a oiding
non- a ge o gans. Such selec i i y is key o an i umo al d ugs
because o hei ex eme cy o oxici y. By ehicula ing he d ug
using a nanoca ie , he dis ibu ion o he d ug is con olled by
he physicochemical p ope ies o he nanopa icle [4,17,81]
a he han hose o he d ug. No mally, small molecule d ugs ( he
majo i y o d ugs) ha e a sho plasma hal -li e and a e apidly
clea ed by he enal sys em [18], which he eby g ea ly educes he
d ug’s cu a i e e ec . Also, i escaping he kidney, NPs a e easily
endocy osed/phagocy osed, gene ally by ci cula ing monocy es o
ixed mac ophages, which leads o hei elimina ion om
ci cula ion and hei simul aneous concen a ion in o gans wi h
high phagocy ic ac i i y. Al hough se e al ac o s such as co e size
and su ace composi ion in luence he a e o he NPs inside he
o ganism, di e en ypes o NPs ha e been epo ed o be clea ed
wi hin minu es om he bloods eam wi h a ypical inal
biodis ibu ion in he spleen, li e , and kidneys [20,45,67,81–
83]. No only he NPs’ physicochemical p ope ies (e.g., size,
shape, and su ace composi ion) de e mines he inal a e o he
NPs, bu an impo an ole is played by he a ay o se um p o eins
a ac ed by he NPs ha o m he well-known p o ein co ona [11]
which ul ima ely con e s hei biological p ope ies [84]. In his
ega d, many s udies indica e ha pa icle size and su ace
chemis y (coa ing) go e n ansloca ion ac oss epi helial and
endo helial cell laye s. In pa icula , ega ding ansloca ion o
NPs, he s udies summa ized by Meh a e al. [85] and hose
pe o med by Heckel e al. [86] using in a enous adminis a ion
o albumin-coa ed gold nanopa icles in oden s demons a ed
ecep o -media ed anscy osis (albumin-binding p o eins). Simi-
la ly, polys y ene pa icles o 240 nm ansloca ed ac oss he
al eolus-capilla y ba ie when coa ed wi h leci hin, whe eas
uncoa ed pa icles did no ansloca e [87].
In ou expe imen s, g oups o 15 SCID human- umo -bea ing
mice we e used o assay he biodis ibu ion o bo h he ehicle and
he d ug. The same amoun o a high dose o ee o AuNP-bound
cispla in was adminis a ed o he mice (4 mg P (kg mice)
–1
) ia
in ape i oneal injec ion. This ou e acili a es he a ic o
pa icles om he pe i oneal ca i y o he lympha ic sys em be o e
he pa icles inally en e sys emic ci cula ion [88], om whe e
hey a e dis ibu ed o he di e en o gans. The amoun s o bo h
P and Au in blood, li e , spleen, hea , b ain, lung, kidney, o a y,
and umo we e measu ed using ICP-MS a 30 min, and 3, 6, and
24 hou s a e he injec ion (Figu e 6). I is wo h no ing ha om
he c ude eadings i is impossible o de e mine i he obse ed P
is s ill ac i e, inac i e bound o p o eins, o a ached o he NPs
wai ing o be ac i a ed. F ee cispla in is known o be emo ed
om he ci cula ion in wo s eps: an ini ial apid enal clea ance
(less han 1 h) ollowed by a slow loss om he ci cula ion o he
cispla in bound o plasma p o eins (hou s o days) [46], wi h less
han abou 3% eaching he umo al cells’ DNA. Since he long-
ci cula ing cispla in is mainly bound o p o ein and consequen ly
deac i a ed [46], nei he signi ican oxici y no he apeu ic
bene i s a e expec ed om i [47]. This apid clea ance o ee
cispla in, which is also obse ed om all o gans in ou wo k, di e s
conside ably om wha is obse ed in he case o he cispla in
bound o AuNPs. The amoun o P in blood se a eleased om
he AuNP-cispla in conjuga es (AuNPs we e emo ed by cen i-
uga ion be o e measu ing) was ini ially negligible (52.1261.9 mg
L
–1
). Howe e , once he conjuga es we e p ocessed, mainly by
phagocy ic o gans ( ide in a), he e was a slow delayed elease o
P o blood ha eached 932.256343.4 mgL
–1
a 24 h. In he es
o he o gans i is common o see an ini ial dec ease in P
concen a ion, om 30 min o 3 hou s and hen an inc ease a 6 h
and 24 h, likely o he non-conjuga ed o m as he changes o he
Au/P a ios in o gans wi h ime seem o indica e (Figu e 7); his
shows how he Au and cispla in spli and ollow di e en pa hways
a e being p ocessed by cells. The highe he a io, he less ee
cispla in is p esen . Al hough he signal om conjuga ed and non-
conjuga ed cispla in is di icul o decon olu e, wo di e en
beha io s a e clea ly obse ed: o gans whe e he a io p og es-
si ely dec eases ( hose indica ed by dashed lines) and o gans whe e
he a io inc eases ( hose indica ed by con inuous lines). No e ha
he o gans whe e his a io inc eased a e phagocy ic o gans whe e
NPs accumula e. F om hese esul s we belie e ha some
Figu e 4. Cispla in elease s. pH in physiological condi ions.
(high-ionic-s eng h media wi h 20% BSA; BSA = bo ine se um albu-
min). A neu al pH he elease was almos negligible bu i inc eased a
acidic pH alues.
doi:10.1371/jou nal.pone.0047562.g004
Nanopa icles o De oxi ying An i umo al D ugs
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Figu e 5. Cell in e naliza ion and accumula ion, and he apeu ic bene i s
in i o
.(a) Rep esen a i e TEM images showing he
in e naliza ion o pa icles a 1, 3, and 24 h. I is clea ly shown ha AuNPs we e en apped in esicles ha p og essi ely use. The cell nucleus
emained ee o AuNPs. Scale ba s ep esen 4 mm. (b, le ) Cells show g ea e amoun s o P when i en e s a ached o AuNPs han when in ee
cispla in, especially a sho ime pe iods. (b, igh ) The acidic pH alue in he endosomes p omo es he elease o cispla in. Consequen ly he DNA
a ge ing was also conside ably imp o ed by AuNP–cispla in. (c) The colloidal s abili y is gua an eed in blood whe e he p esence o agg ega es is
excluded by DLS. The 12 nm inc ease is due o he o ma ion o a so p o ein co ona. (d) Inc ease in bioluminescence measu ed by IVIS which is
p opo ional o he numbe o li ing cells in he umo . 1.5 mg cispla in (kg mouse)
–1
injec ions we e used in bo h ea men s (day 0, 19, and 34).
A ows indica e injec ion days. E o s a e s anda d e o o he mean (n = 5).
doi:10.1371/jou nal.pone.0047562.g005
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