Detoxifying antitumoral drugs via nanoconjugation: the case of gold nanoparticles and cisplatin

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 PLOS ONE | www.plosone.o g 4 Oc obe 2012 | Volume 7 | Issue 10 | e47562 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 PLOS ONE | www.plosone.o g 5 Oc obe 2012 | Volume 7 | Issue 10 | e47562 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 Nanopa icles o De oxi ying An i umo al D ugs PLOS ONE | www.plosone.o g 6 Oc obe 2012 | Volume 7 | Issue 10 | e47562 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 Nanopa icles o De oxi ying An i umo al D ugs PLOS ONE | www.plosone.o g 7 Oc obe 2012 | Volume 7 | Issue 10 | e47562 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 PLOS ONE | www.plosone.o g 8 Oc obe 2012 | Volume 7 | Issue 10 | e47562 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. 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