Challenges and Contradictions of Metal Nano-Particle Applications for Radio-Sensitivity Enhancement in Cancer Therapy

In e na ional Jou nal o Molecula Sciences A icle Challenges and Con adic ions o Me al Nano-Pa icle Applica ions o Radio-Sensi i i y Enhancemen in Cance The apy E a Pagáˇco á1, Lenka Š e anˇcíko á1,2, F anz Schmid -Kale 3, Geo g Hildenb and 3,4 , Tomáš Viˇca 5, Daniel Depeš 1, Jin-Ho Lee 3, Felix Bes a e 6, Sand ine Lacombe 2, E ika Po cel 2, S éphane Roux 7, F ede ik Wenz 4, Olga Kopeˇcná1, I a Falko á1, Michael Hausmann 3,* and Ma in Falk 1,* 1Czech Academy o Sciences, Ins i u e o Biophysics, . .i., K alo opolska 135, 612 65 B no, Czech Republic; [email p o ec ed] (E.P.); [email p o ec ed] (L.S.); [email p o ec ed] (D.D.); [email p o ec ed] (O.K.); [email p o ec ed] (I.F.) 2Ins i u e des Sciences Moléculai es d’O say (ISMO), Uni e si éPa is Saclay, Uni e si éPa is Sud, CNRS, 91405 O say Cedex, F ance; [email p o ec ed] (S.L.); [email p o ec ed] (E.P.) 3Ki chho -Ins i u e o Physics, Uni e si y o Heidelbe g, Im Neuenheime Feld 227, 69120 Heidelbe g, Ge many; [email p o ec ed] (F.S.-K.); [email p o ec ed]g.de (G.H.); [email p o ec ed] (J.-H.L.) 4Depa men o Radia ion Oncology, Uni e si ä smedizin Mannheim, Medical Facul y Mannheim, Heidelbe g Uni e si y, 68159 Mannheim, Ge many; F ede ik.W[email p o ec ed]g.de 5B no Uni e si y o Technology, Depa men o Biomedical Enginee ing, Technická3082/12, 61600 B no, Czech Republic; [email p o ec ed] 6Ge man Cance Resea ch Cen e (DKFZ), Im Neuenheime Feld 280, 69120 Heidelbe g, Ge many; [email p o ec ed] 7Ins i u e UTINAM, UMR CNRS 6213-Uni e si éde Bou gogne F anche-Com é, 25020 Besançon Cedex, F ance; [email p o ec ed] *Co espondence: [email p o ec ed] (M.H.); [email p o ec ed] (M.F.); Tel.: +49-6221-549824 (M.H.); +420-541517116 (M.F.) Recei ed: 17 Janua y 2019; Accep ed: 24 Janua y 2019; Published: 30 Janua y 2019   Abs ac : F om he e y beginnings o adio he apy, a c ucial ques ion pe sis s wi h how o a ge he adia ion e ec i eness in o he umo while p ese ing su ounding issues as undamaged as possible. One p omising app oach is o selec i ely p e-sensi ize umo cells by me allic nanopa icles. Howe e , hough he “physics” behind nanopa icle-media ed adio-in e ac ion has been well elabo a ed, p ac ical applica ions in medicine emain challenging and o en disappoin ing because o limi ed knowledge on biological mechanisms leading o cell damage enhancemen and e en ually cell dea h. In he p esen s udy, we analyzed he in luence o di e en nanopa icle ma e ials (pla inum (P ), and gold (Au)), cance cell ypes (HeLa, U87, and SKB 3), and doses (up o 4 Gy) o low-Linea Ene gy T ans e (LET) ionizing adia ion ( γ - and X- ays) on he ex en , complexi y and epa abili y o adia ion-induced γ H2AX + 53BP1 oci, he ma ke s o double s and b eaks (DSBs). Fi s ly, we sensi i ely compa ed he ocus p esence in nuclei du ing a long pe iod o ime pos -i adia ion (24 h) in spa ially ( h ee-dimensionally, 3D) ixed cells incuba ed and non-incuba ed wi h P nanopa icles by means o high- esolu ion immuno luo escence con ocal mic oscopy. The da a we e compa ed wi h ou p elimina y esul s ob ained o Au nanopa icles and ecen ly published esul s o gadolinium (Gd) nanopa icles o app oxima ely he same size (2–3 nm). Nex , we in oduced a no el supe - esolu ion app oach—single molecule localiza ion mic oscopy (SMLM)— o s udy he in e nal s uc u e o he epai oci. In hese expe imen s, 10 nm Au nanopa icles we e used ha could be also isualized by SMLM. Al oge he , he da a show ha di e en nanopa icles may o may no enhance adia ion damage o DNA, so mul i-pa ame e e ec s ha e o be conside ed o be e in e p e he adiosensi iza ion. Based on hese indings, we discussed on conclusions and In . J. Mol. Sci. 2019,20, 588; doi:10.3390/ijms20030588 www.mdpi.com/jou nal/ijms In . J. Mol. Sci. 2019,20, 588 2 o 25 con adic ions ela ed o he e ec i eness and p esump i e mechanisms o he cell adiosensi iza ion by nanopa icles. We also demons a e ha SMLM o e s new pe spec i es o s udy in e nal s uc u es o epai oci wi h he goal o be e e alua e po en ial di e ences in DNA damage pa e ns. Keywo ds: me al nanopa icles; cance adio he apy; umo cell adiosensi iza ion; DNA damage; DNA epai ; DNA double s and b eaks (DSBs); supe - esolu ion mic oscopy; single-molecule localiza ion mic oscopy (SMLM); DNA epai oci; damage o lysosomes 1. In oduc ion Mo e han a hal o all cance pa ien s a e cu en ly ea ed wi h adio he apy [ 1 ] ha , oge he wi h chemo he apy, s ill ep esen s he mos e icien cu a i e app oach o many cance ypes. The he apeu ic window o adio he apy (and chemo he apy) [ 2 ] is based on di e en capaci ies o no mal and cance cells o epai DNA damage. Because o de ec s in cell cycle checkpoin s and/o epai pa hways [ 3 ], cance cells mo e o less su e om genomic ins abili y and a e mo e suscep ible han no mal cells o DNA-damaging agen s. Some umo s a e highly adio esis an hough, making hem di icul o e adica e while p ese ing he su ounding no mal issues undes oyed [ 4 – 6 ]. A c ucial pa o cance ea men de elopmen he e o e conce ns a ques ion o how o deli e he adia ion e ec i eness in o he umo while p ese ing he no mal su ounding issues as much as possible. This issue becomes o undamen al impo ance o adio esis an umo s and/o umo s loca ed in close p oximi y o i al o gans o s uc u es. An illus a i e example could be he mos agg essi e and adio esis an umo [ 7 ] s a ing in he b ain—glioblas oma—leading us o selec U87 glioblas oma cells as a model in he p esen s udy. HeLa cells, an o en used model in bio-medical esea ch, we e included in he p esen s udy as a di e en cance cell ype o hei lowe adio esis ance and di e en o igin. SkB 3 cells [ 8 ] we e in ol ed as a model o b eas cance wi h He 2/neu up- egula ion, on which he adia ion e ec s a e s udied in combina ion wi h an ibody and/o chemo- ea men [9]. Se e al p omising s a egies a e con inuously being de eloped o imp o e adio he apy. Fo ins ance, spa ial dose ac iona ion, ime dose ac iona ion, mic o/mini-beam i adia ion, hea y-ion i adia ion [ 10 – 17 ], and applica ion o no mal cell adio-p o ec an s [ 18 , 19 ] and/o umo cell adiosensi ize s [ 20 ] could al eady be used in p ac ice and e en ually combined. One o he adiosensi izing app oaches p oposed is o selec i ely po en ia e adia ion oxici y o umo cells by me al nanopa icles [ 21 – 28 ]. Due o hei high elec on con en and pho oelec ic abso p ion c oss-sec ion, me al (high a omic numbe = high-Z ma e ial) nanopa icles emi showe s o seconda y elec ons upon i adia ion [ 29 , 30 ]. Launched elec ons hen gene a e clouds o high ioniza ion densi ies, capable o enhancing adia ion-induced cell damage and dea h a es [31]. The cell nucleus and DNA loca ed he ein a e sensi i e o many s esso s [ 32 – 36 ] and can be highly damaged wi h ela i ely low doses o ionizing adia ion [ 37 , 38 ]. As dele e ious e ec s o ionizing adia ion on (cance ) cells a e mos ly media ed h ough agmen a ion o nuclea ch oma in by inse ing double s and b eaks (DSBs) in o he DNA molecule [ 39 ], nanopa icle adiosensi izing e ec s ha e p ima ily been asc ibed o an inc eased numbe and/o complexi y o DSBs gene a ed by adia ion in p esence o nanopa icles [ 23 ]. Clus e ed (complex) DSBs can only be epai ed wi h di icul y [ 16 , 40 , 41 ] and we e ecognized as he main ac o esponsible o he supe io adiobiological e iciency (RBE) o densely ionizing adia ions. Hence, acco ding o his hypo hesis, a a gi en abso bed dose and i adia ion pa ame e s, nanopa icles boos cell killing by locally ampli ying he dose [ 42 ] and, in u n, DNA damage. Indeed, inc eased numbe s ela i e o un ea ed samples o single-s anded b eaks (SSBs) and DSBs we e measu ed in DNA i adia ed in he solu ion wi h a ious me al nanopa icles [ 43 ]. Since nanopa icles a e p e e en ially in e nalized and accumula ed by cance cells, e en passi ely due o mechanisms collec i ely known as he so-called Enhanced Pe meabili y In . J. Mol. Sci. 2019,20, 588 3 o 25 and Re en ion (EPR) e ec , hese enhancemen e ec s o adio he apy could be selec i ely a ge ed o umo s [ 44 – 50 ]. Mo eo e , some nanopa icles exe dual mul iple bene i s in cance ea men a he same ime— hey can be used as con as agen s in he anos ics [ 51 ] and/o ehicles o deli e y o a ious chemo he apeu ics o biological ea men compounds o he umo . Nanopa icles can be also unc ionalized (su ace ma e ial modi ica ion, a ached an ibodies, size, shape, e c.) o be e iden i y and in il a e he umo [ 23 , 48 ]. Mo eo e , nanopa icles can be used as imaging ags especially whe e pho o-bleaching has o be a oided [52,53]. A o emen ioned physical p edic ions on he mechanism o nanopa icle-media ed adiosensi iza ion we e con i med expe imen ally [ 51 ]. As al eady no iced, isola ed DNA showed inc eased agmen a ion a e being i adia ed in p esence o a ious nanopa icles [ 43 ]. In o he expe imen s, nanopa icles also inc eased cell dying when being added o cell cul u es p io o i adia ion [ 21 , 43 , 54 ]. Ne e heless, i is in ac no so easy o explain he nanopa icle-media ed cell adiosensi iza ion, despi e seemingly ideal co espondence be ween he heo e ical p edica ions and expe imen al esul s. The Achilles’ heel o he cu en “mains eam” hypo hesis ollowed om in si u/ in i o expe imen s showing ha nanopa icles, e en hose o e y small dimensions (e.g., o 2–3 nm in diame e , as used in his wo k), pene a e he cells bu no he cell nucleus [ 21 , 23 , 53 , 55 ] unless hey a e speci ically modi ied o his pu pose [ 42 ]. Nanopa icles o di e en ma e ials and sizes, en e ing he cells by pinocy osis ( e iewed in [ 56 ]), hus emain e ained inside he cy oplasm, whe e hey accumula e especially in endoplasmic esicles (endosomes) and lysosomes [ 21 , 55 , 57 ]. Unde some ci cums ances, nanopa icles may co-localize p e e en ially wi h he endoplasmic e iculum (ER) [ 23 , 58 ] and Golgi appa a us ( e iewed in [ 56 ]). In e es ingly, mi ochond ia, he only cy oplasmic o ganelles in human cells ha con ain hei own DNA, do no ep esen a p ima y a ge o nanopa icles, hough some nanopa icles can also be a ge ed o hese s uc u es ( e iewed in [ 56 ]). These indings pu in o play a ple ho a o a ious cellula p ocesses po en ially pa icipa ing in nanopa icle-media ed umo cell adiosensi iza ion. I is he e o e possible ha di e en nanopa icles do no sha e a common mode o ac ion, bo h in e ms o he ype o cell damage and i s unde lying mechanism ( e iewed in [59]). In he p esen wo k, we analyzed o di e en me al nanopa icles, whe he hei ex a- nuclea [ 21 – 23 , 55 ] p esence in cells can, by i sel o upon cell i adia ion, enhance damage o he nuclea DNA. In addi ion, we ollowed in de ail nanopa icle e ec s on he kine ics and e iciency o DNA epai in cells exposed o low-Linea Ene gy T ans e (LET) ionizing adia ion ( γ - and X- ays). We used high- esolu ion immuno luo escence con ocal mic oscopy (ICM) and single molecule localiza ion mic oscopy (SMLM) [ 38 , 60 ] o quan i y γ H2AX/53BP1 DSB epai oci o ma ion [ 61 ] and disassembly du ing a long pe iod o ime pos -i adia ion (PI) in cells exposed o di e en doses o γ /X- ays a e being o being no incuba ed wi h pla inum nanopa icles (P -NPs). On he basis o hese da a, p elimina y da a o gold nanopa icles (Au-NPs), and ou ea lie da a o gadolinium nanopa icles (Gd-NPs) [ 21 ], we discussed he e on wha is known abou me al nanopa icle e ec s on cells and po en ial mechanisms o nanopa icle-media ed adiosensi iza ion. In gene al, he aim o he ollowing s udy was o e i y whe he cell adiosensi iza ion by me al nanopa icles is co ela ed wi h escala ion o DNA damage and/o a ec ion o DNA damage epai capaci y. The esul s should con ibu e o a be e unde s anding o he mechanism by which a ious nanopa icles (di e en ma e ials and sizes) adiosensi ize cells wi h u u e a emp o a ionally design he apeu ically mo e e icien nanopa icles. 2. Resul s 2.1. Expe imen al Condi ions and App oaches We explo ed how pla inum (P ) and gold (Au) nanopa icles in luence DNA DSB induc ion and epai in h ee di e en cance cell ypes, U87 glioblas oma cells, HeLa ce ix cance cells and SkB 3 b eas cance cells, exposed o γ -( 137 Cs) o X- adia ion. U87 glioblas oma cells we e selec ed o hei high esis ance o adio he apy. HeLa cells, showing ela i ely lowe esis ance o adia ion, In . J. Mol. Sci. 2019,20, 588 4 o 25 we e hen in ol ed in o he s udy o explo e how umo cell ypes o di e en adiosensi i i ies and o igins espond o nanopa icle up ake and nanopa icle up ake ollowed by i adia ion. The SkB 3 model is known o be mo e adio esis an in compa ison o HeLa cells and was especially aken o supe - esolu ion localiza ion mic oscopy. The mechanism and kine ics o nanopa icle in e naliza ion we e in de ail e alua ed in ou p e ious s udies wi h Gd-NPs (3 ± 1 nm) [ 21 , 55 ] and Au-NPs (10 nm) [ 53 ] o a compa able size o P -NPs (2.6 nm) and Au-NPs (2.4 nm) used in he p esen s udy. We showed ha hese ul a ine Gd-NPs as well as he la ge Au-NPs e icien ly pene a e in o he cell cy oplasm bu emain es ic ed om he cell nucleus. E en sho (2 h) incuba ion wi h nanopa icles was p o ed o be su icien o ensu e hei in e naliza ion and cell adiosensi iza ion upon i adia ion wi h γ /X- ays [ 53 ]. The e o e, we used he compa ible condi ions also he e, hough lowe concen a ion and longe incuba ion pe iod (0.5 mM/6 h) we e p e e ed o ensu e su icien cell accumula ion bu minimize he po en ial cy o oxici y. Mode n, op- ech mic oscopy app oaches we e used in he p esen s udy o analyze DNA damage and epai wi h high p ecision. ICM allowed o quan i ica ion o DSBs du ing a long PI ime pe iod [ 16 ] (Figu e 1). When co-localized γ H2AX and 53BP1 epai oci a e used as DSB ma ke s, he sensi i i y o he me hod is clea ly supe io o e o he mode n me hods, including luo escence COMET assay (single cell gel elec opho esis) on single cells [ 20 ]. Newly de eloped SMLM, used he e o supe - esolu ion ul a-s uc u al analyses o epai oci [ 61 ], o e s e en be e esolu ion (up o 10–20 nm) and sensi i i y han ICM. Ne e heless, since highe numbe s o cells can be cu en ly analyzed by ICM, we ook ad an age o his me hod o de e mine he ex en o DSB induc ion and DSB epai kine ics in s a is ically ele an numbe s o spa ially ( h ee-dimensionally = 3D) ixed cells, s ill wi h a e y high c edibili y and ideli y o analysis. To u he inc ease he c edibili y o ou s udy, we sco ed he epai oci bo h manually and au oma ically. This also allowed us o compa e he posi i es and nega i es o bo h app oaches and o de e mine he in luence o γ H2AX/53BP1 ocus sco ing me hod on he esul s. In . J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 o 25 esolu ion localiza ion mic oscopy. The mechanism and kine ics o nanopa icle in e naliza ion we e in de ail e alua ed in ou p e ious s udies wi h Gd-NPs (3 ± 1 nm) [21,55] and Au-NPs (10 nm) [53] o a compa able size o P -NPs (2.6 nm) and Au-NPs (2.4 nm) used in he p esen s udy. We showed ha hese ul a ine Gd-NPs as well as he la ge Au-NPs e icien ly pene a e in o he cell cy oplasm bu emain es ic ed om he cell nucleus. E en sho (2 h) incuba ion wi h nanopa icles was p o ed o be su icien o ensu e hei in e naliza ion and cell adiosensi iza ion upon i adia ion wi h γ/X- ays [53]. The e o e, we used he compa ible condi ions also he e, hough lowe concen a ion and longe incuba ion pe iod (0.5 mM/6 h) we e p e e ed o ensu e su icien cell accumula ion bu minimize he po en ial cy o oxici y. Mode n, op- ech mic oscopy app oaches we e used in he p esen s udy o analyze DNA damage and epai wi h high p ecision. ICM allowed o quan i ica ion o DSBs du ing a long PI ime pe iod [16] (Figu e 1). When co-localized γH2AX and 53BP1 epai oci a e used as DSB ma ke s, he sensi i i y o he me hod is clea ly supe io o e o he mode n me hods, including luo escence COMET assay (single cell gel elec opho esis) on single cells [20]. Newly de eloped SMLM, used he e o supe - esolu ion ul a-s uc u al analyses o epai oci [61], o e s e en be e esolu ion (up o 10–20 nm) and sensi i i y han ICM. Ne e heless, since highe numbe s o cells can be cu en ly analyzed by ICM, we ook ad an age o his me hod o de e mine he ex en o DSB induc ion and DSB epai kine ics in s a is ically ele an numbe s o spa ially ( h ee-dimensionally = 3D) ixed cells, s ill wi h a e y high c edibili y and ideli y o analysis. To u he inc ease he c edibili y o ou s udy, we sco ed he epai oci bo h manually and au oma ically. This also allowed us o compa e he posi i es and nega i es o bo h app oaches and o de e mine he in luence o γH2AX/53BP1 ocus sco ing me hod on he esul s. Figu e 1. The abili y o immuno luo escence con ocal mic oscopy o quan i y DSBs (Double S and B eaks) in cells incuba ed wi h nanopa icles o incuba ed wi h nanopa icles and consecu i ely i adia ed. DSBs we e quan i ied by he means o immuno luo escence de ec ion o co-localized γH2AX (g een) and 53BP1 ( ed) epai oci, he DSB ma ke s. The nucleus o an illus a i e U87 cell exposed o 2 Gy o γ- ays and spa ially ( h ee-dimensionally = 3D) ixed a 2 h pos -i adia ion (PI) is shown as: (A) a maximum in ensi y p ojec ion o 40 con ocal slices (0.3 µm hick; “maximum image”) o (B) a single con ocal slice (0.3 µm hick) in e sec ing he indica ed (whi e a ow) γH2AX/53BP1 ocus. Images a e displayed in all h ee (in he x-y, x-z and y-z) planes, and ch oma in is coun e s ained wi h TO-PRO-3 (a i icially blue). (C) An example o compu a ional de ec ion o co- localized (yellow) γH2AX (g een) and 53BP1 ( ed) epai oci in 3D space (Aqua ium So wa e). 2.2. P -NP and Au-NP Sho -Te m Geno oxici y— he E ec on Nuclea DNA in Non-I adia ed Cells Fi s ly, we analyzed po en ial nega i e in luence o 2.6 nm P -NPs and 2.4 nm Au-NPs on he nuclea DNA o U87 and HeLa cells be o e i adia ion. Cells we e cul u ed wi h P -NPs o Au-NPs in he concen a ion o 0.5 mM o 6 h and po en ial induc ion o γH2AX/53BP1 (DSB) oci was s udied as an indica o o nanopa icle-media ed geno oxici y. The epai oci ha e been p esen in bo h U87 and HeLa cell ypes al eady p io o incuba ion wi h nanopa icles and γH2AX oci mos ly Figu e 1. The abili y o immuno luo escence con ocal mic oscopy o quan i y DSBs (Double S and B eaks) in cells incuba ed wi h nanopa icles o incuba ed wi h nanopa icles and consecu i ely i adia ed. DSBs we e quan i ied by he means o immuno luo escence de ec ion o co-localized γ H2AX (g een) and 53BP1 ( ed) epai oci, he DSB ma ke s. The nucleus o an illus a i e U87 cell exposed o 2 Gy o γ - ays and spa ially ( h ee-dimensionally = 3D) ixed a 2 h pos -i adia ion (PI) is shown as: ( A ) a maximum in ensi y p ojec ion o 40 con ocal slices (0.3 µ m hick; “maximum image”) o ( B ) a single con ocal slice (0.3 µ m hick) in e sec ing he indica ed (whi e a ow) γ H2AX/53BP1 ocus. Images a e displayed in all h ee (in he x-y, x-z and y-z) planes, and ch oma in is coun e s ained wi h TO-PRO-3 (a i icially blue). ( C ) An example o compu a ional de ec ion o co-localized (yellow) γH2AX (g een) and 53BP1 ( ed) epai oci in 3D space (Aqua ium So wa e). In . J. Mol. Sci. 2019,20, 588 5 o 25 2.2. P -NP and Au-NP Sho -Te m Geno oxici y— he E ec on Nuclea DNA in Non-I adia ed Cells Fi s ly, we analyzed po en ial nega i e in luence o 2.6 nm P -NPs and 2.4 nm Au-NPs on he nuclea DNA o U87 and HeLa cells be o e i adia ion. Cells we e cul u ed wi h P -NPs o Au-NPs in he concen a ion o 0.5 mM o 6 h and po en ial induc ion o γ H2AX/53BP1 (DSB) oci was s udied as an indica o o nanopa icle-media ed geno oxici y. The epai oci ha e been p esen in bo h U87 and HeLa cell ypes al eady p io o incuba ion wi h nanopa icles and γ H2AX oci mos ly co-localized wi h 53BP1 p o ein. This obse a ion poin s o a pe manen exis ence o DSBs in U87 and HeLa cells, which is in acco dance wi h hei umo ous na u e associa ed wi h genomic ins abili y (Figu e 2). U87 cells ca ied highe numbe s o he oci han HeLa cells, wi h he mean alues o 3.47 and 2.03, espec i ely. Figu es 3–5(0 min pos i adia ion (PI) in all g aphs) show ha he a e age/median numbe s o he oci pe nucleus we e almos iden ical (manual analysis, Figu e 3) o inc eased sligh ly (au oma ic analysis, Figu es 4and 5) a e incuba ion o cells wi h P -NPs. The mean numbe s o co-localized γ H2AX/53BP1 oci pe nucleus, p o ided by he au oma ic analyses, we e 4.34 o U87 (Figu e 3) and 3.88 HeLa cells (Figu e 5). Such a di e ences, s a is ically signi ican hough (U87: p= 0.010; HeLa: p= 0.003), a e no suppo i e o biologically mo e ele an geno oxici y o he nanopa icles s udied (2.6 nm P -NPs, and 2.4 nm Au-NPs; Figu e 6), a leas in e ms o inc eased DNA agmen a ion, consequen ly leading o genome ea angemen s. Ne e heless, ou s udies limi ed o DSB induc ion canno exclude a “milde ” e ec o nanopa icles on he DNA molecule, mani es ed o ins ance as oxida i e base modi ica ions. This kind o DNA damage may appea due o nanopa icle-media ed p oduc ion o eac i e oxygen species (ROS), which was equen ly epo ed in he li e a u e as he main cause o nanopa icle cy o oxici y. Mo eo e , especially in he con ex o wha will ollow, a nega i e po en ial o cy oplasmically localized nanopa icles may be p e e en ially o e en exclusi ely a ge ed o he cy oplasmic s uc u es. To summa ize, ou obse a ions did no e eal mo e p ominen geno oxici y o 2.6 nm pla inum nanopa icles a e sho - e m (6 h) incuba ion wi h U87 and HeLa cells, bu mo e expe imen s a e needed o comp ehend po en ial cy o oxic e ec s o hese nanopa icles in a mo e comp ehensi e way. P elimina y esul s seem o con i m his conclusion also o 2.4 nm Au-NPs. 2.3. DSB Induc ion and Repai in U87 Cance Cells T ea ed o No -T ea ed wi h Me al Nanopa icles P io o I adia ion A e excluding he possibili y ha he s udied 2.6 nm P -NPs and 2.4 nm Au-NPs ma kedly inc ease γ H2AX/53BP1 ocus (DSB) o ma ion e en by hemsel es, i.e., al eady in non-i adia ed cells, we analyzed whe he hese nanopa icles can enhance DSB induc ion o a ec DSB epai capaci y o U87 and HeLa cells upon i adia ion. The si ua ion was compa ed o wo γ - ay doses, 2 Gy and 4 Gy. We decided o a 2 Gy dose since his exposu e is equen ly used in clinical p ac ice as a single ac ion dose deli e ed o pa ien s du ing a ac iona ed he apy. The highe dose o 4 Gy was applied in o de o gene a e la ge numbe s o DSBs and explo e di e ences be ween samples wi h be e sensi i i y (since he di e ences in DSB numbe s pe nucleus may be only small o low doses and he e o e dis inguishable om na u al a iabili y only wi h di icul y). Figu e 2compa es he γ H2AX/53BP1 ocus (DSB) o ma ion and epai kine ics o U87 cells ea ed o no - ea ed wi h 2.6 nm P -NPs p io o i adia ion wi h 4 Gy o γ - ays. Rep esen a i e cell nuclei o bo h cell popula ions a e displayed o di e en pe iods o ime PI up o 48 h PI. Independen ly o he nanopa icle ea men , i is e iden om Figu e 2 ha γ H2AX oci a e only incomple ely o med in U87 cells ea ly a e i adia ion (5–30 min PI) and also hei co-localiza ion wi h 53BP1 epai p o ein is e y low. Co espondingly, he backg ound signals (i.e., he p opo ions o γ H2AX and especially 53BP1 molecules ou side oci) a e o en high. A simila “pic u e” has also been epo ed o U87 cells exposed o hea y ions [ 62 , 63 ]. Wi h ongoing ime a e i adia ion, γ H2AX and 53BP1 oci g ow bo h in numbe and size and hei mu ual co-localiza ion inc eases oo. Fo bo h cell ypes (U87, and HeLa) and adia ion doses (4 Gy, and 2 Gy), he numbe o co-localized γ H2AX and 53BP1 oci eached he maximum be ween 30 min and 1 h PI. La e on, he numbe o oci s a ed o dec ease, In . J. Mol. Sci. 2019,20, 588 6 o 25 while he size o oci g adually inc eased and he ex en o co-localiza ion be ween γ H2AX and 53BP1 emained e y high. Impo an ly (as quan i ied la e ), we did no obse e any isual di e ence be ween nanopa icle- ea ed cells and hei un ea ed coun e pa s wi h ega d o he ex en o DSB induc ion and epai kine ics. In . J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 o 25 Figu e 2. γH2AX/53BP1 oci (DSB) o ma ion and epai kine ics in U87 cells incuba ed o no incuba ed wi h 2.6 nm pla inum nanopa icles (P -NPs; 0.5 mM o 6 h) and consequen ly i adia ed wi h 4 Gy o γ- ays. Maximum images (see Figu e 1) a e displayed o ep esen a i e nuclei o cells ha we e spa ially (3D) ixed in he indica ed pe iods o ime PI. Fo he nucleus ixed a 2 h PI, γH2AX oci (inse ed G- channel panel) and 53BP1 oci (inse ed R-channel panel) a e also shown sepa a ely o demons a e hei mu ual co-localiza ion. γH2AX (g een), 53BP1 ( ed), and ch oma in coun e s ained wi h TO-PRO-3 (a i icially blue). None-IR igu es co espond o non-i adia ed cells. The quan i a i e esul s ob ained o di e en ways o analysis (i.e., manual and au oma ed) and wo adia ion doses (4 Gy and 2 Gy) a e summa ized in Figu es 3–5. Figu e 3 compa es he a e age/median numbe s o γH2AX/53BP1 oci pe nucleus oge he wi h he ocus numbe dis ibu ions as gained by manual analysis o U87 cells exposed o 4 Gy o γ- ays in p esence and absence o P -NPs, espec i ely. Excep o wo la e ime poin s PI (4 h and 24 h PI), all s a is ical cha ac e is ics (means, medians, and dis ibu ions) a e almos iden ical o nanopa icle- ea ed and un ea ed cells. Figu e 2. γ H2AX/53BP1 oci (DSB) o ma ion and epai kine ics in U87 cells incuba ed o no incuba ed wi h 2.6 nm pla inum nanopa icles (P -NPs; 0.5 mM o 6 h) and consequen ly i adia ed wi h 4 Gy o γ - ays. Maximum images (see Figu e 1) a e displayed o ep esen a i e nuclei o cells ha we e spa ially (3D) ixed in he indica ed pe iods o ime PI. Fo he nucleus ixed a 2 h PI, γ H2AX oci (inse ed G-channel panel) and 53BP1 oci (inse ed R-channel panel) a e also shown sepa a ely o demons a e hei mu ual co-localiza ion. γ H2AX (g een), 53BP1 ( ed), and ch oma in coun e s ained wi h TO-PRO-3 (a i icially blue). None-IR igu es co espond o non-i adia ed cells. In . J. Mol. Sci. 2019,20, 588 7 o 25 In . J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 o 25 Figu e 3. Manual analysis o he ex en o γH2AX+53BP1 ocus (DSB) induc ion and epai kine ics in U87 glioblas oma cells i adia ed wi h 4 Gy o γ- ays compa ed wi h cells ea ed (0.5 mM o 6 h) and no ea ed p io o i adia ion wi h 2.6 nm pla inum nanopa icles (P -NPs). The a e age and median numbe s o co-localized γH2AX + 53BP1 epai oci (i.e., DSBs) pe nucleus a e shown o di e en pe iods o ime PI, oge he wi h he ocus numbe dis ibu ions in each cell popula ion. The boxes include 50% o he alues (25 h o 75 h pe cen ile) cen e ed on he median ( he ho izon al line h ough he box). The mean alues a e ep esen ed by he squa es wi hin he boxes. The ou lie s we e iden i ied acco ding o he 1.5*IQR me hod (IQR = in e qua ile ange). P —samples ea ed wi h pla inum nanopa icles, m— he pe iod o ime a e i adia ion in minu es, 0 m—non-i adia ed samples. (a) (b) Figu e 4. So wa e analysis o he ex en o γH2AX+53BP1 ocus (DSB) induc ion and epai kine ics in U87 glioblas oma cells i adia ed wi h 4 Gy (a) o 2 Gy (b) o γ- ays compa ed wi h cells ea ed (0.5 mM o 6 h) o no ea ed p io o i adia ion wi h 2.6 nm pla inum nanopa icles (P -NPs). The a e age and median numbe s o co-localized γH2AX + 53BP1 epai oci (i.e., DSBs) pe nucleus a e P P P P P P P P -------- 0 m 5 m 30 m 1 h 2 h 4 h 24 h 48 h 0 50 100 Time pos -i adia ion (PI) T ea men wi h nanopa icles Numbe o oci 25%~75% Range wi hin 1.5IQR Median Line Mean ### P P P P P P P P -------- 0 m 5 m 30 m 1 h 2 h 4 h 24 h 48 h 0 50 100 Time pos -i adia ion (PI) T ea men wi h nanopa icles Numbe o oci 25%~75% Range wi hin 1.5IQR Median Line Mean ### P P P P P P P P -------- 0 m 5 m 30 m 1 h 2 h 4 h 8 h 24 h 0 50 Time pos -i adia ion (PI) T ea men wi h nanopa icles Numbe o oci 25%~75% Range wi hin 1.5IQR Median Line Mean ### Figu e 3. Manual analysis o he ex en o γ H2AX+53BP1 ocus (DSB) induc ion and epai kine ics in U87 glioblas oma cells i adia ed wi h 4 Gy o γ - ays compa ed wi h cells ea ed (0.5 mM o 6 h) and no ea ed p io o i adia ion wi h 2.6 nm pla inum nanopa icles (P -NPs). The a e age and median numbe s o co-localized γ H2AX + 53BP1 epai oci (i.e., DSBs) pe nucleus a e shown o di e en pe iods o ime PI, oge he wi h he ocus numbe dis ibu ions in each cell popula ion. The boxes include 50% o he alues (25 h o 75 h pe cen ile) cen e ed on he median ( he ho izon al line h ough he box). The mean alues a e ep esen ed by he squa es wi hin he boxes. The ou lie s we e iden i ied acco ding o he 1.5*IQR me hod (IQR = in e qua ile ange). P —samples ea ed wi h pla inum nanopa icles, m— he pe iod o ime a e i adia ion in minu es, 0 m—non-i adia ed samples. The quan i a i e esul s ob ained o di e en ways o analysis (i.e., manual and au oma ed) and wo adia ion doses (4 Gy and 2 Gy) a e summa ized in Figu es 3–5. Figu e 3compa es he a e age/median numbe s o γ H2AX/53BP1 oci pe nucleus oge he wi h he ocus numbe dis ibu ions as gained by manual analysis o U87 cells exposed o 4 Gy o γ - ays in p esence and absence o P -NPs, espec i ely. Excep o wo la e ime poin s PI (4 h and 24 h PI), all s a is ical cha ac e is ics (means, medians, and dis ibu ions) a e almos iden ical o nanopa icle- ea ed and un ea ed cells. The au oma ed image analysis (Figu e 4a) o he same cells ha we e p e iously e alua ed manually p o ided much lowe numbe s o γ H2AX/53BP1 oci compa ed o ha in he manual analysis, especially a he ea ly pe iods o ime PI (up o 1 h PI). The maximum numbe s o oci pe nucleus we e de ec ed a 1 h PI in all samples, i espec i e o he nanopa icle ea men and he way o analysis. Du ing his pe iod o ime, abou 50 oci pe nucleus we e coun ed manually while his alue dec eased o abou 35 wi h he au oma ed analysis. Taking in o accoun p e ious epo s showing ha 1 Gy o γ - ays gene a es ~9–35 γ H2AX oci pe nucleus on a e age, depending on he cell ype, he esul s o he manual analysis (mean = 12.5 oci/nucleus/Gy) can be conside ed as mo e ealis ic in e ms o absolu e numbe s. A lowe sensi i i y o au oma ic analysis ollows om he ac ha compu a ional pa ame e s o ocus sco ing we e se e y s ic ly, jus o de ec only well-de eloped oci wi h an ex ensi e o e lap be ween γ H2AX and 53BP1. The eason o his se ing was o elimina e po en ial unce ain y wi h iden i ica ion o small and/o imma u e oci since hese oci could no be o en easily sepa a ed om he backg ound signal. Consis en ly, mo e p ominen di e ences In . J. Mol. Sci. 2019,20, 588 8 o 25 be ween he manual and au oma ed analysis appea ed a he sho e ime poin s PI (up o 1 h PI), i.e., du ing he pe iod o ime when he ep esen a ion o imma u e oci was high, especially in U87 cells. Unde such condi ions, au oma ic so wa e analysis is s ill ex emely di icul and manual analysis p omises mo e p ecise esul s, especially in e ms o coun ing he absolu e ocus numbe s. On he o he hand, compu a ional analysis ensu es de ec ion o only p ecisely speci ied oci and he e o e high ep oducibili y and objec i i y o esul s, independen ly o he obse e expe ience. In . J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 o 25 Figu e 3. Manual analysis o he ex en o γH2AX+53BP1 ocus (DSB) induc ion and epai kine ics in U87 glioblas oma cells i adia ed wi h 4 Gy o γ- ays compa ed wi h cells ea ed (0.5 mM o 6 h) and no ea ed p io o i adia ion wi h 2.6 nm pla inum nanopa icles (P -NPs). The a e age and median numbe s o co-localized γH2AX + 53BP1 epai oci (i.e., DSBs) pe nucleus a e shown o di e en pe iods o ime PI, oge he wi h he ocus numbe dis ibu ions in each cell popula ion. The boxes include 50% o he alues (25 h o 75 h pe cen ile) cen e ed on he median ( he ho izon al line h ough he box). The mean alues a e ep esen ed by he squa es wi hin he boxes. The ou lie s we e iden i ied acco ding o he 1.5*IQR me hod (IQR = in e qua ile ange). P —samples ea ed wi h pla inum nanopa icles, m— he pe iod o ime a e i adia ion in minu es, 0 m—non-i adia ed samples. (a) (b) Figu e 4. So wa e analysis o he ex en o γH2AX+53BP1 ocus (DSB) induc ion and epai kine ics in U87 glioblas oma cells i adia ed wi h 4 Gy (a) o 2 Gy (b) o γ- ays compa ed wi h cells ea ed (0.5 mM o 6 h) o no ea ed p io o i adia ion wi h 2.6 nm pla inum nanopa icles (P -NPs). The a e age and median numbe s o co-localized γH2AX + 53BP1 epai oci (i.e., DSBs) pe nucleus a e P P P P P P P P -------- 0 m 5 m 30 m 1 h 2 h 4 h 24 h 48 h 0 50 100 Time pos -i adia ion (PI) T ea men wi h nanopa icles Numbe o oci 25%~75% Range wi hin 1.5IQR Median Line Mean ### P P P P P P P P -------- 0 m 5 m 30 m 1 h 2 h 4 h 24 h 48 h 0 50 100 Time pos -i adia ion (PI) T ea men wi h nanopa icles Numbe o oci 25%~75% Range wi hin 1.5IQR Median Line Mean ### P P P P P P P P -------- 0 m 5 m 30 m 1 h 2 h 4 h 8 h 24 h 0 50 Time pos -i adia ion (PI) T ea men wi h nanopa icles Numbe o oci 25%~75% Range wi hin 1.5IQR Median Line Mean ### Figu e 4. So wa e analysis o he ex en o γ H2AX+53BP1 ocus (DSB) induc ion and epai kine ics in U87 glioblas oma cells i adia ed wi h 4 Gy ( a ) o 2 Gy ( b ) o γ - ays compa ed wi h cells ea ed (0.5 mM o 6 h) o no ea ed p io o i adia ion wi h 2.6 nm pla inum nanopa icles (P -NPs). The a e age and median numbe s o co-localized γ H2AX + 53BP1 epai oci (i.e., DSBs) pe nucleus a e shown o di e en pe iods o ime PI, oge he wi h he ocus numbe dis ibu ions in each cell popula ion. The boxes include 50% o he alues (25 h o 75 h pe cen ile) cen e ed on he median ( he ho izon al line h ough he box). The mean alues a e ep esen ed by he squa es wi hin he boxes. The ou lie s we e iden i ied acco ding o he 1.5*IQR me hod (IQR = in e qua ile ange). P —samples ea ed wi h pla inum nanopa icles, m— he pe iod o ime a e i adia ion in minu es, 0 m—non-i adia ed samples. Ano he mo i a ion o es ic he au oma ed analysis selec i ely on well-de eloped oci ollowed om he ques ion whe he nanopa icles in i adia ed cells may di e en ly in luence gene a ion o epai o small and la ge γ H2AX oci ( he smalle oci we e sco ed as DSBs by he manual analysis bu no au oma ed analysis). Excep as desc ibed, bo h app oaches p o ided e y simila esul s despi e o he di e en cha ac e s o manual and au oma ed ocus coun ing. Impo an ly, as o he manual analysis, he a e age numbe s, medians, and dis ibu ions o γ H2AX/53BP1 oci a ied only inapp eciably be ween U87 cells i adia ed (4 Gy) wi h P -NPs p esen o absen . Ve y simila esul s o nanopa icle- ea ed and un ea ed samples we e ound also a 4 h PI and 24 h PI (Figu e 4a), making he di e ences ob ained o hese ime poin s by he manual analysis a he a de ia ion om o he wise igh ly “o e lapping” γ H2AX/53BP1 ocus p o iles in ime PI han a biologically ele an esul . Fo he lowe adia ion dose o 2 Gy o γ - ays (equi alen o a common single daily dose in ac iona ed adio he apy), he same esul s as o he highe dose o 4 Gy we e acqui ed (Figu e 4b). Again, e y simila numbe s o γH2AX+53BP1 oci pe nucleus we e coun ed in i adia ed U87 cells, i espec i e o hei incuba ion wi h P -NPs. Sligh ly highe mean numbe s o γ H2AX oci pe nucleus we e ecognized in nanopa icle- ea ed cells compa ed o hose in un ea ed ones only a 8 h and 24 h PI; howe e , compa able medians o he compa ed samples do no suppo exis ence o signi ican di e ences be ween P -NP-con aining cells and con ols e en a hese pe iods o ime. In . J. Mol. Sci. 2019,20, 588 9 o 25 In . J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 8 o 25 shown o di e en pe iods o ime PI, oge he wi h he ocus numbe dis ibu ions in each cell popula ion. The boxes include 50% o he alues (25 h o 75 h pe cen ile) cen e ed on he median ( he ho izon al line h ough he box). The mean alues a e ep esen ed by he squa es wi hin he boxes. The ou lie s we e iden i ied acco ding o he 1.5*IQR me hod (IQR = in e qua ile ange). P —samples ea ed wi h pla inum nanopa icles, m— he pe iod o ime a e i adia ion in minu es, 0 m—non- i adia ed samples. The au oma ed image analysis (Figu e 4a) o he same cells ha we e p e iously e alua ed manually p o ided much lowe numbe s o γH2AX/53BP1 oci compa ed o ha in he manual analysis, especially a he ea ly pe iods o ime PI (up o 1 h PI). The maximum numbe s o oci pe nucleus we e de ec ed a 1 h PI in all samples, i espec i e o he nanopa icle ea men and he way o analysis. Du ing his pe iod o ime, abou 50 oci pe nucleus we e coun ed manually while his alue dec eased o abou 35 wi h he au oma ed analysis. Taking in o accoun p e ious epo s showing ha 1 Gy o γ- ays gene a es ~9–35 γH2AX oci pe nucleus on a e age, depending on he cell ype, he esul s o he manual analysis (mean = 12.5 oci/nucleus/Gy) can be conside ed as mo e ealis ic in e ms o absolu e numbe s. A lowe sensi i i y o au oma ic analysis ollows om he ac ha compu a ional pa ame e s o ocus sco ing we e se e y s ic ly, jus o de ec only well- de eloped oci wi h an ex ensi e o e lap be ween γH2AX and 53BP1. The eason o his se ing was o elimina e po en ial unce ain y wi h iden i ica ion o small and/o imma u e oci since hese oci could no be o en easily sepa a ed om he backg ound signal. Consis en ly, mo e p ominen di e ences be ween he manual and au oma ed analysis appea ed a he sho e ime poin s PI (up o 1 h PI), i.e., du ing he pe iod o ime when he ep esen a ion o imma u e oci was high, especially in U87 cells. Unde such condi ions, au oma ic so wa e analysis is s ill ex emely di icul and manual analysis p omises mo e p ecise esul s, especially in e ms o coun ing he absolu e ocus numbe s. On he o he hand, compu a ional analysis ensu es de ec ion o only p ecisely speci ied oci and he e o e high ep oducibili y and objec i i y o esul s, independen ly o he obse e expe ience. Figu e 5. Au oma ed analysis o he ex en o γH2AX + 53BP1 ocus (DSB) induc ion and epai kine ics compa ed o HeLa cells i adia ed wi h 4 Gy o γ- ays in p esence (0.5 mM o 6 h) o absence o 2.6 nm P -NPs. The a e age and median numbe s o co-localized γH2AX+53BP1 oci (i.e., DSBs) pe nucleus a e shown o di e en pe iods o ime PI, oge he wi h he ocus numbe dis ibu ions in each cell popula ion. The boxes include 50% o he alues (25 h o 75 h pe cen ile) cen e ed on he median ( he ho izon al line h ough he box). The mean alues a e ep esen ed by he squa es wi hin he boxes. The ou lie s we e iden i ied acco ding o he 1.5*IQR me hod (IQR = in e qua ile ange). P P P P P P P P -------- 0 m 5 m 30 m 1 h 2 h 4 h 24 h 48 h 0 50 Time pos -i adia ion (PI) T ea men wi h nanopa icles Numbe o oci 25%~75% Range wi hin 1.5IQR Median Line Mean ### Figu e 5. Au oma ed analysis o he ex en o γ H2AX + 53BP1 ocus (DSB) induc ion and epai kine ics compa ed o HeLa cells i adia ed wi h 4 Gy o γ - ays in p esence (0.5 mM o 6 h) o absence o 2.6 nm P -NPs. The a e age and median numbe s o co-localized γ H2AX+53BP1 oci (i.e., DSBs) pe nucleus a e shown o di e en pe iods o ime PI, oge he wi h he ocus numbe dis ibu ions in each cell popula ion. The boxes include 50% o he alues (25 h o 75 h pe cen ile) cen e ed on he median ( he ho izon al line h ough he box). The mean alues a e ep esen ed by he squa es wi hin he boxes. The ou lie s we e iden i ied acco ding o he 1.5*IQR me hod (IQR = in e qua ile ange). P —samples ea ed wi h pla inum nanopa icles, m— he pe iod o ime a e i adia ion in minu es, 0 m—non-i adia ed samples. In . J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 10 o 25 inco po a ion o hese la ge Au-NPs, incuba ion o cells was hold o 16 h p io o i adia ion (Figu e 6). Fo y- i e minu es a e i adia ion, he specimens we e ixed and subjec ed o SMLM ollowed by so wa e analysis o he H2AX labelling ags and hei mu ual dis ances. In Figu e 7, ypical nex - neighbo densi y images a e shown. In con as o aw SMLM images showing jus he posi ioning o luo och omes wi h high p ecision (10–20 nm), hese images encode he densi y o nex neighbo s in a 1000 nm en i onmen by in ensi y. A a i s glimpse, i seems ha he cells wi h inco po a ed Au-NPs o m mo e in ensi e oci, i.e., oci wi h mo e poin signals han he cells ha we e i adia ed wi h he same dose bu wi hou Au-NPs. In he case o he non-i adia ed con ol, a andom dis ibu ion may be suppo ed by he isual imp ession, which con as s wi h signal clus e ing in all i adia ed cells. A mo e quan i a i e analysis based on Ripley’s K- and L- alues [38] e ealed a non- andom dis ance dis ibu ion in all i adia ed cell samples as i is shown o a case a e 500 mGy adia ion exposu e wi hou Au-NPs (Figu e 8). This esul indica es ha , in all cases, clus e ing o γH2AX labelling ags can be expec ed. The e o e, we u he s udied he dis ance equencies in o de o ind ou whe he he gene al γH2AX pa e n is di e ing o he adia ion doses and/o nanopa icle ea men condi ions (Figu e 9). In all i adia ed cells, he a e age dis ance be ween γH2AX poin s was be ween 20 and 25 nm. Impo an ly, his γH2AX pa e n did no change in specimens ea ed wi h Au-NPs. Figu e 6. An illus a i e Single Molecule Localiza ion Mic oscopy (SMLM) image o an SkB 3 cell a e up ake o 10 nm Au-NPs in he cy osol. The Au-NPs show a luo escen blinking a e lase illumina ion a 594 nm. Each poin hus ep esen s a single Au nanopa icle. Whe eas he cy osol seems o be ull o nanopa icles, he nucleus is emp y. The poin s o low in ensi y seemingly co e ing he nucleus in he image ei he a e he backg ound o belong o ou -o - ocus image planes abo e o below he nucleus. Scale ba 1 µm. The numbe s o γH2AX labelling ags can be used o de e mine he dependence o DNA damage ex en on adia ion dose and p esence o nanopa icles [61]. Hence, we cons uc ed p elimina y dose– e iciency cu es o X- ay doses up o 4 Gy and compa ed he numbe s o γH2AX signal poin s in cells incuba ed o no incuba ed wi h Au-NPs (Figu e 10). A sligh linea inc ease in he numbe o γH2AX poin s was egis e ed up o 2 Gy. In his dose in e al, he cu es we e compa able o cells wi h and wi hou Au-NP inco po a ion. In e es ingly, a s eep inc ease o he cu e appea ed be ween 2 Gy and 4 Gy a e Au-NP inco po a ion, which was no obse ed in he con ol. In addi ion, he SMLM da a indica e ha he dose enhancemen e ec s, as indica ed by γH2AX signals, may be small, especially in dose anges up o 2 Gy, which suppo s he da a ob ained abo e by ICM and oci coun ing. Howe e , u he expe imen s wi h o he cell lines seem o be necessa y o making he inal conclusions. Figu e 6. An illus a i e Single Molecule Localiza ion Mic oscopy (SMLM) image o an SkB 3 cell a e up ake o 10 nm Au-NPs in he cy osol. The Au-NPs show a luo escen blinking a e lase illumina ion a 594 nm. Each poin hus ep esen s a single Au nanopa icle. Whe eas he cy osol seems o be ull o nanopa icles, he nucleus is emp y. The poin s o low in ensi y seemingly co e ing he nucleus in he image ei he a e he backg ound o belong o ou -o - ocus image planes abo e o below he nucleus. Scale ba 1 µm. In . J. Mol. Sci. 2019,20, 588 16 o 25 li e span o animals bea ing umo (9L cell gliosa coma in b ain) o he inhibi ion o umo g ow h (A375sc melanoma in lank) when he animals we e ea ed by adio he apy a e in a enous (9L gliosa coma) o in a umo al (melanoma) injec ion whe eas he majo i y o he nanopa icles in he umo we e suspec ed o be ou side he cells. Mo eo e , we also obse ed in p elimina y expe imen s ha he numbe o γ H2AX is almos he same when i adia ion is pe o med in p esence o in absence o he adiosensi izing nanopa icles (unpublished esul s). Hence, hese nanopa icles seem o sensi ize cells o adia ion h ough cy oplasmic e ec s ha a e independen o DNA damage and/o epai . While ou indings do no exclude he possibili y ha some ypes o nanopa icles suppo adia ion cell killing h ough he “classic” DNA damage-based mechanism, hey open he doo o exi ing esea ch o new mechanisms ha could be dominan unde some ci cums ances, as o ins ance ch oma in opology- ela ed e ec s and e-a angemen s o compac ion o ms. Fu he mo e, accumula ion o nanopa icles in endosomes and lysosomes as e ealed in ou ea lie epo s [ 21 , 55 ] could esul in damage o hese s uc u es wi h impo an consequences. While lysosomes we e o iginally hough only as cellula dus bins, ecen s udies in ol e lysosomes in impo an cell signaling pa hways, e en ually ini ia ing apop osis (see [ 32 ] and ci a ions he ein). In addi ion, e en simple dis up ion o a la ge amoun o lysosomes due o hei memb ane damage by locally ampli ied adia ion e ec s, media ed by in a-lysosomal nanopa icle accumula ions, may esul in massi e leakage o ly ic enzymes om hese “suicide bags” [ 65 , 66 ] and ex ensi e cy oplasmic damage. This can also ini ia e cell dea h. Indeed, he des abiliza ion o lysosomes ia lysosomal memb ane pe meabiliza ion (LMP), leading o elease o hei agg essi e con en in o he cy oplasm, is cu en ly in ensi ely s udied as a po en ially e icien way o he apeu ic cell dea h igge ing [56]. Cy oplasmically loca ed nanopa icles may also in luence o ganelles o s uc u es which hey do no co-localize wi h. Fo ins ance, inc eased p oduc ion o ROS has been equen ly epo ed in he li e a u e as a main cause o nanopa icles’ cy o oxici y. The e o e, ROS gene a ed by nanopa icles in ex ensi e amoun s upon i adia ion may damage o ganelles loca ed in close p oximi y o nanopa icle loca ion si es, o ins ance mi ochond ia. Among o he cy oplasmic a ge s, mi ochond ia a e especially a ac i e since hey a e c i ical o cell su i al (ene gy me abolism) and ep esen he only ex acellula s uc u es ha ing hei own DNA. The e o e, nanopa icle-media ed agmen a ion o mi ochond ial DNA may ep esen an elegan modi ica ion o he “classic” DNA damage-based hypo hesis on cell adiosensi iza ion by nanopa icles, e u ning his idea in o he game. I should also be no ed ha ROS a e e ec i e signaling molecules wi h a s ong po en ial o di ec ly in luence biochemical cellula pa hways. The Endoplasmic Re iculum (ER) may ep esen ano he a ge o nanopa icle e ec s. While he e icien unc ioning o he ER is essen ial o mos cellula ac i i ies and su i al, i may be unde some modi ica ions also in aded by nanopa icles [ 23 ]. Mo eo e , ER plays an impo an ole in he esponse o oxida i e s ess-induced damage and is qui e sensi i e o ROS [ 67 ]. Hence, i adia ed nanopa icles may exe cy o oxic e ec s on cells by modula ing ER s ess [ 67 ]. Fo ins ance, Ag-NPs esul ed in cy o oxici y and cell dea h by apop o ic, which was associa ed wi h (seconda y) DNA agmen a ion [ 67 ]. This obse a ion no only explains how nanopa icles may ini ia e cell dea h h ough dis u bing unc ions o ER, bu also s esses he impo ance o ime when in e p e ing he nanopa icle-media ed DNA e ec s. In his ligh , i is possible ha in some s udies he nanopa icle-media ed e ec s on DNA can a he e lec his seconda y apop o ic DNA agmen a ion han p ima y enhancemen o DSB induc ion by adia ion. The mechanism, how ER s ess can lead o apop osis, has been desc ibed by [ 68 ]. A dis up ion o ER unc ion leads o accumula ion and agg ega ion o un olded p o eins accompanied wi h s ess signaling. The s ess signals a e de ec ed by ansmemb ane ecep o s, which in u n ini ia e he un olded p o ein esponse (UPR) ying o es o e no mal ER unc ions. Howe e , i he s ess pe sis s oo long, apop o ic cell dea h ensues [68]. Al oge he , we show ha he adiosensi izing e ec o a leas some me al nanopa icles may ely on cy oplasmic p ocesses a he han DNA damaging e en s. Based on he a ailable li e a u e, we also ou line he way o how damage o he mos ele an cy oplasmic s uc u es may ini ia e cell In . J. Mol. Sci. 2019,20, 588 17 o 25 dea h. Though we did no obse e di e en esponses o nanopa icles o i adia ion in p esence o nanopa icles o he wo s udied cell ypes (U87, and HeLa), we emphasize he necessi y o analyze in de ail each pa icula combina ion o nanopa icles and he cell ype planned o be he apeu ically a ge ed. This impe a i e ollows om ex ensi e con o e sies ha a e s ill p esen in he li e a u e on he nanopa icle-media ed i adia ion e ec s. Fo ins ance, Au-NPs induced apop osis in MCF-7 and N87 cance cell lines by dis up ing lysosomes and mi ochond ia, bu his e ec did no appea in no mal Chinese hams e o a y (CHO) and 293T cell lines. This obse a ion u he suppo s ou conclusion ha nanopa icle-media ed cell killing enhancemen may be loca ed in he cy oplasm, bu mo e impo an ly gi es a pe spec i e o selec i e nanopa icle oxici y o umo cells [ 64 , 69 ]. In e es ingly, om he opposi e poin o iew, some adio-p o ec i e chemicals (ami os ine) p o ec no mal cells om adia ion e ec s bu delay DSB epai in umo cells [20]. The inal ques ion emains whe he i is in p inciple a good o bad massage inding ha nanopa icles damage he cells wi hou a ec ing DNA. On he one hand, i could be bene icial since nanopa icles loca ed ou side he umo will no inc ease he isk o genome damage and seconda y malignancies induc ion in no mal issues su ounding he umo . On he o he hand, he adiosensi izing mechanism ope a ing h ough DNA damage could be mo e e icien . A solu ion o his dilemma could be based on selec i e a ge ing o nanopa icles o speci ic genome sequences, like oncogenes, using app op ia ely designed oligo-nucleo ides as being a ailable o adio-emi e s [ 70 ]. Wi h echniques o COMBO-FISH [ 71 , 72 ] and PNA p obe combina ions [ 73 ], NPs may be ans e ed o cell nuclei and speci ically add essed o gi en ch oma in a ge s. This could be achie ed by adding a nuclea localiza ion signal (NLS) pep ide mo i and a speci ic PNA oligonucleo ide p obe o he su ace o nanopa icles [ 74 ]. Using such sophis ica ed app oaches o speci ic a ge ing o genome abe a ions like mul iple gene copies would open new aspec s in umo he apy. 4. Ma e ials and Me hods 4.1. Cells and Cell Cul u ing Th ee cance cell ypes we e s udied in he p esen s udy: highly adio esis an U87 glioblas oma cance cells, adio esis an SkB 3 b eas cance cells and ela i ely less adio esis an HeLa ce ix cance cells. U87 and HeLa cells we e ob ained om ATCC (Ame ican Type Cul u e Collec ion, Manassas, VA, USA). SkB 3 was comme cially a ailable and used o se e al SMLM s udies in ou labo a o y. U87 and HeLa cells we e g own in Dulbecco’s modi ied essen ial medium (The mo Fishe Scien i ic, Wal ham, MA, USA) supplemen ed wi h 10% hea -inac i a ed e al cal se um (The mo Fishe Scien i ic, Wal ham, MA, USA), 100 U/mL penicillin (PAA), 100 µ g/mL s ep omycin (PAA), and 1% NEAA (The mo Fishe Scien i ic, Wal ham, MA, USA). Cell cul u es we e kep in T-25 cell lasks a 37 ◦C in a humidi ied a mosphe e wi h 5% CO2. Fo he expe imen s wi h SMLM, SkB 3 cells we e p epa ed as desc ibed in de ail elsewhe e [ 72 ]. SkB 3 cells we e g own in McCoy’s 5A cell medium, con aining 10% e al bo ine se um (FBS) and 1% penicillin/s ep omycin. Cells we e cul i a ed and main ained a 37 ◦ C in a humidi ied a mosphe e a 95% ai /5% CO 2 . Then, he cells we e ypsinized and ans e ed o co e slips, pu in o six-well pla es and u he cul i a ed (abou h ee passages, i.e., abou 38 h) un il 80% con luence. 4.2. Nanopa icles and Incuba ion o Cells wi h Nanopa icles Pla inum nanopa icles (P -PEG-17, e e ed o as P -NPs) we e p epa ed as explained in he ecen ly submi ed F ench pa en (FR 1900008). B ie ly: P -NPs we e syn he ized by γ - ay wa e adiolysis o P con aining sal and embedded wi h polye hylene glycol (PEG) o inc ease hei biocompa ibili y. P -NPs we e mainly sphe ical wi h an a e age pla inum co e diame e o 2.6 nm. P elimina y esul s we e ob ained also o gold nanopa icles (Au-NPs) which a e composed o a Au co e o 2.4 nm encapsula ed by he di hiola ed polyaminoca boxyla e (DTDTPA) shell. Fo SkB 3 In . J. Mol. Sci. 2019,20, 588 18 o 25 Au-NPs inco po a ion, 8 µ L o 10 nm-sized gold pa icles (Au ion, Wageningen, The Ne he lands) we e added o he medium in each well 16 h p io o i adia ion in o de o ob ain a maximum up ake in he cell cy oplasm ia di usion (Figu e 6) [ 53 , 75 ]. In o he expe imen s 2.6 nm P -NPs o 2.4 nm Au-NPs we e added o he medium 6 h be o e i adia ion a 0.5 mM concen a ion. 4.3. Cell I adia ion Cells we e i adia ed in 6-well cul u e pla es con aining a cul u e medium and 2.0 × 10 4 –2.0 × 10 5 cells pe well. Consecu i ely, cells we e exposed o 2 o 4 Gy o γ - ays (1 Gy/min), deli e ed by a 137 Cs i adia o a oom empe a u e (RT). Du ing i adia ion, he samples we e kep in he mo-isola ing boxes o p e en sample in ec ion and empe a u e changes, and hen immedia ely e u ned o he incuba o (37 ◦ C, 5% CO 2 ) un il aken o he expe imen . Fo SMLM expe imen s, 10 nm Au-NPs we e incuba ed 16 h be o e i adia ion. Then, he cells we e simul aneously exposed wi h and wi hou Au-NPs using a 6 MeV Linac adia ion sou ce (A is e, Siemens, E langen, Ge many). The exposu e doses o 0.5, 1, 2 and 4 Gy we e ob ained by changing he i adia ion ime a he same dose a e. 4.4. Immunode ec ion o γH2AX/53BP1 Foci and Double S and B eak Quan i ica ion DNA DSBs we e quan i ied in spa ially ( h ee-dimensionally = 3D) ixed cells by he means o high- esolu ion ICM de ec ion o co-localized γ H2AX and 53BP1 epai oci as desc ibed ea lie [ 16 , 20 ]. B ie ly, cells we e ixed wi h 4% pa a o maldehyde (10 min, a oom empe a u e RT) p io o i adia ion (0 min PI, non-i adia ed con ols) and in se e al ime poin s PI co e ing a long (48 h) PI pe iod (5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h and 48 h PI). Cells we e pe meabilized in 0.2% T i on X-100/PBS (15 min, RT) and immunoassayed wi h mouse an iphospho-H2AX (se ine 139) (Me ck, Da ms ad , Ge many, ca . no.: 05-636) and abbi an i-53BP1 (Cell Signaling Technology, Dan e s, MA, USA, ca . no.: 4937) p ima y an ibodies o simul aneously de ec he γ H2AX and 53BP1. An iphospho-H2AX an ibody was isualized wi h he seconda y FITC-conjuga ed donkey an i-mouse an ibody and an i-53BP1 an ibody wi h Cy3-conjuga ed donkey an i- abbi an ibody (bo h Jackson Labo a o y, Wes G o e, PA, USA, ca . no.: 715-095-150 and 711-165-152). Ch oma in was coun e s ained wi h 1 µ M TO-PRO-3 (Molecula P obes, Eugene, OR, USA) p epa ed in 2 × saline sodium ci a e (SSC). A e b ie washing in 2 × SSC, Vec ashield medium (Vec o Labo a o ies, Bu ling on, On a io, Canada) was used o sample moun ing. 4.5. Fixa ion and Immunos aining o γH2AX o Single Molecule Localiza ion Mic oscopy 45 min a e i adia ion, he cells we e ixed in o de o ob ain an ea ly esponse o he biological sys em o damage. The cells we e washed in 1 × Phospha e-Bu e ed Saline (PBS) wi h MgCl 2 (0.901 mM)/CaCl 2 (0.493 mM) and ixed in 3.7% o maldehyde (in 1 × PBS + Mg/Ca; eshly p epa ed om pa a o maldehyde) o 20 min a RT. A e washing wice wi h 1 × PBS + Mg/Ca, he cells we e s o ed in 3.7% o maldehyde (in 1 × PBS + Mg/Ca) a 4 ◦ C. A e 4-weeks s o age, he o maldehyde was eplaced by 1 × PBS (+ 0.1% sodium azide). A e emo ing he sodium azide om he co e slips, he cell memb anes we e pe meabilized by 0.2% T i on-X100 h ee imes o 5 min. A e washing h ee imes in 1 × PBS (+ Mg/Ca), he cells we e blocked in 2% bo ine se um albumin (BSA) o hal an hou and incuba ed in 100 µ L o he p ima y an ibody solu ion (mouse an i-phospho-his one H2A.X (Se 139) an ibody; Me ck Chemicals, Da ms ad , Ge many; dilu ion: 1:500) a 37 ◦ C o 18 h in a humidi ied chambe . The ea e he co e slips wi h he cells we e washed h ee imes o 5 min wi h 1 × PBS (+ Mg/Ca) o emo e he emaining, unbound p ima y an ibodies. A e wa ds he seconda y AlexaFluo 647 goa an i-mouse an ibody was incuba ed in a humidi ied chambe a 37 ◦ C o 30 min. Then, he cells we e ixed in 2% o maldehyde (in 1 × PBS (+ Mg/Ca) a 37 ◦ C o 10 min. Finally, he cells we e coun e s ained wi h 4 0 19,6-diamidin-2-phenylindol (DAPI; Sigma Ald ich, now Me ck, Da ms ad , Ge many) o 5 min in da kness and we e, a e washing wice wi h 1 × PBS (+ Mg/Ca) o 5 min each, embedded in 20 µ L P oLong Gold embedding medium (The moFishe Scien i ic, Wal ham, In . J. Mol. Sci. 2019,20, 588 19 o 25 MA, USA, P oLong Gold An i ade Moun an , P36930). The specimen was sealed and s o ed a 4 ◦ C in comple e da kness un il SMLM applica ion. 4.6. Con ocal Mic oscopy An au oma ed high- esolu ion con ocal luo escence mic oscopic sys em Leica DM RXA [ 76 – 78 ], equipped wi h a CSU10a Nipkow disc (Yokogawa, Tokyo, Japan), an oil imme sion Plan Fluo a objec i e (100 × /NA1.3), a CoolSnap HQ CCD came a (Pho ome ix, Tucson, AZ, USA), and an A /K lase (Inno a 70C Spec um, Cohe en , San a Cla a, CA, USA), was used o image acquisi ion [ 79 , 80 ]. Abou 40 indi idual con ocal slices wi h 0.3 µ m z-s ep inc emen s ac oss he nuclei we e cap u ed o each cell. Ob ained images we e analyzed using Acquia ium so wa e [ 80 ] which enabled he h ee-dimensional econs uc ion o images and inspec ion o indi idual γ H2AX and 53BP1 oci in 3D space. Co-localized γ H2AX/53BP1 oci we e conside ed as DSBs o inc ease he p ecision o DSB de ec ion, especially in he ea ly-s age PI (wi h a highe backg ound o signals) and also he p obabili y ha only un epai ed DSBs a e s ill e alua ed in la e and e y la e pe iods o ime PI. 4.7. Single Molecule Localiza ion Mic oscopy As desc ibed in de ail elsewhe e [ 61 , 81 ], he localiza ion mic oscope used was equipped wi h ou lase s o exci e di e en luo opho es. The wa eleng h and he in ensi y o illumina ion we e chosen by an acous o–op ical unable il e (AOTF). Fo ou expe imen s, he 642 nm lase wi h 140 mW ou pu powe was used o s imula e he dye molecules o blink. A 100x/NA 1.46 oil imme sion objec i e was used. The luo escence o he specimen was sepa a ed om he illumina ion ligh by wo quadband in e e ence il e s and was eco ded by an EMCCD came a (Ando iXon Ul a 897, Bel as , UK). The EMCCD came a was ope a ed a a gain o 100 and a se ies o 2000 up o 6000 image ames was eco ded o each cell nucleus. P io o he SMLM measu emen , a wide ield image was aken in he DAPI channel and he γ H2AX channel wi h 10% lase in ensi y. The ea e , he γ H2AX image s ack was eco ded a 70% illumina ion in ensi y. Cells we e chosen o ha e consis en size and o m, a dis inc i e edge, a good s aining signal- o-backg ound a io and a ce ain minimal dis ance o he nex cell. The acqui ed da a s acks we e e alua ed as desc ibed in de ail in [ 61 ]. γ H2AX labelling molecules we e coun ed and dis ances be ween each poin we e de e mined. Da a displayed in box g aphs (Figu e 9) show he equency dis ibu ions o dis ances o γ H2AX-labelling molecules. The boxes include 50% o he alues (25 h o 75 h pe cen ile) cen e ed on he median ( he ho izon al line h ough he box). The mean alues a e ep esen ed by he squa es wi hin he boxes. The e ical lines begin a he 5 h pe cen ile and end a he 95 h pe cen ile. 4.8. Da a Analysis and S a is ical E alua ion a e Con ocal Mic oscopy The SigmaPlo 14.0 (Sys a So wa e Inc., San Jose, CA 95131 USA) and O igin 2018b (O iginLab Co po a ion, No hamp on, MA 01060, USA) we e used o da a analysis and p ocessing. The Mann– Whi ney ank sum es was employed o compa e γ H2AX/53BP1 ocus (DSB) numbe s in un ea ed and nanopa icle- ea ed cells a all he pa icula pe iods o ime PI. The esul s we e conside ed as s a is ically signi ican a p< 0.05. The oci numbe s we e quan i ied bo h manually and au oma ically. In manual analysis, a ound 100 nuclei in each single expe imen we e blind-inspec ed (no in o ma ion abou he sample ea men ) by eye by an expe ienced e alua o . Fo compu a ional analysis, be ween 100 and 250 nuclei we e sco ed. Because he e is no a sui able ool ul illing ou demands on au oma ic γ H2AX/53BP1 oci coun ing wi h ou speci ic da a, a cus om p og am o as and accu a e oci coun ing, calib a ed o ou da a, has been de eloped. The p og am wo ks in a semi-au oma ic manne , whe e i allows o a isual inspec ion wi h he possibili y o make quick manual adjus men s and co ec ions, i necessa y. The algo i hm is composed o 3 s eps—nucleus segmen a ion, oci segmen a ion and inal oci classi ica ion, in o de o elimina e alse de ec ions. Con olu ion Neu al Ne wo k (wi h SegNe opology) was ained o obus nuclei segmen a ion, ollowed by spli ing o ouching nuclei wi h wa e shed ans o m applied on he dis ance ans o m In . J. Mol. Sci. 2019,20, 588 20 o 25 o he segmen ed bina y image. Inside a bounding box o each nucleus, he oci a e segmen ed wi h a maximally s able ex emal egion de ec o , which is as and in a ian o image in ensi y alues. The de ec o is se o high ecall in o de o ob ain all possible oci o he classi ie . Classi ica ion o ue oci is done wi h Suppo Vec o Machine classi ie s on some ex ac ed ea u es (e.g., oci mean in ensi y and oci size). The p og am allows use o adjus he classi ie bias alue ( o se classi ie sensi i i y), because he p ope ies o oci a e e y he e ogeneous be ween di e en samples and measu emen s. Besides he coun o oci, i also allows expo ing some o he ea u es o ollowing analysis (cell size, oci sizes, oci in ensi ies, e c.) ( he ull desc ip ion o he so wa e will be published sepa a ely). In Figu es 3–5, he da a a e displayed as box g aphs also showing he dis ibu ions o DSBs oci pe nucleus. The boxes include 50% o he alues (25 h o 75 h pe cen ile) cen e ed on he median ( he ho izon al line h ough he box). The mean alues a e ep esen ed by he squa es wi hin he boxes. The e ical lines begin a he 5 h pe cen ile and end a he 95 h pe cen ile. 5. Conclusions In he p esen s udy, we demons a e ha ul a ine (2–10 nm) pla inum and gold nanopa icles do no escala e DNA damage o comp omise DSB epai in i adia ed umo cells o di e en ypes. This con i ms ou ecen indings o 2.0 nm gadolinium nanopa icles [ 21 ]. Howe e , 10 nm Au-NPs may po en ially in luence he cha ac e o DNA damage a he nanoscale, as i was disco e ed by using SMLM [ 61 , 72 , 81 ]. Some indica ions in his sense ha e been ob ained also by ICM o 2 nm P -NPs. While hese indings a e di icul o be in e p e ed in e ms o biological ele ance, con adic ions s ill pe sis in he li e a u e on he enhancemen o nuclea DNA damage in cells i adia ed in p esence o me al nanopa icles. A he cu en s age o knowledge, i is easonable o conclude ha di e en mechanisms, in ol ing an enhancemen o DNA damage on he one side and cy oplasmic e ec s on he o he side, pa icipa e in adiosensi iza ion exe ed by me al nanopa icles. Mo e mechanisms p obably con ibu e o he inal adiosensi izing e ec , in ol emen o which depends on he nanopa icle cha ac e is ics (ma e ial, size, composi ion, and su ace unc ionaliza ion), cell ype and expe imen al condi ions. The e o e, many ques ions on nanopa icle-media ed adiosensi iza ion emain open, emphasizing he impo ance o mo e sys ema ic u u e esea ch. Me hodologically, we demons a e cu en possibili ies and use ulness o he newly de eloped supe - esolu ion mic oscopy echnique (SMLM) ha oge he wi h app op ia e nano-p obing echnologies has a po en ial o shi ou s udies on DNA damage and epai o nanoscale dimensions. Mu ual compa ison o mic o- and nano-scale esul s may p o ide a clue on many impo an p ocesses aking pa in cells and hei molecula mechanisms. Au ho Con ibu ions: Resea ch concep ualiza ion, M.F., M.H., G.H., L.S. and S.L; me hodology de elopmen , M.F., M.H., S.L., L.S. and S.R.; expe imen pe o mance, E.P. (E a Pagáˇco á), L.S., O.K., I.F., F.S.-K., J.-H.L. and S.R.; so wa e de elopmen , T.V.; alida ion, D.D., E.P. (E a Pagáˇco á), J.-H.L., G.H., M.H. and M.F.; o mal analysis, D.D, E.P. (E a Pagáˇco á), I.F., M.H., M.F.; in es iga ion, E.P. (E a Pagáˇco á), S.L., M.H. and M.F.; esou ces, F.B., F.W., M.H. and M.F.; da a cu a ion, M.F., D.D., E.P. (E a Pagáˇco á), T.V.; w i ing o he o iginal d a p epa a ion, F.S.-K., M.F. and M.H.; w i ing o e iew and edi ing, G.H., S.L., M.F. and M.H.; isualiza ion, E.P. (E a Pagáˇco á), F.S.-K., D.D., I.F., O.K.; supe ision, E.P. (E ika Po cel), S.L., M.H. and M.F.; p ojec adminis a ion, M.F. and M.H.; unding acquisi ion, M.H. and M.F. Funding: The wo k was suppo ed by he Minis y o Heal h o he Czech Republic (AZV g an no. 16-29835A), he Czech Science Founda ion (p ojec 16-12454S), he Heidelbe g Uni e si y Mobili y G an o In e na ional Resea ch Coope a ion wi hin he excellence ini ia i e II o he Deu sche Fo schungsgemeinscha (DFG) o M.H., and om he g an s om he Czech Republic o he Join Ins i u e o Nuclea Resea ch, Dubna (P ojec s o he Czech Plenipo en ia y and he 3 + 3 P ojec s). The inancial suppo by Deu sche Fo schungsgemeinscha and Rup ech -Ka ls-Uni e si ä Heidelbe g wi hin he unding p og am Open Access Publishing is g a e ully acknowledged. The esea ch leading o hese esul s has also ecei ed unding om he People P og amme (Ma ie Cu ie Ac ions) o he Eu opean Union’s Se en h F amewo k P og amme (FP7/2007–2013) unde REA G an Ag eemen No [624370]. In . J. Mol. Sci. 2019,20, 588 21 o 25 Acknowledgmen s: The au ho s hank Emanuel Maus (Ki chho -Ins i u e o Physics) o p o iding an SMLM image. The au ho s acknowledge Daniela Salado (Ins i u e des Sciences Moléculai es d’O say, Uni e si éPa is Sud 11, O say Cedex, F ance) and Hynd Remi a (Labo a oi e de Chimie Physique, O say, F ance) o syn hesizing pla inum and 2.4 nm gold nanopa icles. Fu he mo e, we hank Jin-Hau Ewwe , Ins i u e o Resea ch Ra ing and Enhancemen (IRRE), Al enbu schla, Ge many, and Paul I. M. P inz Zippl, Uni e si y o Vienna, Aus ia, o always inding he igh way o haziness in cons uc i e discussions. Con lic s o In e es : The au ho s decla e no con lic s o in e es . The unde s had no ole in he design o he s udy; in he collec ion, analyses, o in e p e a ion o da a; in he w i ing o he manusc ip , and in he decision o publish he esul s. Abb e ia ions DSB double s and b eak SMLM single molecule localiza ion mic oscopy ICM immuno luo escence con ocal mic oscopy P -NPs pla inum nanopa icles Au-NPs gold nanopa icles PI pos -i adia ion EPR enhanced pe meabili y and e en ion Re e ences 1. 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