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
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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
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