Bioimaging
An Indocyanine G een-Based Nanop obe o In Vi o De ec ion o
Cellula Senescence
And ew G. Bake , Muhamad Ha ono, Hui-Ling Ou, And ea Bis o ićPopo ,
Emma L. B own, James Joseph, Monika Golinska, Es ela González-Gualda, Da id Macias,
Jian eng Ge, Ma y Denholm, Sami Mo sli, Chandan Sanghe a, Thomas R. Else,
Hea he F. G ee , Aude Ve ne , Sa ah E. Bohndiek, Daniel Muñoz-Espín,* and
Ljiljana F uk*
Abs ac : The e is an u gen need o imp o e con en ional cance - ea men s by p e en ing de imen al side e ec s,
cance ecu ence and me as ases. Recen s udies ha e shown ha p esence o senescen cells in issues ea ed wi h
chemo- o adio he apy can be used o p edic he e ec i eness o cance ea men . Howe e , al hough he
accumula ion o senescen cells is one o he hallma ks o cance , su p isingly li le p og ess has been made in
de elopmen o s a egies o hei de ec ion in i o. To add ess a lack o de ec ion ools, we de eloped a biocompa ible,
injec able o ganic nanop obe (NanoJagg), which is selec i ely aken up by senescen cells and accumula es in he
lysosomes. The NanoJagg p obe is ob ained by sel -assembly o indocyanine g een (ICG) dime s using a scalable
manu ac u ing p ocess and cha ac e ized by a unique spec al signa u e sui able o bo h pho oacous ic omog aphy
(PAT) and luo escence imaging. In i o, ex i o and in i o s udies all indica e ha NanoJaggs a e a clinically
ansla able p obe o de ec ion o senescence and hei PAT signal makes hem sui able o longi udinal moni o ing o
he senescence bu den in solid umo s a e chemo he apy o adio he apy.
In oduc ion
Cellula senescence is a esponse o i epa able damage and
s ess esul ing in s able cell cycle a es , which can p e en
he expansion o al e ed, po en ially pa hological, cells. This
igge s he sec e ion o a complex mix u e o in lamma o y
and issue emodeling ac o s (senescence-associa ed sec e-
o y pheno ype, known as SASP) o aid he epai o he
su ounding issue. Unde no mal condi ions, he SASP can
ec ui in lamma o y cells o d i e senescen cell clea ance
ul ima ely execu ed by phagocy ic cells.[1,2] Howe e , pe sis-
en damage and s ess can esul in dys egula ion o
immunosu eillance and he accumula ion o senescen cells
in issues. Such an accumula ion p omo es he onse and
p og ession o mul iple age- ela ed and ch onic diso de s
including ca dio ascula diseases, ib osis, neu ological dis-
o de s, obesi y, in lamma o y synd omes and cance .[1,3] The
impo an ole o senescence in he de elopmen o a ie y
o pa hologies has been acknowledged by i s inclusion wi hin
he hallma ks o bo h ageing and cance .[4,5]
Al hough senescence can be a con ex -dependen an ag-
onis ic esponse, s udies ha e shown ha he emo al o
senescen cells by pha macogene ic app oaches can signi i-
[*] D . A. G. Bake , M. Ha ono, D . H.-L. Ou, D . E. González-Gualda,
D . J. Ge, D . M. Denholm, D . S. Mo sli, D . D. Muñoz-Espín
Ea ly Cance ins i u e, Depa men o Oncology, Uni e si y o
Camb idge, Hills Road, Camb idge CB2 0XZ, UK
E-mail: [email p o ec ed]
D . A. G. Bake , M. Ha ono, D . A. B. Popo , D . C. Sanghe a,
D . L. F uk
Depa men o Chemical Enginee ing and Bio echnology, Uni e si y
o Camb idge, Philippa Fawce D i e, Camb idge CB3 0AS, UK
E-mail: [email p o ec ed]
D . E. L. B own, D . J. Joseph, D . M. Golinska, T. R. Else,
D . S. E. Bohndiek
Depa men o Physics, Uni e si y o Camb idge, JJ Thomson
A enue CB3 0HE, Uni ed Kingdom
D . E. L. B own, D . J. Joseph, D . M. Golinska, T. R. Else,
D . A. Ve ne , D . S. E. Bohndiek
Cance Resea ch UK Camb idge Ins i u e, Robinson Way, Cam-
b idge CB2 0RE, UK
D . J. Joseph
School o Science and Enginee ing, Uni e si y o Dundee, Dundee
DD1 4HN Sco land, UK
D . D. Macias
Ins i u o de Biomedicina de Se illa, IBIS/Hospi al Uni e si a io
Vi gen del RocioUni e sidad de Se illa, A da.D . Fed iani/>,
Se illa 41009, Spain
D . H. F. G ee , D . D. Muñoz-Espín, D . L. F uk
Yusu Hamied Depa men o Chemis y, Uni e si y o Camb idge,
Lens ield Road, Camb idge CB2 1EW, UK
© 2024 The Au ho s. Angewand e Chemie In e na ional Edi ion
published by Wiley-VCH GmbH. This is an open access a icle unde
he e ms o he C ea i e Commons A ibu ion License, which
pe mi s use, dis ibu ion and ep oduc ion in any medium, p o ided
he o iginal wo k is p ope ly ci ed.
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How o ci e: Angew. Chem. In . Ed. 2024,63, e202404885
doi.o g/10.1002/anie.202404885
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can ly p omo e (by ~30%) he heal hspan and li espan in
p oge oid and na u ally-aged mice.[6,7] These landma k
s udies accele a ed de elopmen o he apeu ic app oaches
such as senoly ic compounds o e adica e senescen cells,
and senomo phics and senos a ics o egula ion o he
in lamma o y SASP.[3,8–12] In case o cance , combina ion o
bo h p o- and an i-senescence ea men s e e ed o as he
“one- wo punch” app oach has shown pa icula ly p omising
esul s and has been highligh ed as a p omising u u e
s a egy wi hin p ecision oncology.[11–14]
Conside ing he impo an ole o cellula senescence in
disease de elopmen as well as he ecen ad ances in he
ield o seno he apies, su p isingly li le p og ess has been
made in he design o con as agen s and ools o in i o
de ec ion o he senescence bu den. This can pa ially be
explained by he lack o an uni e sal bioma ke o
senescence, hus, senescence de ec ion o en equi es a
mul is ep o algo i hmic app oaches based on an a ay o
bioma ke s.[15,16] Mos commonly employed a e he exp es-
sion o umo supp esso p53 and cell cycle inhibi o
p o eins p16 and/o p21 o asce ain he cell cycle a es ; and
senescence-associa ed β-galac osidase (SA-β-gal) pheno ype,
a colo ime ic eac ion based on inc eased lysosomal β-
galac osidase enzyma ic ac i i y in senescen cells. Al hough
widely used, enhanced SA-β-gal ac i i y is no a dis inc i e
ea u e o only senescen cells, bu i is also p esen in o he
cell ypes such as os eoclas s, neu ons and mac ophages.[17]
Despi e limi ed speci ici y, enhanced ac i i y o lysosomal β-
galac osidase was employed in he design o small molecule
luo escen senop obes.[18–20] Howe e , luo escence-based
s a egies a e no well sui ed o clinical applica ions because
o hei poo pene a ion dep h,[10] limi ing he imaging o
in e nal o gans which equi es a pene a ion dep h o up o
se e al cen ime e s.[21,22] To add ess his limi a ion, wo
posi on emission omog aphy (PET) p obes based on SA-
β-gal ac i i y ha e ecen ly been epo ed o de ec ion o
senescence.[23] Al hough PET is widely employed as a
clinical imaging s a egy, i equi es he use o adioac i e
aces, sophis ica ed imaging sys ems, as well as specialized
in as uc u e. In addi ion o PET, magne ic esonance
imaging (MRI), and compu ed omog aphy (CT), pho o-
acous ic omog aphy (PAT) has ecen ly eme ged as a
powe ul in i o imaging echnique[24] wi h signi ican
po en ial o ea ly de ec ion o cance .[25–28]
PAT employs a pulsed lase o exci e he app op ia e
con as agen , esul ing in con e sion o abso bed ligh in o
acous ic ene gy. As acous ic wa es sca e o a lesse ex en
han ligh , PAT images can be cons uc ed om la ge
pene a ion dep h compa ed o ligh mic oscopy echniques.
In con as o CT and PET, PAT does no equi e ionizing
adia ion, he acquisi ion imes a e sho e , and he ins u-
men s mo e a o dable and easie o ope a e.[25] Impo an ly,
by acqui ing images a se e al wa eleng hs, PAT allows o
mul iplexed imaging, esul ing in da a se s ha e eal he
dis ibu ion o se e al ch omopho es.[26]
Bo h endogenous molecules such as hemoglobin, and
exogenous nea -in a ed (NIR) abso bing dyes can be used
as PAT con as agen s.[29] B oadly speaking, an ideal PAT
con as agen should ha e a high mola ex inc ion coe -
icien and exhibi a dis inc , well-de ined abso p ion spec-
um o maximize he ligh abso p ion and acili a e di e -
en ia ion om o he ch omopho es.[29] In addi ion, i should
be pho os able while main aining a low quan um yield o
ensu e e icien con e sion o ligh ene gy in o hea .[30]
These p e equisi es make nano-based p obes excep ionally
well-sui ed, gi en hei unable abso p ion spec a, high
ex inc ion coe icien s and a la ge su ace a ea ha allows
o u he (bio) unc ionaliza ion wi h s abilizing and a ge -
ing agen s.[31–33]
He ewi h, we desc ibe design and alida ion o a no el
PAT nanop obe o in i o de ec ion o senescen cells. The
nanop obe was p epa ed by exploi ing sel -assembly o
indocyanine g een (ICG) dime s in o J-agg ega e s uc u es
unde mild and scalable condi ions. J-agg ega es, ini ially
desc ibed nea ly 100 yea s ago, a e sup amolecula assem-
blies o o ganic dyes such as cyanines, po phy ins and
pe ylene bisimides, cha ac e ized by unique op ical and
pho ophysical p ope ies.[34,35] A numbe o nanos uc u ed
J-agg ega es, anging in sizes om 90 o 130 nm, ha e been
p epa ed o da e, mainly o umo imaging and pho o-
he mal he apy.[36–40] He ewi h, we desc ibe nanosized J-
agg ega e p obe (NanoJagg) cha ac e ized by high pu i y,
ema kable physiochemical p ope ies, long- e m s abili y
unde con en ional s o age condi ions and biocompa ibili y.
NanoJaggs can be manu ac u ed in a scalable and ep odu-
cible way and employed o de ec ion o chemo he apy-
induced senescence in i o,ex i o and in i o using bo h
luo escence and PAT imaging.
Resul s and Discussion
Syn hesis and Cha ac e iza ion o NanoJagg P obes
Pu e NanoJaggs we e p epa ed by s i ing an aqueous
solu ion o indocyanine g een (ICG) dye a 65°C o 24 h
(Figu e 1a), and subsequen ly pu i ied by dialysis and ul a-
cen i uga ion (Figu e 1b). The cen i uga ion s ep is c ucial,
as p e ious s udies ha omi ed his pu i ica ion s ep
ob ained a complex mix u e o J-agg ega es and side
p oduc s (Figu e 1S).[36–39] This aises conce ns abou he
eliabili y o he conclusions d awn om hese ea lie
s udies, as J-agg ega es and hei indi idual building blocks
likely ha e di e en impac on cells. Assembly o NanoJaggs
was moni o ed by measu ing he abso bance o J-agg ega es
a λmax =895 nm and ICG a λmax =780 nm (Figu e 1c). Bo h
NMR and LC–MS/MS analyses we e pe o med o be e
unde s and he s uc u al composi ion o he pelle and
supe na an ob ained a e cen i uga ion (Figu e S2 o S4).
A single peak wi h 751 m/z was obse ed in he ch oma o-
g am o he pelle (Figu e S3b), while he ch oma og am o
he supe na an con ained mul iple peaks, some o which
ha e been iden i ied as ICG deg ada ion p oduc s (Fig-
u e S4b o d).
The disappea ance o he 1H NMR peak a 6.60 ppm
co esponding o he hyd ogen in he ca bonyl chain o ICG
indica es he o ma ion o he b idging bond be ween wo
ICG molecules wi hin NanoJagg s uc u e (Figu e 1d). This
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was in line wi h p e ious s udies epo ing o ma ion o ICG
dime s.[41,42] The 1H NMR s udy o he supe na an con-
i med he p esence o mul iple compounds (Figu e S4),
which amoun ed o 54% o he o al yield by mass.
Unlike p e iously epo ed J-agg ega e nanos uc u es,
NanoJaggs we e chemically pu e wi h 97% dime composi-
ion as demons a ed by HPLC (Figu e S5). While dime iza-
ion o ICG has been no ed when J-agg ega es o med in
hin ilms o nanoemulsions,[43] o he bes o ou knowledge,
his is he i s J-agg ega e nanopa icle epo ed ha is
en i ely composed o dime ic ICG.
To gain a be e unde s anding o he size dis ibu ion
and shape o he ob ained NanoJaggs, c yo-elec on mic o-
scopy (c yo EM) imaging was pe o med showing semi-
sphe ical NPs wi h an a e age diame e o 34�4.8 nm
(Figu e 1b and Figu e S6a). Fu he , ene gy dispe si e X- ay
(EDX) analysis demons a ed he p esence o sul u s em-
ming om he sul ona e g oup on he ICG building blocks
(Figu e S6b).
The abso bance spec um o NanoJaggs displays a ed
shi in compa ison o ICG (λmax =895 nm and λmax =780,
espec i ely Figu e 1c), while he luo escence spec a e eal
quenching o ICG dime s wi hin he agg ega es (λem =
810 nm) (Figu e S6c). Such quenching phenomenon is
ad an ageous o pho oacous ic imaging as i esul s in a
highe p opo ion o abso bed ene gy being con e ed in o
acous ic ene gy, hus enhancing he PAT signa u e.[44] In
ac , when no malized o he same abso bance maximum,
NanoJaggs exhibi a ema kable h ee- old enhancemen in
he o e all gene a ed pho oacous ic signal (Figu e S6d),
which is in line wi h p e ious epo s on pho oacous ic
p ope ies o J-agg ega es.[45] In addi ion, he ex inc ion
coe icien was calcula ed o be 1.92�0.07×109M1cm1
(Figu e S6e), which esembles ha o me allic plasmonic
nanopa icles,[29,46] e en hough NanoJaggs a e composed
en i ely o o ganic molecules.
In e es ingly, when we compa ed NanoJaggs o ICG
dime s in Dulbecco’s Modi ied Eagle medium (DMEM)
wi h 10% e al bo ine se um (FBS) used o main aining
cell cul u es, we obse ed ha dime s apidly assemble in o
NanoJaggs (Figu e S7a). This assembly was empe a u e-
dependen and inhibi ed a low empe a u e (Figu e S7 b
and c). The e we e also no disce nible changes in he
abso bance spec a o NanoJaggs when assessed in di e en
biologically ele an media (PBS bu e a pH 7.4, DMEM,
DMEM wi h 10% FBS and FBS only) o e a span o 7 days
indica ing hei ema kable colloidal s abili y (Figu e S8).
Rema kably, hese nanopa icles can be s o ed o mo e
han a yea as lyophilized powde , o o a leas 8 mon hs as
aqueous suspension, which signi ican ly enhanced hei
po en ial o clinical use.
Accumula ion o NanoJaggs in Senescen Cells
P e iously i was shown ha ICG dye unde goes e icien
up ake in o he lysosomes o cance cells lines.[47,48] The e-
o e, we hypo hesized ha ICG-con aining NanoJaggs
would le e age he bene i s o ICG imp o ed lysosomal
up ake. Compa ed o cance cells and no mal (di e -
en ia ed) cells, senescen cells a e cha ac e ized by an
enla ged lysosomal compa men , e iden in bo h an
inc eased size and a g ea e quan i y o lysosomes.[49–52]
Consequen ly, signi ican ly mo e NanoJagg p obes would be
expec ed o accumula e in senescen cells. To es his
hypo hesis, we i s pe o med cell up ake and colocaliza ion
s udies in human lung adenoca cinoma (A549) and melano-
ma (SK-MEL-103) cance cell lines demons a ing ha
NanoJaggs, simila ly o ICG, indeed a ge he lysosomal
compa men (Figu e S9).
Nex , we wan ed o explo e he speci ici y o NanoJaggs
owa ds senescen cells. Gi en ha senescence is a he e oge-
neous esponse ha depends on he igge , cellula ype
and he con ex , a ious models o senescence we e
employed in his s udy. Speci ically, senescence was induced
in SK-MEL-103 and A549 cance cells by ea men wi h
chemo he apeu ic Palbociclib, which selec i ely inhibi s
cyclin dependen kinases 4 and 6 (CDK4/6). In addi ion,
DNA in e cala ing chemo he apy agen Cispla in was
employed in A549 cance cells, and DNA-damaging adia-
ion was applied o human WI-38 ib oblas s o se e as
adio he apy-induced senescence model. The implemen a-
ion o senescence s a us in hese models was e alua ed
using es ablished p o ocols including inc eased SA-β-gal
ac i i y, p oli e a ion assays and enhanced exp ession o
p21 cell cycle inhibi o . In addi ion, dec ease in exp ession
le els o phospho yla ed e inoblas oma (Rb) p o ein, which
is a ma ke o an ac i e cell cycle p og ession, was
de e mined by wes e n blo ing (Figu e S10). Once senes-
cence models we e success ully es ablished, con ocal mic o-
scopy was used o image he accumula ion o NanoJaggs in
cells showing a subs an ial inc ease in all senescen cell
models compa ed o hei non-senescen coun e pa s (Fig-
u e 2a and b; Mo ie 1 and Mo ie 2). I should be no ed ha
ela i e luo escence uni s we e no malized by he o al cell
Figu e 1. Syn hesis and cha ac e iza ion o NanoJagg p obe. (a) Nano-
Jaggs a e p epa ed by s i ing o an aqueous solu ion o indocyanine
dye (ICG) a 65°C o 24 h. (b) C yo-EM o he esul ing sphe ical
nanopa icle indica ed an a e age size o 34�4.8 nm. (c) Cha ac e is ic
ed abso bance shi om 780 (ICG) o 895 nm is obse ed upon
NanoJagg o ma ion. (d) 1H NMR spec a indica e he p esence o ICG
dime s as he main NanoJagg building blocks.
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a ea (μm2) o accoun o abe an mo phology and enla ged
size o senescen cells, hus, ensu ing ha he ob ained
a e age signal is compa able ac oss di e en cell ypes.
The la ges NanoJagg up ake was obse ed in senescen
SK-MEL-103 and A549 cells (9.4× and 5.2×, espec i ely,
when compa ed o hei non-senescen cance cell coun e -
pa s), while adia ion-induced senescen WI-38 ib oblas s
showed only 2.7× highe luo escen signal han he co e-
sponding non-i adia ed cells (Figu e 2b). These esul s
we e addi ionally con i med by low cy ome y s udies
(Figu e S11). While senescen cells a e known o ha e
inc eased au o luo escence,[53] we ha e no obse ed any
in e e ence s emming om au o luo escence wi hin Nano-
Jagg’s exci a ion and emission ange (Figu e S12 a and b).
Unlike o cance cells, he accumula ion o NanoJaggs
in senescen cells was ound o be dose dependen and
esul ed in ele a ed signal le el, e en a he lowes
concen a ion es ed (0.4 mg/mL) (Figu e S13). This was in
con as o ICG dye, which showed signi ican up ake in
bo h he con ol and senescen cells (Figu e S14). We also
e alua ed he up ake o ICG dime s, howe e hei apid
assembly in o J-agg ega es esul ed in he simila up ake
pa e n as obse ed o NanoJaggs (Figu e S8 and S15).
Colocaliza ion s udies showed ha NanoJaggs a e
la gely accumula ed in lysosomes si ua ed in he pe inuclea
space (Figu e 2b and Figu e S16 and S17) while no no ice-
able accumula ion was obse ed in mi ochond ia (Fig-
u e S16 and S18) o endoplasmic e iculum (Figu e S16 and
S19).
Addi ionally, biocompa ibili y o NanoJaggs was as-
sessed by CellTi e -Blue iabili y assays in bo h senescen
and non-senescen human lung adenoca cinoma (A549) and
melanoma (SK-MEL-103) cell lines ea ed wi h wide ange
o NanoJagg concen a ions (0.1–100 μg/mL). As i can be
seen in supplemen a y Figu e S20, no signi ican oxici y was
obse ed wi hin his ange con i ming ha NanoJaggs a e
no oxic and can be sa ely used as a e sa ile senop obe o
de ec mul iple senescen pheno ypes by luo escence mic o-
scopy.
In addi ion o luo escence mic oscopy, we also showed
ha con en ional op ical o ligh mic oscopy can be used o
moni o selec i e accumula ion o NanoJaggs in senescen
cells (Figu e S21). This capabili y ende s NanoJaggs com-
pa able o comme cially used SA-β-galac osidase ac i i y
ki s in e ms o selec i i y, bu wi h he ad an age o being
applicable o li e cells due o absence o oxici y.
Finally, NanoJagg de ec ion po en ial was alida ed
using wo addi ional senescen cell models employing non-
cance ous cell lines. Senescen human umbilical ein
endo helial cells (HUVECs), a p ima y endo helial cell line,
we e ob ained a e Palbociclib ea men , while mouse
emb yonic ib oblas s (MEFs) a high passage numbe o
eplica i e cycles we e employed as a model o age- ela ed
eplica i e senescence. Bo h he con en ional SA-β-gal and
ou no el NanoJagg s aining we e pe o med using hese
cell lines. Palbociclib- ea ed HUVECs showed an inc eased
numbe o SA-β-gal posi i e cells and a signi ican inc ease
in he up ake o NanoJaggs (Figu e S22). The same was
obse ed wi h MEFs, which showed inc eased numbe o
SA-β-gal posi i e cells a eplica i e passage 9, a which
poin a signi ican inc ease in NanoJagg up ake was also
obse ed (Figu e S23). This esul indica es ha besides
being used as p obes o chemo- and adio he apy induced
senescence, NanoJaggs can also be employed o de ec ion
o age- ela ed senescence.
Mechanism o NanoJagg Up ake in Senescen Cells
A e we obse ed a signi ican inc ease in NanoJaggs
accumula ion in a ious senescence models, we wan ed o
gain mo e insigh s in o he unde lying mechanism espon-
sible o cellula up ake. Gene ally, ac i e endocy osis pa h-
ways can be g ouped in o mac opinocy osis (o phagocy o-
sis), pinocy osis, ecep o -media ed endocy osis (also known
as cla h in-media ed endocy osis), and ecep o -independen
endocy osis (in ol ing choles e ol-binding p o ein ca eolin
plasma memb ane buds).[54] As ecen s udies ha e iden i ied
al e ed endocy osis pa hways in senescence,[55] we se ou o
explo e NanoJagg up ake in p esence o p e- and pos -
cellula up ake inhibi o s (Figu e 3a). P e-up ake endocy o-
sis inhibi o s ei he block cell memb ane-based pa hways
such as mac opinocy osis (phopha idylinos i ol-3-kinase in-
hibi o Ly294002) and cla h in-coa ed pi o ma ion (Pi s op
2), o inhibi he o ma ion o dynamin p o ein (Dyngo4a)
in ol ed in cla h in- and ca eolin- media ed endocy osis
(s uc u es o hese inhibi o s a e shown in Figu e S24).[56–59]
Pos -up ake inhibi o s in e e e wi h p ocesses wi hin he
cell such as la e s age o cla h in-coa ed esicle ission om
he memb ane (p ochlo pe azine, PCZ)[56,60] and au ophagy
(Chlo oquine).[61]
Figu e 2. Selec i i y and accumula ion o NanoJaggs in senescen cells
in i o. (a) Con ocal images o non-senescen and senescen SK-MEL-
103 cells induced by Palbociclib and ea ed wi h 50 μg/mL NanoJaggs.
(b) Con ocal quan i ica ion o ela i e luo escen uni s (RFU)/μm2o
he NanoJagg signal in he di e en cell lines. Da a ep esen as
mean�SD (n=3 biological eplica es), and a Two ailed - es was
used o calcula e he signi icance (*p<.05, **p<.01, ***p<.001). All
examples used show a signi ican inc ease in NanoJagg up ake
compa ed o con ol cells and adjus ed o cell a ea.
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Signi ican dec ease in NanoJagg up ake (p<0.0001)
was obse ed when senescen SK-MEL-103 and A549
cance cells we e ea ed wi h inhibi o s o cla h in-media ed
endocy osis (Pi s op 2 and Dyngo4a) (Figu e 3b and c,
Figu e S25 and S26), wi h smalle changes obse ed upon
adminis a ion o mac opinocy osis inhibi o Ly294002 (p<
0.01) (Figu e 3b and c, Figu e S25 and S26).
In con as , non-senescen , con ol SK-MEL-103 and
A549 cells ea ed wi h p e-up ake endocy osis inhibi o s
showed no signi ican di e ences in in acellula accumula-
ion o NanoJaggs (Figu e S27 and S28) indica ing ha
inc eased endocy ic up ake is a senescence-dependen
e ec .
This aligns wi h p e ious s udies ha ha e shown an up-
egula ion o endo-lysosomal machine y as well as he
ac i a ion o mac opinocy osis (as a su i al mechanism
h ough nu ien sca enging) and phagocy osis (employed as
a o m o cell cannibalism) in senescen cells.[62–66] Indeed,
due o hei capaci y o engul o he cells and syn hesized
ma e ials,[62,63,67] senescen cells ha e o en been compa ed o
mac ophages.[67]
Con a y o p e-up ake endocy osis inhibi o s, pos -
up ake inhibi o s PCZ and Chlo oquine, esul ed in in-
c eased NanoJagg luo escence in bo h senescen and non-
senescen cell lines (Figu e 3b and c, Figu e S25 o S29). In
addi ion, signi ican ly lowe co-localiza ion wi h lysosomes
was obse ed a e PCZ ea men , which could be a ib-
u ed o PCZ-media ed dis up ion o he usion be ween
cla h in-coa ed esicles and lysosomes.[56] Chlo oquine, on
he o he hand, is an inhibi o o au ophagy ha induces
lysosome dila ion and a ec s a ious p ocesses downs eam
o lysosome o ma ion, hus no impac ing he colocaliza ion
(Figu e S25 o S29).[68]
Toge he , hese indings sugges ha NanoJaggs a e
in e nalized h ough an ac i e mechanism, likely in ol ing
bo h cla h in-media ed endocy osis and mac opinocy osis.
While his phenomenon has been p e iously no ed wi hin
he con ex o cell engul men ,[62] i s applica ion in de elop-
men o de ec ion s a egies o o imp o ed d ug deli e y
emains la gely unexplo ed. Gi en ha ICG has been
obse ed o bind o he cance cell memb ane and
subsequen ly unde go endocy osis,[47] i is emp ing o
hypo hesize ha NanoJaggs could po en ially ecognize
speci ic molecules on he senescen su aceome hus ena-
bling selec i e a ge ing. The iden i ica ion o he senescen
su aceome ep esen s an eme ging a ea wi h signi ican
po en ial o a ge ing senescen cells, and i wa an s
u he in es iga ion. Fu u e s udies will del e deepe in o
he mechanism o up ake o gain a mo e comp ehensi e
unde s anding.
Ex Vi o and In Vi o Assessmen o Senescence in
Chemo he apy-Induced Xenog a Using NanoJaggs
To assess he e ec i eness o NanoJaggs o selec i e up ake
in o senescen cells, we u ilized immunocomp omised mice
bea ing umo xenog a s es ablished using SK-MEL-103
melanoma cells ea ed wi h senescence-inducing
chemo he apy.[13] A e he o ma ion o umo s, mice we e
subjec ed o daily ea men wi h Palbociclib o e 7 days
(Figu e 4a). This egimen led o high le els o in e umo al
senescence as indica ed by an inc ease in SA-β-gal ac i i y
(Figu e 4b), he absence o he p oli e a i e ma ke Ki-67,
and a dec ease in phospho yla ed Rb p o ein le els.[13] A
single injec ion o ehicle (DMEM, phenol ed- ee), Nano-
Jaggs (200 μL a 1 mg/mL) o an equi alen amoun o ICG
dye we e adminis e ed o he mice, and he luo escen
le els analyzed a e 6 h by IVIS luo escence imaging
(Figu e 4a). Signi ican le els o NanoJaggs luo escence
Figu e 3. Mechanism o NanoJagg up ake in senescen cells. (a)
Schema ic ep esen a ion o endocy osis in senescen cells and he
inhibi o s used in his s udy. Th ee p e-up ake inhibi o s we e used
(Pi s op 2, Dyngo4a and Ly294002), as well as wo pos -up ake
inhibi o s (PCZ and Chlo oquine). (b) Con ocal images o NanoJagg
up ake in senescen SK-MEL-103 cells ea ed wi h p e- and pos -up ake
inhibi o s o endocy osis. Lysosomes a e s ained wi h Lyso acke g een
and nuclei wi h Hoechs (blue). (c) Con ocal quan i ica ion o ela i e
luo escen uni s (RFU/μm2) o SK-MEL-103 and A549 cells. Con ol
cells no ea ed wi h inhibi o s a e shown in black. Pi s op 2 and
Dyngo4a bo h esul in a signi ican inhibi ion o NanoJagg up ake in
bo h cell lines (p<0.0001), while Ly294002 caused lowe bu s ill
signi ican educ ion (p<0.01 o SK-MEL-103 and p<0.05 o A549).
Bo h pos -up ake inhibi o s esul in an inc ease o NanoJagg signal:
p ochlo pe azine’s inc ease p<0.01 o SK-MEL-103, and p<0.05 o
A549; Chlo oquine causes p<0.01 o SK-MEL-103 and p<0.0001 o
A549. Da a ep esen mean�SD (n=3 biological eplica es) and a
Two- ailed - es was used o calcula e he signi icance (*p<0.05,
**p<0.01, ***p<0.001, and ****p<0.0001).
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we e de ec ed in senescen umo s ea ed wi h Palbociclib
(n=4) bu no in con ol (p oli e a i e) umo s (n=4) (p=
0.0077) (Figu e 4c). In con as , ICG luo escence was
p edominan ly obse ed (p=0.0438) in non-senescen u-
mo s (n=4) (Figu e 4d), which aligns wi h he p e iously
epo ed endency o ICG dye o accumula e in agg essi e
solid umo s.[48,69]
Negligible backg ound luo escence signal in ehicle-
ea ed mice con ol (Figu e S30a) con i med ha obse ed
luo escence can be p edominan ly a ibu ed ei he o
NanoJaggs o ICG wi hin he umo s. Mo eo e , subsequen
ex i o biodis ibu ion s udies e ealed ha NanoJaggs
luo escence was ha dly de ec able 6 h a e adminis a ion
in o gans o he e iculoendo helial sys em such as he li e
and spleen, in bo h un ea ed and Palbociclib- ea ed mice.
In bo h mice g oups, a conside able luo escence signal was
obse ed in he kidneys, sugges ing ha enal clea ance is a
p e e en ial ou e o elimina ion (Figu e S30b).
Con ocal imaging o issue slices was conduc ed o
p o ide addi ional alida ion o he accumula ion o Nano-
Jaggs in he senescen umo s (Figu e 4e, p=0.0028).
To u he assess he e ec i eness o NanoJaggs in
a ge ing senescen cells, we also u ilized immunocomp o-
mised mice bea ing umo xenog a s es ablished using A549
adenoca cinoma cells ea ed wi h cispla in. A e he
o ma ion o umo s, mice we e subjec ed o in ape i oneal
cispla in o ehicle injec ions h ee imes a week o 14 days
(Figu e S31a). This egimen led o high le els o in a-
umo al senescence as indica ed by an inc ease in SA-β-gal
ac i i y and a educed umo g ow h a e (Figu e S31b and
e). Upon NanoJaggs adminis a ion ia he ail ein
injec ion, he signal- o-backg ound a io was signi ican ly
inc eased in cispla in- ea ed umo s (n=6) when compa ed
o he ehicle con ol (n=6) (p<0.05.). In addi ion, he
signal- o-backg ound luo escen a io signi ican ly in-
c eased (p<0.001) when cispla in- ea ed mice we e com-
pa ed p e- and pos -NanoJagg adminis a ion. This longi u-
dinal moni o ing clea ly demons a es ha he signal
inc ease s emmed solely om he accumula ion o he
nanop obe wi hin chemo he apy-induced senescen umo s.
NanoJaggs as Con as Agen s o Pho oacous ic (PA) Imaging
o Senescen Cell Bu den In Vi o
Following he encou aging esul s ob ained om
luo escence imaging, a subsequen s udy was conduc ed o
explo e he po en ial o NanoJaggs o be used as pho o-
acous ic (PA) con as agen s. PA is an eme ging imaging
echnique wi h subs an ial po en ial o ansla ional appli-
ca ions in cance and o he diseases, as i enhances he
imaging dep h, all he while main aining spa ial and
empo al esolu ion.[25,28,29,70] Two in a ed (IR) dyes, ICG
and ce uximab-800CW, a e cu en ly employed as PAT
agen s in clinical s udies o lymph node mapping in
melanoma (NCT05467137) and ce ical cance
(NCT03923881), espec i ely. Mo eo e , se e al PAT nano-
pa icle con as agen s, bo h me al nanopa icles and
o ganic s uc u es, ha e been epo ed and success ully
alida ed in p eclinical s udies.[29]
To e alua e NanoJaggs po en ial o PAT, we employed
immunocomp omised emale nude mice bea ing SK-MEL-
103 melanoma umo xenog a s, di ided in o Palbociclib- o
ehicle- ea ed g oups. NanoJaggs o ICG we e adminis-
e ed ia ail ein injec ion and PAT imaging ca ied ou a
in e als o 6 and 24 h pos -injec ion (Figu e 5a). Induc ion
o senescence wi hin umo s was con i med a e in i o
imaging by SA-β-gal ac i i y assays and immunohis ochemis-
y (IHC) s aining o pRB and Ki-67 (Figu e S32a). As
expec ed, he Palbociclib- ea ed g oup showed an inc eased
lysosomal SA-β-gal ac i i y and a dec eased nuclea s aining
Figu e 4. Fluo escence de ec ion o NanoJaggs ex i o. (a) Schema ic
o e iew o he in i o expe imen . The up ake o bo h ICG and he
NanoJaggs we e compa ed in Palbociclib- ea ed umo s s un ea ed
umo s. (b) SA-β- gal s aining o un ea ed and Palbociclib- ea ed
umo s. (c) IVIS luo escence images o dissec ed con ol (n=4) and
senescen umo s (n=4) a e ea men wi h NanoJaggs (le ).
Quan i ica ion o he a e age adian e iciency compa ing NanoJagg
luo escen signal (p=0.0077) is shown on he igh . (d) IVIS images
o dissec ed con ol and senescen umo s a e ea men wi h ICG o
6 h (le ). Quan i ica ion o he a e age adian e iciency compa ing
ICG luo escen signal (p=0.0438) is shown on he igh . (e) Con ocal
images and quan i ica ion o NanoJagg-posi i e cells compa ing he
un ea ed o Palbociclib ea ed umo s (n=4). Da a ep esen
mean�SD, and a Two- ailed - es was used o calcula e he
signi icance. (*p<0.05, **p<0.01, ***p<0.001).
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o pRB and Ki-67, indica ing a educed p oli e a i e
capaci y and he implemen a ion o he senescen p og am
(Figu e 5b). In addi ion, umo s ea ed wi h Palbociclib
showed a ma ked educ ion in p oli e a ion a es, he eby
con i ming he onse o cell cycle a es cha ac e is ic o
senescence (Figu e S32c o e).
As i can be obse ed in Figu e 5c, he NanoJaggs signal
was p ominen ly dis ibu ed h oughou he senescen umo
o Palbociclib- ea ed mice. In con as , in he con ol (non-
senescen , ea ed wi h ehicle) p oli e a i e umo s, he
signal was concen a ed a he umo edges. The signal- o-
backg ound a io (SBR) was de e mined by di iding he
signal om he umo egion o in e es (ROI) by he
backg ound signal ob ained h ough spec al unmixing o
he NanoJagg PAT spec um (Figu e 5c). This SBR was
compu ed o e e y axial plane o bo h con ol and
Palbociclib- ea ed umo s, p o iding a ep esen a i e Nano-
Jagg signal ac oss he en i e umo olume. A no ably highe
SBR was obse ed in he senescen , Palbociclib- ea ed
umo s (n=4), in compa ison o he p oli e a i e umo
coun e pa s (n=2) (p=0.0135). This obse a ion was
co obo a ed by compa able alues ob ained h ough quan-
i ica ion based on con as - o-noise a ios (Figu e S33a).
One o he ad an ages o PAT imaging lies in i s abili y
o acqui e and analyze mul iple da a s eams, including
pa ame e s such as blood oxygena ion and he co-colocaliza-
ion o blood essels using dis inc signals om de-oxy-
gena ed (Hb) and oxygena ed (HbO2) hemoglobin. Linea
spec al unmixing applied o hemoglobin (Hb), deoxyhemo-
globin (HbO2), and NanoJagg/ICG spec a (Figu e S33b),
e ealed ha in non-senescen umo s, NanoJaggs colocalise
wi h Hb and HbO2indica ing hei dis ibu ion wi hin
ascula u e (Figu e 5d). Howe e , in case o Palbociclib-
ea ed (senescen ) xenog a s, NanoJaggs we e ound o
in il a e he umo issue, and he e was no o e lap wi h Hb
and HbO2signals indica ing ha hey we e no co- localized
wi h ascula u e. To alida e ha he inc ease in NanoJagg
signal is no due o inc eased blood pe usion wi hin he
Figu e 5. Pho oacous ic imaging o senescen cells in li e mice employing NanoJagg p obe. (a) Expe imen al design using wo g oups o xenog a
mice ansplan ed wi h SK-MEL-103 cells: ea ed wi h Palbociclib o induce senescence, and a con ol ehicle. NanoJaggs we e injec ed in o bo h
g oups h ough ail ein. (b) Assessmen o senescence in umo s pos -in i o imaging. Palbociclib- ea ed, senescen umo s show s ong SA-β-
Gal ac i i y and dec ease in nuclea pRB and Ki-67 le els compa ed o con ol umo s. Scale ba s=100 mm. (c) Rep esen a i e axial images ( op)
o con ol ( ehicle+NanoJaggs, n=2) and senescen umo s (Pablociclib+NanoJaggs, n=4) ea ed wi h NanoJaggs. Signi ican di e ence can
be obse ed in signal o backg ound a io (SBR, bo om) o PAT signal in con ol and senescen umo . (d) PAT Images ob ained by spec al
unmixing o NanoJagg (yellow), Hb (blue) and HbO2( ed). Bo om image ep esen s scale ba o bo h (c) and (d). (e) Rep esen a i e con ocal
images o biopsied issue sec ions pos -in i o PAT imaging. In bo h cases mul iple umo s we e used, as well as imaged in di e en pa s o each
slice. Quan i ica ion o NanoJagg-posi i e cells indica es signi ican di e ences be ween senescen and con ol mice. Da a ep esen mean�SD,
and a Two- ailed - es was used o calcula e he signi icance (*p<0.05, **p<0.01, ***p<0.001).
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umo , a compa ison o HbO2and Hb signals was conduc ed
be ween senescence and non-senescen umo s (Fig-
u e S33c). This analysis e ealed no signi ican di e ences
be ween wo g oups (p=0.5099 and p=0.2926 espec i ely).
To u he con i m ha he NanoJagg signal is associa ed
wi h senescence, a co ela ion analysis was conduc ed using
pa ame e s such as he umo size and g ow h a e (Fig-
u e S33d). Rema kably, we ound ha he pho oacous ic
NanoJagg signal s ongly co ela es wi h he a es ed umo
g ow h a e ( 2=0.875, p=0.0061), bu no he umo size
( 2=0.015, p=0.817, ns). This co ela ion wi h g ow h a e
was no obse ed in he case o endogenous hemoglobin
HbO2( 2=0.112, p=0.516, ns, Figu e S33e).
The p esence o NanoJaggs in senescen umo s was
addi ionally con i med by luo escence imaging o he ex
i o umo slices (Figu e 5e). The o al numbe o Nano-
Jagg-posi i e cells is signi ican ly inc eased in senescen
umo s compa ed o con ol coun e pa s (p=0.0002).
Mo eo e , hese posi i e cells wi hin he senescen umo s
displayed a signi ican ly inc eased NanoJagg luo escence
signal when compa ed o he un ea ed con ols (p=0.0301,
Figu e S33 ).
Finally, PA imaging was also employed o moni o
NanoJaggs biodis ibu ion and ou es o elimina ion con-
i ming he da a ob ained om ex i o s udies (Fig-
u e S34a). S ong PAT signal in kidneys sugges s enal
clea ance ha is in line wi h he luo escence ex i o and in
i o da a. Con a y o he ex i o images, NanoJagg
accumula ion can also be obse ed in he spleen and li e ,
possibly due o he imp o ed sensi i i y o he imagng
echnique. Howe e , impo an ly, NanoJaggs signal in he
li e , spleen and kidneys dec eases o e ime, and becomes
signi ican ly educed a e 24 h (Figu e S34a and b). Fu -
he mo e, no disce nible his ological di e ences in issue
a chi ec u e be ween mice ea ed wi h he ehicle and hose
ea ed wi h NanoJaggs we e e ealed by hema oxylin and
eosin (H&E) s aining, indica ing an absence o hepa ic and
enal damage, as well as he lack o in lamma ion in spleen
(Figu e S34c).
Conclusions
The o ganic nanos uc u ed J-agg ega es (NanoJaggs) de-
sc ibed in his s udy enable he de ec ion o a ious ypes o
chemo he apy-induced senescence using ange o echniques
such as low cy ome y, con ocal imaging, in i o
luo escence, and PA imaging ac oss di e en se ings,
including in i o, ex i o, and in i o models. NanoJagg
syn hesis is highly ep oducible and scalable and deli e s a
p obe, which is s able in wa e and biologically ele an
media. The p obe is also sui able o lyophiliza ion, which
signi ican ly imp o es s o age ime and anspo . Unlike
p e iously epo ed J-agg ega e nanos uc u es, ou p obe
has high pu i y and we ha e shown ha i is composed
comple ely o ICG dime s. Al hough u he esea ch is
needed o ho oughly unde s and he mechanism o Nano-
Jagg up ake and lysosomal a ge ing, we p o ide e idence
ha he p e e en ial up ake o NanoJaggs in senescence is
egula ed, a leas in pa , by cla h in-media ed endocy osis
and mac opinocy osis. We also demons a e ha he
enhanced accumula ion o he p obe in senescen cells
co ela es wi h hei inc eased lysosomal con en .
The simple syn hesis, speci ici y owa ds senescen cells
and s ong PAT signal, makes NanoJaggs sui able o la ge-
scale p epa a ion and clinical ansla ion. Howe e , u he
s udies a e needed, including a ho ough biodis ibu ion and
biodeg ada ion s udy in heal hy mice as well as he
in es iga ion o o he models such as senescence- ela ed
ch onic diso de s, oncogene-induced senescence, and na u-
ally aged mice.
Ou s udy has demons a ed ha NanoJaggs can e ec-
i ely se e as selec i e con as agen s o luo escence and
PA imaging o senescen cells. The p ope ies o NanoJaggs
coupled wi h enhanced issue pene ance o PA imaging,
o e a p ac ical a enue o ansla ion o his senop obe
in o human se ings. The in i o de ec ion and moni o ing
o he senescen bu den using NanoJaggs could no only
o e aluable insigh s in o he ex en o issue dys unc ion
bu could also g ea ly impac isk s a i ica ion and diagnos-
ic e iciency o pa ien s ac oss a ious diseases including
cance . Assessmen o senescen bu den could aid di e -
en ia ion o pa ien s esponding well o he he apy and
hose a isk o umo elapse and me as asis. In addi ion, in
i o de ec ion ool could p o ide da a on he e iciency o
de eloped senoly ic in e en ions aimed a a ge ed emo al
o senescen cells. Indeed, he demons a ed inc ease in
NanoJaggs up ake by senescen cells could lead o he
design o he anos ic ools o selec i e nanos uc u ed
senoly ics ha do no equi e addi ional unc ionaliza ion
wi h a ge ing species.
Suppo ing In o ma ion
The au ho s ha e ci ed addi ional e e ences wi hin he
Suppo ing In o ma ion (Re . [30,31]).
Acknowledgemen s
The F uk labo a o y is suppo ed by Cance Resea ch UK
(CRUK) g an C67674/A27887. AGB was suppo ed by he
W. D. A ms ong S uden ship. MH was suppo ed by he
Ga es Camb idge Schola ship. AB was suppo ed by he
Enginee ing and Physical Sciences Resea ch Council Cen e
In e disciplina y Resea ch Collabo a ion G an (EPSRC
IRC, EP/S009000/1). Munoz-Espin labo a o y is suppo ed
by he Cance Resea ch UK (CRUK) Camb idge Cen e
Ea ly De ec ion P og amme (RG86786), by a CRUK
P og amme Founda ion Awa d (C62187/A29760), by a
CRUK Ea ly De ec ion OHSU P ojec Awa d (C62187/
A26989), by a Medical Resea ch Council (MRC) New
In es iga o Resea ch G an (NIRG) (MR/R000530/1) and
by a Royal Socie y Resea ch G an (RG160806). EGG was
holde o a “La Caixa” Founda ion Schola ship o pos -
g adua e s udies a Eu opean Uni e si ies and unded by
he CRUK Camb idge Cen e Ea ly De ec ion P og amme.
Angewand e
Chemie
Resea ch A icles
Angew. Chem. In . Ed. 2024,63, e202404885 (8 o 10) © 2024 The Au ho s. Angewand e Chemie In e na ional Edi ion published by Wiley-VCH GmbH
15213773, 2024, 25, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/anie.202404885 by Csic O ganización Cen al Om (O icialia Mayo ) (U ici), Wiley Online Lib a y on [07/11/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
DM was unded by a New In es iga o Resea ch G an
(NIRG) (MR/R000530/1) and a CRUK P og amme Foun-
da ion Awa d (C62187/A29760). HLO is unded by a
CRUK Ea ly De ec ion OHSU P ojec Awa d (C62187/
A26989) and a Da ley/Sands Downing College Fellowship
(G109261). JG was unded by a Da ley/Sands Downing
College Fellowship (G109261). SM is unded by a CRUK
P og amme Founda ion Awa d (C62187/A29760). MD was
unded by he CRUK Camb idge Cen e Clinical Resea ch
T aining Fellowship. CS was suppo ed by he Camb idge
Uni e si y Hospi als NHS ounda ion T us . ELB, JJ, MG,
TRE and SEB we e suppo ed by Cance Resea ch UK
(C9545/A29580). JJ and SEB we e also suppo ed by
EPSRC (EP/R003599/1). The TEM was unded h ough he
EPSRC Unde pinning Mul i-use Equipmen Call (EP/
P030467/1).
We would also like o hank Gemma C onshaw and all
he eam in he biological esou ce uni (BRU) o he
Cance Resea ch UK Camb idge Ins i u e o hei suppo
wi h he animal models and day o day main enance. Fu he
we would like o hank And ew Mason, Duncan Howe, and
Pe e Gie h o he Yusu Hamied Depa men o Chemis y,
Uni e si y o Camb idge, NMR acili y, and Asha Boodhun,
Robe o Canales, and Dijana Ma ak-Vinko ic o he
Depa men o Chemis y Mass Spec ome y o hei
con inued suppo .
Compe ing in e es
AGB, DME and LF ha e iled a pa en o he p esen ed
echnology.
Da a A ailabili y S a emen
The da a ha suppo he indings o his s udy a e a ailable
in he supplemen a y ma e ial o his a icle.
Keywo ds: Cellula senescence ·cance ·endocy osis ·ICG
nanop obe ·pho oacous ic omog aphy (PAT)
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Angewand e
Chemie
Resea ch A icles
Angew. Chem. In . Ed. 2024,63, e202404885 (9 o 10) © 2024 The Au ho s. Angewand e Chemie In e na ional Edi ion published by Wiley-VCH GmbH
15213773, 2024, 25, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/anie.202404885 by Csic O ganización Cen al Om (O icialia Mayo ) (U ici), Wiley Online Lib a y on [07/11/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License