dezemb o de 2021
UMinho | 2021
Uni e sidade do Minho
Escola de Medicina
Mónica Alexand a da Cos a Ce quei a
T ea men o iple-nega i e b eas cance
(TNBC) h ough ex e nally igge ed a ge -
less d ug ca ie s
T ea men o iple-nega i e b eas cance (TNBC)
h ough ex e nally igge ed a ge -less d ug ca ie s
Mónica Alexand a da Cos a Ce quei a
DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR
TERCEIROS
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dezemb o de 2021
Uni e sidade do Minho
Escola de Medicina
Mónica Alexand a da Cos a Ce quei a
T ea men o iple-nega i e b eas cance
(TNBC) h ough ex e nally igge ed a ge -
less d ug ca ie s
Disse ação de Mes ado
Mes ado em Ciências da Saúde
T abalho e e uado sob a o ien ação de
Dou o a Ma ia de Fá ima Monginho Bal aza
e de
Dou o Manuel Bañob e-López
iii
DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR
TERCEIROS
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i
ACKNOWLEDGEMENTS
Após a conclusão de mais uma e apa impo an e na minha ida, não posso deixa de ag adece às
pessoas ma a ilhosas que o na am udo is o possí el. Em p imei o luga , ag adeço à p o esso a Fá ima
po oda a disponibilidade ao longo des e ano, dedicação, empenho e pa ilha de conhecimen o. Ao
Dou o Manuel Bañob e, gos a ia de enal ece oda a con iança que deposi ou em mim enquan o eu
es a a no INL, es ando semp e disponí el pa a me ajuda a c esce p o issionalmen e. À Dou o a Ma a
Cos a, não podia deixa de ag adece oda a dedicação, bondade, simpa ia e empenho que e e comigo.
Ob igada pelas con e sas, so isos, apoio, ajuda e opo unidades que me p opo cionas e. Ao E es e à
Raquel, ou “The Team”. Ob igada po odos os ca és e jan a es o a de ho as. À Céline, ob igada po
oda a pa ilha de conhecimen o e con e sas in e miná eis du an e os longos dias no bio é io. Às minhas
Ana e Ma alda, ob igada, meninas, pelos almoços e companhia pa a o ca é, e pela linda amizade que
c iámos. As minhas meninas, Ca a ina, Ri a, Na acha e Sónia, ob igada pelo lindo g upo de desaba o
“We Social, We Sha e Meals”. Ju, Gui, Cunha e Cá ia, ocês são os melho es amigos que pode ia pedi .
Po im, que o ag adece aos meus pais e ao Vasco. Vocês são as pessoas mais impo an es pa a mim,
a minha amília, o meu po o segu o. Ob igada po odo o apoio, pelas pala as na ho a ce a, pelo
ca inho e amo que ansmi em. Po ocês e a ocês, SEMPRE. Amo- os.
Ag adeço ao P o esso Nuno Sousa, P esiden e da Escola de Medicina da Uni e sidade do Minho;
P o esso Jo ge Co eia-Pin o, Di e o do Ins i u o do In es igação de Vida e Saúde (ICVS); P o esso a
Fá ima Bal aza , Coo denado a do Domínio de Ciências Ci ú gicas, e à P o esso a Pa ícia Maciel,
Di e o a do Mes ado em Ciências da Saúde pelo apoio ins i ucional.
FUNDING
The wo k p esen ed in his hesis was pe o med in he Li e and Heal h Sciences Resea ch Ins i u e
(ICVS), Uni e si y o Minho, and In e na ional Ibe ian Labo a o y o Nano echnology. Financial suppo
was p o ided by g an s om he Founda ion o Science and Technology (FCT) - p ojec PTDC/BTM-
SAL/31142/2017, ICVS Scien i ic Mic oscopy Pla o m, membe o he na ional in as uc u e PPBI -
Po uguese Pla o m o Bioimaging (PPBI-POCI-01-0145-FEDER-022122; by he p ojec NORTE-01-0145-
FEDER-000055, suppo ed by No e Po ugal Regional Ope a ional P og am (NORTE 2020), unde he
PORTUGAL 2020 Pa ne ship Ag eemen , h ough he Eu opean Regional De elopmen Fund (ERDF),and
by Na ional unds, h ough he Founda ion o Science and Technology (FCT) - p ojec UIDB/50026/2020
and UIDP/50026/2020.

STATEMENT OF INTEGRITY
I he eby decla e ha ing conduc ed his academic wo k wi h in eg i y. I con i m ha I ha e no
used plagia ism o any o m o undue use o in o ma ion o alsi ica ion o esul s along he p ocess
leading o i s elabo a ion.
I u he decla e ha I ha e ully acknowledged he Code o E hical Conduc o he Uni e si y o
Minho.
i
ABSTRACT
Cance s ill anks as a p incipal public heal h p oblem wo ldwide, being esponsible o 9.9 million
ela ed-dea hs in 2020. B eas cance is he mos commonly diagnosed cance - ype and he p incipal
cause o cance - ela ed mo ali y in women, om which he iple-nega i e b eas cance (TNBC) sub ype
is he mos le hal. TNBC ea men include con en ional chemo he apy (
e.g.,
doxo ubicin/DOX), adia ion
and su ge y, bu esponse is poo , jus i ying he low su i al a es o hese pa ien s. Thus, he e is an
u gen need o mo e speci ic and a ge ed app oaches o ea ly diagnosis, con ol, and ea men . The
main objec i e o his hesis was he p eclinical alida ion o a biocompa ible he anos ic p obe ac i a ed
in he umo si e h ough a speci ic ex e nal s imulus, a magne ic ield. The nanosys em is based on
magne ic solid lipid nanopa icles (mSLNs) loaded wi h a chemo he apeu ic d ug (DOX) and magne ic
nanopa icles (Fe3O4), combining he syne gis ic e ec o he mochemo he apy wi h non-in asi e MR
imaging.
The combina o y e ec o a dual ea men was explo ed in a i s sc eening h ough IC50 de e mina ion
in TNBC cells, whe e he empe a u e- esponsi e DOX-loaded mSLNs showed powe ul an icance
p ope ies agains cance cells, associa ed o a highe cy o oxic ou come in compa ison o ee DOX.
Th ough mo e speci ic
in i o
s udies in 2D monolaye , mSLNs, as d ug ca ie s, we e able o inhibi cell
p oli e a ion and mig a ion, in a ime-dependen manne , being i s ly in e nalized in o he cell cy oplasm
and, wi h an inc ease o incuba ion ime, we e able o elease he d ug in o he cell nuclei. In he 3D
sphe oid model and he
ex i o
Chick Cho ioallan oic Memb ane (CAM) model, he dual- ea men
enhanced he pene a ion o he chemo he apeu ic agen in o he cance cells, wi h imp o ed he apeu ic
p ope ies in compa ison o ee DOX. Then,
in i o
s udies we e pe o med using an o ho opic xenog a
NSG mice model. The unloaded nano o mula ions demons a ed o be sa e, wi hou any de ec ed oxic
e ec s on animal-wellbeing and i al o gan his ology and p omo ed a T2-MRI con as enhancemen .
Addi ionally, he DOX-loaded mSLNs demons a ed, wi hin wo weeks pos - ea men , a signi ican capaci y
o inhibi umo g ow h when combined wi h hype he mia, in compa ison o he o he ea men
condi ions.
In conclusion, he syne gis ic po en ial b ough by he combina ion o ac i e a ge ing wi h
he mochemo he apy, p o ided by empe a u e-sensi i e DOX-loaded mSLN, agains TNBC cells, e ealed
he anos ics capabili ies, cons i u ing a p omising op ion as con as agen s o ea ly diagnos ics, coupled
wi h an imp o emen in he he apeu ic index in compa ison o con en ional chemo he apy.
Key wo ds: Cance , T iple-nega i e b eas cance , The anos ic, Nanopa icles, Hype he mia
ii
RESUMO
O canc o é ainda o p incipal p oblema de saúde pública mundial, endo sido esponsá el po 9,9
milhões de mo es em 2020. O canc o da mama é o mais diagnos icado e a p incipal causa de
mo alidade po canc o em mulhe es, sendo o canc o da mama iplo-nega i o (CMTN) o sub ipo mais
le al. O a amen o do CMTN inclui a quimio e apia con encional (po exemplo, doxo ubicina/DOX),
adiação e ci u gia, mas a má espos a à e apêu ica jus i ica as baixas axas de sob e i ência dos
pacien es. Assim, há uma necessidade iminen e de abo dagens mais especí icas e di ecionadas pa a o
seu diagnós ico p ecoce, con olo e a amen o. O p incipal obje i o des e abalho é a alidação p é-
clínica de sondas e anós icas e biocompa í eis, a i adas no local do umo a a és de um es ímulo
ex e no especí ico, um campo magné ico. O nanossis ema é baseado em nanopa ículas magné icas
solido-lipídicas (mSLNs) ca egadas com um á maco quimio e apêu ico (DOX) e nanopa ículas
magné icas (Fe3O4), combinando o e ei o siné gico da e moquimio e apia com imagem não in asi a.
O e ei o combinado do a amen o duplo oi explo ado numa p imei a iagem a a és da
de e minação do IC50, onde as mSLNs esponsi as à empe a u a e ca egadas com DOX exibi am
p op iedades an icance ígenas con a células do CMTN, associado a um esul ado ci o óxico supe io ao
da DOX li e. A a és de es udos
in i o
usando células em monocamada, as mSLNs ca egadas com
á maco o am capazes de inibi a p oli e ação e mig ação celula , de o ma dependen e do empo, sendo
inicialmen e in e nalizadas no ci oplasma celula e, com o aumen o do empo de incubação, con i mou-
se a libe ação do á maco no núcleo. Com o modelo 3D de es e óide e no modelo
ex i o
da Memb ana
Co ioalan oide do emb ião de galinha (CAM), obse ou-se que o a amen o duplo aumen ou a pene ação
dos agen es quimio e apêu icos nas células cance ígenas, com um aumen o signi ica i o das
p op iedades e apêu icas em compa ação com a adminis ação da DOX li e. Es udos
in i o
o am
ealizados usando o modelo o o ópico em mu ganhos (NSG). As nano o mulações sem á maco
demons a am se segu as pa a o bem-es a do animal, sem se em de e ados, po his ologia, e ei os
óxicos em ó gãos i ais, com um aumen o do con as e em T2-RMI. Além disso, mSLNs ca egadas com
DOX demons a am, a é duas semanas após a amen o, uma capacidade de inibição signi ica i a do
c escimen o umo al a a és da combinação e moquimio e apêu ica.
Em conclusão, o po encial siné gico da combinação e moquimio e apêu ica das mSLNs sensí eis à
empe a u a e ca egadas com DOX e elou e capacidades e anós icas, cons i uindo uma opção
p omisso a pa a o uso como agen es de con as e em diagnós ico p ecoce, associado a uma melho ia
do índice e apêu ico em elação à quimio e apia con encional.
Pala as-cha e: Canc o, Canc o da mama iplo-nega i o, Te anós ica, Nanopa ículas, Hipe e mia
iii
INDEX
Di ei os De Au o E Condições De U ilização Do T abalho Po Te cei os ............................................... iii
Acknowledgemen s ............................................................................................................................. i
Funding .............................................................................................................................................. i
S a emen O In eg i y ..........................................................................................................................
Abs ac .............................................................................................................................................. i
Resumo............................................................................................................................................. ii
Index ................................................................................................................................................ iii
Lis O Abb e ia ions And Ac onyms .................................................................................................... xi
Lis O Figu es .................................................................................................................................... xi
LIST OF TABLES .................................................................................................................................... xiii
1| INTRODUCTION ........................................................................................................................... 1
1. Cance ............................................................................................................................. 2
1.1 B eas Cance ................................................................................................................. 4
1.2 B eas Cance Sub ypes .................................................................................................. 6
1.3. B eas Cance T ea men ................................................................................................. 8
1.3.1. Doxo ubicin T ea men .................................................................................................... 14
2. Nanomedicine In Cance The anos ics ................................................................................... 15
2.1. Cance Physiobiological Cha ac e is ics And Nanopa icle Fea u es – Enhanced
Pe meabili y, Re en ion E ec And Tumo Nanopa icle Accumula ion ........................................ 16
2.2. Magne ic Nanopa icles ................................................................................................. 18
2.2.1. Nanopa icle Composi ion .......................................................................................... 18
2.3. Solid Lipid Nanopa icles ............................................................................................... 19
2.3.1. Nanopa icle Composi ion .......................................................................................... 20
2.4. Magne ic Solid Lipid Nanopa icles ................................................................................. 21
2.4.1. Su ace Modi ica ions................................................................................................. 22
2.4.1.1. Pegyla ion Fo Nanopa icle Func ionaliza ion ......................................................... 23
2.4.1.2. Func ionaliza ion Wi h Cell Pene a ing Pep ides ..................................................... 24
2.4.1.3. Diels-Alde Reac ion ............................................................................................... 24
2.4.2. Biomedical Applica ions ............................................................................................. 25
2.4.2.1. Magne ic Resonance Imaging (M i) As A Diagnos ic Tool ......................................... 25
2.4.2.3. D ug Deli e y ......................................................................................................... 28
xiii
LIST OF TABLES
Table I. B eas cance s a is ics, in 2018. ................................................................................. 5
Table II. TNM ana omic pa ame e s based on AJCC s aging sys ems o b eas cance .............. 9
Table III. Classes o chemo he apeu ic d ugs. ....................................................................... 13
Table IV. Magne ic solid lipid nanopa icles (mSLNs) used in he p esen wo k. ........................ 33
Table V. Pa ame e o acquisi ion o elaxa ion ime. .............................................................. 41
Table VI. IC50 alues o he mSLNs and Doxo ubicin o Hs578 and MDA-MB-231 cance cell line
.............................................................................................................................................. 49
Table VII. IC50 alues o he mSLNs and Doxo ubicin o Hs578 cance cell line ....................... 53
Table VIII. Deg ees o syne gism o combina ion he apies...................................................... 55
Table IX. IC50 alues o he mSLNs and Doxo ubicin o THP-1 cance cell line .......................... 58

1
1| INTRODUCTION
2
1. CANCER
Cance is a disease cha ac e ized by a se o he e ogeneous diso de s wi h di e en p ognosis,
o igin si es, kine ics, and bioma ke s (1). As a esul o epigene ic, gene ic modi ica ions, and
ch omosomal abe a ions combined wi h e olu ion and clonal selec ion, occu s he ans o ma ion
o heal hy cells in o malignan pheno ype wi h a de egula ion on he pa hways ha coo dina e he
no mal cell p oli e a ion and homeos asis (Figu e 1) (2–4). In cance , he e is an uncon olled
and apid g ow h o abe an and un unc ional cells wi h he capaci y o me as asize o dis an pa s
o he body (2). Li es yle beha io s ha e a c ucial in luence on he onse o cance , including
physical inac i i y, smoking, high sola exposu e, poo die , and ep oduc i e changes (
e.g.,
la e
age o i s child) (5,6).
This he e ogeneous disease s ill anks as he p incipal public heal h p oblem wo ldwide (7),
being he second leading cause o dea h wo ldwide, p eceded by ca dio ascula diseases (8). In
2020, i was egis e ed 19.2 million o newly diagnosed cance cases, and 9.9 million dea hs om
cance - ela ed disease in all coun ies (9). Mo eo e , acco ding o he Wo ld Heal h O ganiza ion
(WHO), in he nex 2 decades, an inc ease o 60% o new global cance incidence is expec ed (10).
Rega ding Po ugal, he s a is ics e ealed ha , in 2018, i aised 58 199 o new cases and 28
960 o dea hs (9).
Figu e 1. T ans o ma ion o no mal cells in o cance cells. A e he exposu e o possible ca cinogenic
ac o s, gene ic, epigenic modi ica ions and ch omosomal abe a ions may occu . The cells ha acqui e e olu iona y
ai s a e selec ed ob aining he umo .
3
Due o di e en ypes and sub ypes ha could be es ablished wi hin he same o gan, cance
is conside ed a complex disease, whe e umo igenesis is conside ed as a mul is ep e en . In o de
o simpli y he cha ac e is ics o cance , in 2000, Hanahan and Weinbe g de ined an o e all o se
o ai s acqui ed by cance cells o su i e, p oli e a e, and dissemina e, as hallma ks o cance .
In his a icle, 6 hallma ks o cance whe e p oposed, including sel -su iciency in g ow h signals,
insensi i i y o g ow h-inhibi o y (an ig ow h) signals, e asion o p og ammed cell dea h (apop osis),
limi less eplica i e po en ial, sus ained angiogenesis, issue in asion and me as asis (4).
One decade la e , he same au ho s e ised and upda ed he concep o hallma ks o cance ,
conside ing new ‘eme ging’ cha ac e is ics: abili y o e ade immune des uc ion, o al e and
ep og am cellula me abolism, in lamma ion h ough inna e immune cells, and genomic mu a ion
and ins abili y (Figu e 2) (2). Mo e ecen ly, he loss o di e en ia ion was s a ed by Floo and co-
wo ke s (11).
These hallma k capabili ies a e unc ional ai s acqui ed by cance cells h ough de egula ion
and ep og amming o di e en signaling pa hways a dis inc imes du ing umo igenesis,
combined wi h he in e connec ions and c oss alk be ween he indi idual sub-ci cui s (2). By
de ec s in signals esponsible o g ow h, cell cycle, apop osis, and cell mig a ion, he cance cells
Figu e 2. Hallma ks o cance . Schema ic illus a ion o he ad an ageous ai s acqui ed by cance cells essen ial
o umo g ow h and p og ession (adap ed om [2]).
4
could decide hei own a e, wi hou main enance o cell homeos asis. This dynamic sys em is a
p incipal eason o he di icul y in eaching a speci ic ea men o cance (12,13).
1.1 BREAST CANCER
B eas cance (BC) is he second mos commonly occu ing cance wo ldwide (Figu e 3), and
i is conside ed he mos diagnosed cance - ype and he p incipal cause o cance - ela ed mo ali y
in women, wi h an es ima ion o 626 679 dea hs in 2018 (14). In 2012, GLOBOCAN es ima ed an
incidence o 1.7 million o bo h sexes, which al eady ansla es in o high incidence. Howe e , in
2018 he incidence numbe inc eased o 2.1 million cases (Figu e 3), and o he yea o 2040,
3 million new cases a e es ima ed, poin ing ou he need o de elop ea ly-diagnosis s a egies.
Conce ning he male popula ion, male b eas cance has a a e occu ence, wi h less han 1% o
all-cance cases in men, and app oxima ely 1% o all BCs a ound he wo ld (15–17). Despi e his,
since 1975, male b eas cance mo ali y a e emained unchanged, wi h diagnosis in a la e s age
whe e i is obse ed la ge umo size and lymph node in ol emen (17).
The p obabili y o occu a elapse is highe , as could be consul ed on Table I whe e he
p e alence o BC up o 5-yea s a e he i s diagnosis is expec ed (14). Con adic o y o wha
would be expec ed, he highes BC incidence esides in coun ies wi h high Human De elopmen
Index (HDI), al hough he scena io in low HDI ha e p opensi y o agg a a e un il 2030, expec ing
an occu ence o 22.2 million o all-cance cases (18).
Figu e 3. Wo ldwide s a is ics. Wo ldwide es ima ed numbe o cance incidences, in bo h sexes, in 2018 [14].
5
Table I. B eas cance s a is ics, in 2018. Es ima ed incidence and p e alence o bo h sexes in Eu ope and
Uni ed S a es o Ame ica (USA), in 2018 (14).
*p opo ions by 100.000
In Po ugal, o bo h sexes, b eas cance had an incidence and mo ali y o 7 041 and 1 864
cases, espec i ely, in 2020 (9). Due o i s high incidence, b eas cance cons i u es a conce n o
he scien i ic communi y, leading o inc easing sea ch o new a ge ed compounds wi h po en ial
o diagnose and ea , known as he anos ic agen s.
BC ini ia ion and p og ession is associa ed o amily medical his o y, en i onmen al ac o s,
ho monal he apy, gene ical diseases, and inhe i ed mu a ion in key genes, like
BRCA1
and
BRCA2
–
wo high-pene ance umo supp esso genes, whose p o eins ha e a ole in DNA double-s and
b eak epai (19,20).
An e icien BC p e en ion enhances he p obabili y o a be e ea men esponse,
emphasizing he need o an e ec i e medical sc eening p ocedu e, such as mammog aphy
(21). Sc eening by mammog aphy is a gold s anda d me hod used o he iden i ica ion o ea ly BC
in asymp oma ic women, whe e small umo s ha p ecede BC p og ession could be iden i ied
(22). A educ ion o 19% in BC mo ali y was co ela ed o he implemen a ion o mammog aphy
as a p e en ion ool, highligh ing he co ela ion be ween an ea ly diagnosis and a be e p ognosis
(21). Howe e , oge he wi h he possible ha m ul e ec s (
i.e
., pain, anxie y, and adia ion
isk), his me hod may p oduce alse posi i es (benign umo s) and alse nega i es, p omo ing an
ine icien de ec ion o BC, being some cases de ec ed in ad anced s ages o he disease (21–25).
Ea ly de ec ion and moni o ing o b eas cance a e poin ed ou as main ool o he success
o b eas cance ea men (26), in which di e se imaging echniques ha e eme ged as
majo app oaches o diagnosis and assessmen o esponse o b eas cance
he apy. Di e se s udies indica ed ha magne ic esonance imaging (MRI), posi on-emission
omog aphy (PET), compu ed omog aphy (CT), ul asound and single-pho on emission compu ed
omog aphy (SPECT) could be employed as e icien ools o de ec BC, whe e mammog aphy,
ul asound and MRI is he iple-modali y cu en ly applied (27–29). A s udy om 2015 indica ed
a highe p opensi y o MRI o de ec p e-in asi e and in asi e cance s in women wi h a amily
Popula ion
Incidence
P e alence
1-yea (p op.*)
3-yea (p op.*)
5-yea (p op.*)
Eu ope
522 513
477 849 (124.3)
1 327 794 (345.5)
2 054 887 (534.7)
USA
234 087
226 698 (137.4)
632 622 (383.4)
983 907 (596.3)

6
medical his o y and highe BC isk, compa a i ely o o he imaging ools (28). Though, he imaging
echniques ha e some associa ed d awbacks, such as being expensi e (28), acing he need o
use no el ools o cance diagnosis. The use o he exp ession o biochemis y bioma ke s (
e.g
.,
enzymes, ho mones, mRNA exp ession p o ile, miRNAs, and exosomes) appea s as new
complemen a y diagnos ic and he apeu ic ools o pa ien s wi h BC a a ious s ages (27–31).
1.2 BREAST CANCER SUBTYPES
BC is a complex disease whe e he epi helial cells lining he milk duc s a e poin ed as he o igin
spo . B eas cance - ype exposes a ema kable deg ee o in e - and in a- umo al a iabili y,
e ealing a high he e ogenei y ha is associa ed wi h ei he he ana omic si e o pheno ypically.
Fu he unde s anding o BC he e ogenei y, and he complex in e play be ween he umo cells and
di e en s oma elemen s will allow an app op ia e diagnosis, a be e insigh o umo p og ession,
and he choice o an accu a e a ge ed he apy (32).
In he his ological pe spec i e (Figu e 4), duc al ca cinoma is he mos common BC (50-70%
o pa ien s), ollowed by lobula ca cinoma (5-15% o pa ien s) (19,33). While his ological
s a i ica ion is a common p ac ice in BC diagnosis, echnological ad ances e eal u he b eas
cance complexi y wi h di e en clinicopa hological p o iles (34). Th ough gene ic sequencing and
molecula analysis o b eas cance wi h he use o high- h oughpu gene exp ession mic oa ay
and nex -gene a ion sequencing, Pe ou
e al.
selec ed 496 genes using he c i e ia o signi ican ly
supe io and minimum a ia ions be ween di e en umo s and pai ed samples, espec i ely (35).
The genes and samples we e agg ega ed acco ding o hei simila i y, whe e in he clus e analysis
subse i is possible o obse e wo main b anches, clinically desc ibed as ER-
posi i e
o ER-
nega i e
(es ogen ecep o ) (35,36). Mo eo e , using immunohis ochemis y (IHC) and luo escen
in si u
hyb idiza ion, he au ho s could also di ide in o mo e g oups, PR-
posi i e
and PR-
nega i e
(PR, p oges e one ecep o ) and can assess HER2 (human epi helial g ow h ac o ecep o 2)
exp ession. Al oge he , i e di e en BC sub ypes we e iden i ied: Luminal A, Luminal B, no mal-
like, HER2-en iched, and Basal-like o T ipe-nega i e b eas cance (TNBC) (Figu e 4) (19,33,35).
Howe e , his app oach could no be enough o clea ly iden i y he BC sub ype, making he di e en
agg essi eness o umo sub ypes indis inguishable. Taking his in o accoun , S . Gallen 2013 (37–
39) classi ica ion also includes he assessmen o p oli e a ion- ela ed genes, such as Ki67,
7
highligh ing ha sub ypes which ha e high le els o Ki67 exp ession esul s in a wo se p ognosis
(36).
The medical goal is he co ela ion o di e en BC sub ypes wi h clinical ou comes and
esponse o a ge ed he apies. Di e en ly om o he BC sub ypes, TNBC was dis inguished in o
6 sub ypes by Lehmann
e al.
(37) h ough he analysis o he gene exp essing o 386 umo s. The
sub ypes included wo basal-like (BL1 and BL2), an immunomodula o y, a mesenchymal, a
mesenchymal s em-like, and a luminal and ogen ecep o sub ype. The accu a e desc ip ion o
biological ai s o BC sub ypes enables i) a co ec diagnosis o di e en BC sub ypes, ii) a
comp ehension o he p ognosis, isk o p og ession and occu ence o me as asis, and iii) a design
o e ec i e and a ge ed he apies, in e ms o su ge y, adio he apy, o ad anced a ge ed
he apies, leading o highe disease-speci ic su i al a es (38).
Figu e 4. B eas cance ana omy and di e en sub ypes. O e iew o mos common local o appea he BC
and BC molecula sub ypes (19,33,35).
8
1.3. BREAST CANCER TREATMENT
The p esc ip ion o a BC ea men o indi idual pa ien s is cen e ed on di e se ac o s including
gene al heal h s a e o he pa ien , s age, mo phology, umo g ade, umo size, p esence o lymph
nodes and me as asis, as well as exp ession o bioma ke s (38). BC s aging is a suppo i e ool o
clinicians o de e mine he p ognosis and design a speci ic ea men plan o indi idual pa ien s.
The Ame ican Join Commi ee on Cance (AJCC) s aging sys ems o BC has assigned ana omical
s ages (0 o IV) based on umo size (T), lymph node in ol emen (N) and he p esence o absence
o dis an me as asis (M), o ming he adi ional TNM ana omic pa ame e s (Table II) (40).
9
Table II. TNM ana omic pa ame e s based on AJCC s aging sys ems o b eas cance . TMN
s a i ica ion based on umo size (T), lymph node in ol emen (N) and he p esence/absence o me as asis (M).
Adap ed om (40).
S age 0
▪ Cance cells s ays wi hin a duc ;
▪ No e idence o su ounding a y b eas issue in asion;
▪ Cance ha e no sp ead o lymph nodes o dis an si es.
▪ This is called duc al ca cinoma in si u (DCIS), a non-in asi e b eas cance .
S age IA
▪ Tumo is 2 cm ac oss o less;
▪ Cance has no sp ead o lymph nodes o dis an si es.
S age IB
▪ Tumo is 2 cm ac oss o less (o is no ound);
▪ Mic ome as ases in 1 o 3 axilla y lymph nodes (wi h a size no la ge han 2 mm);
▪ Tumo has no sp ead o dis an si es.
S age IIA
▪ Tumo is 2 cm o less (o is no ound), has no sp ead o dis an si es, and one o he ollowing is
applied:
▪ Tiny amoun s o cance a e ound in in e nal mamma y lymph nodes;
▪ Cance has sp ead o 1 o 3 lymph nodes unde he a m and o in e nal mamma y lymph
nodes;
▪ Tumo is la ge han 2 cm and less han 5 cm ac oss, wi hou sp ead o lymph nodes
S age IIB
▪ Tumo is la ge han 2 cm and less han 5 cm ac oss;
▪ I has sp ead o 1 o 3 axilla y lymph nodes and/o iny amoun s o cance a e ound in in e nal
mamma y lymph nodes.
OR
▪ Tumo is la ge han 5 cm ac oss wi hou g ow in o he ches wall o skin;
▪ Tumo has no sp ead o lymph nodes.
S age IIIA
▪ Tumo has no sp ead o dis an si es, and:
▪ Tumo is no mo e han 5 cm ac oss (o canno be ound), and i has sp ead o 4 o 9 axilla y
lymph nodes;
OR
▪ Tumo is la ge han 5 cm ac oss, wi hou any g ow h in o he ches wall o skin. Mo eo e , i
has sp ead o 1 o 9 axilla y nodes, o o in e nal mamma y nodes.
S age IIIB
▪ Tumo has g own in o he ches wall o skin wi hou me as asis in o dis an si es, and one o he
ollowing is applied:
▪ I has no sp ead o lymph nodes;
▪ I has sp ead o 1 o 3 axilla y lymph nodes and/o iny amoun s o cance a e ound in in e nal
mamma y lymph nodes;
▪ I has sp ead o 4 o 9 axilla y lymph nodes o i has expanded he in e nal mamma y
lymph nodes.
S age IIIC
▪ Tumo a any size (o canno be ound), wi hou me as asis a dis an si es, and one o he ollowing
is applied:
▪ Tumo has sp ead o 10 o mo e axilla y lymph nodes;
▪ Tumo has sp ead o he lymph nodes unde o abo e he cla icle;
▪ Tumo in ol es axilla y lymph nodes;
▪ Cance has sp ead o bo h 4 o mo e axilla y lymph nodes and in o in e nal mamma y lymph
nodes.
S age IV
▪ Cance can ha e any size and could sp ead o nea by lymph nodes;
▪ I has sp ead o dis an o gans o o lymph nodes a om he b eas (mos common o gans a e bone,
li e , b ain, o lung).
16
o hei unique physical and op ical p ope ies, and chemical s abili y, nanopa icles can o e come
a ious biological ba ie s ha ee d ugs s uggle wi h (92). Fu he mo e, su ace unc ionaliza ion
can g an selec i i y o d ugs o speci ic body/o gan/ issue a ge ing, and e en o indi idual
ecogni ion and a ge ing o single cance cells. Hence, he nanopa icle cha ac e is ics can bene i
he bioac i i y o he compounds h ough he educ ion o he concen a ion needed o he same
pheno ypic ou come; po en ially inc easing hei he apeu ic index and pha macokine ic p ope ies,
and al oge he educing hei po en ial side e ec s on heal hy issues (84,92,93). Many nano-
o mula ions ha e been in es iga ed p e-clinically, ye only a mino i y ha e p og essed o clinic
s ages (94). Cu en ly, he app o ed by he Uni ed S a es Food and D ug (U.S. FDA) and Eu opean
Medicines Agency (EMA) (20) nano o mula ions include: Ab axane/pacli axel (95),
Doxil/doxo ubicin (81), and ONPATTRO/Pa isi an (96). These o mula ions y o ul ill he need
o c ea e new sys ems ha imp o e d ug selec i i y and deli e y, wi h g ea e e iciency and ha
help p omo e an ea ly diagnosis o cance .
The de elopmen o cance diagnosis ools is ano he esea ch a ea in he nanomedicine ield.
WHO’s 2018 wo ld cance epo p edic ed an inc ease o 60% in cance incidence by 2040.
Cu en ly, ea ly de ec ion is he mos e ec i e way o inc ease he p obabili y o success ul
ea men o mos cance s. Fo TNBC, ea ly de ec ion is undamen al al hough sc eening is usually
complica ed due o low incidence o mic ocalci ica ions and o he p esence o duc al ca cinoma
in si u which educes he mammog aphy e ec i eness (97,98). These malignancies equi e a non-
in asi e, as , and p ecise diagnos ic sys em able o p o ide he loca ion and he size o he umo s,
in addi ion o he localiza ion and size o o he me as a ic masses (99). Addi ionally, he
moni o iza ion o cance - esponse o he ea men is a possibili y, enabling an indi idual design o
a la e he apeu ic s a egy, implemen ing a mo e di ec ed ea men wi h minimal side e ec s
(100).
2.1. CANCER PHYSIOBIOLOGICAL CHARACTERISTICS AND NANOPARTICLE
FEATURES – ENHANCED PERMEABILITY, RETENTION EFFECT AND
TUMOR NANOPARTICLE ACCUMULATION
Di e en nano- o mula ions, including lipid-based nanopa icles and polyme -based
nanopa icles (Figu e 8) (101–104), could each he umo h ough he leaky ascula iza ion, a
p ocess called Enhanced Pe meabili y and Re en ion (EPR) e ec (105). In 1986, Ma sumu a
e

17
al.
ound ha p o eins (15 – 70 kDa as molecula ange) could p e e en ially accumula e in he
umo egion o longe pe iods o ime as a consequence o he enhanced ascula pe meabili y
wi h poo lympha ic d ainage a ound he umo (106). O e he las h ee decades, EPR e ec
became he p incipal dogma o he design o nanopa icles-based an icance d ug deli e y in o de
o inc ease he e iciency o umo he apy (105,107–111).
Tumo g ow h equi es a con inuously supply o nu ien s and oxygen o sus ain he
p oli e a ion. The malignan cells sec e e g ow h ac o s, as VEGF, and p o eins, inducing he build
o new blood essels om he exis ing ones, a p ocess called angiogenesis and well-known as one
hallma k o cance (Figu e 2) (107–109). The apid gene a ion o new capilla ies agg ega ed o a
lack o basal memb ane ( ascula u e suppo i e issue) p omo es he o ma ion o abno mal
essels a chi ec u e wi h enes a ion h ough he endo helium wi h 200 nM o 2 µM o diame e
(107). The nanopa icles in ci cula ion can easily ex a asa e o he umo egion h ough he
enes a ion loca ed in he su ounded blood essels, due o hei cha ac e is ic small size (<200
nm) compa ed o he po e size (107,110). Conjuga ed o his enhanced pe meabili y, an enhanced
e en ion is also obse ed due o de iciency o he lympha ic sys em. Nanopa icles, cha ac e ized
o a la ge hyd odynamic size, a e incapable o e u n o he su ounded capilla ies, inc easing
he e en ion ime in he umo (106,107,109).
Figu e 8. Nanomedicine applica ion on iple nega i e b eas cance . Di e en nano- o mula ions ha e been
s udied in o de o imp o e he p ognosis o ho mone nega i e cance and, consequen ly, o e a a ge ed ea men
o TNBC (101–104).
18
EPR e ec enables a highe d ug accumula ion in umo compa ed o ee d ugs, dec easing
he side e ec s associa ed o chemo he apeu ic d ug and inc easing he e iciency o he umo
ea men (105,107–111).
2.2. MAGNETIC NANOPARTICLES
Magne ic Nanopa icles (MNPs) a e being widely s udied now-a-days in many a eas (such as
in he biomedical ield), because hey o e a ple ho a o oppo uni ies (97). Thei physicochemical
p ope ies, supe pa amagne ic beha io , small size, and capabili y o p omo e biological
in e ac ions a he cellula and molecula le el (97,112), allows MNPs o be employed as d ug
deli e y sys ems (112,113), magne ic esonance imaging con as enhance s (114), and
hype he mia induce s (115) o he ea men o cance . The supe pa amagne ic p ope ies o
MNPs enable a deg ee o con ol h ough he applica ion o an al e na ing magne ic ield (AMF).
He e, selec i e applica ion o he AMF can o ce he MNP o gene a e local hea and p omo e di ec
umo abla ion and/o d ug elease in o he desi ed egion, ul ima ely a oiding in asi e diagnos ic
and he apeu ic echniques (116,117).
MNPs ha e also he po en ial o be used as he anos ic pla o ms in cance he apy. A
he anos ic pla o m combines diagnos ic and he apeu ic capabili ies in he same o mula ion,
enabling an e icien umo a ge ing, ea men , and moni o iza ion o he apy esponse (o image-
guided he apeu ics; isualiza ion o issue images be o e, du ing and a e he ea men ) (117).
This combina ion can help ailo he he apy equi emen s o each pa ien wi hin an indi idualized
he apeu ic s a egy design, wi h a g ea e p obabili y o a posi i e ou come and, a he same ime,
educed side e ec s (100). MNP pe o mance is dependen on hei composi ion, mo phology,
su ace coa ing, and size o he ino ganic co e, all o which in luence hei
in i o
beha io (97) and
a po en ial oxici y (118). S udies pe o med in a mouse model wi h MNPs coa ed wi h DMSA
(dime cap o-succnic acid) e ealed accumula ion in he li e , spleen, and lungs, wi hou side e ec s
(119). Hence, he unc ionaliza ion o he o mula ions’ su ace wi h a ge ed ligands can be a
s a egy o educe oxici y in un a ge ed o gans, whils also inc easing he he apeu ic e icacy in
a ge ed ones (118).
2.2.1. NANOPARTICLE COMPOSITION
MNPs a e composed wi h me allic, e i es (MFe2O4, CoFe2O4, NiaZn(1-a)Fe2O4, MnaZn(1-a)Fe2O4),
alloyed me allic (FeCo, alnico, and pe malloy), and magne ic elemen s (hema i e (α- Fe2O3),
19
magne i e (Fe3O4) and maghemi e (γ-Fe2O3)) (118). The mos commonly used nanopa icles in he
biomedical ield a e supe pa amagne ic i on oxide nanopa icles (SPIONs), such as Fe3O4 and γ-
Fe2O3, which p esen high biocompa ibili y and lowe oxici y compa ed o o he me al s uc u es
(
e.g
., quan um do s, gold nanopa icles and ca bon nano ubes (CNTs) may p esen lowe
biodeg ada ion and body-elimina ion issues (97), oge he wi h inc eased cy o oxici y) (116,118).
Su ace unc ionaliza ion is a c i ical equi emen in o de o inc ease hei colloidal s abili y and
biocompa ibili y (120).
Bo h magne i e and maghemi e p esen simila p ope ies, whe eas he s uc u al
di e ences be ween hem a e di icul o dis inguish wi h he usual cha ac e iza ion echniques.
Magne i e is cha ac e ized by a highe sa u a ion magne iza ion compa ed o maghemi e (92-100
e sus
60-80 A.m2/Kg) (121). Due o hei supe pa amagne ic beha io (pa icle sizes < 20-25
nm), bo h i on-based nanopa icles can be he mally ac i a ed h ough he applica ion o an
ex e nal magne ic ield (122). Typically, hese MNPs can be designed in wo main con igu a ions,
(i) co e-shell s uc u es whe e he i on co es a e coa ed wi h di e en o ganic o ino ganic
compounds (
e.g
., hyd ophilic polyme s, such as PEG o silica shells), (ii) he SPIONs a e
inco po a ed in di e en ma ices (
e.g.,
polyme ic, lipidic). The las con igu a ion is in p inciple
mo e sui able o he anos ic pu poses, since in addi ion o he d ug encapsula ion e iciency o he
o ganic ma ix he inal sys em bene i s om he physicochemical p ope ies o he inco po a ed
SPIONs (123).
2.3. SOLID LIPID NANOPARTICLES
O e he las decades, he heal h- ela ed scien i ic ield has es ablished and imp o ed di e en
d ug deli e y sys ems, including MNPs. As a o emen ioned, he nanoca ie ’s success depends on
hei capaci y o a ge a speci ic si e, o e coming any ana omical ba ie s ha block he ac ion o
he ee d ug, and ul ima ely allowing a selec i e, con olled, and sus ained d ug elease. The
composi ion o he nanoca ie s (o ganic, ino ganic, o hyb id), in addi ion o hei sizes, shapes,
and su ace modi ica ions can modula e hei o e all physicochemical p ope ies (124). These
modi ica ions can u he a ec hei biological p ope ies, such as oxici y and s abili y, as well as
he loading o d ugs and nanopa icles in he sys em. One o hese nanoca ie s ha ha e been
ex ensi ely s udied as a d ug deli e y sys em and which cons i u e he ocus o his wo k a e he
solid lipid nanopa icles (SLNs) (125–128).
20
SLNs we e i s ema ked in he ea ly 1990s (129–132) as an upg ade o he al e na i e
polyme ic, ino ganic and liposomic nanopa icles adi ionally used un il hen as ca ie s (128).
SLNs o mula ions a e al eady app o ed by FDA o by GRAS (Gene ally Recognized As Sa e), and
a e ecognized as sa e o be adminis e ed
ia
di e en ou es including in anasal, (133) inhala ion
(134), in a enous (135), subcu aneous (136), ec al (137), o al (138), ocula (139), and
in amuscula (140). SLN design empowe s he biodis ibu ion and pha macokine ics o he
in ended d ugs, enhancing he d ug ea men e ec i eness by o e coming he MDR (141) which
imp o es hei bioa ailabili y, p o ec ion om clea ance by he e iculoendo helial sys em (RES),
and con olling he d ug elease a e (125). Addi ionally, he possibili y o modi y he SLNs’ su ace
enhance he capabili y o o e come he biological ba ie s, o a ge he cance cells wi h minimal
side-e ec s (126), and dec ease he ini ial apid d ug elease, called “bu s e ec ” (125) (majo
d awback o he con olled elease sys em since hey could expose he pa ien o d ug o e dose
(142)). Iden ical o wha happens wi h MNPs, coa ing he SLNs wi h PEG a oids he apid immune
sys em cell up ake o hese nanoca ie s and inc ease hei ci cula ion ime (125,126,141,143).
2.3.1. NANOPARTICLE COMPOSITION
SLNs nanoca ie s a e colloidal pa icles composed o a lipid ma ix, solid a bo h oom and
body empe a u es, and su ac an s used as s abilizing and sol a ing agen s. Di e en lipid and
su ac an composi ions can con ol he size, polydispe si y, su ace cha ge, s abili y, and d ug
elease p o ile o he o mula ions (85). The selec ion o he lipid can also in luence he
biodeg adabili y, s abili y, and embedmen o he d ugs and o he elemen s (me als, dyes, e c.).
Commonly, a y acids, mono-, di-, and iglyce ides, a y alcohols, and waxes a e used o he
p epa a ion o SLNs (144). The small size o he o mula ions ( anging om 10 o 1000 nm), he
la ge su ace- o- olume a io, and he high d ug encapsula ion e iciency a e he key ad an ages o
he SLNs.
SLNs enable he encapsula ion o se e al d ugs, bo h hyd ophobic and hyd ophilic
(p e e en ially hyd ophobic), ha could be inco po a ed in ou di e en ways: i) dispe sed
homogeneously in he lipid ma ix, ii) dispe sed h oughou he shell, iii) inco po a ed in he co e,
and i ) dispe sed on he ex e nal shell (86,145). As a o emen ioned, chemo he apeu ic d ugs a e
di ided in o di e en classes ha could make di icul hei encapsula ion in o nanoca ie s.
Howe e , i is al eady demons a ed ha SLNs e icien ly inco po a e di e en chemo he apeu ic
d ug classes and could be used in he ea men o a wide ange o cance s (82,146–149).
21
2.4. MAGNETIC SOLID LIPID NANOPARTICLES
As a o emen ioned, SLNs p esen a b oad a ie y o ad an ages o he ea men o cance .
Se e al esea ch g oups ha e ocused on he de elopmen o hese pla o ms ying o exploi and
maximize hese bene i s (82,147–149). Howe e somewha su p isingly, he magne ic ma e ial
inco po a ion in he SLNs was no explo ed un il qui e ecen ly.
Di e en me als and me al de i a i es such as i on oxide, gold and gadolinium
(84,89,93,100,150) ha e been inco po a ed in he nano o mula ions, p oducing no el pla o ms
wi h g ea po en ial in cance he apy and issue imaging. In pa icula , encapsula ed i on oxide
and gadolinium ha e been s udied abundan ly as magne ic deli e y sys ems ha can be guided o
umo egions and/o ac i a ed o con olled d ug- elease and cell abla ion (magne ic
hype he mia) ia an ex e nal magne ic ield, o by pH changes (151–153). In pa icula , i on oxide
nanopa icles a e conside ed biocompa ible and sa e ma e ials, ha despi e being able o cause
some cy o oxic e ec s om he gene a ion o ROS ( eac i e oxygen species) ia Fen on eac ion,
which can lead o he damage o DNA, lipids, p o eins, and ca bohyd a es (154).
Magne ic solid lipid nanopa icles (mSLNs) ep esen a new class o unc ional
nanopla o ms, which usually consis o ino ganic magne ic nanopa icles and solid lipid
nanopa icle mix u es, ha ha e g ea applicabili y in he cance ield (155,156). The p elimina y
small size and high en apmen e iciency o he mSLNs managed o use he bene i s o bo h ypes
o nanoca ie s (SLNs and MNPs) and o e come hei independen applica ion issues. mSLN
shown an enhanced colloid and chemical s abili y and caused lowe oxici y
in i o
and
in i o
models,
compa ed o he MNPs alone, as desc ibed by Mülle and colleagues (157).
mSLNs syn hesis can be achie ed h ough di e en me hodologies, including he
emulsi ica ion dispe sion-ul asonic me hod (158), he emulsi ica ion-di usion me hod ollowed by
sonica ion (159), chemical co-p ecipi a ion (54,160), and he sol en e apo a ion echnique (161).
The cha ac e iza ion o he esul ing mSLNs can hen iden i y he s uc u e o he o mula ion,
whe e he magne ic nanopa icles can be embedded in he co e and/o su ace as desc ibed by
se e al au ho s (159–162). On he one hand, a ep esen a ion o magne ic nanopa icles
embedded in he co e can be seen in Figu e 9, whe e he MNPs’ hyd ophobic su ace show
chemical a ini y by he lipid ma ix o yield mSLNs. Fo he mSLN su ace, di e en su ac an s
can be used du ing he syn hesis o con e colloidal s abili y and sol a ion in wa e .

22
Al oge he , mSLNs ha e demons a ed o be a p omising ool due o hei good biocompa ibili y
(154–159), imp o emen o he mo esponsi eness compa ed o SLNs (156), e iciency in
a ge ing umo s (156–161), and hei high d ug encapsula ion e iciency. Fu he mo e, hese
nanosys ems allow he applica ion o magne ic hype he mia as a mean o p o ide he mal he apy
and con ol d ug elease (159–162), in addi ion o being used as MRI con as agen s (156). S ill,
he e a e only ew s udies in ol ing es s
in i o
, so highligh ing he need o alida e he
pe o mance o hese nanoca ie s in mo e biological complex sys ems.
2.4.1. SURFACE MODIFICATIONS
The applica ion o nanopa icles in he biomedical ield is a challenging ask whe e i is
equi ed o conside he nanopa icle biocompa ibili y and he capaci y o cellula in e naliza ion by
a ge cells (163,164). No mally, he sys emically adminis e ed nanopa icles passi ely accumula e
in he umo issue h ough he EPR e ec (165,166). Howe e , an e ec i e nanopa icle
biodis ibu ion is in ima ely ela ed wi h he nanopa icle-blood componen s’ in e ac ions. Di e en
biological ac o s could in luence he e icien d ug deli e y om nanopa icles, including he
mechanism o clea ance om blood ci cula ion. Thus, he immune sys em ecognizes he
Figu e 9. Schema ic s uc u e o magne ic solid lipid nanopa icles (mSLNs) and hei applica ion in
cance he anos ics. Due o he p ope ies o magne ic nanopa icles (MNPs), mSLNs can be used o diagnos ic
pu poses (e.g., MRI applica ion), and cance he apy, ia magne ic hype he mia. Mo eo e , magne ic hype he mia
in mSLNs o e s an ex a le el o con ol o e he d ug elease in o he egion o in e es , ul ima ely inc easing he
cy o oxici y o cance cells, in compa ison wi h SLNs o MNPs alone.
23
nanopa icles (p e e en ially he hyd ophobic ones) as o eign, ac i a ing mac ophages om he
mononuclea phagocy e sys em (MPS) (165–170).
The biophysiological unc ionali y is achie ed h ough he unc ionaliza ion o he
nanoca ie , allowing i s applica ion as d ug deli e y sys em, con as agen o he anos ic p obe
(165–167). The su ace unc ionaliza ion is a s a egy o p e en he nanopa icle phagocy osis
h ough opsoniza ion, inc ease he blood ci cula ion ime, imp o e he in acellula d ug deli e y
and, consequen ly, educe he d ug dose adminis a ion (165,166). A b oad ange o ligands can
be used o coa he nanopa icles’ su ace, including su ac an s, polyme s, small molecules,
pep ides, an ibodies, and oligonucleo ides (163).
2.4.1.1. PEGyla ion o nanopa icle unc ionaliza ion
The hyd ophobic su ace o nanopa icles inc ease he opsoniza ion which consecu i ely
ac i a e he MPS ac ion educing he bloods eam ci cula ion (164,167,168). A physiochemical
app oach o a oid he inal ou come is o coa he nanopa icles’ su ace wi h hyd ophilic polyme s,
such as polye hylene glycol (PEG), which esis s in e ac ion wi h blood componen s, impa ing
“s eal h” p ope ies o he nanopa icles (164,166–172).
The biocompa ible hyd ophilic PEG possesses he capaci y o minimize he binding o
opsonins, limi ing he immune sys em esponse and, subsequen ly, he coa ed nanopa icles
emain longe pe iods o ime in he bloods eam, enhancing he oppo uni y o passi ely
accumula e in he desi ed egion (166–173).
PEGyla ion is widely used, as speci ic- a ge ing ool, in he cance he anos ic ield. The su ace
modi ica ion o nanopa icles wi h PEG chains p o ides a a ge ed accumula ion in umo egion
due o EPR e ec , wi h minimal side-e ec s o non- a ge cells (165,167). A e ex a asa ion om
capilla ies o he umo en i onmen , PEG chains a e clea ed by he umo -associa ed
mic oen i onmen componen s, such as ma ix me allop o einases (MMP), whose concen a ion is
inc eased in umo a ea (164,166). Thus, nanopa icle accumula ion enables a clea ecogni ion
o umo egion o diagnos ic p oposes, as well as an imp o emen o a ge ed d ug deli e y, o
ea men pu poses (166–173).
24
2.4.1.2. Func ionaliza ion wi h Cell Pene a ing Pep ides
In cance ea men , he chemo he apeu ic d ugs place o ac ion is in he in acellula egion,
as cy oplasm, nuclei, o mi ochond ia. Howe e , due o impe meable ba ie o he cell memb ane,
he he apeu ic d ugs can be ba ed om pene a ing cells. As a s a egy o o e come his p oblem,
he conjuga ion be ween PEGyla ion and Cell Pene a ing Pep ides (CPP) has been used o enhance
he d ug in acellula up ake h ough umo cells, aking ad an age o a s eal h e ec (80,174).
Su ace unc ionaliza ion wi h CPP is he mos ly used pla o m o p omo e he lipid-based
nanopa icle pene a ion in o a ge cells, due o he posi i e cha ge o CPP (165,168,174–176).
Pep ides, as HIV-1 de i ed T ansac i a o o T ansc ip ion (TAT), a e speci ic sho pep ides (less
han 40 amino acids) capable o deli e ca gos ac oss he cell memb ane, enhancing he d ug
deli e y by endocy osis o di ec pene a ion. Also, TAT- unc ionaliza ion imp o es d ug deli e y,
and he e icacy o a s anda d d ug dose by o e coming d ug esis ance, associa ed wi h an
a enua ion o ad e se e ec s (168,175,176).
2.4.1.3. Diels-Alde eac ion
The applica ion o nanopa icles in he biomedical ield is always accompanied wi h he
pu pose o enhance hei biocompa ibili y, being c ucial o employ chemical p ocedu es ha a e
ep oducible and easy o manage (163,164). Click chemis y is an inc easingly used echnique wi h
g ea applicabili y on su ace unc ionaliza ion o nanopa icles, speci ically o a con olled- elease
sys ems (177,178). In 2001, Kolb and co-wo ke s in oduced he concep o “click chemis y” – a
eac ion di e en om he adi ional ones – cha ac e ized by a gene a ion o ino ensi e inal
p oduc s and s able unde biophysiological condi ions (179).
Due o he cha ac e is ics desc ibed by Kolb and colleagues, only some chemical
ans o ma ions ul ill he c i e ia, in pa icula Diels-Alde (DA) cycloaddi ion cha ac e ized by hei
he mal e e sibili y is one o hem (180). This one-s ep eac ion o ms a biocompa ible cycloadduc
wi hou side p oduc s, conside ing an ideal click eac ion (180–183).
A e eaching he pa hological si e, d ug elease occu s in esponse o mic oen i onmen (
e.g
.,
pH) o ex e nal s imuli, as hea , adio equency, o ul asound. DA eac ion can be explo ed o
nanopa icle su ace deco a ion, enhancing he e iciency o d ug elease sys ems. Speci ically,
magne i e nanopa icles o e he possibili y o ac i a ing DA he mal e e sibili y aking ad an age
o hei magne ic p ope ies o induce a empe a u e inc ease (181–183). Ex e nally ac i a ed DA-
based igge ed mechanisms in conjuga ed sys ems enhance hei applicabili y o deli e y o
25
in e nalized molecules wi h a decline o oxic e ec s and p omo e he conjuga ion o he anos ic
ools wi h he con olled- elease esponse – conside ed he nex gene a ion nanopa icles
(182,183).
2.4.2. BIOMEDICAL APPLICATIONS
2.4.2.1. Magne ic Resonance Imaging (MRI) as a diagnos ic ool
WHO’s 2018 wo ld cance epo p edic ed an inc ease o 60% in cance incidence by 2040.
Cu en ly, ea ly de ec ion is he mos e ec i e way o inc ease he p obabili y o success ul
ea men o mos cance s. Fo TNBC, ea ly de ec ion is undamen al once sc eening being usually
la e and di icul (97). De ec ion o malignancy ideally equi es a non-in asi e, as , and p ecise
diagnos ic sys em able o p o ide he posi ion, size, and cha ac e is ics o he p ima y umo , in
addi ion o he localiza ion and size o me as a ic masses (99).
MRI is a diagnos ic ool used in he clinical se ing o umo de ec ion and acking, classi ied
as a non-in asi e, sa e, and painless echnique ha uses magne ism and adio pulses o p oduce
eal- ime images om di e en angles all a ound he body. The esul is usually a clea depic ion
o so issues, including umo s (7,28,118).
The echnique is based on he nuclea magne ic esonance (NMR) p inciple, cen e ing on he
p ope ies o some a oms o abso b ene gy in he o m o adio wa es when unde a magne ic
ield, and consequen ly p oduce an elec ic signal (117,184). Such e en causes a spin pola iza ion
ha can induce a signal in a adio equency coil ha can hen be de ec ed by a nea by
an ennae/de ec o . Usually, hyd ogen nuclei consis ing o a single p o on, a e used o c ea e he
signals. Hyd ogen is na u ally abundan in all o ms o li e, and hence can be used o c ea e a
mac oscopic pola iza ion o hyd ogen- ich issues ( ich in wa e and a ) (28,99). The pulses o
adio wa es exci e he nuclea spin ene gy ansi ion whils he magne ic ield g adien s localize
he pola iza ion in space. A e exci a ion, his echnique measu es he elaxa ion
1
ime o he nuclei
in he longi udinal (
T1
- spin-la ice elaxa ion), and ans e se planes (
T2
- spin-spin elaxa ion)
(99,117,185). The image o med he e is dependen on he issue’s local a omic densi y and
associa ion o hyd ogen o o he a oms (28). The e o e, he pulse sequence can gene a e di e en
con as s be ween issues acco ding o he di e en chemical en i onmen o adjacen issues. MRI
enables apid
in i o
imaging acquisi ion wi h high spa ial esolu ion (~100 µM), wi hou exposing
1
The e m elaxa ion e e s o he mo emen o nuclea spins o hei ini ial low-ene gy s a e when he
adio equency wa e is emo ed (118).
32
1. MAGNETIC SOLID LIPID NANOPARTICLES USED IN THE PRESENT
WORK
The nanopa icles s udied o hei an icance po en ial in he p esen esea ch wo k a e based
on he magne ic solid lipid nanos uc u es. Nanomedicine g oup a he In e na ional Ibe ian
Nano echnology Labo a o y (INL) de eloped d ug-loaded magne ic (mSLNs) om a comme cially
a ailable ege al wax, which was simul aneously loaded wi h a chemo he apeu ic d ug (DOX) and
magne ic i on oxide nanopa icles (MNPs). Addi ionally, a su ace unc ionaliza ion was ca ied ou
using a mix u e o wo di e en esponsi e ligands. One o hem was a empe a u e esponsi e
PEGyla ed ligand ha ende ed mSLNs wi h s eal hy p ope ies, whe eas he o he was a cell
pene a ing pep ide (CPP)-modi ied ligand. Thus, by local and ex e nal applica ion o an AMF on
he a ea o in e es (magne ic hype he mia, MH), a he mal-induced sheddable PEGyla ion is
induced ha exposes he CPP ligand and igge s on-si e TNBC in e naliza ion h ough a ge -less
magne ic ca ie s. Con ols mSLNs o mula ions we e also p epa ed o e alua e he di e en
componen s in he inal mSLNs design in o de o accomplish he expec ed pe o mance
in i o
.
The unc ionalized su ace cha ac e is ics o he di e en samples used in his esea ch a e
desc ibed in Table IV.

33
2. CELL CULTURE AND CELL LINES
The human basal b eas cance cell lines Hs578 (basal-like), MDA-MB-231(basal-like), and
he human mac ophage cell line THP-1 we e ob ained om Ame ican Type Cul u e Collec ion
(ATCC, Vi ginia, USA). Hs578 and MDA-MB-231 cell lines was cul u ed in Dubelcco’s Modi ied
Eagle Medium, 4.5 g/L glucose (DMEM, Bioch om), while THP-1 cells we e cul u ed in Roswell
Nanopa icles
Componen s
Func ion
DA-DOX-TAT-PEG-mSLNs
(Nomina ed as EBR85)
▪ Cell-pene a ing
pep ide (TAT-
unc ionaliza ion)
▪ PEGyla ion
▪ Diels-alde bond
▪ Ca nauba Wax
▪ Tween89 (co e-shell)
▪ Doxo ubicin
▪ Magne i e (Fe3O4)
Applica ion on MRI diagnosis and
TNBC he apy ( he anos ic
p ope ies), o e ing in a combina ion
wi h a dual ea men capabili y
(chemo he apy plus hype he mia)
DOX-TAT-PEG-mSLNs
(Nomina ed as EBR86)
▪ Cell-pene a ing
pep ides (TAT-
unc ionaliza ion)
▪ PEGyla ion
▪ Ca nauba Wax
▪ Tween89 (co e-shell)
▪ Doxo ubicin
▪ Magne i e (Fe3O4)
Con ol o Diels-Alde (DA) ac i a ion
cha ac e iza ion
DOX-TAT-mSLNs
(Nomina ed as EBR52)
▪ Ca nauba Wax
▪ Tween89 (co e-shell)
▪ Doxo ubicin
▪ Magne i e (Fe3O4)
Con ol o ligands ac i i y e iciency
assessmen
DA-TAT-PEG-mSLNs
(Nomina ed as EBR89
▪ Cell-pene a ing
pep ides (TAT-
unc ionaliza ion)
▪ PEGyla ion
▪ Diels-alde bond
▪ Ca nauba Wax
▪ Tween89 (co e-shell)
▪ Magne i e (Fe3O4)
Con ol o doxo ubicin (DOX) cell
in e naliza ion and he mo-
chemo he apeu ic syne gis ic
e iciency
Table IV. Magne ic solid lipid nanopa icles (mSLNs) used in he p esen wo k. Schema ic ep esen a ion o he mSLN
o mula ions used in his wo k, oge he wi h iden i ica ion o hei main unc ion.
34
Pa k Memo ial Ins i u e (RPMI, Gibco), bo h supplemen ed wi h 10% hea ing ac i a ed Fe al Bo ine
Se um (FBS, Sigma-Ald ich) and 1% o an ibio ic solu ion (Penicillin-S ep omycin, Gibco).
Cells we e g own in a humidi ied incuba o a 37 ºC and 5% CO2. Sub-cul u ing was pe o med
using 80% con luence cul u e lasks. Then, he cells we e eaped by washing he T75 lasks wi h
phospha e-bu e saline (PBS 1x) and de ached om he lasks using ypsin (T yplexTM Exp ess,
Gibco) a 37 ºC. A e he con i ma ion ha he cells we e no longe adhe ed, 10% FBS medium
was added o he lasks o inac i a e ypsin and hen, cells we e collec ed and cen i uga ed du ing
5 min, a 1200 pm, 20 ºC. The cul u e medium was disca ded and cells esuspended in medium
o de e mine he numbe o iable cells, T ypan blue exclusion me hod (T ypan Blue Solu ion, 4%,
Gibco) and Neubaue chambe we e u ilized.
3. 2D MONOLAYER CELL CULTURE
3.1. RESAZURIN ASSAY
Resazu in dye has been used in se e al cy o oxici y s udies as indica o o he cell iabili y. In
his app oach, ae obic espi a ion o me abolically ac i e cells is esponsible o he educ ion o
esazu in (blue compound) o eso u in (pink colo ). The eso u in luo escence de ec ed a 560
nm exci a ion / 590 nm emission is ela ed o he numbe iable cells (199,200). A mic opla e
eade (Bio ek Syne gy H1) was used o measu e he luo escence o he gene a ed eso u in.
3.1.1. CELL VIABILITY ASSAY WITHOUT MAGNETIC HYPERTHERMIA APPLICATION
To de e mina e he cy o oxici y e ec s o he s udied compounds o bo h TNBC and
mac ophage cell lines, wi hou MH applica ion, he cells we e pla ed in 96-well pla es a a densi y
o 5x103 cells/100 µL o 1x104 cells/100 µL, espec i ely, and incuba ed wi h supplemen ed
medium.
TNBC cells we e allowed o adhe e o e nigh a 37 ºC unde 5% o CO2 en i onmen and, in
he nex day, he medium was disca ded and cells we e ea ed wi h inc easing concen a ions o
he medium con aining he co esponden compound. Rela i ely o THP-1 cell line (g own in
suspension), cells we e ea ed wi h medium con aining compounds on he same day ha hey
we e pla ed. The nega i e con ol was a g oup o cells no ea ed wi h he compounds.
35
Then, he cul u ed cells we e incuba ed o 24, 46 and 72 h, and subsequen ly 10% o
Resazu in (10x concen a ed) was added in each well and incuba ed o 4 h. Spec opho ome ical
measu emen s we e pe o med in a mic opla e eade (Bio ek Syne gy H1), using 560 nm
exci a ion / 590 nm emission se s. A leas , h ee independen expe imen s we e pe o med, each
one in iplica e, and he s a is ical analysis so wa e G aphPad P ism 7 was used o de e mine he
IC50 (hal -maximal inhibi o y concen a ion) alues o each compound, applying a sigmoidal dose-
esponse ( a iable slope) non-linea eg ession a e loga i hmic ans o ma ion.
The he apeu ic gain (TG) was calcula ed in compa ison wi h he IC50 alue o ee DOX when
no hype he mia was applied (Equa ion 1).
𝑇𝐺 (%)=100 𝑥(𝐼𝐶50𝐷𝑂𝑋
𝐼𝐶50𝑚𝑆𝐿𝑁𝑃
-1
) Equa ion 1
3.1.2. CELL VIABILITY ASSAY WITH MAGNETIC HYPERTHERMIA APPLICATION
The o e hea ing cha ac e is ic o MH ea men is widely used as an adju an in cance
he apy (193,194). Wi h he applica ion o an AMF, MNPs can locally ans o m he elec omagne ic
ene gy in o hea , inc easing he local cance cell’s empe a u e and esul ing in umo g ow h
inhibi ion (193).
To selec he equency and ampli ude o he ex e nal AMF, i is necessa y o conside he
B ezo ich limi (
H0
< 4.85x108 A.m-1.s-1), which speci y he uppe limi o he magne ic ield ha
can be applied o human issues a oiding undesi able hea ing o su ounded issues (201). Fi s ly,
he sample is placed inside a helical induc ion coil, whe e he selec ed ampli ude o he AMF is
achie ed. The e o e, an al e na ed cu en low along he coil changing, a chosen equency, he
magne ic lux. The equipmen is also composed by a empe a u e senso (op ical ibe ), connec ed
o a compu e ha eco ds he empe a u e changes along ime (202,203).
In his wo k, he cy o oxici y e ec o he syne gis ic he mal ea men was e alua ed by
pe o ming he cell iabili y assay a e MH applica ion, whe e Hs578 cells we e seeded in a
dedica ed Pe i dish (di ided in 4 compa men s) a a densi y o 2.5x104 cells/500 µL. Cells we e
incuba ed o e nigh a 37 ºC unde 5% o CO2 a mosphe e. The ea e , he medium was emo ed
and he cells we e ea ed wi h inc easing concen a ions o he medium con aining he mSLNs o
in e es and subjec ed o an AMF a a ield and equency o 23 mT and 174.5 kHz, espec i ely,
36
o 1 h, in a NAN201007 Li e Cell Exposu e module om NanoThe ics. Fo con ol pu pose, one
g oup o cells did no ecei e u he ea men .
Cells we e incuba ed again o 24, 48 and 72 h and, a e each ime-poin , 10% Resazu in
was added o each compa men and incuba ed o 4 h. Then, he solu ion om each compa men
was ans e ed on o a 96-well pla e and spec opho ome ic analysis was pe o med in a
mic opla e eade , using 560 nm exci a ion / 590 nm emission se s. A leas , h ee independen
expe imen s we e pe o med, each one in iplica e, and he s a is ical analysis so wa e G aphPad
P ism 7 was used o de e mine he IC50 alues o each compound, applying a sigmoidal dose-
esponse ( a iable slope) non-linea eg ession a e loga i hmic ans o ma ion.
The ac i i y gain (AG) by hype he mia was calcula ed by i s ly no malizing o he DOX IC50
alue (wi hou hype he mia applica ion), and hen compa ing he DOX IC50 wi h he mSLNs IC50
a e hype he mia applica ion (Equa ion 2).
𝐴𝐺 (%)=100 𝑥 (𝐼𝐶50
𝐷𝑂𝑋
𝐼𝐶50
𝑚𝑆𝐿𝑁𝑃 𝐼𝐶50
𝐷𝑂𝑋 & 𝑀𝐻
𝐼𝐶 50
𝑚𝑆𝐿𝑁𝑃 & 𝑀𝐻
⁄ −1) Equa ion 2
The deg ee o syne gism o he dual- ea men was calcula ed using he Equa ion 3, whe e
MHT is he MH and Chemo is he chemo he apy. Fo a α equal o 1 he ea men applied had an
addi i e e ec ; α<1, he dual- ea men is an agonis , and α>1 ep esen a syne gis ic e ec o he
dual- ea men .
𝛼= 𝐼𝐶50
𝑀𝐻𝑇.𝐼𝐶50
𝐶ℎ𝑒𝑚𝑜 𝐼𝐶50
𝑀𝐻𝑇+𝐶ℎ𝑒𝑚𝑜
⁄ Equa ion 3
3.2. CONFOCAL MICROSCOPY IMAGING
To ollow he cellula pheno ype o b eas cance cells upon hei in e ac ion wi h mSLN
o mula ions, con ocal mic oscopy was used. Con ocal mic oscopy o e s se e al ad an ages ace
o con en ional op ical mic oscopy whe e, he p incipal poin is he elimina ion, o educ ion, o
backg ound “noise” om he ocal plane (204).
The con en ional op ical mic oscopy uses a beam o ligh o di ec ly illumina e he en i e ield
o he sample, being all pa s o he sample exci ed a same ime. Howe e , he inal image is
composed by bo h in- ocus ligh a he ocal plane o he lens and ou -o - ocus ligh coming om
37
o he ocal planes, p omo ing a dec ease o signal o noise (204,205). In con as , he con ocal
mic oscopy uses a pinhole a he image plane o p e en ha he ou -o - ocus ligh does no each
he de ec o , sca e ing he o he ligh . Though, in o ma ion abou a unique poin o he sample is
p o ided and he e o e, i is necessa y o mo e he specimen o al e he exci ing ligh o ob ain
he inal image [195]. In his way, wo-dimensional images could be acqui ed plane o plane, a
di e en dep h, enabling he h ee-dimensional econs uc ion by using a mic oscopy decon olu ion
so wa e (z-s ack). This echnique allows he de ec ion o di e en sec ions o he sample due o
dyes ha u n luo escen when s imula ed wi h ligh , named luo opho es. By using a speci ic
wa eleng h o exci e he specimen, i enables he exci a ion o speci ic luo opho es (206).
In his s udy, he con ocal mic oscope LSM780 om Zeiss was used o isualize he
in e ac ions be ween Hs578 li e cells and he ea ed compounds. On he day be o e, 6x103 cells
we e seeded in Pe i dish di ided in 4 compa men s, and hen incuba ed o e nigh a 37 ºC unde
5% CO2. The ea e , he medium was emo ed and he cells we e ea ed wi h medium con aining
he compounds. The cells we e again incuba ed o 2 di e en ime poin s, 2 h and 6 h. A e he
incuba ion ime, he cells we e washed wi h 1x PBS and Hoechs 33342 was added (1:1000, ca .
no. ab139481, Abcam). The cells we e incuba ed o 10 min, washed again wi h 1x PBS, and hen
DMEM phenol ed- ee was added. The inhe en luo opho e (DiO) inco po a ed in mSLNs
memb anes pe mi ed o ack he designed nanopa icles, and he DOX inhe en luo escence was
ollowed and co-localized wi h a nuclea s ain (Hoechs 33342), using he exci a ion lase o 488
nm o DOX and DiO, and 405 nm o Hoechs 33342.
3.3. CELL PROLIFERATION ASSAY
The cell p oli e a ion is a colo ime ic immunoassay e alua ed using 5-b omo-2’-deoxyu idine
(B dU) (Cell P oli e a ion ELISA, B dU, Roche®). B dU is a py imidine analog which could be
inco po a ed in o cells DNA du ing he syn hesis p ocess.
Acco ding o manu ac u e 's ins uc ions, Hs578 cells we e pla ed in 96-well pla es, in
iplica e (8000 cells/well), and allowed o adhe e o e nigh in comple e DMEM medium, a 37 ºC
in a 5% CO2 humidi ied a mosphe e. A e medium emo al, he adhe en cells we e ea ed wi h
espec i e EBR85, EBR89 and DOX a IC50 and 1⁄2 IC50 dosages o 24 and 48 h o incuba ion.
The con ols we e ea ed wi h a comple e DMEM medium. A e incuba ion, B dU dilu ed in 1%
FBS cul u e medium was added o he cells ( inal B dU concen a ion o 10 μM) and eincuba ed

38
o 6 h. Du ing his incuba ion s ep, B dU was inco po a ed (in hymidine place) in o DNA o
p oli e a i e cells. A e B dU labeling, cells we e ixed, and DNA was dena u ed h ough incuba ion
wi h 200 μL o FixDena solu ion o 30 minu es a oom empe a u e. A e emo al o his solu ion,
100 μL o An i-B dU-POD an ibody (1:100 dilu ion) we e added, which will bind o ecen ly
inco po a ed B dU in he cells’ DNA. Following 90 min o incuba ion a oom empe a u e, he wells
we e insed h ee imes wi h PBS 1x. Nex , 100 μL o subs a e solu ion ( e ame hyl-benzidine)
was added a oom empe a u e o 5 min un il colo de elopmen . The colo ime ic eac ion was
s abilized by adding 25 μL/well o 1 M H2SO4. The eac ion p oduc was quan i ied by measu ing
he abso bance a 450 nm (The mo Scien i ic Va ioskan® Flash). A blank con ol was used in each
expe imen al ime poin , wi hou cells. Pe cen age o cell p oli e a ion (no malized o con ol
condi ion) om a leas h ee independen expe imen s was e alua ed wi h he G aphPad P ism 7
so wa e.
3.4. WOUND-HEALING ASSAY
Cell mig a ion abili y was app aised by he wound-healing assay in o de o mimic he mig a ion
e en ha occu s in i o. The basic s eps consis in pe o ming a sc a ch in a con luen well pla e,
simula ing a wound, cap u ing images a he beginning o he expe imen and a di e en egula
ime-poin s o, a he end, compa e he images and quan i y he cell mig a ion a e in di e en
ea men s (207).
Hs578 cell line was seeded in 6-well pla es a a densi y o 9.0x105 cells/well/2 mL and
incuba ed o e nigh a 37 ºC unde 5% o CO2 a mosphe e. A ime poin ze o, cul u e medium was
emo ed and wo sc a ches we e execu ed wi h a 200 µL pipe e ip on he cell monolaye . The
cell deb is we e de ached by gen ly washing wi h 500 µL 1x PBS, and he cells ea ed wi h
espec i e compounds ( ee DOX, EBR85, and EBR89) a he espec i e IC50 alue o medium only
o con ol pu poses, o 48 h. A ime-poin s o 0, 12, 24, 36 and 48 h o ea men , eigh speci ic
“wounded” a eas pe condi ion we e pho og aphed a 100x magni ica ion by phase con as
mic oscopy (Olympus® IX51). The mig a ion dis ances om a leas h ee independen assays
we e assessed using he beWound - Cell Mig a ion Tool (Ve sion 1.5), and he pe cen age o cell
mig a ion a e no malized o he con ol condi ion was e alua ed wi h he G aphPad P ism 7
so wa e.
39
4. 3D BIOMODELS (SPHEROIDS)
4.1. DEVELOPMENT OF SPHEROIDS
Al hough he 2D monolaye cell cul u e o e s many bene i s, due o hei simple p ocedu e,
hey could no mimic he eal cell mic oen i onmen , esul ing in a poo ep oduc ion o he
nanopa icle-cell in e ac ions. The cells no mally li e in 3D en i onmen essen ial o hei
me abolism, g ow h, and cell-cell in e ac ions. Fo d ug sc eening, he use o 3D cell cul u e, as
sphe oids, is impo an since i conside s he cell mo phology, p oli e a ion, was e p oduc ion, and
d ug up ake. Mo eo e , cellula esponse o d ug he apies in 3D cul u e displayed esul s mo e
simila o wha happens
in i o
(208,209).
In he cu en wo k, Hs578 sphe oids we e ob ained om Hs578 cell line cul u e. Fi s ly,
1x104 cells/100 µL we e seeded in 96-well pla es, con aining 100 µL o aga ose 1% in each well.
Then, he pla es we e cen i uged a 1600 pm o 30 min o allow he cells o se le by g a i y.
Subsequen ly, he pla es we e incuba ed a 37 ºC unde 5% CO2. Fo 1 week, he sphe oids’
medium was eplaced e e y 2 days. On he second week, sphe oids we e seeded in a Pe i dish
wi h 4 compa men s con aining aga ose 1% (4 o 5 sphe oids pe quad an ), o ming he
polysphe oids, and he medium was changed e e y 2 days.
4.2. EVALUATION OF 3D POLYSPHEROID GROWTH
In o de o e alua e he esponse o Hs578 polysphe oids o a single (only DOX-con aining
mSLNs) and dual he apy (DOX plus MH), sphe oid g ow h was assessed along 3 weeks and
compa ed o ee DOX ea men .
A e sphe oid o ma ion, ea men was conduc ed by adding 10 µM o he co esponding
compound. Then, he Pe i dishes planned o dual ea men we e subjec ed o AMF a 23 mT o
ield and 174.5 kHz o equency, o 1 h. The same ea men was pe o med e e y 2 days o 1
week. The ea e , du ing he ollowing 2 weeks, he medium was changed e e y 2 days.
To assess polysphe oid g ow h, pho og aphs we e aken e e y 2 days, ep esen a i e o each
s udy condi ion, s a ing 3 days be o e he beginning o he ea men . Th ough he pho og aphs,
and using he RS udio p og am, i was possible o measu e he sphe oid daily g ow h, by i s ly
no malizing he g ow h pe sphe oid (Equa ion 4),
𝐺𝑛 (%)=100 𝑥 𝐴𝑟𝑒𝑎𝑑𝑎𝑦
𝐴𝑟𝑒𝑎𝑑𝑎𝑦=1 Equa ion 4
40
whe e he A eaday is he sphe oid a ea co esponding o each day and he A eada=1 is he
measu ed sphe oid a ea a he beginning o he ea men . Then, using he Equa ion 5, we ob ain
he no malized daily g ow h enabling he compa ison o di e en condi ions es ed independen ly
o ime.
𝐷𝐺𝑛 (%)=∑𝐺𝑛
𝐷𝑎𝑦𝑠= ∑𝐺𝑛
19 Equa ion 5
4.3. MAGNETIC RESSONANCE IMAGING (MRI) STUDIES
The magne ic p ope ies o i on oxide nanopa icles ha e made hem in e es ing p obes in MRI.
These p ope ies enable he easy co-localiza ion o he MNP when passi ely o selec i ely
accumula ed in umo s, whils in u n hey can simul aneously p o ide a he apeu ic e ec (
i.e
.
d ug deli e y, MH) – he anos ics (119).
In o de o e alua e he
T2
-MRI con as enhancemen capabili y o he de eloped mSLNs,
samples we e placed inside he scanne bo e, whe e an oscilla e magne ic ield is c ea ed,
p omo ing he alignmen o he p o ons in same di ec ion
B0
. By a se o g adien coils, he
adio equency magne ic ield is swi ched om on o o , a an app op ia e esonance equency,
and he p o on alignmen al e s, ealigning wi h he magne ic ield. As a esul o he signal coming
om he exci ed a oms and he changes in x, y and z di ec ion caused by his, a MR image is
gene a ed. Then, he de ec ed signal is ans o med by Fou ie T ans o m, ampli ied, and p esen ed
in a compu e . MR images could be acqui ed wi h di e en se s o pulse sequences and g adien s
which esul s in di e en image appea ance.
In his assay, a e inishing he 3D sphe oid g ow h, MRI s udies we e pe o med o alida e
he
in i o
T2
con as capabili y o mSLNs o MRI applica ions. Thus, a change in elaxi i y was
analyzed a e he in e naliza ion o mSLNs in Hs578 polysphe oids. MR imaging was pe o med
in a MR Solu ions Bench op scanne (Guild o d, UK), unde a clinical magne ic ield o 3.0 T
ho izon al bo e, a oom empe a u e.
To measu e he samples, polysphe oids we e placed in a 200 µL o Milli-Q wa e in a Pe i dish
(each compa men o each condi ion). The pla e was se in o a cus om p in ed MRI holde and
posi ioned in he cen e o he MRI scanne bo e. Subsequen ly, a imaging acquisi ion ook place
using a as spin-echo (FSE) sequence, wi h pa ame e s men ioned in Table V. Pos -p ocessing
was comple ed using he ImageJ so wa e (1.46 , NIH, USA), o de e mine he elaxa ion ime o
41
each condi ion, using he “MRI analysis calcula o ” plugin. To calcula e he ans e se elaxi i y ( 2)
o he mSLNs, he Equa ion 6 was used. 𝑟2=1/𝑇2 Equa ion 6
Table V. Pa ame e o he as spin-echo (FSE) sequence used o imaging acquisi ion. Using he as
spin-echo (FSE) sequence, he elaxa ion ime o each condi ion was acqui ed wi h he pa ame e s shown in his able.
Pa ame e s
Values
Ma ix
4096 x 2048
Field o iew (FOV)
40 x 40 mm
Slices
6
Slice hickness
1.5 mm
Echo ime (TE)
15 ms
Repe i ion ime (TR)
1500 ms
Numbe o a e ages (NA)
5
To al acquisi ion ime (AT)
581 ms
4.4. CONFOCAL MICROSCOPY IMAGING
The con ocal mic oscope LSM780 om Zeiss was used o isualize compound in e naliza ion
in o Hs578 sphe oids. A e sphe oid o ma ion, one sphe oid/well/500 µL we e placed in a Pe i
dish di ided in 4 compa men s. The sphe oids we e ea ed wi h 10 µM o espec i e compounds,
and hen incuba ed o e nigh a 37 ºC unde 5% o CO2. The ea e , sphe oids we e washed wi h
1x PBS and Hoechs 33342 was added, a a concen a ion o 0.1% o o al olume. The sphe oids
we e incuba ed o 10 min and isualized by con ocal mic oscopy. The inhe en luo opho e (DiO)
inco po a ed in mSLNs memb anes pe mi ed o ack he designed nanopa icles, and
addi ionally, he DOX inhe en luo escence was ollowed and co-localized wi h a nuclea s ain
(Hoechs 33342), using he exci a ion lase o 488 nm o DOX and DiO, and 405 nm o Hoechs
33342.
4.5. INDUCTIVELY COUPLED PLASMA-OPTICAL EMISSION SPECTROSCOPY
Induc i ely coupled plasma-op ical emission spec oscopy (ICP-OES) is a b oadly used
analy ical echnique o de e mina e and quan i y ace elemen s in complex sample o ma s (0.1 –
100 ng.mL-1) (210,211). ICP-OES can simul aneously de ec up o 70 elemen s, being widely used
in nanomedicine o quan i y he pe cen age o speci ic elemen s (
e.g.,
Fe) in nanopa icles. This
48
3| RESULTS AND DISCUSSION

49
1. DOSE RESPONSE CURVES AND IC50 DETERMINATION
The he apeu ic e ec o he de eloped mSLNs p obes (Table IV) was s udied by de e mining
he IC50 (concen a ion ha inhibi s 50% o cell iabili y) using he esazu in assay. Hs578 and
MDA-MB-231 cance cell lines we e ea ed wi h 14 di e en concen a ions o di e en mSLNs:
100, 50, 25, 12.5, 5, 2.5 1.25, 0.63, 0.31, 0.17, 0.08, 0.04, 0.02, and 0.01 µM ela i e o DOX
con en , wi hou an ex e nal magne ic s imulus, o 24, 48 and 72 h o incuba ion. DOX, a cy o oxic
d ug used in b eas cance ea men , was used as e e ence compound o compa e he cy o oxic
e ec o his comme cial d ug wi h DOX-loaded mSLNs. The esul s om he IC50 de e mina ions
o bo h cell lines a e summa ized in Table VI and he dose esponse cu es a e ep esen ed in
Figu e 13 and Figu e 14 o Hs578 and MDA-MB-231 cell lines, espec i ely.
Table VI. IC50 alues o ee DOX and DOX-loaded mSLNs o Hs578 and MDA-MB-231 TNBC cell lines.
The IC50 alues we e de e mined o h ee di e en imes o incuba ion, 24, 48 and 72 h.
Compound[a]
Hs578
MDA-MB-231
24 h
IC50 (µM) ± SD[b]
48 h
IC50 (µM) ± SD[b]
72 h
IC50 (µM) ± SD[b]
24 h
IC50 (µM) ± SD[b]
48 h
IC50 (µM) ± SD[b]
72 h
IC50 (µM) ± SD[b]
DOX
8.88 ± 2.20
4.47 ± 0.997
2.26 ± 0.29
12.21 ± 1.258
3.40 ± 0.19
3.06 ± 0.25
EBR52
3.55 ± 0.64
3.31 ± 0.39
2.27 ± 0.54
3.75 ± 0.21
3.10 ± 0.22
2.58 ± 0.51
EBR85
3.73 ± 0.40
2.35 ± 1.08
1.82 ± 0.60
2.98 ± 0.24
1.35 ± 0.06
1.13 ± 0.19
EBR86
3.49 ± 0.35
2.29 ± 0.64
1.98 ± 0.43
2.28 ± 0.14
1.74 ± 0.51
1.23 ± 0.19
EBR89
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
[a]: Each ea men was es ed, a leas , in iplica e and he da e a e p esen ed as mean alues. [b]: S anda d
De ia ion. n.d.: non-de e mined
50
Figu e 13. Dose esponse cu es and IC50 compa isons. E ec o mSLNs (EBR52, EBR85, EBR86, and EBR89)
and e e ence compound ( ee DOX) on he cell iabili y o Hs578 cance cells, o o al cell biomass, a 24, 48, and
72 h o incuba ion. Resul s a e exp essed as a mean ± SD.
a
: Resul s signi ican ly di e en om EBR52, EBR85,
EBR86, and EBR89 (
p
<0.0001);
b
: Resul s signi ican ly di e en om DOX, EBR52, EBR85, and EBR86 (
p
<0.0001);
c: Resul s signi ican ly di e en om EBR85, and EBR86 (
p
<0.05).
51
Figu e 14. Dose esponse cu es and IC50 compa isons. E ec o mSLNs (EBR52, EBR85, EBR86, and EBR89)
and e e ence compound ( ee DOX) on he cell iabili y MDA-MB-231 cance cells, o o al cell biomass, a 24, 48,
and 72 h o incuba ion. Resul s a e exp essed as a mean ± SD.
a
: Resul s signi ican ly di e en om EBR52, EBR85,
EBR86, and EBR89 (
p
<0.05);
b: Resul s signi ican ly di e en om DOX, EBR85, EBR86, and EBR89 (
p
<0.05);
c:
Resul s signi ican ly di e en om DOX, EBR52, EBR85, and EBR86 (
p
<0.0001);
d
: Resul s signi ican ly di e en om
DOX, EBR85, and EBR86 (
p
<0.0001)
52
The unc ionalized nanopa icles (EBR85 and EBR86) we e he mos ac i e molecules, in bo h
cell lines, wi h a capaci y o educe he cell iabili y in a dose-dependen manne and displayed he
lowes IC50 alue a 72h o incuba ion, in compa ison wi h DOX- ea ed cells, which could be
indica ing a sus ained d ug elease along ime. Addi ionally, i was possible o obse e ha he
nano o mula ions wi hou d ug (EBR89) a e non- oxic o cance cells wi hin he whole ange o
d ug concen a ions es ed. Fo a sa e biological applica ion o nanoca ie s in human issues,
hei oxici y should be lowe o negligible. The esazu in luo ome ic assay demons a ed ha
EBR89- unc ionalized nanopa icle p esen ed a good biocompa ibili y a he highes concen a ion
es ed (0.014 mgFe.ml-1). Acco ding o he li e a u e, hese esul s a e consis en wi h o he
epo ed esul s, e ealing only cy o oxici y e ec s o highe Fe concen a ions (219,220).
The syne gis ic oxici y o chemo he apy in combina ion magne ic hype he mia combina ion
was also assessed using he esazu in assay, in Hs578 cells. The objec i e o his dual ea men
was o inc ease he he apeu ic index o chemo he apeu ic ea men alone, hus allowing he use
o educed d ug doses and a oiding he ha m ul side e ec s associa ed o s anda d chemo he apy
ea men s.
The e o e, Hs578 cells we e ea ed wi h 6 di e en concen a ions, 0.1, 0.3, 0.6, 1.2, 2 and
5 µM ela ed o DOX con en , ollowed by 1 h o an ex e nal magne ic s imulus applica ion and 72
h o incuba ion. The dose esponse cu e and he g aph ep esen a ion o he IC50 alues ob ained
wi h o wi hou magne ic hype he mia applica ion a e p esen ed in Figu e 15 and he IC50 alues
a e summa ized on Table VII. Addi ionally, using he IC50 alues ob ained o he cells- ea ed wi h
o wi hou magne ic hype he mia applica ion, he ac i i y gain o each o mula ion was calcula ed
o he dual ea men using he Equa ions 1 and 2 (Chap e 2, sec ion 3).
53
Table VII. IC50 alues o he mSLNs and Doxo ubicin o Hs578 cance cell line. The IC50 alues we e
de e mined o 72 h o incuba ion a e ea he cells wi h espec i e compound plus 1 h o hype he mia.
T ea men [a]
IC50 (µM) ± SD[b]
Only Hs578 cells + HT
n.d.
DOX + HT
2.1 ± 0.11
EBR52 + HT
0.62 ± 0.0.7
EBR85 + HT
0.21 ± 0.16
EBR86 + HT
0.60 ± 0.02
EBR89 + HT
1.84 ± 0.26
[a]: Each ea men was es ed, a leas , in iplica e and he da e a e p esen ed as mean alues. [b]: S anda d
de i a ion. HT: Hype he mia applica ion. n.d.: non-de e mined.
Figu e 15. Cy o oxic e ec s o di e en mSLNs on Hs578 cells. A. E ec o mSLNs (EBR52, EBR85, EBR86,
and EBR89) and e e ence compound ( ee DOX) plus 1 h o hype he mia on Hs578 cance cells, o o al cell
biomass, a e 72 h om ea men . B. Compa ison o he IC50 alues ob ained o he cells- ea ed wi h and wi hou
he applica ion o a magne ic s imuli. Resul s a e exp essed as a mean ± SD.

54
The esul s demons a ed ha he main di e ence be ween he o mula ions can be obse ed
unde dual- ea men applica ion o he cance cells, wi h signi ican cance cell iabili y dec ease.
EBR89 o mula ion displayed he highes ac i i y gain and, o he wise, he highes IC50 alue, in
compa ison o he o he nanopa icles (Figu e 15 (B)). This is an expec ed esul , as his
o mula ion does no con ain DOX. MH e ec is inhe en o he p esence o magne i e inside o he
nanopa icles, whe e i s ac i i y as nanohea e s is con olled by applying an AMF (221), being
conside ed he MH i sel as an an icance p omo e (222). Indeed, he inc eased ac i i y obse ed
on EBR89- ea ed cells is in insic o he MH ac i i y. Howe e , he majo an icance ac i i y is
obse ed unde he mo-chemo he apy ea men , whe e he EBR85- unc ionalized nanopa icle
displayed he lowes IC50 alue (0.212 µM) wi h a highes pe cen age o ac i i y gain among he
DOX-con aining o mula ions (>80%), in Hs578 cells, indica ing a s ong po en ial o i s applica ion
as a combina o ial ea men . The esul s ob ained in he p esence o an AMF showed ha he
EBR85- unc ionalized nanopa icles a e e ec i ely anspo ing and deli e ing he d ug and
p omo ing a cell iabili y dec ease compa ed o he expe imen al condi ion in which no
hype he mia was applied (0.21 ± 0.16 µM
s
1.82 ± 0.60 µM).
Mic oscopically, he e ec o dual he mo-chemo he apy
e sus
mono he apy (chemo he apy
alone) was obse ed by mo phological al e a ions in he Hs578 cells, a e 72 h o incuba ion a e
ea men s, wi h and wi hou MH, using he espec i e IC50 DOX concen a ion o he di e en
mSLNs (Table VII) (Figu e 16). The combined he mo-chemo he apy ea men induced cell
pheno ypic al e a ions and appa en cell dea h, as obse ed in Figu e 16 (C).
Chemo he apy is associa ed wi h ad e se side e ec s o he pa ien s, such as ca dio oxici y
in he case o DOX ea men s (64,76), being u gen o disco e new s a egies o o e come hese
Figu e 16. Mo phological al e a ions in Hs578 cance cells a e di e en ea men ypology.
Pheno ype o Hs578 cance cells ea ed wi h only medium
(A), EBR85 a espec i e IC50 concen a ion (B)
, and wi h
EBR85 a espec i e IC50 concen a ion and 1 h o MH applica ion
(C), a 100X o magni ica ion.
A
C
B
55
issues. One possibili y would be inc easing he he apeu ic index o he ea men in such a way
ha lowe chemo he apeu ic doses could be adminis e ed o he pa ien , hus p ese ing no mal
issues om damage. The syne gis ic an icance e ec o he he mo-chemo he apy ea men was
de e mined by he analysis o he co esponding syne gis ic deg ee (Table VIII) calcula ed om
Equa ion 3 (Chap e 2, sec ion 3). As illus a ed in Table VIII, in e ac ions be ween EBR85-
unc ionalized nanopa icles and hype he mia showed he highes alue o syne gis ic e ec o
he Hs578 cance cell line, a esul al eady obse ed in Figu es 15 and 16. This indica es ha
he dual ea men using he no el unc ionalized mSLNs p obes enhance he an icance
he apeu ic index ace o chemo he apy o MH alone, indica ing a possible b eak h ough o u u e
he apies on TNBC wi h a possible educ ion o con en ional chemo he apy side e ec s.
Table VIII. Deg ees o syne gism o di e en he apy combina ion. Alpha (α) alues calcula ed wi h Equa ion
3 (Chap e 2, Sec ion 2) o di e en mSLNs o mula ions in combina ion wi h an al e na ing magne ic ield.
T ea men
α
Resul
DOX
1.08
Addic i e e ec
EBR52
3.66
Syne gis ic e ec
EBR85
8.67
Syne gis ic e ec
EBR86
3.30
Syne gis ic e ec
2. EFFECT OF NANOPARTICLE SURFACE FUNCTIONALIZATION ON
IMMUNE SYSTEM RESPONSE
In he sys emic adminis a ion o nanopa icle-based ea men s, he opsoniza ion p ocess
s a s when he nanopa icles a e injec ed in he bloods eam, ini ia ing a apid MPS ac ion. As a
esul , he clea ance o he nanoca ie om he ci cula ion occu s wi hin a ew hou s pos -injec ion.
He ein, he biophysiological unc ionali y o he nanoca ie is achie ed h ough he mSLNs su ace
unc ionaliza ion wi h a empe a u e- esponsi e biocompa ible hyd ophilic polyme (PEG)-
con aining ligand. This speci ic su ace modi ica ion esis s o he immune cell in e ac ion, a oiding
opsoniza ion and, consequen ly, i s elimina ion om he blood ci cula ion (172,173). In addi ion,
56
o a oid he immune esponse, PEGyla ion enhances he p obabili y o i s passi e accumula ion in
he umo egion.
THP-1 cell line was used as model o monocy e cells which a e in ol ed in he inna e immune
esponse, esponsible o ecognize and c ea e a esponse agains o eign pa hogens (223). In o de
o s udy he esponse o monocy es cells agains nanopa icles, a cell iabili y assay was conduc ed
using he esazu in colo ime ic assay. THP-1 cells we e ea ed wi h 6 di e en concen a ions
using a ange o concen a ions 0.1, 0.3, 0.6, 1.2, 2 and 5 µM ela ed o doxo ubicin con en ,
wi hou hype he mia applica ion, o 24, 48 and 72 h o incuba ion. The dose esponse cu e and
he g aphic ep esen a ion o he IC50 alues ob ained a e illus a ed in Figu e 17 and summa ized
in Table IX.
57
Figu e 17. Dose esponse cu es and IC50 compa isons. E ec o mSLNs (EBR52, EBR85, EBR86, and EBR89)
and e e ence compound ( ee DOX) on he cell iabili y o THP-1 cells, o o al cell biomass, a 24, 48, and 72 h o
incuba ion. Resul s a e exp essed as a mean ± SD.
a
: Resul s signi ican ly di e en om EBR85, EBR86, and EBR89
(
p
<0.05);
b: Resul s signi ican ly di e en om DOX, EBR85, EBR86, and EBR89 (
p
<0.05); b
: Resul s signi ican ly
di e en om DOX, EBR52, EBR85, and EBR86 (
p
<0.0001)
64
ag eemen wi h he li e a u e whe e i is desc ibed a sus ained d ug elease along ime om he
nanopa icles, hwa ing he “bu s e ec ”, con a y o an immedia e exposu e o a o al DOX
concen a ion (84,93,94,97,125,141,142).
Wi h an inc ease in incuba ion ime (6h pos - ea men ), he cell nuclei exhibi a highe DOX-
ed luo escence, p incipally on EBR85- ea ed cells. A e nanopa icles we e in e nalized in o
Hs578 cance cells, a con inuous DOX libe a ion om he nanopa icle o mula ions was p omo ed
whe e, i was i s eleased in o he cell cy oplasm and wi h inc easing incuba ion ime, could each
he cell nuclei and consequen ly induce cance cell dea h.
6. EFFECT ON 3D HS578T POLYSPHEROID GROWTH
The monolaye cell cul u e (o 2D cul u e) is a me hod o en used in he s udy o cellula
esponses o d ug es ing, p e-clinical
in i o
assays, including o cance esea ch. The high
ep oducibili y, low cos , simplici y, and o his well-es ablished model enhances i s a ac i i y o
in i o
s udies, being he mos commonly used me hod o cell cul u e. Though, di e en s udies
poin ed ou la ge disc epancies be ween
in i o
and
in i o
esul s, highligh ing he di icul y o
eplica e he complex umo mic oen i onmen and he d ug esis ance obse ed in solid umo s
(208,209).
3D cul u e models ha e come o ill he gap be ween
in i o
and
in i o
sys ems. Sphe oids
a e conjuga ion o cell agg ega es in he o de o mic o-sized, gene a ed om a cance cell line ha
ha e eme ged as a new ool o cance esea ch. Di e en d ug s udies ha e used sphe oids as
models o di e en cance ypes, such as b eas , colon, lung, panc eas, li e (226,227).
The d ug esis ance de ec ed on solid umo s is inhe en o he ailu e o subsc ibed
chemo he apies. Due o cell agglome a ion es ablished on 3D umo sphe oids, he physical
communica ion and signaling pa hways could be mimicked, a ea u e ha plays a ole in he
chemo he apy esis ance obse ed in solid umo s (226). Wi hin he cells’ agglome a e, di e en
ECM cons i uen s (e.g., collagen laminin, ib onec in) a e deposi ed, suppo ing ECM-cell
in e ac ions which a e esponsible o a ba ie o ma ion ha limi s he pene a ion, dis ibu ion,
and ac ion o an icance d ugs (227). Addi ionally, as occu s in solid umo s, 3D umo sphe oids
a e di ided in o di e en laye s esponsible o impai he he apeu ic e icacy o an icance d ugs
(228). The ex e nal laye is cons i u ed by highly p oli e a i e cells, while he middle con ains
quiescen cells and he inne co e is o med by nec o ic cells, whe e he loca ion o he cells is
co ela ed o hei capaci y o cap u e nu ien s and oxygen (Figu e 23) (226–228).

65
Di e en epo s demons a ed di e en ou comes o d ug up ake and ac ion in 2D cell laye s
and 3D sphe oids (226,227). Taking his in o conside a ion, he mSLNs e ec on 3D umo g ow h
was e alua ed and compa ed o he 2D monolaye esul s. Hs578 polysphe oids we e ea ed wi h
2xIC50 concen a ion o he de eloped nanopa icles (EBR52, EBR85, EBR86, and EBR89) and
e e ence compound (DOX) plus 1 h o AMF e e y second day o 1 week (Figu e 24 (A)). Then,
umo g ow h was assessed du ing he ollowing 2 weeks wi h pho og aphic egis a ion. The
no malized polysphe oid g ow h was ob ained using he Equa ion 4 (Chap e 2, sec ion 4) and
plo ed on Figu e 24 (B).
Figu e 23 3D sphe oids schema ic ep esen a ion. Sphe oids a e di ided in o h ee main laye s (p oli e a ion
zone, quiescen zone, and nec o ic zone), caused by a g adien o oxygen, nu ien s, pH, CO2, and me abolic was e,
simila o wha is obse ed in solid umo s.
66
Polysphe oids ea ed wi h a combina ion o e e ence compound (DOX) plus 1h o MH
displayed an inhibi o y g ow h e ec un il day 8 o he expe imen , bu o e ime, he sphe oids
eini ia e an exponen ial g ow h (Figu e 24 (B)). P e ious epo s al eady demons a ed ha
b eas cance cell lines cul u ed in 3D condi ions showed mo e chemo he apeu ic esis ance in
Figu e 24. E ec o dual- ea men on polysphe oids g ow h. (A) Ch onological ep esen a ion o he
polysphe oids ea men o h ee weeks
. (B) G aphical ep esen a ion
o he sphe oid umo g ow h a e ea ed wi h
2xIC50 concen a ion o espec i e compound plus 1 h o hype he mia o h ee days in one week. Resul s a e p esen ed
as mean ± SD o a leas h ee independen expe imen s
. (C)
G aphical ep esen a ion o no malized polysphe oids
daily g ow h a e pe o med a ea men 3 imes pe week wi h espec i e compounds and 1 h o hype he mia.
Resul s a e p esen ed as mean ± SD o a leas h ee independen expe imen s
.
a: DOX and EBR52 signi ican ly
di e en om EBR85, EBR86, and EBR89 (
p
<0.0001); b: EBR85 signi ican ly di e en om DOX, EBR52, EBR86,
and EBR89 (
p
<0.0001); c: EBR86 signi ican ly di e en om DOX, EBR52, EBR85, and EBR89 (p<0.0001); d: EBR89
signi ican ly di e en om DOX, EBR52, EBR85, and EBR86 (
p
<0.0001).
67
compa ison o hose cells in 2D condi ions (79). Simila ly, EBR52- ea ed cells displayed he same
g ow h beha io as DOX- ea ed cells, highligh ing he key ole o he su ace modi ica ions on he
sus ained d ug elease ha enables a mo e p olonged ea men . In ag eemen wi h 2D cell
monolaye esul s, EBR89- ea men seems o exhibi he leas e ec on Hs578 sphe oid g ow h.
This means ha he mono he apy applica ion (only hype he mia) did no display a cy o oxic e ec
on cance cells, enhancing he eliabili y o hei use o diagnos ic p oposes.
The unc ionalized nanopa icles, EBR85 and EBR86, we e he nanopa icles wi h he bes
g ow h inhibi o y capaci y. Fo a be e isual app ecia ion, he daily g ow h o he umo s we e
plo ed on Figu e 24 (C). Posi i e alues ep esen he posi i e g ow h o he polysphe oids,
whe e he compounds did no demons a e a he apeu ic e ec , whe eas nega i e alues
co espond o he sphe oids which we e suscep ible o he ea men .
As p e iously men ioned, sphe oids can ep oduce he mechanisms o d ug esis ance
obse ed in solid umo s, being a easible me hodology o he s udy o new he apeu ic
app oaches. In Figu e 24 (C), i was shown ha unc ionalized mSLNs- ea men po en ia es he
e ec o he chemo he apeu ic d ug along ime wi h a highe inhibi ion on umo daily g ow h, in
compa ison o DOX- ea men alone, enhancing he cell sensi i i y o chemo he apeu ic ea men ,
as p e iously da ed (79).
As obse ed in Figu e 24 (C), he unc ionalized mSLNs displayed he bes inhibi o y e ec
on polysphe oids daily g ow h, whe e he EBR86- ea ed g oup showed a highe inhibi ion on
polysphe oid umo g ow h in compa ison o EBR85- ea ed g oup. The applica ion o an AMF is
supposed o selec i ely ac i a e he magne i e o he nano o mula ions (Figu e 25). This
gene a es ene gy in he o m o hea ha consequen ly b eaks he he mo-suscep ible Diels-Alde
bond, exposing he pene a ing pep ide o he cance cells (177,181,183). Then, he nanopa icles
could be easie and as e in e nalized in o cance cells, accele a ing he deli e y o he
chemo he apeu ic d ug and also o he magne i e nanopa icles (used as a con as agen o MRI)
o Hs578 polysphe oids. Once inside, he cells will deg ade he magne ic solid lipid nanopa icles
o libe a e he chemo- and he mo-deadly ca go. This will damage he a ec ed cells and diminish
hei p oli e a ion a es (180,182). Howe e , he e ec o he empe a u e- esponsi e Diels-Alde
bond con ained in he PEGyla ed ligand on he sphe oid p oli e a ion a e was simila o ha o he
empe a u e-insensi i e con ol o mula ion, wha poin s o an e ec i e sphe oid pene a ion o he
68
mSLNs e en when he a ge ing mechanism ough he CPP is no he mally ac i a ed (Figu e 24
(C)).
On he o he hand, MH ac ion is in insically dependen on he concen a ion o magne i e
nanopa icles inside he mSLNs, in such a way ha a highe concen a ion o magne i e is
co ela ed o a highe gene a ion o hea . In his way, he concen a ion o magne i e inside he
di e en mSLNs could be behind he p e ious indings ega ding he umo g ow h. Since low
magne i e concen a ions inside he mSLNs unc ionalized wi h he empe a u e-sensi i e
PEGyla ed ligand could no be enough hea as o be able o ac i a e he CPP-media ed a ge ing
e ec , wha would jus i y he non-imp o ed sphe oid g ow h inhibi ion obse ed o EBR85
pa icles. To cla i y his issue, and o s udy he nanopa icle in e naliza ion in o TNBC Hs578 cells,
Fe con en was quan i ied by ICP-OES a he end o he polysphe oid g ow h a e expe imen s
(Figu e 26).
Figu e 25. Diels-Alde bond a e hype he mia applica ion. The hype he mia applica ion will ac i a e he
magne i e ha , consequen ly, will gene a e an enough amoun o hea o b eak he he mo-suscep ible diels-alde
bond. A e wa ds, he cell-pene a ing pep ide ligand will be exposed o he cance cells, acili a ing he nanopa icle
cell in e naliza ion.
69
As obse ed in Figu e 26, he amoun o Fe was measu ed in he DOX- ea ed polysphe oid.
The alues ob ained, named as eal alues, co espond o he amoun o Fe p esen in cells and
medium. Once his alue was sub ac ed o he eal alues achie ed in each ea ed polysphe oid
g oup, he esul ing Fe con en accoun s o ha ac ually in e nalized oge he wi h he mSLNs
nano o mula ions. Las ly, Figu e 27 compa es he measu ed Fe con en coming om he mSLNs
sphe oid cell in e naliza ion o he ac ual Fe con en coming om he mSLNs nano o mula ion
dispe sions.
Figu e 26. Fe in e naliza ion in Hs578 polysphe oids a e ea men wi h compounds plus 1 h o MH.
A he end o he polysphe oid g ow h a e expe imen s, Fe (magne i e) in e naliza ion in o polysphe oids was assessed
by quan i a i e analysis o Fe h ough ICP-OES. Resul s a e p esen ed as mean ± SD o a leas h ee independen
expe imen s.
Figu e 27. Co ela ion be ween he Fe concen a ion o he s ock nano o mula ions and he Fe con en
amoun in e nalized in o he polysphe oid cance cells. (A) Fe concen a ion o s ock solu ions
e sus
Fe
con en o polysphe oid cance cells, and
(B)
linea eg ession o he p e ious plo ed esul s. The linea eg ession
has he ollow equa ion: µg Fe = 0.011 [Fe o nano o mula ion s ock] + 0.057 wi h 0.929 alue o 2.

70
On Figu e 27 (A) and (B), a clea s ong ela ionship be ween he Fe concen a ion inside o
mSLNs dispe sions and he Fe con en in e nalized in o he polysphe oids is app ecia ed. These
pa ame e s show a linea and posi i e dependency. As a inal ou come, i was obse ed ha
EBR85- ea ed polysphe oids had a lowe Fe con en , in compa ison o EBR86- ea ed cells, which
could be a possible explana ion o he unexpec ed esul s ega ding he lowe polysphe oid g ow h
a e obse ed o EBR85- ea ed polysphe oid. Following his, he polysphe oid g ow h a e was
e alua ed aking in o conside a ion he quan i ied Fe con en inside he polysphe oid cance cells
(Figu e 28).
Using ICP-OES, i was possible o de e mine he Fe con en in e nalized in o polysphe oids
s udy i s e ec on polysphe oids’ g ow h a e. As obse ed in Figu e 28, he EBR85- ea ed g oup
displayed a signi ican inhibi ion on umo daily g ow h in compa ison o EBR86- ea ed g oup wi h
he same amoun o Fe in e nalized. When conside ing he in e nalized magne i e, he e ec o he
Diels-Alde -media ed a ge ing ac i a ion was e idenced, which enhances he nano o mula ion
ac i i y agains cance cells. This e ec seems o be Fe dose-dependen , whe e a highe Fe con en
po en ia es he inhibi o y e ec a he umo g ow h le el, an ou come al eady obse ed by Hayashi
e
al.
whe e hey demons a ed ha an enhanced accumula ion o nanopa icles inc eases he
magne ic elaxi i y (229). Al oge he , EBR85- unc ionalized mSLNs induced a highe inhibi o y
e ec on umo g ow h in compa ison o he o he mSLNs o mula ions, when he e ec i s
no malized o he amoun o Fe in e nalized, hus o e coming a possible d ug esis ance obse ed
Figu e 28. E ec o compounds on umo daily g ow h a e conside ing he Fe con en in e nalized in o
polysphe oid cance cells. G aphical ep esen a ion o no malized polysphe oids daily g ow h pe mg/ml o
in e nalized Fe. Resul s a e p esen ed as mean ± SD o a leas h ee independen expe imen s. *
p
<0.05
71
o pa ien s ea ed wi h chemo he apy and maximizing he DOX ac ion on umo cells. These
esul s ein o ce he po en ial use o EBR85 as a new he apeu ic app oach o TNBC ea men
aced o con en ional chemo he apy.
7. FUNCTIONALIZED NANOPARTICLES AS T2-CONTRAST AGENTS FOR
MRI
The MRI echnique is employed o a apid
in i o
image; howe e , he associa ed low sensi i i y
challenges i s use o diagnos ic p oposes, which appeals o he use o magne ic CAs o e come
his issue (118). Fe3O4 nanopa icles made i in o he clinical se ing as T2 CAs, wi h associa ed
limi a ions ega ding he magne ic suscep ibili y a i ac s which may no clea ly di e en ia e he
highe om he lowe o MRI signal le els a ising om adjacen issues (230). In his con ex , he
e icacy o he de eloped mSLNs was e alua ed in e ms o MRI pe o mance as T2 CA. The
de eloped nano o mula ions ha e he nanosized magne i e inco po a ed in he o ganic ma ix, so
T2-MRI beha io (da k con as enhancemen ) is expec ed (85). In his way, he magne ically-
labelled polysphe oids used in he p e ious expe imen we e u ilized o s udy he MRI con as
enhancemen e ec o he mSLNs in compa ison o DOX- ea ed cells, using a p eclinical 3 T MRI
scanne unde T2 acquisi ion modes using as spin-echo (FSE) sequences (Figu e 29).
72
Su ace unc ionaliza ion ac s as an imp o emen o nanopa icle in e naliza ion in o cance
cells, whe e i s aul will signi ican ly diminish he endosomal in e naliza ion o nanopa icles and,
consequen ly, dec ease in acellula magne i e con en . A T2-MRI beha io is co ela ed o a long
ange o magne ic in e ac ion wi h wa e molecules induced by he supe pa amagne ic co e unde
a magne ic ield, which esul s in hype in ense changes in he esonance signal in MRI imaging
(85,113,115). Thus, he MRI con as enhancemen is a concen a ion dependen phenomenon,
Figu e 29. MRI con as enhancemen e ec o magne ically-labelled polysphe oid cells. In (A) images
ob ained using p eclinical 3 T MRI scanne unde T1 and T2 acquisi ion modes using as spin-echo (FSE) sequences o
he ea ed polysphe oids. Using he ImageJ wi h MRI-T2 calcula ion plugin, i was ob ained he g aphic ep esen ed in
(B) o he T2 alues (s, a 3 T 37 ºC) o he di e en ea men s applied. Resul s a e p esen ed as mean ± SD o a leas
h ee independen expe imen s. S a is ical analysis using he ANOVA es s whe e *
p
<0.05, ***
p
<0.001,****
p
<0.0001
in compa ison o he o he compounds- ea ed polysphe oids.
73
he highe he concen a ion o magne ic nanopa icles, he lowe he elaxa ion ime, and he
da ke MRI con as e ec , as obse ed in Figu e 29.
The unc ionalized nanopa icles EBR85, EBR86, and EBBR89 (all con aining magne i e
nanopa icles in he o ganic ma ix) showed an enhancemen o he T2-MRI con as in compa ison
o he o he nano o mula ions wi h EBR85 p obe displaying he lowes T2 alue, wi hou signi ican
di e ences among he ea men s (Figu e 29). As p e iously obse ed, EBR85- ea ed
polysphe oids had lowe magne i e, meaning ha magne ic dipola in e ac ions and ela ed
clus e ing e ec s coming om he con inemen o he MNPs in he wax ma ices could be playing
a majo ole in he inal elaxa ion imes obse ed, as al eady demons a ed in o he epo s (231).
Addi ionally, i is impo an o highligh ha he ans e se elaxi i y 2 – igu e o me i ha
quan i ies he e iciency o MNPs as CAs – calcula ed as he slope o he in e se o he elaxa ion
ime (T2 alues om Figu e 29, Equa ion 6 om chap e 2, sec ion 4)
e sus
he concen a ion
o he ac i e magne ic elemen o he unc ionalized-EBR85 mSLNs was o 120 mM-1s-1, alue ha
ou weighs ha o Fe idex® ( 2 = 93 mM-1s-1, a 3 T, 37 ºC), supe pa amagne ic i on oxide
nanopa icle al eady app o ed by FDA as nega i e CAs (188).
Al oge he , EBR85 showed a ema kable e ec on he educ ion o he T2-MRI elaxa ion ime,
becoming p omising as T2-MRI con as agen able o o e a nonin asi e imaging capabili y o
diagnosis and moni o iza ion du ing cance ea men .
8. EFFECT OF THE NANOPARTICLES ON INVASION CAPACITY OF
HS578T SPHEROIDS
A malignan umo is cha ac e ized o i s po en ial o in ade su ounding issues and sp ead
o dis an o gans, being esponsible o he majo i y o he cance - ela ed dea hs o ela ed o highe
p obabili y o cance elapse wi hin a pe iod o 5-yea s (2,19). This b ings a new pe cep ion o he
alida ion o no el he apies. The implemen a ion o no el an icance d ugs should pass h ough
an
in i o
es ha allows he iden i ica ion o an inhibi o y capabili y o his key hallma k o
cance . A 3D umo sphe oid in asion assay was pe o med as a no el app oach, being a apid
and highly ep oducible echnique ha has an associa ed capaci y o ep oduce a umo egion
wi h abili y o c ea e mic o-me as asis (232,233). This ask s a ed wi h he de elopmen o single
Hs578 sphe oids and, 4 days pos -de elopmen , he in asion assay was ini ia ed. Ma igel was
added di ec ly o each well o p o ide a semi-solid ma ix which enabled he in asion o cance
80
In published s udies, Hs578 cells we e used o
ex i o
angiogenesis s udies o e alua e he
e ec o he unc ionalized zeoli es as p oangiogenic o an iangiogenic agen s. I was demons a ed
ha he unc ionalized zeoli es dec eased he ec ui men and o ma ion o blood essels o he
umo egion ace o non- unc ionalized p obes (236). The poo ou come and he p esence o highe
le els o angiogenesis ha e been associa ed wi h di e en cance ypes, as o b eas cance , enal
cance , p os a e cance , colon cance and melanoma (237). Thus, he e is a need o alida e he
no el he apies o hei abili y o inhibi o dec ease angiogenesis and, consequen ly, umo
p og ession.
A e 4 days o umo de elopmen , he o ma ion o blood essels is no iceable on he con ol
g oup (as obse ed in Figu e 33 (A) and in Figu e 34), whe eas he ea ed g oups showed a
signi ican dec ease in he numbe o blood essels, in compa ison o he con ol. Al hough EBR85-
ea men induced a highe dec ease in angiogenesis in compa ison o DOX, sugges ing an
imp o emen in blood essel o ma ion, his di e ence was no signi ican (
p
>0.05).
Fo u he cha ac e iza ion o he nanopa icles’ an icance ac i i y, Ki67
immunohis ochemis y analysis we e pe o med. Ki67 p o ein is in ol ed in all cell cycle phases
(G1, S, G2, and M), wi h an absen exp ession du ing he cell es ing phase (G0) (238). The
exp ession o his p o ein is s ongly co ela ed wi h ac i e cell p oli e a ion o in insic cell
popula ions in umo cells, allowing i s use as a bioma ke o umo agg essi eness. In in asi e
b eas cance s, Ki67 bioma ke is s ongly applied o s a i y good and poo p ognos ic
Figu e 34. E ec o nanopa icles EBR85 and DOX on Hs578 umo angiogenesis. G aphical
ep esen a ion o he numbe o blood essels o ma ion in esponse o a di e en ea men . Da a was analyzed by
one-way ANOVA and he esul s a e ep esen a i e o he mean o a leas 15 eggs pe g oup;***
p
<0.0001 *
p
<0.05
in compa ison o he con ol g oup.

81
classi ica ions (16,22). The p oli e a ion s a us o b eas cance s is also a guiding decision o he
cance esponse o he p esc ibed chemo he apy whe e low-p oli e a i e umo s a e less sensi i e
o such he apies. In his s udy, Ki67 exp ession analysis was pe o med on sec ions om o malin-
ixed, pa a in-embedded CAM cance samples. Sco ing was assigned as 0 o 0% o posi i e cells,
1 o <10% o posi i e cells, 2 o 10-50% o posi i e cells and 3 classi ica ions o >50% o posi i e
cells. (Figu e 35). Ki67 sco es a e used in he clinics as bioma ke o he esponse o
chemo he apy in TNBC (239).
A high p oli e a i e index was de ec ed by Ki67 immunos aining in he con ol- ea ed g oup,
whe e a la ge numbe o b own cells was de ec ed, which usually co ela es o TNBC
agg essi eness. A s udy wi h 613 b eas ca cinoma samples was conduc ed and ollowed-up o
10 yea s, being obse ed ha a Ki67 high exp ession was co ela ed o a signi ican ly highe isk
Figu e 35.. E ec o EBR85 and DOX on angiogenesis o Hs578 cance cell line
ex i o
, de ec ed by
chick cho ioallan oic memb ane (CAM) assay a 96 h pos - ea men .
In (A)
ep esen a i e images o he
excised CAM issue sec ions o he con ol, ea ed (DOX and EBR85) condi ions, immunos ained o Ki67. Posi i e
immuno eac ions o Ki67 in malignan cells a e shown in b own (DAB s aining). O iginal magni ica ion a 100x and
200x (inse s).
(B)
is a g aphic ep esen a ion o he a ibu ed sco es o each sec ion cu s, whe e 0 was assigned o
0% o posi i e cells, 1 o <10% o posi i e cells, 2 o 10-50% o posi i e cells and 3 classi ica ions o >50% o posi i e
cells.
Resul s a e exp essed as he mean ± SEM o one expe imen . *
p
<0.05 ela i e o con ol.
82
o sho e umo - ee pa ien su i al (240). These indings highligh he impo ance o disco e
new d ugs ha ha e cell an i-p oli e a i e e ec s o imp o e TNBC p ognosis. The unc ionalized
nanopa icle EBR85 showed p omising esul s a his le el, e ealing a signi ican educ ion in
umo cell p oli e a ion in compa ison o con ol g oup (0.3±0.5
e sus
1.25±.1.0,
p
<0.05). Ki67
quan i ica ion o DOX- ea men e ealed a highe p oli e a i e ac i i y, demons a ing i s lowe
e ec i eness compa ed o EBR85 mSLNs. Al oge he , hese ini ial esul s sugges ha EB85-
ea men induces an impo an educ ion in cance cell p oli e a ion in TNBC.
11.
IN VIVO
THERANOSTIC EFFECT OF FUNTIONALIZED NANOPARTICLES
USING ORTHOTOPIC TNBC XENOGRAFTS
Pa I - Toxici y and diagnosis
Based on he p omising
in i o
p ope ies o he DA-TAT-PEG-mSLNs (EBR89), which e ealed
no oxici y o bo h 2D monolaye cells, 3D Hs578 sphe oids, and
ex i o
CAM model, he
de eloped nanop obes we e e alua ed
in i o
on Hs578 umo bea ing immunocomp omised
emale NSG mice. The mice we e inocula ed o ho opically wi h Hs578 cells (650 000 cells pe
mice) in a mamma y a pad. T ea men s a ed 3 days pos - umo implan a ion and mice we e
andomly di ided in o h ee g oups. Mice-bea ing Hs578 umo s we e injec ed in a enously e e y
2 days o 1 week wi h: T ea men 1 (saline solu ion), T ea men 2 (EBR89 a 0.05 mgFe.Kg-1 o
mice weigh ), and T ea men 3 (EBR89 a 0.05 mgFe.Kg-1 o mice weigh plus 1 h o an AMF using
he ollowing se ings: 15.4 A o cu en , 15 V o ol age, and 181 kHz o equency. Tumo
p og ession was e alua ed once a week a e inishing he ea men (Figu e 11, Chap e 2,
sec ion 6) The animal su i al a e was 100% wi h all animals eaching he p ede e mined 43-day
endpoin .
83
T ea men wi h EBR89 did no display any di e ence in umo g ow h in compa ison o he
con ol g oup, indica ing ha EBR89 pa icles do no p omo e any an icance ac i i y (Figu e 36
(A)). The same ou come was obse ed o he g oup which bene i ed om he hype he mia
applica ion (EBR89 + HT), whe e he e was no signi ican umo g ow h inhibi ion e ec compa ed
o he o he g oups (
p
>0.05). This goes in ag eemen wi h he
in i o
esul s whe e i was
demons a ed ha EBR89 does no display di e en complemen a y mechanisms o p omo e
cance cell selec i e oxici y, e en in he p esence o an ex e nal magne ic s imuli.
Addi ionally, mice body weigh was moni o ed o assess he o e all e ec o he umo s and
ea men on he animals (Figu e 36 (B)). No signi ican body weigh loss was obse ed du ing
he expe imen al pe iod in nei he g oup. On sac i ice day, he umo s we e collec ed om he
animals and we e weighed. As obse ed on Figu e 36 (C), no di e ences we e obse ed be ween
he ea men s, suppo ing he ine icacy o hese nano o mula ions
in i o
. Al oge he , he
de eloped nanopa icles hemsel es appea o be sa e ehicles in sys emic ci cula ion, no a ec ing
he cance cell g ow h, which suppo hei applica ion in ea ly diagnosis.
Figu e 36. E alua ion o nanopa icle oxici y on mice-bea ing Hs578 umo s. The 3 g oups o mice we e
ea ed wi h saline solu ion, EBR89, o EBR89 plus 1 h o hype he mia, h ee imes o one week and humanely
eu hanized 35 days pos - umo implan a ion. (
A) e ec o he ea men on umo g ow h along ime, (B)
g aphical
ep esen a ion o he body weigh o he animals, and
(C) weigh o umo s collec ed 43 days pos -implan a ion.
84
The e icacy o he nano o mula ions o speci ically a ge he cance cells and p o ide a MRI
con as enhancemen
in i o
was e alua ed
pos -mo em
in NSG mice bea ing Hs578 xenog a
umo s. The speci ici y o he nanop obes was e alua ed by compa ing he con as enhancemen
o he umo egions o mice ecei ing he combina ion o EBR89 wi h an AMF and hose ecei ing
only he EBR89 nanopa icle a he same concen a ion, as well as compa ing he con as
enhancemen wi h he g oup which ecei ed he ehicle.
In i o
T2*-weigh ed MRI images (Figu e
37) we e aken 43 days pos - umo implan a ion (30 days a e he las ea men day (Figu e
11, Chap e 2, sec ion 6).
Figu e 37.
Pos -mo em
T2*-weigh ed MRI images aken om a NSG mice bea ing Hs578 umo s. (A)
On he op, a e he ep esen a i e
in i o
T2 MRI images ob ained om a p eclinical 3 T MRI scanne unde T1 and T2
acquisi ion using as spin-echo (FSE) sequences and, a he bo om, a e he colo -mapped images ob ained om MRI-
T2 calcula ion plugin o ImageJ p og am, 30 days a e in a enous injec ion o EBR89 nanopa icles, EBR89 ollowed
by hype he mia (HT), o only ehicle in o a NSG mice bea ing Hs578 cells (650 000 cell injec ion). The ed a ows
de ine he umo loca ion in he di e en ep esen a i e MR images and he g een ones demons a e he same loca ion
on ea ed images ( h ough ImageJ). The colo -scale was ob ained h ough ImageJ. The g aphic ep esen ed in (B)
was ob ained using he ImageJ wi h MRI-T2 calcula ion plugin o he T2 alues (s, a 3 T 37 ºC) conce ning he di e en
ea men s applied. Resul s a e p esen ed as mean ± SD. S a is ical analysis using he ANOVA es s whe e *
p
<0.05,
***
p
<0.001,****
p
<0.0001 in compa ison o ehicle.
85
Di e en a icles assessed he use o i on-based nanopa icles
in i o
as MRI con as agen s.
The usual p ocedu e in ol es he e alua ion o
in i o
T2-weigh ed images o mice-bea ing umo
models a 0, 2, 4, 8, 24, and 48 h pos -nanopa icles injec ion ( e iewed in (241)). Only a ew
s udies ocused on umo de ec ion by MR imaging a a longe ime pos -injec ion, being 14 days
he maximum s udied ime, whe e he nanopa icles could s ill be de ec ed in he umo egion
(242,243).
The ocus o his pa o he
in i o
s udies was o i) assess he nanopa icle oxici y; and ii)
e alua e,
pos -mo em,
he nanopa icles’ e icacy as T2-con as agen s o diagnos ic applica ions
and moni o iza ion o umo esponse o he apy. As obse ed in Figu e 37, he unc ionalized
nanopa icles wi hou d ug - EBR89 - we e able o signi ican ly diminish he T2 alue in compa ison
o he con ol g oup, demons a ing i s e icacy as T2-con as agen o MRI applica ion. A be e T2
p o ile was obse ed in he p esence o hype he mia applica ion. These esul s can be co ela ed
o he ac i a ion o Diels-Alde bonds which would igge enhance he nanopa icle in e naliza ion
in o cance cells.
To imp o e he MRI inal image, a highe concen a ion o nanopa icles inside he cells is
equi ed, which will p omo e a supe io da k con as . In a published s udy, he au ho s poin ed o
60 mgFe.Kg-1 o mice as he bes i on concen a ion o ge good quali y images, being conside ably
supe io o he concen a ion used he e (13.5 mgFe.Kg-1 as accumula i e dose) (242). Though, he
concen a ion es ed he e seems o be enough o MRI applica ion. Al oge he , hese esul s a e in
ag eemen wi h hose obse ed
in i o
, whe e he in luence o he su ace ac i a ion by an ex e nal
s imulus was obse ed. This con i ms ha , hese new nanopa icles ha e he po en ial o ac as
p obes in ea ly umo diagnosis.
A he end o he expe imen , hea , lungs, kidneys, li e , and umo we e collec ed om each
animal o u he chemical analysis. His ological slices om he collec ed o gans o he con ol
and ea ed animals we e s ained wi h hema oxylin and eosin (H&E s aining). As obse ed in
Figu e 38, no o gan damage was obse ed in e ms o his ology, in compa ison o he con ol
g oup, suppo ing a sa e p o ile. Though, an analy ical con i ma ion is s ill equi ed. Li e and
kidney unc ion is co ela ed o physiological oxici y, whe e he e alua ion o li e enzymes could
be used as a key bioma ke o unde s and he ex ension o li e cell damage (87). So, in u u e
wo k, i will be in e es ing o know he c ea inine alues in he animal se um.

86
Figu e 38. H&E s aining o he excised o gans and umo s o he mice-bea ing Hs578 umo .
Rep esen a i e his ological images using H&E s ain o isualizing he umo , hea , kidney, li e and lung issues om
each g oup. Scale ba = 100 µM.
87
Pa II - The apeu ic e icacy
The second pa o he expe imen was o e alua e he
in i o
an i umo e icacy o he DOX
loaded mSLN (EBR85), which seems o display he bes ea u es. The abo emen ioned umo
implan a ion p ocedu e was ollowed.
Mice-bea ing Hs578 umo s we e andomly di ided in o 6 g oups, which ecei ed speci ic
ea men s ia i. . injec ion: G oup 1 ecei ed a saline solu ion, G oup 2 was ea ed wi h EBR85
(1 mgDOX.Kg-1), G oup 3 ecei ed he EBR89 nanopa icle (0.012 mgFe.Kg-1), he e e ence
compound DOX (1 mgDOX.Kg-1) was adminis e ed o G oup 4, and G oups 5 and 6 ecei ed
EBR85 (1 mgDOX.Kg-1) and EBR89 (0.012 mgFe.Kg-1), espec i ely, ollowed by 1 h o
hype he mia. DOX belongs o class III o chemo he apeu ic d ugs being cha ac e ized o i s high
solubili y and low pe meabili y. So, he in a ascula injec ion is he p e e en ial ou e o
adminis a ion o a oid ca dio ascula pa hology. Due o he comp omised immune sys em o NSG
mice, he DOX dose was chosen ca e ully and based on di e en a icles. As demons a ed in
Wunde lich, M.,
e al
., Mohan y, S.,
e al
, Si, Y.,
e al
., and Fa eau-Lessa d, A.,
e al
., 3 mgDOX.Kg-
1 mice o accumula i e dose seems o be he bes op ion o obse e a he apeu ic e ec wi hou
a ec ing he animals wellbeing (78,216–218). Tumo p og ession was e alua ed once a week a e
inishing he ea men (Figu e 12, Chap e 2, sec ion 6). The animal su i al a e was 100% wi h
all animals eaching he p ede e mined 39-day endpoin .
88
Figu e 39. E alua ion o nanopa icles’ he apeu ic e ec on mice-bea ing Hs578 umo s. The 6 g oups
o mice we e ea ed wi h ehicle, ee DOX, EBR85, EBR89, o 1h o hype he mia plus EBR85 o EBR89, h ee imes
once pe week and humanely eu hanized 39 days pos - umo implan a ion. (
A)
G aphical ep esen a ion o umo
e olu ion un il wo weeks a e he las day o ea men , and
(B)
e ec o he ea men on umo g ow h along ime,
un il he las day o expe imen .
(C) G aphical ep esen a ion o umo daily g ow h, on (D)
is ep esen ed he
pe cen age o ascula iza ion on umo egion o each animal- ea ed. On
(E)
is shown he weigh o umo s collec ed
39 days pos -implan a ion, and on
(F)
is he g aphical ep esen a ion o he body weigh o he animals h oughou
he expe imen . *
p
<0.05, **
p
<0.01, ***
p
<0.001
89
Tumo olume was e alua ed o 22 days pos - ea men h ough VEVO3100 ul asound. A
he end o he expe imen , no signi ican di e ences we e obse ed be ween ea ed g oups, as
obse ed Figu e 39 (B) and Figu e 40. The umo olume inc eased along ime in all
expe imen al g oups. Once again, hype he mia ea men did no inhibi he umo g ow h (as
obse ed on EBR89+HT g oup), a esul p e iously indica ed in he i s
in i o
ask. The
combina ion o he mochemo he apy esul ed in a lowe umo olume along he expe imen
(al hough no s a is ically signi ican a day 39 endpoin ), which indica es a possible eco e y o he
umo g ow h some days a e ea men . When looking a umo olume only wo weeks a e he
las day o ea men (Figu e 39 (A)), a signi ican di e ence on umo olume o DOX-loaded
EBR85+HT g oup is obse ed in compa ison o unloaded EBR89+HT g oups (
p
<0.001), ehicle
(
p
<0.05), and EBR85 (
p
<0.05). The dual- ea men , un il 11-days pos - ea men , showed a
signi ican inhibi o y e ec on umo g ow h, sugges ing mSLNs e icacy o con e he
elec omagne ic ene gy om MH in o hea ene gy, which enhances he he apeu ic e ec .
Addi ionally, MH applica ion imp o ed he suscep ibili y o cance cells o chemo he apy (al eady
s a ed in (195)), as obse ed among DOX-loaded EBR85- ea men and he mochemo he apy
EBR85- ea men (
p
<0.05). A e his ime poin , cance cells we e able o eco e hei oncogenic
Figu e 40. Illus a ion o mice-bea ing Hs578 umo s ob ained h ough VEVO LAB so wa e o 39-
day endpoin . In he i s ow is illus a ed a B-mode image o Hs578 umo s a he end o he expe ience. The
umo bounda y con ou s a e ma ked a blue on each image. The second ow shows he 3D umo essels (iden i ied
a ed) o he same animal, using a opaci y o 2%. The 3D umo olume images ( hi d ow) was econs uc ed by he
so wa e, enabling i s compa ison wi h he eal umo s images ( ou h ow).
96
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