Ci a ion: Albuque que, T.; Ne es,
A.R.; Paul, M.; Biswas, S.; Vuel a, E.;
Ga cía-Tuñón, I.; Sánchez-Ma in, M.;
Quin ela, T.; Cos a, D. A Po en ial
E ec o Ci cadian Rhy hm in he
Deli e y/The apeu ic Pe o mance
o Pacli axel–Dend ime
Nanosys ems. J. Func . Bioma e . 2023,
14, 362. h ps://doi.o g/10.3390/
j b14070362
Academic Edi o : Syed Nasi Abbas
Bukha i
Recei ed: 13 June 2023
Re ised: 5 July 2023
Accep ed: 9 July 2023
Published: 11 July 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
Jou nal o
Func ional
Bioma e ials
A icle
A Po en ial E ec o Ci cadian Rhy hm in he Deli e y/The apeu ic
Pe o mance o Pacli axel–Dend ime Nanosys ems
Tânia Albuque que 1,†, Ana Raquel Ne es 1,†, Milan Paul 2, Swa i Biswas 2, Elena Vuel a 3,4,5 ,
Ignacio Ga cía-Tuñón4, Manuel Sánchez-Ma in 3,5,6 , Telma Quin ela 1,7 and Diana Cos a 1,*
1CICS-UBI—Heal h Sciences Resea ch Cen e, Uni e sidade da Bei a In e io , A enida In an e D. Hen ique,
6200-506 Co ilhã, Po ugal; [email p o ec ed] (T.A.); [email p o ec ed] (A.R.N.);
[email p o ec ed] (T.Q.)
2Depa men o Pha macy, Nanomedicine Resea ch Labo a o y, Bi la Ins i u e o Technology & Science-Pilani,
Hyde abad Campus, Jawaha Naga , Medchal, Hyde abad 500078, Telangana, India;
[email p o ec ed] (M.P.); [email p o ec ed] (S.B.)
3Se icio de T ansgénesis, Nucleus, Uni e sidad de Salamanca, 37008 Salamanca, Spain;
[email p o ec ed] (E.V.); [email p o ec ed] (M.S.-M.)
4IBSAL, Ins i u o de In es igación Biomédica de Salamanca, 37007 Salamanca, Spain;
[email p o ec ed]
5Depa amen o de Medicina, Uni e sidad de Salamanca, 37008 Salamanca, Spain
6Unidad de Diagnós ico Molecula y Celula del Cánce , Ins i u o Biología Molecula y Celula del
Cánce (USAL/CSIC), 37007 Salamanca, Spain
7UDI-IPG-Unidade de In es igação pa a o Desen ol imen o do In e io , Ins i u o Poli écnico da Gua da,
6300-559 Gua da, Po ugal
*Co espondence: [email p o ec ed]
†These au ho s con ibu ed equally o his wo k.
Abs ac :
The ci cadian clock con ols beha io and physiology. P esen ly, he e is clea e idence o a
connec ion be ween his iming sys em and cance de elopmen /p og ession. Mo eo e , ci cadian
hy hm conside a ion in he he apeu ic ac ion o an icance d ugs can enhance he e ec i eness
o cance he apy. Nanosized d ug deli e y sys ems (DDS) ha e been demons a ed o be sui able
enginee ed pla o ms o d ug a ge ed/sus ained elease. The in es iga ion o he ch onobiology-
nano echnology ela ionship, i.e., iming DDS pe o mance acco ding o a pa ien ’s ci cadian hy hm,
may g ea ly imp o e cance clinical ou comes. In he p esen wo k, we syn hesized nanosys ems
based on an oc a-a ginine (R8)-modi ied poly(amidoamine) dend ime conjuga ed wi h he an icance
d ug pacli axel (PTX), G4-PTX-R8, and i s physicochemical p ope ies we e e ealed o be app op ia e
o
in i o
deli e y. The in luence o he ci cadian hy hm on i s cellula in e naliza ion e iciency
and po en ial he apeu ic e ec on human ce ical cance cells (HeLa) was s udied. Cell-in e nalized
PTX and caspase ac i i y, as a measu e o induced apop osis, we e moni o ed o six ime poin s.
Highe le els o PTX and caspase-3/9 we e de ec ed a T8, sugges ing ha he in e naliza ion o
G4-PTX-R8 in o HeLa cells and apop osis a e ime-speci ic/- egula ed phenomena. Fo a deepe
unde s anding, he clock p o ein Bmal1— he main egula o o hy hmic ac i i y, was silenced by
Clus e ed Regula ly In e spaced Sho Palind omic Repea s (CRISPR) echnology. Bmal1 silencing
was e ealed o ha e an impac on bo h PTX elease and caspase ac i i y, e idencing a po en ial ole
o ci cadian hy hm on d ug deli e y/ he apeu ic e ec media ed by G4-PTX-R8.
Keywo ds:
apop osis; Bmal1 silencing; cance he apy; caspases; ci cadian hy hm; nano-deli e y
sys ems; PAMAM
1. In oduc ion
Al hough he unde s anding o cance has inc eased o e he pas decades, i con inues
o be a leading cause o dea h wo ldwide and one o he majo heal h scou ges o he 21s
cen u y [
1
]. Fo decades, he main goal o oncology esea ch has been he de elopmen
J. Func . Bioma e . 2023,14, 362. h ps://doi.o g/10.3390/j b14070362 h ps://www.mdpi.com/jou nal/j b
J. Func . Bioma e . 2023,14, 362 2 o 20
o e icien he apies o elimina e cance cells by he induc ion o p og ammed cell dea h.
The apop osis p ocess can occu h ough wo di e en pa hways (in insic and ex insic)
bo h leading o he ac i a ion o caspases, p o eins esponsible o ini ia e and con ol his
mechanism [2].
Mo e ecen ly, nano echnology has been in ensely explo ed o cance ea men .
Unlike con en ional chemo he apeu ics, nanoscale deli e y sys ems ha e shown many
ad an ages, such as imp o ed s abili y and biocompa ibili y, p ecise a ge ing o umo
cells, inc eased cellula up ake, educ ion in side e ec s, and d ug esis ance [
3
,
4
]. The e-
o e, esea ch in he nano echnology ield o e s sa e and mo e e icien possibili ies o
he cons an imp o emen o cance he apy [
5
]. Se e al nanoca ie s wi h dis inc phys-
iochemical p ope ies and composi ions ha e been designed o an i umo d ug elease.
Some o hose include polyme -based ec o s, liposomes, micelles de i ed om polyme s,
ca bon nano ubes, solid lipid nanopa icles, and magne ic nanopa icles [
6
]. To be signi i-
can ly e ec i e, hey should be able o o e pass biological ba ie s and a ge umo issues
wi hou losing subs an ial ac i i y upon blood ci cula ion [
7
]. La e , a e cellula up ake,
hey should ideally be capable o endosomal escape and speci ic o ganelle a ge ing o he
he apeu ic agen s [8].
Dend ime s a e syn he ic polyme ic nanoma e ials applied in bo h gene and d ug
deli e y [
9
]. They a e ecognized as e sa ile ca ie s due o hei unique s uc u al ea u es:
a sphe ical polyme ic co e su ounded by mul iple well-o de ed geome ical b anching
molecules, whe e d ugs can be encapsula ed, and a pe iphe y ha can be unc ionalized
wi h di e en ligands o enhance hei s abili y, a ge ing abili y, and issue pene a ion [
10
].
These h ee-dimensional ee-like s uc u es can be classi ied in o “gene a ions” (G) con-
ce ning he numbe o he b anching in he dend ime [
11
]. Wi h an inc ease in gene a ions,
he molecula weigh and numbe o eac i e su ace g oups inc ease as well, c ea ing
dend ime s wi h di e en p ope ies and possibili ies o applica ions. Poly (amido amine)
dend ime s (PAMAM), in pa icula , ha e been one o he mos commonly used and
ha e shown p omising esul s o he deli e y o poo ly soluble an icance d ugs such
as PTX [
12
], me ho exa e (MTX) [
13
], doxo ubicin [
14
], and doce axel [
15
]. To u he
imp o e he cellula in e naliza ion o such cons uc s, Cell-Pene a ing Pep ides (CPPs)
can be added. CPPs a e small pep ides o igina ing om di e en sou ces ha possess
ema kable p ope ies o cell pene a ion, acili a ing he ansloca ion o he apeu ic agen s
in o cells [
16
,
17
]. Ca ionic pep ides a e a class o CPPs ha easily in e ac wi h he phos-
pholipids, nega i e in cha ge, in he cell memb ane, acili a ing memb ane ansloca ion.
Fo ins ance, oc a-a ginine (R8), a sho -chain pep ide wi h eigh a ginine esidues was
ound o media e in acellula up ake o he nano-cons uc s leading o he in acellula
elease o a high amoun o PTX p omo ing i s an icance e icacy [
12
,
18
]. Ch ono he apy
has also gained much a en ion in he cance esea ch ield as an eme gen s a egy o
enhance cu en cance ea men s. I is based on he p inciple ha he sa e y and e icacy o
chemo he apeu ics can be dependen on he ime o hei adminis a ion [
19
]. So a , se e al
clinical ials ha e applied ch ono he apeu ic p o ocols o cance ea men and ha e
con i med an op imal iming o he apy, imp o ing i s e icacy, diminishing d ug oxici y,
and enhancing pa ien su i al [
20
,
21
]. In ac , i is well documen ed ha he ci cadian
hy hm exe s a ole in cance p og ession since i s dys unc ion was associa ed wi h he
ac i a ion o in acellula in lamma o y and oncogenic signaling pa hways [
22
,
23
]. On he
o he side, ci cadian hy hms modula e many cellula and physiological p ocesses includ-
ing d ug me abolism, abso p ion, anspo , dis ibu ion, de oxi ica ion, and subsequen
oxici y and e icacy o he ea men . The s udy o ci cadian luc ua ions o he molecules
ha a e in ol ed in hese p ocesses allows o he es ima ion o a mo e sui able ime o
ea men adminis a ion. The coo dina ion o ci cadian hy hms is con olled by biological
clocks ound in nea ly e e y cell in he body. A molecula clock machine y consis s o
clock genes/p o eins ha in eg a e egula o y eedback loops in which hey egula e hei
own exp ession. The main componen s o he clock include b ain and muscle ARNT-like1
(BMAL1), ci cadian locomo o ou pu cycles p o ein kapu (CLOCK), he p o eins pe iod
J. Func . Bioma e . 2023,14, 362 3 o 20
(PER1, PER2, and PER3), and c yp och ome (CRY1 and CRY2). The ansc ip ion ac o
CLOCK:BMAL1 o ms a dime o p omo e he exp ession o PER and CRY, which, in
u n, ac as ep esso s o CLOCK:BMAL1 in he nucleus. The ou come is he ci cadian
oscilla ion o many genes and p o eins in ol ed in se e al physiological p ocesses, wi hin
a pe iod o abou 24 h [
24
]. A co ela ion be ween clock gene exp ession and he apop o ic
e ec o an icance agen s has been epo ed [
25
]. Sla e al. ound ha he an icance
d ug emozolomide (TMZ) a ained a maximum e ec close o he daily peak o Bmal1
exp ession. Mo eo e , he dele ion o he co e clock gene abolished he hy hmici y in
TMZ-induced apop osis
in i o
[
25
]. Ano he s udy using
in i o
and in silico models
linked i ino ecan cy o oxici y, a d ug o colo ec al cance , o clock gene Bmal1 exp ession,
pu ing in e idence a ole o ci cadian hy hms in cance ea men [26].
An a ac i e app oach o o e come he limi a ions o cu en chemo he apeu ics
would be he combina ion o ch onobiology wi h nano echnology; howe e , ew wo ks
ha e add essed he in luence o he ci cadian clock in he e iciency o nano o mula ions,
namely, he cell en y o he ec o , i s elease pa e n and he apeu ic e ec . In a p e ious
wo k, he e ec o he ci cadian clock on he cellula up ake o nanopa icles based on
polye hylenimine (PEI) o deli e MTX and p53-plasmid DNA o HeLa and C33A cell
lines was in es iga ed [
27
]. The esul s e ealed a speci ic ime poin , a e cell synch o-
niza ion, o he high pe o mance o he d ug/gene co-deli e y sys em. In his wo k, we
in ended o con inue he esea ch on his opic and b ing ad ances o he ch onobiology-
nano echnology ela ionship. Pu suing his challenge, we syn hesized a PAMAM den-
d ime o gene a ion 4 (G4), in which PTX and R8 we e coupled (G4-PTX-R8). A e he
physicochemical cha ac e iza ion o he dend ime nanosys em, i was es ed whe he he
ime poin o deli e y a ec ed he amoun o bo h cell-associa ed PTX and caspase ac i i y
in HeLa cells. Fu he mo e, we success ully pe o med Bmal1 silencing on HeLa cells
o cla i y he impac o he ci cadian clock on cellula up ake and apop osis. Ou da a
demons a ed signi ican di e ences, in he p o ile o hese phenomena, be ween wild- ype
and knockou cells, sugges ing a po en ial ole o he ci cadian clock in he pe o mance o
a G4-PTX-R8 deli e y sys em.
2. Ma e ials and Me hods
2.1. Ma e ials
Dulbecco
´
s Modi ied Eagle
´
s Medium wi h Ham’s F-12 Nu ien Mix u e (
DMEM-F12
)
wi h L-glu amine was pu chased om CORNING (New Yo k, NY, USA). Ce ical cance
HeLa cells we e ob ained om ATCC (Manassas, VA, USA). 3-(4,5- dime hyl hiazol-2-
yl)-2,5-diphenyl e azolium b omide (MTT) was supplied by Sigma-Ald ich (S . Louis,
MO, USA). PTX was pu chased by MedChemExp ess (Monmou h Junc ion, NJ, USA). Fo
G4-PTX-R8 syn hesis, he dend ime o ou h gene a ion con aining e hylenediamine co e
g oups and su aced amino g oups (G4 PAMAM) was acqui ed om Dend i ech (Midland,
MI, USA). N-hyd oxysuccinimide and N-(3-Dime hylaminop opyl)-N-e hyl ca bodiimide
hyd ochlo ide (EDC. HCl, 98%) we e supplied by Sigma Ald ich Chemicals (S . Louis,
MO, USA). Finally, he cellulose dialysis memb ane (MWCO 3.5 KDa) was ob ained om
Spec um Labo a o ies, Inc. (Dominguez, CA, USA). All o he comme cially pu chased
sol en s and chemicals we e o analy ical g ade.
The eagen s equi ed o CRISPR-Cas9 echnology (single guide RNAs (c RNAs),
ATTO-labelled ac RNA, and Cas9) we e ob ained om IDT (IA, Co al ille, IA, USA);
Bu e R and he p ime s o Bmal1 we e pu chased om In i ogen (Ca lsbad, CA, USA).
The ApoAle TM Caspase-3 Colo ime ic assay ki was pu chased om Clon ech Lab
(O ange, CA, USA), and Caspase-Glow®9 Assay om P omega (Madison, WI, USA).
2.2. P epa a ion o PTX-2-Hemisuccina e (PTX-SA)
The s eps o he o ma ion o he G4 dend ime conjuga e a e illus a ed in Figu e 1.
The PTX-2
´
-hemisuccina e was syn hesized by ac i a ing he 2
0
-OH g oup o PTX using
succinic anhyd ide (SA). B ie ly, o 25 mg o PTX in Dichlo ome hane (DCM, 2 mL),
4.4 mg
J. Func . Bioma e . 2023,14, 362 4 o 20
o SA was added, in he p esence o d y py idine, a a mol a io o 1:1.5. This eac ion was
con inuously mixed o 3 days [
28
]. Nex , he PTX-SA was ex ac ed om he aqueous
eac ion mix u e using e hyl ace a e, and he sol en was e apo a ed unde acuum,
esul ing in a whi e powde wi h an ~85% yield. The p oduc was iden i ied by hin-
laye ch oma og aphy using silica-coa ed pla e and sol en sys em. Dichlo ome hane:
me hanol. 85:15.
J. Func . Bioma e . 2023, 14, x FOR PEER REVIEW 5 o 21
Figu e 1. Illus a ion ep esen ing he main h ee s eps in he p epa a ion o he mul i unc ional G4-
PTX-R8 complex.
2.5. Nuclea Magne ic Resonance (NMR) Spec oscopy o PAMAM G4-PTX-R8 Complexes
NMR was employed o cha ac e ize he de eloped conjuga es, using an NMR Spec-
ome e (400 MHz, B uke , Bille ica, MA, USA). The samples o G4, PTX-SA, and G4-
PTX-R8 (10 mg/mL) we e dissol ed in he sol en CDCl3 and he spec a we e ob ained
a e 128 scans [30].
2.6. Ci cula Dich oism (CD) Spec oscopy
The seconda y s uc u e o R8 and G4-PTX-R8 was analyzed, unde a ni ogen a -
mosphe e using a CD spec ome e (Shimadzu, Japan) wi h a scanning ange o 200–260
nm. The spec um was compa ed wi h he s anda d R8 o con i m he p esence o R8 in
he o mula ion [31].
2.7. Ze a Po en ial and Pa icle Size
The a e age ze a po en ial and pa icle size o he G4-PTX-R8 we e de e mined by
he p inciple o dynamic ligh sca e ing using Ze asize 3600 (Mal e n Ins umen s L d.,
Mal e n, UK). The ze a po en ial, pa icle size, and polydispe si y index (PdI) we e meas-
u ed be o e and a e lyophilizing he sample. The sample was dilu ed in Milli-Q wa e
be o e analysis [32].
2.8. Mo phological Analysis Using SEM
Fo he s udy o he G4-PTX-R8 su ace mo phology, Scanning Elec on Mic oscopy
(SEM, NOVA NANOSEM 450) analysis was pe o med.
Fo sample p epa a ion, a hin laye was e enly dis ibu ed and ixed o an aluminum
s ub wi h an adhesi e ca bon ape. Then, he s ub was coa ed wi h he desi ed gold hick-
ness and analyzed wi h a 20 kV accele a ing ol age [33].
2.9. Cell Cul u e
Fo he in i o expe imen s, ce ical cance HeLa cells we e g own in 25 cm
3
T- lasks
as desc ibed be o e [27]. HeLa cells (including knockou HeLa cells) we e g own wi h
DMEM-F12 supplemen ed wi h 10% hea -inac i a ed e al bo ine se um (FBS) and 1%
Figu e 1.
Illus a ion ep esen ing he main h ee s eps in he p epa a ion o he mul i unc ional
G4-PTX-R8 complex.
2.3. P epa a ion o PAMAM G4-PTX
The c oss-linking agen 1-e hyl-3-(3-dime hylaminop opyl)-ca bodiimide/
N-hyd oxysuccinimide (EDC/NHS) was used o ac i a e he PTX-2-hemisuccina e in 2 mL
o Dime hyl o mamide (DMF). The eac ion occu ed o 6 h a oom empe a u e [29].
Following ha , 50 mg o G4 PAMAM Dend ime in DMF was ans e ed o he
ac i a ed PTX-NHS es e , d opwise unde ni ogen.
The PAMAM G4: PTX-SA mol a io was 1:4. The eac ion con inued o e nigh . Nex ,
using a acuum, he DMF was apo ized. Then, he solu ion was added o a dialysis
memb ane (3500 Da MWCO) and dialyzed o 48 h.
A e wa d, he solu ion was lyophilized, and a whi e powde was ob ained. A yield
o ~75% was ob ained.
2.4. P epa a ion o PAMAM G4-PTX-R8
In o he solu ion o R8 (9.87 mg, 0.0078 mmol) and ime hylamine (20
µ
L) in DMF,
EDC/NHS (3 mol equi alen o he R8) G4-PTX (40 mg, 0.0026 mmol) was combined ol-
lowing he ac i a ion s ep. The eac ion was con inued o e nigh , ollowed by e apo a ion
o DMF. Finally, using a cellulose es e memb ane (MWCO. 3.5 KDa), he c ude p oduc
was dialyzed agains wa e , and he inal mix u e was lyophilized. A pu e p oduc was
ob ained wi h a yield o ~82%. An illus a ion wi h he main s eps o he p epa a ion o he
dend ime complex is ep esen ed in Figu e 1.
J. Func . Bioma e . 2023,14, 362 5 o 20
2.5. Nuclea Magne ic Resonance (NMR) Spec oscopy o PAMAM G4-PTX-R8 Complexes
NMR was employed o cha ac e ize he de eloped conjuga es, using an NMR Spec-
ome e (400 MHz, B uke , Bille ica, MA, USA). The samples o G4, PTX-SA, and G4-PTX-
R8 (10 mg/mL) we e dissol ed in he sol en CDCl3 and he spec a we e ob ained a e
128 scans [30].
2.6. Ci cula Dich oism (CD) Spec oscopy
The seconda y s uc u e o R8 and G4-PTX-R8 was analyzed, unde a ni ogen a mo-
sphe e using a CD spec ome e (Shimadzu, Japan) wi h a scanning ange o 200–260 nm.
The spec um was compa ed wi h he s anda d R8 o con i m he p esence o R8 in he
o mula ion [31].
2.7. Ze a Po en ial and Pa icle Size
The a e age ze a po en ial and pa icle size o he G4-PTX-R8 we e de e mined by
he p inciple o dynamic ligh sca e ing using Ze asize 3600 (Mal e n Ins umen s L d.,
Mal e n, UK). The ze a po en ial, pa icle size, and polydispe si y index (PdI) we e mea-
su ed be o e and a e lyophilizing he sample. The sample was dilu ed in Milli-Q wa e
be o e analysis [32].
2.8. Mo phological Analysis Using SEM
Fo he s udy o he G4-PTX-R8 su ace mo phology, Scanning Elec on Mic oscopy
(SEM, NOVA NANOSEM 450) analysis was pe o med.
Fo sample p epa a ion, a hin laye was e enly dis ibu ed and ixed o an aluminum
s ub wi h an adhesi e ca bon ape. Then, he s ub was coa ed wi h he desi ed gold
hickness and analyzed wi h a 20 kV accele a ing ol age [33].
2.9. Cell Cul u e
Fo he
in i o
expe imen s, ce ical cance HeLa cells we e g own in 25 cm
3
T- lasks as
desc ibed be o e [
27
]. HeLa cells (including knockou HeLa cells) we e g own wi h DMEM-
F12 supplemen ed wi h 10% hea -inac i a ed e al bo ine se um (FBS) and 1% (V/V) o a
penicillin/s ep omycin solu ion (100
µ
g/mL). Cells we e incuba ed a
37 ◦C
, unde a 5% CO
2
humidi ied a mosphe e, o p omo e cellula g ow h un il eaching con luency.
2.10. Cy o oxici y S udy
G4-PTX-R8 dend ime powde was dissol ed in DMSO:wa e 0.8:0.2 o pos e io s udies.
To e alua e he cy o oxici y o ee PTX and he G4-PTX-R8 dend ime on HeLa, an
MTT assay was pe o med. HeLa cells we e seeded a a densi y o 5
×
10
3
cells/well
in 96-well pla es, and he day be o e he expe imen s, he cul u e medium was changed
o DMEM-F12 supplemen ed wi h 10% FBS wi hou an ibio ics o enhance he cellula
up ake [34,35].
Di e en o mula ions o PTX and G4-PTX-R8 anging om 0 o 100 ug/mL we e
p epa ed and added o he cells du ing 6 h. A e wa d, medium was eplaced, and cells
we e allowed o g ow a 37
◦
C and 5% CO
2
o 48 h. A e ha pe iod, MTT was added o
each well a a inal concen a ion o 0.5 mg/mL and incuba ed o a u he 4 h. Fu he mo e,
he cul u e medium was disca ded and dime hyl sul oxide (DMSO, 100
µ
L) was added
o dissol e he o mazan c ys als. The ea e , he pla es we e shaken and a pu ple colo
was p oduced.
The abso bance was hen ead a 570 nm using a Benchma k Mic opla e Reade (BioRad,
Vienna, Aus ia). As a nega i e con ol o he expe imen , wells wi h no ea ed cells we e
conside ed and cells ea ed only wi h DMSO we e used as posi i e con ol. The medium
wi hou cells was se up as ze o abso bance and used o spec opho ome e calib a ion.
The cell iabili y (%) was he e o e calcula ed in ela ion o he con ol wells by he
o mula: [A] es /[A]con ol
×
100, [A] es being he abso bance o he es sample and
[A]con ol he abso bance o he posi i e con ol sample.

J. Func . Bioma e . 2023,14, 362 6 o 20
Mo eo e , he hal maximal inhibi o y concen a ion (IC
50
) was assessed o ee PTX
and he G4-PTX-R8 complex.
2.11. In Vi o S udies o Acess Ci cadian Rhy hms Impac on Cellula Up ake o he Nanosys em
Fo
in i o
s udies, HeLa cells we e seeded a a densi y o 5
×
10
4
cells/3.8 cm
2
in 12-
well mic opla es and g own un il eaching 70–90% o con luence. Be o e he expe imen s,
cells we e synch onized o 2 h wi h 0.1
µ
M o dexame hasone. Then, he medium was
emo ed and cells e u ned o no mal condi ions [
27
]. Cells we e incuba ed wi h ee PTX
o he G4-PTX-R8 complex a he IC50 concen a ion, calcula ed o G4-PTX-R8.
The deli e y was pe o med a T0 (immedia ely a e cell synch oniza ion) and e e y
4 h
du ing 24 h (T4; T8; T12; T16; and T20). A e 6 h o incuba ion, cells we e ei he
collec ed a he di e en ime poin s o he cell-associa ed PTX abso bance measu emen o
cells e u ned o usual cul u e condi ion and le o g ow du ing 48 h o subsequen assays.
2.12. Cell-Associa ed PTX
To e alua e he cellula up ake o PTX in HeLa cells a he di e en ime poin s, cell-
associa ed PTX was measu ed. A e collec ing he cells, he pelle was insed in phospha e-
bu e ed saline (PBS) and collec ed. Then, cell lysis was pe o med by incuba ion wi h a
1% T i on X-100 solu ion o 30 min a 37
◦
C. Las ly, he solu ion was pipe ed in o a black
pla e, and in acellula PTX was de e mined by eading he abso bance a 230 nm using a
Shimadzu UV-Vis 1700 spec opho ome e (Bio ad). Cells wi hou ea men we e used as
con ol [34].
2.13. Bmal1 Silencing on Hela Cell Line
The gene edi ing echnique CRISPR-Cas 9 was used wi h he pu pose o s udying
he e ec o Bmal1 dele ion on he e icacy o he G4-PTX-R8 sys em a all he 6 ime
poin s. Two c RNAs (In eg a ed DNA Technology) we e designed o gene a e a small
dele ion (140 bp) a ge ing exon 8 (Supplemen a y Ma e ial, Table S1). Fo he complex
o ma ion, equimola amoun s we e mixed o a inal duplex concen a ion o 44
µ
M. Then,
he mix u e was hea ed o 95
◦
C o 5 min and hen he empe a u e was amped down
o 25
◦
C on a he mocycle . Nex , 18 pmol o Cas9 (In eg a ed DNA Technology) was
added o he ini ial duplex eac ion (22 pmol) o a inal olume o 1
µ
L o elec opo a ion
and he mix u e was se a oom empe a u e (RT) o 1 h. Be o e elec opo a ion, 2
µ
L o
10.8
µ
M o Elec opo a ion Enhance and 9
µ
L o cell suspension we e added o he inal
eac ion. An amoun o 2
×
10
5
cells was elec opo a ed in a Neon TM T ans ec ion Sys em
(In i ogen, Wal ham, CA, USA) ollowing he manu ac u e ’s ins uc ions and using he
elec opo a ion pa ame e s o 1005 V, 35 ms, and 2 pulses.
The day a e ans ec ion, cells we e obse ed unde con ocal mic oscopy o con-
i m he e iciency o elec opo a ion. Subsequen ly, cells we e so ed wi h a FACSa ia
(
BD Biosciences
, San Jose, CA, USA). This s ep was pe o med o selec he gene- a ge ed
cells by sepa a ion o he Bmal1 cell popula ion om he con ol wi h only Cas9. Resul s
we e analyzed using FlowJo so wa e.
A e he so ing, cells we e collec ed, and one single cell was seeded in a new pla e
and le o g ow in o colonies. The con i ma ion o he knockou ha bo ing clones was i s
assessed by con en ional PCR (p ime sequence on Supplemen a y Ma e ial, Table S2).
The AllP ep DNA Ki (Qiagen) was used o he ex ac ion o Genomic DNA acco ding o
he manu ac u e ’s p o ocol. The PCR p oduc s we e cleaned wi h he NZYGelpu e ki
(NZYTech, Lisbon, Po ugal) and sequenced by he Sange me hod [36].
2.14. Caspase-3 and Caspase-9 Ac i i y Assay
Caspase-3 and caspase-9 ac i i y we e measu ed on wild- ype and knockou HeLa
cells ollowing he p o ided ins uc ions. In b ie , a e he incuba ion wi h ee PTX o
G4-PTX-R8, cells we e allowed o g ow o 24 h. An incuba ion wi h 1
µ
M o s au ospo ine,
unde he same condi ions, was used as a posi i e con ol.
J. Func . Bioma e . 2023,14, 362 7 o 20
The caspase-3 ac i i y was de e mined by using a caspase colo ime ic assay Ki
(ApoAle TM), by measu ing he abso bance a 405 nm o p-ni oaniline (p-NA) a e
clea age om he subs a e DEVD-pNA. A Luminescen Assay (Caspase-Glow
®
9) was
applied o he de e mina ion o Caspase-9 ac i i y ollowing he p o ided p o ocol [34].
2.15. S a is ical Analysis
Fo he compa ison be ween he dis inc expe imen al g oups, one-way/ wo-way
analysis o a iance (ANOVA) and Bon e oni es we e employed. The da a we e ana-
lyzed in G aphPad P ism .8.01 so wa e (San Diego, CA, USA). S a is ically signi ican
di e ences we e conside ed o a p- alue below 0.05 (* p
≤
0.05; ** p
≤
0.01; *** p
≤
0.001;
**** p≤0.0001).
The hy hmici y in PTX in acellula up ake and caspase ac i i y was analyzed wi h
Ci cWa e 1.4 analysis so wa e (D . Roelo A. Hu , G oningen, The Ne he lands) by
a ha monic eg ession me hod, assuming a pe iod o 24 h, and wi h alpha se a 0.05.
S a is ically signi ican hy hms we e conside ed o a p- alue below 0.05.
3. Resul s
3.1. P epa a ion and Cha ac e iza ion o Mul i unc ional Dend ime Conjuga e
The mul i unc ional dend ime conjuga e was syn hesized as desc ibed in Figu e 1and
cha ac e ized by NMR spec oscopy, Fou ie ans o m in a ed spec oscopy (FTIR), X- ay
Pho oelec on spec oscopy (XPS), CD Spec oscopy, Gel Pe mea ion Ch oma og aphy
(GPC), Ze a po en ial and Pa icle size de e mina ion, and SEM (de ailed desc ip ion
a ailable in Supplemen a y Ma e ial).
Figu e 2b shows he 1H-NMR spec a o PTX-SA displaying a peak a 2.5–2.8 (
δ
),
which indica es he coupling o succinic acid o he PTX. The spec a o G4 and G4-PTX-R8
p esen a signal a ppm 7–8.5 (
δ
), which co esponds o he a oma ic g oups o he PTX. In
Figu e 2c,d, he signals om he dend ime p o ons a e obse ed a ppm 1.5–3.5 (
δ
). Finally,
he signals isible a ppm 1–1.5 (
δ
) ela e o he me hylene p o ons o R8 [
12
]. Fu he mo e,
FTIR and XPS alida ed he conjuga ion o PTX and R8 o he dend ime (Figu es S1 and
S2 and Table S3, all p esen ed in Supplemen a y Ma e ial)).
J. Func . Bioma e . 2023, 14, x FOR PEER REVIEW 8 o 21
Figu e 2b shows he 1H-NMR spec a o PTX-SA displaying a peak a 2.5–2.8 (δ),
which indica es he coupling o succinic acid o he PTX. The spec a o G4 and G4-PTX-
R8 p esen a signal a ppm 7–8.5 (δ), which co esponds o he a oma ic g oups o he PTX.
In Figu e 2c,d, he signals om he dend ime p o ons a e obse ed a ppm 1.5–3.5 (δ).
Finally, he signals isible a ppm 1–1.5 (δ) ela e o he me hylene p o ons o R8 [12].
Fu he mo e, FTIR and XPS alida ed he conjuga ion o PTX and R8 o he dend ime
(Figu es S1 and S2 and Table S3, all p esen ed in SM).
Figu e 2. Thin-laye ch oma og aphy o PTX-SA conjuga e (a), 1H-NMR spec a o PTX-SA (b), PA-
MAM G4 (c), and G4-PTX-R8 (d).
To u he con i m he p esence o R8 in he o mula ion, CD Spec oscopy was pe -
o med. The alpha-helix and be a-shee alues ob ained o s anda d R8 we e 70.71 ± 0.79
and 10.21%, espec i ely. The R8-conjuga ed polyme , G4-PTX-R8, displayed he alues
as 59.32 ± 0.12 and 14.32%, espec i ely (Figu e 3). The dec ease in he CD signal in he
G4-PTX-R8 conjuga e indica ed he binding o molecules o he pep ide, which induced
i s con o ma ional change. Howe e , he seconda y s uc u e emains he same wi h he
p edominance o alpha-helical s uc u e.
Figu e 2.
Thin-laye ch oma og aphy o PTX-SA conjuga e (
a
), 1H-NMR spec a o PTX-SA
(b), PAMAM G4 (c), and G4-PTX-R8 (d).
To u he con i m he p esence o R8 in he o mula ion, CD Spec oscopy was pe -
o med. The alpha-helix and be a-shee alues ob ained o s anda d R8 we e
70.71 ±0.79
J. Func . Bioma e . 2023,14, 362 8 o 20
and 10.21%, espec i ely. The R8-conjuga ed polyme , G4-PTX-R8, displayed he alues
as 59.32
±
0.12 and 14.32%, espec i ely (Figu e 3). The dec ease in he CD signal in he
G4-PTX-R8 conjuga e indica ed he binding o molecules o he pep ide, which induced
i s con o ma ional change. Howe e , he seconda y s uc u e emains he same wi h he
p edominance o alpha-helical s uc u e.
J. Func . Bioma e . 2023, 14, x FOR PEER REVIEW 9 o 21
Figu e 3. Rep esen a ion o CD spec a o R8 and G4-PTX-R8. The p esence o R8 in he inal o mu-
la ion was con i med, as well as he s uc u al in eg i y ollowing conjuga ion.
The ela i e molecula weigh s o he syn hesized p oduc we e de e mined by using
GPC. The a e age molecula weigh o he dend ime complex was 19313 Da (Table S4,
SM). The GPC da a analysis also indica ed ha 2.48 molecules o R8 we e conjuga ed o
one molecule o G4-PTX (Figu e S3, SM) in he s a ed expe imen al condi ions.
Finally, he ze a po en ial and pa icle size o G4-PTX-R8 o mula ions a e p esen ed
in Table 1. The mo phological analysis using SEM (Figu e 4) displayed uni o m sphe ical
mo phology o he dend ime , and he ze a po en ial e ealed he posi i e su ace cha ge
o he conjuga e.
Table 1. Size, PdI, and ze a po en ial alue ob ained o PAMAM G4-PTX-R8 dend ime .
Ze a Po en ial (mV) PdI Size (nm)
PAMAM G4-PTX-R8 +9.325 0.287 23.74 ± 0.454
Figu e 4. SEM image o PAMAM G4-PTX-R8 dend ime .
3.2. Cy o oxici y S udies
To de e mine he efficacy o he PTX conjuga e in inhibi ing cance cell iabili y, an
MTT assay was pe o med in HeLa cells. Cells we e ea ed wi h ee PTX o G4-PTX-R8
a concen a ions anging om 0 o 100 µg/mL and incuba ed o 48 h, as p esen ed in
Figu e 5. Cells ha did no ecei e he ea men we e aken as a posi i e con ol o
Figu e 3.
Rep esen a ion o CD spec a o R8 and G4-PTX-R8. The p esence o R8 in he inal
o mula ion was con i med, as well as he s uc u al in eg i y ollowing conjuga ion.
The ela i e molecula weigh s o he syn hesized p oduc we e de e mined by using
GPC. The a e age molecula weigh o he dend ime complex was 19313 Da (Table S4,
Supplemen a y Ma e ial). The GPC da a analysis also indica ed ha 2.48 molecules o R8
we e conjuga ed o one molecule o G4-PTX (Figu e S3, Supplemen a y Ma e ial) in he
s a ed expe imen al condi ions.
Finally, he ze a po en ial and pa icle size o G4-PTX-R8 o mula ions a e p esen ed
in Table 1. The mo phological analysis using SEM (Figu e 4) displayed uni o m sphe ical
mo phology o he dend ime , and he ze a po en ial e ealed he posi i e su ace cha ge
o he conjuga e.
Table 1. Size, PdI, and ze a po en ial alue ob ained o PAMAM G4-PTX-R8 dend ime .
Ze a Po en ial (mV) PdI Size (nm)
PAMAM G4-PTX-R8 +9.325 0.287 23.74 ±0.454
3.2. Cy o oxici y S udies
To de e mine he e icacy o he PTX conjuga e in inhibi ing cance cell iabili y, an
MTT assay was pe o med in HeLa cells. Cells we e ea ed wi h ee PTX o G4-PTX-R8 a
concen a ions anging om 0 o 100
µ
g/mL and incuba ed o 48 h, as p esen ed in Figu e 5.
Cells ha did no ecei e he ea men we e aken as a posi i e con ol o cellula iabili y.
The oxici y was accessed by calcula ing he IC
50
alue om he di e en concen a ions
using a nonlinea cu e i ing algo i hm. The esul s o he assay demons a ed ha
he e is e idence o a cellula iabili y dec ease in ime as he PTX concen a ion inc eases
(Figu e 5).
J. Func . Bioma e . 2023,14, 362 9 o 20
J. Func . Bioma e . 2023, 14, x FOR PEER REVIEW 9 o 21
Figu e 3. Rep esen a ion o CD spec a o R8 and G4-PTX-R8. The p esence o R8 in he inal o mu-
la ion was con i med, as well as he s uc u al in eg i y ollowing conjuga ion.
The ela i e molecula weigh s o he syn hesized p oduc we e de e mined by using
GPC. The a e age molecula weigh o he dend ime complex was 19313 Da (Table S4,
SM). The GPC da a analysis also indica ed ha 2.48 molecules o R8 we e conjuga ed o
one molecule o G4-PTX (Figu e S3, SM) in he s a ed expe imen al condi ions.
Finally, he ze a po en ial and pa icle size o G4-PTX-R8 o mula ions a e p esen ed
in Table 1. The mo phological analysis using SEM (Figu e 4) displayed uni o m sphe ical
mo phology o he dend ime , and he ze a po en ial e ealed he posi i e su ace cha ge
o he conjuga e.
Table 1. Size, PdI, and ze a po en ial alue ob ained o PAMAM G4-PTX-R8 dend ime .
Ze a Po en ial (mV) PdI Size (nm)
PAMAM G4-PTX-R8 +9.325 0.287 23.74 ± 0.454
Figu e 4. SEM image o PAMAM G4-PTX-R8 dend ime .
3.2. Cy o oxici y S udies
To de e mine he efficacy o he PTX conjuga e in inhibi ing cance cell iabili y, an
MTT assay was pe o med in HeLa cells. Cells we e ea ed wi h ee PTX o G4-PTX-R8
a concen a ions anging om 0 o 100 µg/mL and incuba ed o 48 h, as p esen ed in
Figu e 5. Cells ha did no ecei e he ea men we e aken as a posi i e con ol o
Figu e 4. SEM image o PAMAM G4-PTX-R8 dend ime .
J. Func . Bioma e . 2023, 14, x FOR PEER REVIEW 10 o 21
cellula iabili y. The oxici y was accessed by calcula ing he IC50 alue om he diffe en
concen a ions using a nonlinea cu e i ing algo i hm. The esul s o he assay demon-
s a ed ha he e is e idence o a cellula iabili y dec ease in ime as he PTX concen a-
ion inc eases (Figu e 5).
Figu e 5. Dose– esponse cu es and cellula iabili y o HeLa ce ical cance cells a e 48 h o ea -
men wi h ee PTX (a) and he G4-PTX-R8 complex (b). Each da a poin ep esen s he mean alue
calcula ed wi h he da a ob ained om independen measu emen s (n = 3, mean ± SD), which we e
analyzed by one-way ANOVA wi h he Bon e oni es (**** < 0.0001 ela i e o con ol).
The de e mina ion o he median inhibi o y concen a ion is used o es ima e how
effec i e a gi en an icance d ug is a educing cellula iabili y. The alue o IC50 is lowe
o G4-PTX-R8, meaning ha a lowe dose is equi ed o p oduce he same oxici y as ee
PTX. The subsequen expe imen s we e conduc ed based on he IC50 alue achie ed o
G4-PTX-R8.
3.3. The Impac o Ci cadian Rhy hm on Cellula Up ake/In e naliza ion
3.3.1. De e mina ion o Cell-Associa ed PTX
Cell synch oniza ion was con i med a e analyzing he exp ession o Bmal1 o e 24
h (Figu e S4). To e alua e he impac o he ci cadian hy hm on PTX cellula up ake/in-
e naliza ion, he eleased PTX in he cy osol o HeLa cells a e incuba ion wi h he G4-
PTX-R8 dend ime o ee PTX was measu ed o each ime poin indica ed abo e. Figu e
6a shows he ob ained esul s. F om he p esen ed esul s, ega ding he cell-associa ed
PTX abso bance measu e, we can in e ha bo h ee d ug and dend ime -associa ed d ug
a e in e nalized by HeLa, howe e , in a diffe en ex ension and diffe en ly o e ime.
Figu e 5.
Dose– esponse cu es and cellula iabili y o HeLa ce ical cance cells a e 48 h o
ea men wi h ee PTX (
a
) and he G4-PTX-R8 complex (
b
). Each da a poin ep esen s he mean
alue calcula ed wi h he da a ob ained om independen measu emen s (n = 3, mean
±
SD), which
we e analyzed by one-way ANOVA wi h he Bon e oni es (**** p< 0.0001 ela i e o con ol).
J. Func . Bioma e . 2023,14, 362 16 o 20
Gi en ou indings, we belie e ha a way o make cance he apy mo e e ec i e should
include he op imiza ion o nanosys em design/de elopmen o ensu e d ug deli e y a
he app op ia e ime o he day, in acco dance wi h he pa ien ´s ci cadian hy hm.
Needless o say, he sui able ime o
in i o
adminis a ion should ake se e al ac o s
in o accoun . Upon adminis a ion, nanopa icles will be exposed o pha macokine ic
phenomena, many o which a e unde ci cadian egula ion. All hese ac o s a e supposed
o impac d ug oxici y and e icacy and mus be conside ed o ch ono he apy applica ions
o enhance cance he apy ou comes.
5. Conclusions
The a ac i e concep o “Ch ono he apy” has been explo ed o enhance cance
he apy ou comes. In pa allel, o o e come he majo obs acles o con en ional he apies, a
a ie y o nanosys ems ha e been conside ed o he a ge ed d ug elease o cance cells.
In his wo k, we de eloped nanocomplexes based on a PAMAM dend ime o gen-
e a ion 4 o PTX deli e y in o HeLa cance cells. Rega dless, hese molecules can also
ace some challenges, such as memb ane pe meabili y. Consequen ly, he R8 pep ide was
success ully conjuga ed on he dend ime su ace. In ou s udy, he e iciency o dend ime
cellula up ake was di ec ly co ela ed wi h highe PTX abso bance ound in he cy osol.
We no ed a supe io PTX in e naliza ion when PTX was coupled wi h PAMAM and R8
a he han when used alone. The e o e, he dend ime is a sui able ehicle o he elease
o poo ly soluble d ugs.
To un eil he e ec o he ci cadian hy hm on he e iciency o cellula in e naliza ion,
we measu ed cell-associa ed PTX and caspase ac i i y on wild- ype HeLa and Bmal1-
silenced cells. The ob ained esul s demons a ed highe deli e y o PTX a T8 and T12 and
supe io caspase-3 ac i i y a T8, and a T8 and T12 o caspase-9 ac i i y. As o caspase
ac i i y, only caspase-3 exp ession was comp omised by he Bmal1 knockdown in HeLa
cells. Also, he knockdown main ained a hy hmic pa e n o PTX in e naliza ion and
caspase ac i i y, howe e , o a much less ex en . We concluded ha Bmal1 appea s o be
necessa y o he e icien up ake o he dend ime and o he execu ion o p og ammed
cell dea h. Bmal1 can ei he p omo e he cellula in e naliza ion o PTX, leading o he
ac i a ion o apop osis, o can inc ease suscep ibili y o he d ug, leading o inc eased
apop osis a a speci ic ime poin . Al hough ou esul s do no co e a ull unde s anding
o he ole o Bmal1, ou wo k s ongly sugges s he in luence o Bmal1 on he pe o mance
o PTX/G4-PTX-G8 cellula up ake, PTX deli e y, and, consequen ly, apop osis induc ion,
and, hus, he apeu ic e ec . The epo ed da a should ins iga e he design o no el
deli e y sys ems displaying a mo e p ecise a ge ing o cance cells, a a mo e a o able
ime. I ansla ed and implemen ed in he clinic, i could ha e an exponen ial impac on
cance he apy.
Supplemen a y Ma e ials:
The ollowing suppo ing in o ma ion can be downloaded a : h ps:
//www.mdpi.com/a icle/10.3390/j b14070362/s1, Figu e S1: FTIR spec a o G4, PTX-SA, G4-
PTX, and G4-PTX-R8; Table S1: c RNA sequences; Figu e S2: XPS spec a o G4, G4-PTX, and
G4-PTX-R8; Table S2: P ime s designed o RT-PCR; Figu e S3: GPC he mog am o G4, G4-PTX, and
G4-PTX-R8; Table S3: XPS da a o PAMAM G4-PTX-R8 dend ime ; Figu e S4: Ci cWa e analysis
o bmal1 hy hmici y a e HeLa cell synch oniza ion; Table S4: GPC da a o PAMAM G4-PTX-
R8 dend ime ; Figu e S5: Genomic map o Bmal1 showing he sequence o he dele ed agmen
om exon 8 in g ey and RT-PCR p oduc s o he isola ed clones; Table S5: S a is ic analyses o
Bon e oni’s mul iple compa isons es o PTX in e naliza ion on HeLa cells; Figu e S6: Wes e n
blo analysis o BMAL1 p o ein exp ession in HeLa cells; Table S6: Resul s o Ci cWa e analysis o
cell-associa ed PTX abso bance o each ime poin ; Figu e S7: Ci cadian oscilla ions a e s a is ically
signi ican as analyzed wi h Ci cWa e o PTX and he G4-PTX-R8 complex; Table S7: S a is ic
analyses o Bon e oni’s mul iple compa isons es o PTX and G4-PTX-R8 in e naliza ion on HeLa
knockou cells; Figu e S8: Ci cadian oscilla ions a e s a is ically signi ican as analyzed wi h Ci cWa e
o caspase-3 and caspase-9 ac i i y a e incuba ion wi h PTX and G4-PTX-R8; Table S8: S a is ic

J. Func . Bioma e . 2023,14, 362 17 o 20
analyses o Bon e oni’s mul iple compa isons es o caspase ac i i y on wild- ype and knockou
a e incuba ion wi h ee PTX o G4-PTX-R8.
Au ho Con ibu ions: Concep ualiza ion, M.S.-M., T.Q. and D.C.; me hodology, T.A., A.R.N., M.P.,
S.B., E.V., I.G.-T., M.S.-M., T.Q. and D.C.; so wa e, T.A., A.R.N., M.P., S.B., E.V., I.G.-T., M.S.-M., T.Q.
and D.C.; alida ion, S.B., E.V., I.G.-T., M.S.-M., T.Q. and D.C.; o mal analysis, T.A., A.R.N., M.P., S.B.,
E.V., I.G.-T., M.S.-M., T.Q. and D.C.; in es iga ion, T.A., A.R.N., M.S.-M., T.Q. and D.C.; esou ces,
S.B., I.G.-T., M.S.-M., T.Q. and D.C.; da a cu a ion, T.A., A.R.N., M.P., S.B., E.V., I.G.-T., M.S.-M., T.Q.
and D.C.; w i ing—o iginal d a p epa a ion, T.A., A.R.N. and D.C.; w i ing— e iew and edi ing,
T.A., A.R.N., S.B., E.V., M.S.-M., T.Q. and D.C.; isualiza ion, T.A., A.R.N., M.P., S.B., E.V., I.G.-T.,
M.S.-M., T.Q. and D.C.; supe ision, S.B., M.S.-M., T.Q. and D.C.; p ojec adminis a ion, S.B., M.S.-M.,
T.Q. and D.C.; unding acquisi ion, S.B., M.S.-M., T.Q. and D.C. All au ho s ha e ead and ag eed o
he published e sion o he manusc ip .
Funding:
D. Cos a acknowledges he Fundação pa a a Ciência e a Tecnologia (FCT) he 2021.03946.CEECIND
Resea che Con ac . T. Albuque que (SFRH/BD/148406/2019) and A. Ne es (2020.08310.BD)
acknowledge hei FCT PhD g an s, co-suppo ed by he Eu opean Social Fund ia P og ama Ope a-
cional Regional Cen o. Swa i Biswas acknowledges he Indian Council o Medical Resea ch (ICMR)
o p o iding unding h ough a esea ch g an (F-20218945). Milan Paul acknowledges ICMR o
p o iding he senio esea ch ellowship (45/11/2022/NAN/BMS). The p esen ed esea ch was
de eloped wi hin he scope o CICS-UBI p ojec s UIDB/00709/2020 and UIDP/00709/2020, inanced
by na ional unds h ough he Po uguese FCT/MCTES.
Da a A ailabili y S a emen :
All da a is p o ided in he p incipal documen o he manusc ip o as
supplemen a y ma e ials.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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