Ci a ion: Re illa, G.; Al Q aish, N.;
Ca uana, P.; Sainz-Ramos, M.;
Lopez-Mendez, T.; Rod iguez, F.;
Paez-Espinosa, V.; Li, C.; Vall e dú,
N.F.; Edwa ds, M.; e al.
Len a inib-Loaded
Poly(lac ic-co-glycolic acid)
Nanopa icles wi h Epide mal
G ow h Fac o Recep o An ibody
Conjuga ion as a P eclinical
App oach o The apeu ically
Imp o e Thy oid Cance wi h
Agg essi e Beha io . Biomolecules
2023,13, 1647. h ps://doi.o g/
10.3390/biom13111647
Academic Edi o : Giuseppe
Pappala do
Recei ed: 28 Sep embe 2023
Re ised: 6 No embe 2023
Accep ed: 8 No embe 2023
Published: 13 No embe 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/).
biomolecules
A icle
Len a inib-Loaded Poly(lac ic-co-glycolic acid) Nanopa icles
wi h Epide mal G ow h Fac o Recep o An ibody Conjuga ion
as a P eclinical App oach o The apeu ically Imp o e Thy oid
Cance wi h Agg essi e Beha io
Gio anna Re illa 1,2,3 , Nuseibah Al Q aish 4,5,6 , Pablo Ca uana 1, My iam Sainz-Ramos 4,5,7,
Tania Lopez-Mendez 4,5,7, F ancisco Rod iguez 1, Ve ónica Paez-Espinosa 8, Changda Li 1,2,3 ,
Nú ia Fucui Vall e dú1, Ma ia Edwa ds 1, An onio Mo al 4,9 , JoséIgnacio Pé ez 9, Juan Ca los Escolà-Gil 1,2,10 ,
JoséLuis Ped az 4,5,7 , Idoia Gallego 4,5,7 , Rosa Co coy 1,3,10 , Ma ía Vi udes Céspedes 1,* ,
Gus a o Pu as 4,5,7 and Eugènia Ma o 1,3,4,*
1Resea ch Biomedical Ins i u e (IIB) San Pau, C/San Quin í77, 08041 Ba celona, Spain;
[email p o ec ed] (G.R.); pca [email p o ec ed] (P.C.); [email p o ec ed] (F.R.); [email p o ec ed] (C.L.);
[email p o ec ed] (N.F.V.); medwa [email p o ec ed] (M.E.); [email p o ec ed] (J.C.E.-G.);
co [email p o ec ed] (R.C.)
2Depa amen o Biochemis y and Molecula Biology, Uni e si a Au ònoma de Ba celona, C/An oni M.
Cla e 167, 08025 Ba celona, Spain
3Depa men o Endoc inology and Nu i ion, Hospi al de la San a C eu i San Pau, 08041 Ba celona, Spain
4
Ne wo king Resea ch Cen e o Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN), C/Mon o e
de Lemos 3-5, 28029 Mad id, Spain; [email p o ec ed] (N.A.Q.); [email p o ec ed] (M.S.-R.);
[email p o ec ed] (T.L.-M.); [email p o ec ed] (A.M.); [email p o ec ed] (J.L.P.);
[email p o ec ed] (I.G.); [email p o ec ed] (G.P.)
5
NanoBioCel Resea ch G oup, Labo a o y o Pha macy and Pha maceu ical Technology, Facul y o Pha macy,
Uni e si y o he Basque Coun y (UPV/EHU), Paseo de la Uni e sidad 7, 01006 Vi o ia-Gas eiz, Spain
6
Pha macy Depa men , College o Pha macy, Amman A ab Uni e si y, P.O. Box 2234, Amman 11953, Jo dan
7Bioa aba, NanoBioCel Resea ch G oup, 01009 Vi o ia-Gas eiz, Spain
8Depa men Clinical Biochemis y, School o Medicine, Pon i icia Uni e sidad Ca ólica del Ecuado (PUCE),
A . 12 de Oc ub e 1076 y Roca, Qui o 17012184, Pichincha, Ecuado ; [email p o ec ed]
9Depa men o Gene al Su ge y, Hospi al de la San a C eu i San Pau, C/San Quin í89,
08041 Ba celona, Spain; jpe [email p o ec ed]
10 CIBER de Diabe es y En e medades Me abólicas Asociadas (CIBERDEM), C/Mon o e de Lemos 3-5,
28029 Mad id, Spain
*Co espondence: [email p o ec ed] (M.V.C.); [email p o ec ed] (E.M.)
Abs ac :
Backg ound: Len a inib, a y osine kinase inhibi o (TKI) app o ed o he ea men o
p og essi e and adioac i e iodine (RAI)- e ac o y di e en ia ed hy oid cance (DTC), is associa ed
wi h signi ican ad e se e ec s ha can be pa ially mi iga ed h ough he de elopmen o no el
d ug o mula ions. The u iliza ion o nanopa icles p esen s a iable op ion, as i allows o a ge ed
d ug deli e y, educing ce ain side e ec s and enhancing he o e all quali y o li e o pa ien s. This
s udy aimed o p oduce and assess, bo h
in i o
and
in i o
, he cy o oxici y, biodis ibu ion, and
he apeu ic e icacy o len a inib-loaded PLGA nanopa icles (NPs), bo h wi h and wi hou deco a ion
using an ibody conjuga ion (ce uximab), as a no el he apeu ic app oach o managing agg essi e
hy oid umo s. Me hods: Poly(lac ic-co-glycolic acid) nanopa icles (NPs), deco a ed wi h o wi hou
an i-EGFR, we e employed as a len a inib deli e y sys em. These NPs we e cha ac e ized o size
dis ibu ion, su ace mo phology, su ace cha ge, and d ug encapsula ion e iciency. Cy o oxici y
was e alua ed h ough MTT assays using wo cellula models, one ep esen ing no mal hy oid
cells (N hy-o i 3-1) and he o he ep esen ing anaplas ic hy oid cells (CAL-62). Addi ionally,
an
in i o
xenog a mouse model was es ablished o in es iga e biodis ibu ion and he apeu ic
e icacy ollowing in agas ic adminis a ion. Resul s: The NPs demons a ed success in e ms o
pa icle size, polydispe si y index (PDI), ze a po en ial, mo phology, encapsula ion e iciency, and
ce uximab dis ibu ion ac oss he su ace.
In i o
analysis e ealed cy o oxici y in bo h cellula
models wi h bo h o mula ions, bu only he deco a ed NPs achie ed an ID50 alue in CAL-62
Biomolecules 2023,13, 1647. h ps://doi.o g/10.3390/biom13111647 h ps://www.mdpi.com/jou nal/biomolecules
Biomolecules 2023,13, 1647 2 o 19
cells. Biodis ibu ion analysis ollowing in agas ic adminis a ion in xenog a ed hy oid mice
demons a ed good s abili y in e ms o in es inal ba ie unc ion and umo accumula ion. Bo h
o mula ions we e gene ally well ole a ed wi hou inducing pa hological e ec s in he examined
o gans. Impo an ly, bo h o mula ions inc eased umo nec osis; howe e , deco a ed NPs exhibi ed
enhanced pa ame e s ela ed o apop o ic/ka yoly ic o ms, mi o ic index, and ascula iza ion
compa ed wi h NPs wi hou deco a ion. Conclusions: These p oo -o -concep indings sugges a
p omising s a egy o adminis e ing TKIs in a mo e a ge ed and e ec i e manne .
Keywo ds: hy oid; nanopa icles; EGFR; len a inib
1. In oduc ion
Well-di e en ia ed hy oid ca cinomas (WDTCs) a e gene ally conside ed malignan
umo s wi h a posi i e p ognosis. Howe e , i is c ucial o ecognize ha a small pe -
cen age, ypically be ween 5% and 15%, may display agg essi e beha io and become
un esponsi e o adioiodine (RAI) ea men , leading o he de elopmen o e ac o y
umo s. Addi ionally, dedi e en ia ed hy oid cance (TC) and anaplas ic hy oid can-
ce (ATC) a e associa ed wi h a poo e p ognosis in compa ison wi h well-di e en ia ed
hy oid cance (WDTC). One o he key cha ac e is ics o dedi e en ia ed and agg essi e
TC ha is e ac o y o RAI ea men is he loss o he abili y o up ake
131
I. This loss o
iodine up ake ep esen s a signi ican su i al a e educ ion in pa ien s (60 o 70%) i e
yea s a e diagnosis [
1
–
3
]. In hese umo s sub ypes, including ATC, poo ly di e en ia ed
hy oid ca cinoma (PDTC), and RAI- e ac o y WDTC pa ien s, he iden i ica ion o he
oncogenic d i e mu a ions, able o ac i a e speci ic kinases, has allowed he use o y osine
kinase inhibi o s (TKIs) as a he apeu ic s a egy [
4
–
7
]. Fo ins ance, he TKI len a inib
demons a ed a signi ican ly longe median p og ession- ee su i al (PFS) compa ed wi h
bo h so a enib and he placebo g oup; PFS ime o len a inib was 18.3 mon hs, whe eas
i was only 3.6 mon hs o he placebo g oup (p< 0.001). In compa ison, so a enib had a
PFS o 10.8 mon hs, and he placebo g oup had one o 5.8 mon hs (bo h wi h p< 0.0001).
This ou s anding pe o mance has posi ioned len a inib as he p e e ed choice among o al
an iangiogenic mul i a ge ed TKIs [8–12].
This mul i a ge ed TKI wo ks agains ascula endo helial g ow h ac o ecep o s
(VEGFRs) 1, 2, and 3; Fib oblas g ow h ac o ecep o s (FGFRs) 1 h ough 4; Pla ele -
de i ed g ow h ac o ecep o alpha (PDGFRA); Re P o o-Oncogene (RET); and he KIT
P o o-Oncogene, Recep o Ty osine Kinase signaling ne wo k, which a e all in ol ed in
umo al angiogenesis p ocesses [
13
–
16
]. Ne e heless, in mos ea ed pa ien s, a dose
educ ion o ea men discon inua ion may be necessa y due o a high equency o
ad e se e en s associa ed wi h VEGF- a ge ed he apies, including hype ension, dia hea,
a igue o as henia, dec eased appe i e, and weigh loss [
12
,
13
,
17
]. Mo eo e , he Epide mal
G ow h Fac o Recep o (EGFR) ep esen s a p omising and alid he apeu ic a ge in solid
umo s, including ATC, ha exhibi s o e exp ession o his ecep o [
18
]. Ce uximab is a
human–mu ine chime ic EGFR- a ge ed monoclonal an ibody wi h inc eased speci ici y o
he ex acellula domain o human EGFR. I s mechanism o ac ion in ol es inhibi ing EGFR
signaling in cells, he eby dis up ing he no mal unc ion o he ecep o [
19
,
20
]. The use o
nanosys ems o deli e d ugs could be conside ed a good s a egy o educing he ad e se
e ec s o hese d ugs. The poly(lac ic-co-glycolic acid) (PLGA) nanosys em is conside ed
one o he mos a o able choices, owing o i s biodeg adabili y and e icien clea ance ia
he pulmona y and enal ou es, as p e iously documen ed [
21
–
24
]. Th ough ou p e ious
in es iga ions wi h so a enib-loaded PLGA-nanopa icles (NPs), we obse ed ha NPs
deco a ed wi h ce uximab exhibi ed a mo e p onounced and sus ained cy o oxic e ec o e
ime in he anaplas ic hy oid cell line CAL-62, compa ed wi h he no mal hy oid cell line
N hy-o i 3-1. These indings sugges a p omising a ge ed s a egy o ea WDTC wi h
poo p ognosis [
25
]. Conside ing he ad an ages o len a inib o so a enib in he e icacy,
Biomolecules 2023,13, 1647 3 o 19
sa e y, and su i al o pa ien s, he objec i e o his s udy was o op imize he o mula ion
o len a inib-loaded PLGA nanopa icles (len a-NPs), bo h wi h and wi hou ce uximab
deco a ion. Fu he mo e, we aimed o assess hei
in i o
cy o oxici y and, as a p oo o
concep , o e alua e hei biodis ibu ion and he apeu ic e ec i eness in a xenog a model
o anaplas ic hy oid ca cinoma.
2. Ma e ials and Me hods
2.1. Ma e ials
The len a inib (E7080) (10 mg) was supplied by Selleckchem (Del aclon, Mad id,
Spain), and he ce uximab (an i-EGFR; 5 mg/mL) was ob ained om Selleckchem, Del a-
clon (Mad id, Spain). The polyme poly (D, L-lac ide-co-glycolide; PLGA, Resome
®
RG
503, Mw 33.900), wi h a copolyme a io o 50:50 (lac ic/glycolic) and an in insic is-
cosi y o 0.8 dL/g in CHCl3, was supplied by Boeh inge Ingelheim K.G. (Ingelheim,
Ge many). Poly inyl alcohol (PVA, MW 30,000–70,000), dichlo ome hane, dime hyl sul-
oxide, 1-e hyl-3-(3-dime hylaminop opyl) ca bodiimide hyd ochlo ide (EDC), and N-
hyd oxysul osuccinimide (sul o-NHS) we e supplied by Sigma Chemical Co. (S . Louis,
MO, USA). 1,1
0
-Dioc adecyl-3,3,3
0
,3
0
-Te ame hylindo ica bocyanine Iodide (DiIC) was
supplied by The mo Fishe Scien i ic (Wal ham, MA, USA). A mic o BCA assay ki was
pu chased om Pie ce by Tekno as (Bilbao, Spain). All o he chemicals we e o analy ical
g ade and we e supplied by Pan eac S.A. (Ba celona, Spain).
2.2. P epa a ion o Len a inib-Loaded PLGA Nanopa icles (Len a-NPs)
The oil-in-wa e (O/W) single emulsion sol en e apo a ion me hod was used in he
p epa a ion o he len a-NPs, wi h a sligh modi ica ion. To gua an ee pe ec solubili y,
200 mg o len a inib was dissol ed in 200
µ
L o dime hyl sul oxide and apidly sonica ed
o 15 s. This solu ion was added o 3.800
µ
L o dichlo ome hane con aining 200 mg o
PLGA o ob ain he inal oil solu ion (5% PLGA, w/ ), which was hen pou ed in o 20 mL o
an 8% poly inyl alcohol aqueous solu ion and sonica ed o 100 s a 50 W (B anson Soni ie
250, B anson Ul asonics
™
, Shanghai, China) o e an ice ba h. To acili a e he e apo a ion
o ola ile o ganic sol en s om he NPs o he ex e nal phase, a 2% isop opanol solu ion
was added, and he sys em was agi a ed o 2 h a oom empe a u e on a magne ic s i
pla e. Ul a-cen i uga ion a 30,000 pm, 4
◦
C o 20 min (So all Legend X1R Cen i uge,
The mo Fishe Scien i ic, Os e ode am Ha z, Ge many) was employed o sepa a e he
NPs, which we e hen washed wi h double dis illed wa e ( he p ocess was epea ed h ee
imes). The eco e ed NP suspension was eeze-d ied wi h 5% ehalose (w/ ) o 24 h
(Lyo Be a 15, Tels a , Ta asa, Spain).
2.3. Cha ac e iza ion o Len a-NPs: Pa icle Size, Polydispe si y Index (PDI), Ze a Po en ial
Measu emen s, and Mo phology S udies (TEM)
The len a-NPs we e analyzed using a Mal e n Ze aize Nano ZS in e ms o pa icle
size, dispe si y index, and ze a po en ial (Mal e n Ins umen , Wo ces e shi e, UK). Dy-
namic ligh sca e ing was used o de e mine he pa icle size based on backsca e de ec ion
op ics a 173
◦
. In b ie , 1 mg o len a-NPs was esuspended in 1 mL o a 0.1 mM NaCl
aqueous solu ion. The e ac i e index (1.33) and iscosi y (0.89) o ul apu e wa e a
25 ◦C
we e u ilized o manual da a analysis. All measu es we e pe o med in iplica e, and
each iplica e was measu ed 11 imes o ob ain he a e age. Cumula i e analysis was used
o de e mine he pa icle size epo ed as he hyd odynamic diame e . The ze a po en ial
was measu ed using lase Dopple elocime y. Fo he ze a analysis, he len a-NPs we e
esuspended using disposable olded capilla y cells, as in he size measu emen p ocedu e.
Simila ly, measu emen s o he ze a po en ial we e pe o med in iplica e using he manual
mode, wi h 20 measu emen s in each un. The Hückel app oxima ion was u ilized o
suppo he ze a po en ial calcula ion. Only da a ha me he so wa e p og am’s . 7.11
(DTS 5.0, Ze asize Nano Sys em) quali y c i e ia we e included in he s udy.
Biomolecules 2023,13, 1647 4 o 19
2.4. T ansmission Elec on Mic oscope (TEM) S udies
TEM examina ion was used o obse e he mo phology o he len a-NPs. B ie ly,
he samples we e adhe ed o glow-discha ged ca bon-coa ed g ids o 60 s. To boos he
con as , he emaining liquid was collec ed by blo ing on a pape il e and dyed wi h 2%
u anyl ace a e o 60 s a a pH o 6.0. Samples we e obse ed using a mic oscope, TECNAI
G2 20 TWIN (FEI, Eindho en, The Ne he lands), ope a ed in b igh - ield image mode a
200 KeV accele a ing ol age. Using an Olympus SIS Mo ada digi al came a, digi al images
we e cap u ed.
2.5. Assessing he Inco po a ion o Len a inib in o Nanopa icles wi h HPLC
Using e e sed-phase HPLC (RP-HPLC), he encapsula ion e iciency o len a inib
in o PLGA NPs was assessed. Len a inib was analyzed on a YMC C18 (4.6
×
150 mm,
5 m) column. We u ilized a mobile phase composed o HPLC-g ade wa e and me hanol
(30/70 / ), wi h he low a e se a 0.6 mL/min, o elu e he len a inib. Unde acuum
il a ion, he mobile phase was il e ed using 0.22-mic on nylon il e s and degassed o
5 min in an ul asonic wa e ba h. As a diluen , he mobile phase was u ilized. The injec ion
olume was 20
µ
L, and he de ec o was calib a ed a 240 nm. Bo h he au o-sample
and he column we e kep a oom empe a u e. The ch oma og aphic p og am un ime
was 7.0 min. The len a inib sample solu ion was p epa ed, and 10 mg o len a inib we e
accu a ely weighed and hen deposi ed in o a olume ic lask con aining 10 mL o clean,
d y olume ic solu ion. Nex , abou 2 mL o diluen (mobile phase) was added, and he
mix u e was sonica ed o dissol e i comple ely. Finally, he olume was b ough up o
he app op ia e ma k using a diluen . An addi ional 10 mL o he a o esaid s ock solu ion
was pipe ed in o a 100 mL olume ic lask and dilu ed wi h diluen o he ma k. Then,
10
µ
L o he s anda d and sample solu ions o he len a inib d ug we e injec ed in o he
ch oma og aphic sys em in iplica e, and he peak a eas o he len a inib d ug we e
measu ed. Using a o mula, he assay pe cen age was calcula ed by compa ing he peak
a ea o he s anda d and sample ch oma og ams. The pe cen age o encapsula ion e iciency
was calcula ed by di iding he esul s o he sub ac ion by he heo e ical len a inib alue,
using he ollowing equa ion:
EE(%) = heo e ical len a inib − ee len a inib
heo e ical len a inib ×100
Len a inib d ug con en was exp essed as he mass o inco po a ed len a inib (
µ
g)
pe each mg o lyophilized len a-NPs.
2.6. In Vi o Len a inib Release S udies
To s udy len a inib elease om NPs, 10 mg o Len a-NPs we e added o es ubes
con aining 1 mL o PBS 0.1 M and 0.01% (w/ ) ween 80 a a pH o 7.4. The suspensions
o NPs we e incuba ed a 37
◦
C wi h cons an o bi al o a ion. The samples we e spun a
30,000 pm, 4
◦
C, o 20 min a egula in e als o up o 38 days. The supe na an s we e
collec ed, and he eleased len a inib was quan i ied using RP-HPLC. The same olume
u ilized o he de e mina ion o len a inib was eplaced wi h a new bu e . The expe imen
was conduc ed wi h h ee dis inc ba ches.
2.7. P epa a ion o Immunonanopa icles (Len a-NPs-Ce uximab)
EDC/sul o-NHS c oss-linking chemis y was used o a ach he ce uximab monoclonal
an ibodies o he su ace o he len a-NPs; b ie ly, 40 mg o len a-NPs was esuspended in
4 mL o 0.1 M, 0.5 M NaCl, pH 5.5 MES bu e . Then, 1.6 mg o EDC ( inal concen a ion
2 Mm) was applied di ec ly o he nanopa icle suspension, yielding a 10- old mola excess
o EDC o PLGA. The apid addi ion o 4.4 mg o sul o-NHS ( inal concen a ion 5 Mm) o
he len a-NP suspension, ollowed by 30 min o agi a ion on a magne ic pla e, allowed he
eac ion o occu . Then, 5
µ
L o 2-me cap oe hanol ( inal concen a ion 20 Mm) was added
o inac i a e he EDC. The sedimen was hen ul acen i uged o 20 min a 30,000 pm
Biomolecules 2023,13, 1647 5 o 19
and 4
◦
C o emo e he un eac ed EDC, sul o-NHS, and 2-me cap oe hanol. The p ocess
was epea ed h ee imes. Each ime he sedimen was washed wi h 1 mL o PBS 0.1 M,
0.15 M NaCl. To dissol e he pelle ob ained a e he las cen i uga ion, 1 mL o an i-EGFR
solu ion (1 mg/mL in PBS 0.1 M) was added, agi a ed o 2 h a oom empe a u e on a
magne ic s i pla e, and incuba ed o e nigh a 4
◦
C. The ollowing day, o emo e any
unconjuga ed ce uximab, he suspension was ul acen i uged o 20 min a 30,000 pm
and 4
◦
C. The p ocess was epea ed h ee imes. Each ime he sedimen was washed wi h
1 mL o PBS 0.1 M, 0.15 M NaCl. The supe na an was collec ed o he de e mina ion
o unconjuga ed an i-EGFR, and he esul ing pelle was lyophilized using an aqueous
solu ion o ehalose (5%, w/ ) o 24 h.
2.8. Cha ac e iza ion o Len a inib-NP-Ce uximab Immunonanopa icles
The esul ing immunonanopa icles we e cha ac e ized in e ms o pa icle size, PDI,
and mo phology, as p e iously desc ibed.
2.9. Quan i ica ion o Ce uximab Conjuga ed on o he Su ace o Nanopa icles
Spec opho ome y was used o quan i y he amoun o unbound an ibodies in he su-
pe na an using a colo ime ic mic oBCA p o ein assay ki (In ini e M200 mic opla e eade ,
Tecan Aus ia GmbH, G ödig, Aus ia). Len a-NP-ce uximab (ce ux) immunonanopa icles
we e ob ained by sub ac ing ee ce uximab om he o al amoun o ce uximab loaded
( heo e ical an i-EGFR). The e o e, he ce uximab con en was ep esen ed as he mass o
ce uximab inco po a ed (g) pe mg o lyophilized nanopa icles. A sample aken om he
supe na an o he len a-NPs wi hou ce uximab was used as a con ol. A s anda d cu e
o he ce uximab solu ion in a concen a ion ange o 5 µg/mL o 100 µg/mL was used o
compa e he esul s.
2.10. P epa a ion o DilC 18 Fluo escen ly Labeled Nanopa icles
To de e mine he cellula up ake o he NPs, he luo escen dye DilC18 was added o
he dime hyl sul oxide (DMSO) solu ion (0.05% w/ ) ins ead o len a inib. A luo escen
PLGA NP p oduced p e iously (desc ibed in Sec ion 2.2) was hen added o ob ain he
DilC luo escen NPs. The esul ing DilC luo escen -loaded and an i-EGFR conjuga ed
DilC NPs we e physiochemically cha ac e ized in e ms o size, PDI, and mo phology
(Sec ion 2.3).
2.11. Cell Lines
The human hy oid ollicula epi helial cell line (N hy-o i 3-1) was p o ided by D .
Pila San is eban (CSIC, Mad id, Spain), and he human epi helial ATC cell line (CAL-62)
was p o ided by Leibniz-Ins i u e DSMZ GmbH (ACC 448). The sho andem epea s
(STRs) o he N hy-o i 3-1 cell line we e analyzed ollowing he manu ac u e ’s ins uc-
ions using he p o ocols o he GeneAmp
®
PCR Sys em 2400 The mal Cycle (Pe kin
Elme /Applied Biosys ems, Wal ham, MA, USA), he ABI PRISM
®
310 Gene ic Analyze
(Pe kin Elme /Applied Biosys ems, Wal ham, MA, USA), and he so wa e package Gen-
emappe 4.1 (Applied Biosys ems, Ca lsbad, CA, USA). The STR o he N hy-o i 3-1 cell
line was ma ched wi h he esul s epo ed in cellosau us da abase (Bai och A., 2018). The
N hy-o i 3.1 cell line was cul u ed in RPMI 1640 (wi h L-glu amine), supplemen ed wi h
10% e al bo ine se um and 2% s ep omycin/penicillin. The CAL-62 was cul u ed in
Dulbecco’s modi ied Eagle’s medium (DMEM; Gibco, In i ogen, Ca lsbad, CA, USA),
supplemen ed wi h 10% hea -inac i a ed e al cal se um (FCS; Gibco), 20 mM L-Glu aMaxI
(Gibco, In i ogen Ca lsbad, CA, USA), and 1% penicillin-s ep omycin (Gibco, In i ogen
Ca lsbad, CA, USA).
2.12. In Vi o Cy o oxici y S udies o Len a-NPs and Len a-NP-Ce ux
The
in i o
cy o oxici y o he len a-NP-ce ux and len a-NP o mula ions was as-
sessed wi h 3-(4,5-dime hyl huazol-2-yl)-2,5-diphenyl e azolium b omide assay (MTT)
Biomolecules 2023,13, 1647 6 o 19
(Sigma, M6494, MO, USA). The cell lines (N hy-o i 3-1 and CAL-62) we e placed in 96-well
pla es wi h 200
µ
L o media a a densi y o 1500 cells pe well. A e 24 h o cul u e, he
media we e emo ed, and esh media wi h he NPs (len a-NPs-ce ux and len a-NPs;
20
µ
M) we e added and incuba ed o 24 h. A he end o he incuba ion pe iod, he media
was gen ly aspi a ed, and wo washes wi h PBS 1
×
a 37
◦
C we e ca ied ou o emo e any
non-in e nalized NPs. Subsequen ly, 50
µ
L o PBS 1
×
a 37
◦
C was added along wi h 10
µ
L
o MTT (12 mM), and he cells we e incuba ed o an addi ional 4 h a 37
◦
C. Subsequen ly,
he o mazan c ys als o med we e dissol ed by shaking wi h 50
µ
L o dime hyl sul oxide
(DMSO; SIGMA D8418) added o each well, mixing ho oughly wi h he pipe e, and
incuba ing a 37
◦
C o 10 min. The abso bance o each well was measu ed a 540 nm using
an ELISA mic opla e eade (xMa k BIORAD). The cy o oxici y a e was calcula ed as a
pe cen age using he ollowing o mula: A ea ed cells/A con ol cells x100, whe e A is
he op ical densi y o he o mazan eleased by he cells. In each expe imen , un ea ed
cells we e used as con ols.
2.13. Biodis ibu ion o NP and NP-Ce ux Nanopa icles in a Mouse Xenog a Model
In o de o e alua e he biodis ibu ion o he nanopa icles, CAL62 cell lines
we e subcu aneously injec ing in o he igh lank o he a hymic Swiss nu/nu mice a
(20
×
10
6
cells /100
µ
L, pe lank) (mice dono s). The umo al g ow h was moni o ed by
measu ing hei size wice a week un il i eached a olume o 500 mm
3
. A his poin , he
mice we e eu hanized, and he umo s we e excised and cu in o small pieces measu ing
2–3 mm3
in size. Subsequen ly, hese issue agmen s we e used o subcu aneous implan-
a ion in o i e-week-old emale a hymic Swiss nu/nu mice, weighing 18–20 g ( ecipien
hos ), ollowing he p ocedu e desc ibed by Céspedes e al. [26].
A e h ee weeks o umo implan a ion, he subcu aneous 300 mm
3
umo -bea ing
mice we e andomized in o h ee g oups: (1) con ol (N= 5), (2) NPs (n = 5), and
(3) NPs-ce ux (n = 5), and he NPs (200
µ
g/100
µ
L) o ehicle (PBS bu e , 100
µ
L) we e
adminis a ed in agas ically (by ga age) h ee imes pe week o 4 weeks. Mice body
weigh s and umo olumes we e eco ded wice pe week. Tumo s we e measu ed using
a calipe and he olumes we e es ima ed acco ding o he o mula V = (ab
2
)/2, wi h a:
leng h o longes diame e , and b: wid h o sho es diame e . Measu emen began on he
i s day o ea men (day 0) and inished on day 22 (when he i s umo in he con ol
g oup eached 1 cm in diame e ). A his ime, all mice we e eu hanized, and he umo
and o gans we e emo ed o analysis.
The ex i o biodis ibu ion luo escen image analysis was conduc ed by measu ing
he luo escen li e ime signals (FLIs) using an IVIS
®
Spec um (Pe kin Elme , Wal ham,
MA, USA), which we e co ela ed wi h he amoun o dyed NPs accumula ed in each o gan
and umo al issue. The signal ob ained was digi alized and no malized by o gan size and
exp essed as adian e iciency ((p/s/cm2/s )/
µ
W/cm
2
). FLI alues we e calcula ed by
sub ac ing he au o luo escence om he nega i e con ol and we e inally exp essed as a
issue/ umo a io o compa e he biodis ibu ion p o ile be ween he di e en NPs.
Da a we e exp essed as a pe cen age o adian e iciency wice a e NP adminis a ion
(5 and 48 h) in each o gan. The exci a ion-emission il e s used o measu e he FLI o each
NP, and nanoconjuga es we e be ween 710–745 and 780–800, espec i ely.
2.14. Mouse Xenog a Model: In Vi o In agas ic Adminis a ion o Nanopa icles wi h
Len a inib and Nanopa icles wi h Len a inib and Deco a ed wi h Ce uximab
Small pieces o umo s p e iously ob ained om animal dono s we e implan ed in o
mice as desc ibed in Sec ion 2.13. The mice we e andomized and di ided in o h ee
g oups (N = 7 mice pe g oup). When he subcu aneous (SC) umo eached app oxima ely
100–120 mm
3
, and hey ecei ed 200
µ
g in agas ic (i.g.) bolus o NPs (len a-NPs) and
len a-NPs-ce ux in PBS bu e h ee imes/week o e 4 weeks; he con ol mice ecei ed an
i.g. bolus o PBS ehicle, based on he esul s ob ained in p e ious biodis ibu ion assays.
The mice we e eu hanized, and hei umo s and o gans we e collec ed o analysis, as
Biomolecules 2023,13, 1647 7 o 19
shown in he g aphical abs ac igu e. The sample size was calcula ed acco ding o ou
expe ience in simila expe imen s [26].
The ca e and use o all mice in his s udy we e unde p ocedu es app o ed by he
IIB San Pau Animal E hics Commi ee egula ions in acco dance wi h he Fede a ion o
Eu opean Labo a o y Animal Science Associa ions (FELASA; p o ocol numbe 11180).
2.15. His opa hological and His omo phome ic Assessmen and Immunohis ochemis y Analysis
o Tumo Nec osis and P oli e a ion Ra es
The umo and non- umo issues we e o malin- ixed, pa a in-embedded, and cu
in sec ions o 5
µ
m hickness, ollowed by hema oxylin–eosin s aining and moun ed in
Pe moun (Fishe Scien i ic). To quan i y he o al his ological nec osis in he umo s, h ee
a eas we e de ined: (1) o al a ea o he umo , (2) acellula egions conside ed nec o ic issue
(appea ing pale pink), and (3) cellula issue. All o hese egions we e measu ed using
he eehand ool in ImageJ so wa e .1.8.0. The pe cen age o nec osis was calcula ed
by di iding he nec o ic a ea by he o al a ea o he umo and mul iplying i by 100.
Apop osis/ka yolisis and mi o ic coun s in he umo s we e pe o med in andom 10 high
powe ields (HPFs) a ×400 magni ica ion.
All da a we e analyzed wi h ImageJ/Fiji so wa e and exp essed in pe cen ages. The
ascula iza ion s a us/index/ a e ( ascula densi y and ascula leng h densi y) analysis was
pe o med using he ImageJ/Fiji 1.46 p og am, which is capable o au oma ically calcula ing
ascula densi y me ics, whe e ascula densi y = essel a ea/ o al a ea * 100%, and ascula
leng h densi y = skele onized essel a ea/ o al a ea * 100% [
27
,
28
]. Immunohis ochemical
analysis o VGFR2 (an i-VEGF ecep o 2 an ibody ab39638) and Ki-67 An igen (Clone
MIB-1, M7240, DAKO) we e pe o med in a DAKO Au os aine Link48 ollowing he
manu ac u e ’s ins uc ions. All he images we e acqui ed using a mic oscopy Olympus
Bx51 wi h a DP72 digi al came a and p ocessed wi h CellD Imaging 3.3 so wa e (Olympus
Co po a ion, Tokyo, Japan).
2.16. Wes e n Blo ing
Wes e n blo analysis was used o e alua e he p o ein exp ession le els o AIF (sc-
13116, San a C uz Bio echnology, Inc., Heidelbe g, Ge many) and Ci oc om C (EPR1327,
Abcam, Camb idge, UK) in o ho opic hy oid umo issue. The umo issue was dis-
pe sed mechanically wi h RIPA bu e (50 mM T is-HCl, pH 7.5; 150 mM NaCl; 1% NP40;
0.5% sodium deoxychola e; 0.1% SDS; 1 mM EDTA) supplemen ed wi h p o ease inhibi o
cock ail (Roche Diagnos ics, Mina o Ci y, Tokyo, Japan), phenylme hylsul onyl luo ide
(PMSF, Sigma, S . Louis, MO, USA), and sodium o ho anada e (Sigma, S . Louis, MO,
USA). The supe na an was collec ed a e cen i uga ion a 12,000
×
g o 15 min a 4
◦
C
and a BCA p o ein assay eagen ki (The moFishe Scien i ic, Wal ham, MA, USA) was
used o ob ain he p o ein concen a ions. A e wa ds, he p o ein ex ac s we e mixed
wi h a 4
×
Laemmli loading bu e and hea ed a 94
◦
C o 4 min. Then, 20
µ
g o p o ein
was size-sepa a ed on a 10% TGX S ain-F ee p ecas gel (Bio-Rad, He cules, CA, USA),
ans e ed o a 0.2
µ
m PVDF memb ane (Bio-Rad, He cules, CA, USA) and blocked wi h
3% d ied milk in T is-bu e ed saline con aining 0.05% o Tween-20 (TBST bu e ) o 15 min.
Finally, memb anes we e incuba ed wi h he p ima y an ibody (bo h a 1/200 dilu ion)
o e nigh a 4
◦
C. The ea e , he memb anes we e washed h ee imes o 10 min wi h
TBST bu e and e-incuba ed wi h he IgG HRP-conjuga ed seconda y an ibody o 1 h.
Finally, he memb anes we e washed h ee imes o 10 min wi h TBST bu e and analyzed
using Immun-S a Wes e n Chemiluminescence Ki (Bio-Rad, He cules, CA, USA). TGX
S ain- ee gels we e ac i a ed o 1 min a e SDS-elec opho esis. Images we e cap u ed us-
ing a ChemiDoc XRS Gel Documen a ion Sys em (Bio-Rad, He cules, CA, USA) and Image
Lab so wa e ( e sion 6.0.1, Bio-Rad, He cules, CA, USA). Da a no maliza ion analysis o
each p o ein band was pe o med wi h he s ain- ee gel image sa ed, and he backg ound
was adjus ed in such a way ha he o al backg ound was sub ac ed om he sum o he
densi y o all he bands in each lane.
Biomolecules 2023,13, 1647 8 o 19
2.17. S a is ical Analysis
All he
in i o
expe imen s we e independen ly epea ed a leas h ee imes. Da a a e
p esen ed as he mean
±
s anda d e o o he mean. Fo he
in i o
expe imen s, - es s
and Mann–Whi ney U- es s we e used o analyze he di e ences in biodis ibu ion be ween
he NP and nanoconjuga e g oups o each es ed o gan. Di e ences be ween g oups we e
conside ed signi ican a p< 0.05. All s a is ical analyses we e pe o med using P ism 8
(G aphPad So wa e Inc., La Jolla, CA, USA).
3. Resul s
3.1. Physicochemical Cha ac e iza ion and In Vi o Release P o ile o Len a-NPs and
Len a-NPs-Ce uximab
The len a-NPs examined he e we e cha ac e ized in e ms o size, PDI, ze a po-
en ial, and mo phology (Figu e 1). I has been shown ha he inco po a ion o len a-
inib in o PLGA NPs esul ed in an inc ease in pa icle size om 222.9 o 262.4 nm
(
Figu e S1A, ba s
). Mo eo e , he addi ion o len a inib esul ed in a sligh inc ease in
dispe si y alue (Figu e S1B) om 0.12 o 0.19. In compa ison wi h he ze a po en ial o
he blank PLGA nanopa icles (
−
16
±
2.1 mV), a small dec ease in he mean ze a po en ial
o he len a-NPs (
−20.9 ±2.9 mV
) was seen a e he addi ion o len a inib o he PLGA
NPs (
Figu e S1A, do s
). Since he de elopmen o he len a-NPs was success ul in e ms
o pa icle size, PDI, ze a po en ial, mo phology, and encapsula ion e iciency, we chose
o include he monoclonal an ibody ce uximab on he su ace o he NPs o a ge cells
ha o e exp ess he EGFR. To ob ain len a-NPs-ce ux, a me hod called co alen bind-
ing, which was based on EDC/sul o-NHS c oss-linking chemis y, was u ilized o adso b
monoclonal an ibody ce uximab on he su ace o he len a-NPs. This eac ion couples
he p ima y amino g oups wi h ca boxyla es o o m s able amide c osslinks. Su ace
plasmon esonance, low cy ome y, and ELISA can all be used o con i m he p esence
o ce uximab on len a-NPs, al hough each has i s own ad an ages. Unbound ce uximab
in he supe na an was de ec ed ia spec opho ome y using a colo ime ic mic oBCA
p o ein assay ki . Acco ding o he indings o ou s udy, 76% o he ce uximab added was
dis ibu ed ac oss he su ace, which is equi alen o 18.1
µ
g o ce uximab pe mg o len a-
NPs (Figu e 2). The dispe sion alue o he len a-NPs-ce ux (Figu e 1B) was e y simila
o ha ob ained o he len a-NPs. This sugges s ha he addi ion o ce uximab did no
esul in a modi ica ion o he NP s uc u e. When obse ed unde TEM, he len a-NPs and
len a-NPs-ce ux (Figu e 1C,D) displayed a sphe ical shape and a uni o mly smoo h su ace,
indica ing ha he addi ion o ce uximab did no esul in a change in he mo phology o
he NPs. Fu he mo e, he TEM obse a ions ag eed wi h hose ob ained using dynamic
ligh sca e ing. Thus, an NP’s
in i o
and
in i o
pe o mance can be in luenced by i s
su ace shape, which should be smoo h and ee o holes. This also indica es ha he sol en
was success ully e apo a ed. The encapsula ion e iciency o len a inib (79
±
6.4%) was
de e mined wi h he RP-HPLC me hod using supe na an ob ained du ing he p epa a ion
p ocess. Unde ou expe imen al condi ions, he componen s p oduced a e dissol ing
he NPs in o ganic sol en s limi ed he de ec ion o he len a inib con en by HPLC due
o in e e ing peaks. Len a inib encapsula ed in PLGA NPs is mainly a ibu ed o he
pa i ion coe icien and is he e o e e ained in he o ganic phase when he mic osphe es
solidi y; howe e , he encapsula ion also depends on many o he aspec s, such as he NPs’
size, he lipophilici y o he d ug inco po a ed, and he p epa a ion me hod. The
in i o
len a inib eleased om he NPs in i o equa ed o 9% a e 24 h (Figu e 3).
Biomolecules 2023,13, 1647 9 o 19
Biomolecules 2023, 13, x 9 o 20
success ully e apo a ed. The encapsula ion e iciency o len a inib (79 ± 6.4%) was de e -
mined wi h he RP-HPLC me hod using supe na an ob ained du ing he p epa a ion
p ocess. Unde ou expe imen al condi ions, he componen s p oduced a e dissol ing
he NPs in o ganic sol en s limi ed he de ec ion o he len a inib con en by HPLC due
o in e e ing peaks. Len a inib encapsula ed in PLGA NPs is mainly a ibu ed o he
pa i ion coe icien and is he e o e e ained in he o ganic phase when he mic osphe es
solidi y; howe e , he encapsula ion also depends on many o he aspec s, such as he NPs’
size, he lipophilici y o he d ug inco po a ed, and he p epa a ion me hod. The in i o
len a inib eleased om he NPs in i o equa ed o 9% a e 24 h (Figu e 3).
Figu e 1. Physicochemical cha ac e iza ion o len a-NPs and len a-NPs-ce ux. (A) Size (ba s) and
ze a po en ial (do s); (B) Dispe si y index and s anda d de ia ion alues o len a-NPs and len a-
NPs-ce ux. Each alue ep esen s he mean ± s anda d de ia ion o h ee independen measu e-
men s; (C) TEM images o len a-NPs. Scale ba s: 500 nm; (D) TEM images o len a-NPs-ce ux. Scale
ba s: 500 nm. Rep esen a i e RP-HPLC ch oma og ams o len a inib: (F) a calib a o sample spiked
wi h 0.5 μg/mL len a inib; (E) a PLGA-len a-NP sample 4 days a e he s a o he elease s udy.
Figu e 2. De e mina ion o ce uximab con en wi h colo ime ic mic o BCA assay.
Figu e 1.
Physicochemical cha ac e iza ion o len a-NPs and len a-NPs-ce ux. (
A
) Size (ba s) and
ze a po en ial (do s); (
B
) Dispe si y index and s anda d de ia ion alues o len a-NPs and len a-NPs-
ce ux. Each alue ep esen s he mean
±
s anda d de ia ion o h ee independen measu emen s;
(
C
) TEM images o len a-NPs. Scale ba s: 500 nm; (
D
) TEM images o len a-NPs-ce ux. Scale ba s:
500 nm. Rep esen a i e RP-HPLC ch oma og ams o len a inib: (
F
) a calib a o sample spiked wi h
0.5 µg/mL len a inib; (E) a PLGA-len a-NP sample 4 days a e he s a o he elease s udy.
Biomolecules 2023, 13, x 9 o 20
success ully e apo a ed. The encapsula ion e iciency o len a inib (79 ± 6.4%) was de e -
mined wi h he RP-HPLC me hod using supe na an ob ained du ing he p epa a ion
p ocess. Unde ou expe imen al condi ions, he componen s p oduced a e dissol ing
he NPs in o ganic sol en s limi ed he de ec ion o he len a inib con en by HPLC due
o in e e ing peaks. Len a inib encapsula ed in PLGA NPs is mainly a ibu ed o he
pa i ion coe icien and is he e o e e ained in he o ganic phase when he mic osphe es
solidi y; howe e , he encapsula ion also depends on many o he aspec s, such as he NPs’
size, he lipophilici y o he d ug inco po a ed, and he p epa a ion me hod. The in i o
len a inib eleased om he NPs in i o equa ed o 9% a e 24 h (Figu e 3).
Figu e 1. Physicochemical cha ac e iza ion o len a-NPs and len a-NPs-ce ux. (A) Size (ba s) and
ze a po en ial (do s); (B) Dispe si y index and s anda d de ia ion alues o len a-NPs and len a-
NPs-ce ux. Each alue ep esen s he mean ± s anda d de ia ion o h ee independen measu e-
men s; (C) TEM images o len a-NPs. Scale ba s: 500 nm; (D) TEM images o len a-NPs-ce ux. Scale
ba s: 500 nm. Rep esen a i e RP-HPLC ch oma og ams o len a inib: (F) a calib a o sample spiked
wi h 0.5 μg/mL len a inib; (E) a PLGA-len a-NP sample 4 days a e he s a o he elease s udy.
Figu e 2. De e mina ion o ce uximab con en wi h colo ime ic mic o BCA assay.
Figu e 2. De e mina ion o ce uximab con en wi h colo ime ic mic o BCA assay.
Biomolecules 2023,13, 1647 16 o 19
in less nega i ely cha ged sys ems. Wi h espec o he
in i o
elease p o ile o len a-NPs,
he e ec de ec ed could be a ibu ed o he di usion o he medica ion ha had been ad-
so bed o weakly a ached o he NPs. In con as , he amoun o d ug inco po a ed in o he
NPs de e mines he a e o p og essi e elease—which was no ed o e 20 days in his s udy.
The low solubili y o len a inib could be esponsible o he slow elease a e obse ed.
An inc eased elease o len a inib was no iced o e he nex 10 days, which was likely
caused by he deg ada ion o he PLGA ma ix h oughou he la e phases. Consequen ly,
he deg ada ion a e and elease p o ile we e ound o be a ec ed by he con en o he
polyme ma ix. By he end o he expe imen , which las ed 38 days, he NPs had eleased
abou 15% o he loaded len a inib—p obably due o he limi ed solubili y o len a inib on
he elease medium. These esul s a e compa able wi h he da a showing he e icacy o
len a inib-elu ing mic osphe es in p eclinical s udies o hepa ocellula ca cinoma [33].
Mo eo e , he cumula i e elease sus ained o e ime de ec ed in len a-NPs could be
impo an o clinical use because i could pa ially a oid he side e ec s associa ed wi h
high concen a ions o he d ug. In epi helial umo al cells, EGFR is equen ly mu a ed
and/o o e exp essed. This e en has been used as a he apeu ic s a egy using speci ic
an ibodies ha block he ligands by binding o he ex acellula domain o his ecep o ,
and, as a consequence, induce a cellula signaling cascade a es . Mo eo e , he umo al
a ge ing and he apeu ic e iciency o an i-EGFR-NPs o deli e ing an icance d ugs ha e
been in es iga ed
in i o
and in p eclinical s udies, wi h good esul s [
34
–
37
]. The esul s
sugges ha o umo s ha o e exp ess speci ic ecep o s, such as EGFR as demons a ed
in glioblas oma, he an ibody-conjuga e in NPs can be ad an ageous in d ug deli e y and
can imp o e he an ip oli e a i e e icacy in he umo cells [
34
].The lack o signi ican
di e ences in he
in i o
cy o oxic e ec o bo h o mula ions, as shown in Figu e 5, can
be a ibu ed o he ela i ely sho du a ion o he assay, which was limi ed o 24 h. This
obse a ion is consis en wi h he esul s o
in i o
d ug elease expe imen s, which in-
dica e ha len a inib is g adually eleased o e a 10-day pe iod due o he p og essi e
deg ada ion o he PLGA ma ix o e ime. I is wo h no ing ha he e a e cu en ly no
a ailable da a ega ding he encapsula ion o his d ug wi hin PGLA nanopa icles. Ne -
e heless, he biodis ibu ion analysis o he nanopa icles e ealed ha he Len a-ce ux
NPs we e mo e e icien ly aken up by umo s 5 h a e adminis a ion compa ed wi h
he Len a-NPs. We mus emphasize ha , despi e he lack o knowledge ega ding he
abso p ion e iciency ia in agas ic adminis a ion o PLGA nanopa icles used in he
in i o
expe imen s, he esul s a e signi ican . This is because he nanopa icles we e able
o main ain s abili y wi hin he s omach and exhibi ed he capabili y o c oss he in es inal
ba ie , po en ially achie ing hei he apeu ic goals [
38
,
39
]. The his ological analysis (apop-
osis/ka yolysis) and he e alua ion o mi osis in he issue samples indica e ha he use o
ce uximab-deco a ed nanopa icles could o e ad an ages compa ed wi h non-deco a ed
nanopa icles. Ne e heless, some epo s demons a e ha he use o ull-size an ibodies
as a ligand does no imp o e hei he apeu ic e ec , owing o hei high immunogenici y
o he in insic umo al esis ance o EGFR inhibi o s, p oposing he deco a ion o NPs
wi h an EGFR-ap ame [
40
,
41
]. Len a inib a ec s he inhibi ion o VEGFRs, p omo ing
hei an i- umo ac i i y by educing he densi y o mic o essels and inc easing umo
nec osis [
42
]. The analysis o neo ascula iza ion ( ascula densi y and ascula leng h den-
si y) in he umo s o he animals ea ed wi h NPs wi h and wi hou ce uximab- a ge ing
showed a signi ican educ ion in hese wo pa ame e s in compa ison wi h he con ols.
These esul s indica e a high deg ee o ischemia in he umo s om animals ea ed wi h
bo h NP o mula ions, al hough he e we e no signi ican di e ences be ween hem. Mo e-
o e , len a inib is an inhibi o o mul iple ecep o y osine kinases capable o inducing
ea ly p ocesses o apop osis and nec osis in Huh-7 cells [
43
]. Fu he mo e, i has been
desc ibed ha PLGA NPs loaded wi h emozolomide conjuga ed wi h ce uximab, designed
o a ge cance s ha o e exp ess EGFR, we e able o p omo e la e p ocesses o apop osis
and nec osis compa ed wi h PLGA NPs loaded wi h emozolomide [
44
] Al hough ou
Ki67 da a did no e eal signi ican di e ences be ween bo h o mula ions in he issue
Biomolecules 2023,13, 1647 17 o 19
a eas a om he nec o ic a eas o he umo s, we mus highligh ha in he analysis o
cells wi h ypical mo phologies o nuclea apop osis (condensa ion and agmen a ion), as
well as he mo phology o ka yolysis, we demons a ed ha he umo al issues ob ained
om animals ea ed wi h deco a ed nanopa icles showed a g ea e pe cen age o hese
cell ypes compa ed wi h hose ea ed wi h undeco a ed nanopa icles. The key ini ial
s ep o he apop osis p ocess is closely associa ed wi h mi ochond ial dys unc ion and he
apid elease o wo p oapop o ic p o eins, alongside he elease o cy och ome c (cy c)
and apop osis-inducing ac o (AIF). Al hough hese wo p o eins ope a e di e en ly in
he apop o ic pa hway (caspase-dependen and caspase-independen , espec i ely), hei
elease om he mi ochond ia igge s ch oma in condensa ion, DNA agmen a ion, and
dea h in cells [
45
,
46
]. In his con ex , ou da a ega ding he mo phology o apop o ic cells
and ka yolysis, in ela ion o he esul s o cy och ome c and AIF p o ein exp ession, indi-
ca e he capabili y o ac i a e bo h mechanisms in umo s ea ed wi h bo h o mula ions.
Howe e , he dis inc pa e n obse ed be ween hem may sugges ha deco a ed NPs
ha e a mo e apid abili y o induce a g ea e le el o cell inju y, wi h a loss o p oapop o ic
p o ein exp ession, e en hough a pe cen age o he umo al issue s ill emains iable.
In summa y, his p oo -o -concep s udy demons a ed ha bo h o mula ions can
main ain hei he apeu ic e ec a e in agas ic adminis a ion, indica ing s abili y in
he diges i e sys em and he abili y o c oss he in es inal ba ie . Howe e , ce uximab-
deco a ed NPs show po en ial ad an ages in up ake capaci y
in i o
, and g ea e abili y o
ini ia e apop osis /nec osis, sugges ing hei g ea e e ec i eness. Fu he in es iga ions
will be necessa y o ully unde s and he speci ic apop osis mechanisms hey p omo e.
Finally, he good ole ance obse ed in animals wi h hese ea men s sugges s ha
hese deli e y sys ems may o e ad an ages o e con en ional me hods and open he doo
o explo ing o he TKI d ugs.
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/biom13111647/s1, Figu e S1: Physicochemical cha ac e iza ion o
blank-NPs and len a-NPs; Figu e S2: O iginal wes e n blo s o Figu e 10.
Au ho Con ibu ions:
Concep ualiza ion, E.M., J.L.P., N.F.V., M.V.C. and G.P.; me hodology, G.R., P.C.,
F.R., C.L., V.P.-E., M.S.-R., T.L.-M., I.G. and N.A.Q.; o mal analysis, G.R. and N.A.Q.;
w i ing—o iginal
d a p epa a ion, G.R., N.A.Q., M.S.-R., T.L.-M. and I.G.; w i ing— e iew and edi ing, A.M., J.I.P.,
J.C.E.-G., R.C., M.V.C. and M.E.; supe ision, J.L.P., M.V.C., G.P. and E.M.; p ojec adminis a ion,
E.M., G.P. and M.V.C.; unding acquisi ion, E.M. All au ho s ha e ead and ag eed o he published
e sion o he manusc ip .
Funding:
This esea ch was unded by he Spanish “Minis e io de Ciencia, Inno ación y Uni e si-
dades” and “Ins i u o de Salud Ca los III” h ough p ojec s FIS PI19/00136 ( o J.C.E.–G. and E.M.)
and PI20/00770 ( o MVC). Addi ionally, G.R. was g an ed a PhD ellowship co- inanced by “Ins i u o
Salud Ca los III, Mad id, Spain” (PFIS) and Fondo Social Eu opeo (FSE), g an FI19/00007. P.C.
ecei ed a PFIS p edoc o al con ac (FI21/00146), and M.V.C is suppo ed by he Miguel Se e
II (CPII20/00007) P og am, bo h om he ISCIII wi h co- unding om he Eu opean Social Fund
(ESF In es ing in You Fu u e), Spain. MVC is also pa o he RICORS Ne wo k, Ins i u o Salud
Ca los III, Mad id, Spain. ME is suppo ed by a p e-doc o al INPhINIT ellowship om La Caixa
(2022, B005830).
Ins i u ional Re iew Boa d S a emen :
The animal s udy p o ocol was app o ed by he IIB San
Pau Animal E hics Commi ee egula ions in acco dance wi h he Fede a ion o Eu opean Labo a o y
Animal Science Associa ion (FELASA; p o ocol numbe 11180).
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : Da a is con ained wi hin he a icle o Supplemen a y Ma e ials.
Biomolecules 2023,13, 1647 18 o 19
Acknowledgmen s:
The au ho s wish o hank ICTS “NANBIOSIS”, speci ically he D ug Fo mu-
la ion Uni (U10) o he CIBER in Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN),
o he in ellec ual and echnical assis ance; SGIke (UPV/EHU) o echnical and human suppo ;
and he Depa men o Educa ion, Uni e si y and Resea ch o he Basque Coun y Go e nmen
(Consolida ed G oups, IT1448-22).
Con lic s o In e es :
The au ho s decla e no con lic o in e es . The unde s had no ole in he design
o he s udy; in he collec ion, analyses, o in e p e a ion o da a; in he w i ing o he manusc ip ; o
in he decision o publish he esul s.
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Disclaime /Publishe ’s No e:
The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual
au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o
people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .
