Lenvatinib-Loaded Poly(lactic-co-glycolic acid) Nanoparticles with Epidermal Growth Factor Receptor Antibody Conjugation as a Preclinical Approach to Therapeutically Improve Thyroid Cancer with Aggressive Behavior

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. Re e ences 1. Wo den, F. T ea men s a egies o adioac i e iodine- e ac o y di e en ia ed hy oid cance . The . Ad . Med. Oncol. 2014 ,6, 267–279. [C ossRe ] [PubMed] 2. Aashiq, M.; Sil e man, D.A.; Na’a a, S.; Takahashi, H.; Ami , M. Radioiodine-Re ac o y Thy oid Cance : Molecula Basis o Redi e en ia ion The apies, Managemen , and No el The apies. Cance s 2019,11, 1382. [C ossRe ] [PubMed] 3. Nixon, I.J.; Whi che , M.M.; Palme , F.L.; Tu le, R.M.; Shaha, A.R.; Shah, J.P.; Ganly, I. The Impac o Dis an Me as ases a P esen a ion on P ognosis in Pa ien s wi h Di e en ia ed Ca cinoma o he Thy oid Gland. Thy oid 2012 ,22, 884–889. [C ossRe ] 4. Lo usso, L.; Pie uzzi, L.; Biagini, A.; Sabini, E.; Vale io, L.; Giani, C.; Elisei, R. Len a inib and o he y osine kinase inhibi o s o he ea men o adioiodine e ac o y, ad anced, and p og essi e hy oid cance . OncoTa ge s The . 2016 ,9, 6467–6477. [C ossRe ] [PubMed] 5. Loca i, L.D.; Pio esan, A.; Du an e, C.; B egni, M.; Cas agna, M.G.; Zo a o, S.; Elisei, R. Real-wo ld e icacy and sa e y o len a inib: Da a om a compassiona e use in he ea men o adioac i e iodine- e ac o y di e en ia ed hy oid cance pa ien s in I aly. Eu . J. Cance 2019,118, 35–40. [C ossRe ] [PubMed] 6. Fe a i, S.M.; Elia, G.; Ragusa, F.; Ru illi, I.; La Mo a, C.; Papa o, S.R.; An onelli, A. No el ea men s o anaplas ic hy oid ca cinoma. Gland Su g. 2020,9, S28–S42. [C ossRe ] 7. Elia, G.; Pa izio, A.; Ragusa, F.; Papa o, S.R.; Mazzi, V.; Bales i, E.; Fe a i, S.M. Molecula ea u es o agg essi e hy oid cance . F on . Oncol. 2022,12, 1099280. [C ossRe ] 8. Killock, D. SELECT—Len a inib in hy oid cance . Na . Re . Clin. Oncol. 2015,12, 189. [C ossRe ] 9. Schlumbe ge , M.; Taha a, M.; Wi h, L.J.; Robinson, B.; B ose, M.S.; Elisei, R.; She man, S.I. Len a inib e sus Placebo in Radioiodine-Re ac o y Thy oid Cance . New Engl. J. Med. 2015,372, 621–630. [C ossRe ] 10. Iwasaki, H.; Toda, S.; Suganuma, N.; Mu ayama, D.; Nakayama, H.; Masudo, K. Len a inib s. pallia i e he apy o s age IVC anaplas ic hy oid cance . Mol. Clin. Oncol. 2020,12, 138–143. [C ossRe ] [PubMed] 11. Iwasaki, H.; Yamazaki, H.; Takasaki, H.; Suganuma, N.; Nakayama, H.; Toda, S.; Masudo, K. Len a inib as a no el ea men o anaplas ic hy oid cance : A e ospec i e s udy. Oncol. Le . 2018,16, 7271–7277. [C ossRe ] 12. Yan, Z.; Yang, M.; Lai, C. Clinical e icacy o len a inib o he ea men o adioiodine- e ac o y hy oid ca cinoma: A sys ema ic e iew and me a-analysis o clinical ials. Clin. Endoc inol. 2021,95, 478–488. [C ossRe ] 13. Cabanillas, M.E.; Takahashi, S. Managing he ad e se e en s associa ed wi h len a inib he apy in adioiodine- e ac o y di e en ia ed hy oid cance . Semin. Oncol. 2019,46, 57–64. [C ossRe ] [PubMed] 14. Zheng, Y.; Huang, C.; Lu, L.; Yu, K.; Zhao, J.; Chen, M.; Zhang, J. STOML2 po en ia es me as asis o hepa ocellula ca cinoma by p omo ing PINK1-media ed mi ophagy and egula es sensi i i y o len a inib. J. Hema ol. Oncol. 2021 ,14, 16. [C ossRe ] [PubMed] 15. Zhu, C.; Ma, X.; Hu, Y.; Guo, L.; Chen, B.; Shen, K.; Xiao, Y. Sa e y and e icacy p o ile o len a inib in cance he apy: A sys ema ic e iew and me a-analysis. Onco a ge 2016,7, 44545–44557. [C ossRe ] [PubMed] 16. Cascinu, S. Len a inib and pemb olizumab in ad anced gas ic cance . Lance Oncol. 2020,21, 1004–1005. [C ossRe ] 17. Sonpa de, G.; Bellmun , J.; Schu z, F.; Chouei i, T.K. The Double Edged Swo d o Bleeding and Clo ing om VEGF Inhibi ion in Renal Cance Pa ien s. Cu . Oncol. Rep. 2012,14, 295–306. [C ossRe ] 18. Simões-Pe ei a, J.; Capi ão, R.; Limbe , E.; Lei e, V. Anaplas ic Thy oid Cance : Clinical Pic u e o he Las Two Decades a a Single Oncology Re e al Cen e and No el The apeu ic Op ions. Cance s 2019,11, 1188. [C ossRe ] 19. Jin, J.; Wu, X.; Yin, J.; Li, M.; Shen, J.; Li, J.; Qu, L. Iden i ica ion o Gene ic Mu a ions in Cance : Challenge and Oppo uni y in he New E a o Ta ge ed The apy. F on . Oncol. 2019,9, 263. [C ossRe ] 20. Ma, Y.; Peng, X.; Wang, L.; Li, H.; Cheng, W.; Zheng, X.; Liu, Y. Ce uximab-conjuga ed pe luo ohexane/gold nanopa icles o low in ensi y ocused ul asound diagnosis abla ion o hy oid cance ea men . Sci. Technol. Ad . Ma e . 2020 ,21, 856–866. [C ossRe ] [PubMed] 21. Rez an alab, S.; D ude, N.I.; Mo a eji, M.K.; Gü ene , N.; Koons, E.K.; Shi, Y.; Kiessling, F. PLGA-Based Nanopa icles in Cance T ea men . F on . Pha macol. 2018,9, 1260. [C ossRe ] [PubMed] 22. Fishe , K.E.; Jani, J.C.; Fishe , S.B.; Foulks, C.; Hill, C.E.; Webe , C.J.; Sha ma, J. Epide mal g ow h ac o ecep o o e exp ession is a ma ke o ad e se pa hologic ea u es in papilla y hy oid ca cinoma. J. Su g. Res. 2013 ,185, 217–224. [C ossRe ] [PubMed] 23. Pé ez-He e o, E.; Fe nández-Meda de, A. Ad anced a ge ed he apies in cance : D ug nanoca ie s, he u u e o chemo he apy. Eu . J. Pha m. Biopha m. 2015,93, 52–79. [C ossRe ] [PubMed] Biomolecules 2023,13, 1647 19 o 19 24. Cabanillas, M.E.; Hab a, M.A. Len a inib: Role in hy oid cance and o he solid umo s. Cance T ea . Re . 2016 ,42, 47–55. [C ossRe ] [PubMed] 25. Pu as, G.M.E. Selec i e An i umo al E ec o So a enib Loaded PLGA Nanopa icles Conjuga ed wi h Ce uximab on Undi e en- ia ed/Anaplas ic Thy oid Ca cinoma Cells. J. Nanomed. Nano echnol. 2015,6, 3. [C ossRe ] 26. Céspedes, M.V.; Guillén, M.J.; López-Casas, P.P.; Sa no, F.; Galla do, A.; Álamo, P.; Mangues, R. Lu binec edin induces deple ion o umo -associa ed mac ophages (TAM), an essen ial componen o i s in i o syne gism wi h gemci abine. Dis. Models Mech. 2016,9, 1461–1471. [C ossRe ] [PubMed] 27. Schneide , C.A.; Rasband, W.S.; Elicei i, K.W. NIH Image o ImageJ: 25 yea s o image analysis. Na . Me hods 2012 ,9, 671–675. [C ossRe ] 28. Teplyi, V.; G ebchenko, K. E alua ion o he sca s’ ascula iza ion using compu e p ocessing o he digi al images. Ski. Res. Technol. 2019,25, 194–199. [C ossRe ] 29. Fe a i, S.M.; La Mo a, C.; Elia, G.; Ragusa, F.; Ru illi, I.; Qua ini, L.; An onelli, A. An ineoplas ic E ec o Len a inib and Vande anib in P ima y Anaplas ic Thy oid Cance Cells Ob ained om Biopsy o Fine Needle Aspi a ion. F on . Endoc inol. 2018 , 9, 764. [C ossRe ] 30. Tohyama, O.; Ma sui, J.; Kodama, K.; Ha a-Sugi, N.; Kimu a, T.; Okamo o, K.; Funahashi, Y. An i umo Ac i i y o Len a inib (E7080): An Angiogenesis Inhibi o Tha Ta ge s Mul iple Recep o Ty osine Kinases in P eclinical Human Thy oid Cance Models. J. Thy oid. Res. 2014,2014, 638747. [C ossRe ] 31. Ye, Z.; Zhang, Y.; Liu, Y.; Liu, Y.; Tu, J.; Shen, Y. EGFR Ta ge ed Ce uximab-Valine-Ci ulline ( c)-Doxo ubicin Immunoconjuga es- Loaded Bo ine Se um Albumin (BSA) Nanopa icles o Colo ec al Tumo The apy. In . J. Nanomed. 2021 ,16, 2443–2459. [C ossRe ] [PubMed] 32. Wu, P.; Zhou, Q.; Zhu, H.; Zhuang, Y.; Bao, J. Enhanced an i umo e icacy in colon cance using EGF unc ionalized PLGA nanopa icles loaded wi h 5-Fluo ou acil and pe luo oca bon. BMC Cance 2020,20, 354. [C ossRe ] [PubMed] 33. Pe, J.; Choi, B.; Choi, H.; Kwon, S.W.; Kim, D.H. P eclinical The apeu ic E alua ion o Len a inib-Elu ing Mic osphe es o T ansca he e A e ial Chemoemboliza ion o Hepa ocellula Ca cinoma. Ca dio asc. In e . Radiol. 2022,45, 1834–1841. [C ossRe ] [PubMed] 34. Kuo, Y.C.; Liang, C.T. Inhibi ion o human b ain malignan glioblas oma cells using ca mus ine-loaded ca anionic solid lipid nanopa icles wi h su ace an i-epi helial g ow h ac o ecep o . Bioma e ials 2011,32, 3340–3350. [C ossRe ] [PubMed] 35. Liao, C.; Sun, Q.; Liang, B.; Shen, J.; Shuai, X. Ta ge ing EGFR-o e exp essing umo cells using Ce uximab-immunomicelles loaded wi h doxo ubicin and supe pa amagne ic i on oxide. Eu . J. Radiol. 2011,80, 699–705. [C ossRe ] [PubMed] 36. Vokes, E.E.; Chu, E. An i-EGFR he apies: Clinical expe ience in colo ec al, lung, and head and neck cance s. Oncology 2006 ,20 (Suppl. S2), 15–25. 37. Egnuni, T.; Ing am, N.; Mi za, I.; Cole a, P.L.; McLaughlan, J.R. E alua ion o he Ta ge ing and The apeu ic E iciency o An i-EGFR Func ionalised Nanopa icles in Head and Neck Cance Cells o Use in NIR-II Op ical Window. Pha maceu ics 2021 , 13, 1651. [C ossRe ] [PubMed] 38. Da e, A.A.; Hanes, J.; Ensign, L.M. Nanopa icles o o al deli e y: Design, e alua ion and s a e-o - he-a . J. Con ol. Release 2016 , 240, 504–526. [C ossRe ] 39. Mo elli, L.; Gimondi, S.; Se ie i, M.; Sal ioni, L.; Guizze i, M.; Colzani, B.; Bigini, P. Moni o ing he Fa e o O ally Adminis e ed PLGA Nano o mula ion o Local Deli e y o The apeu ic D ugs. Pha maceu ics 2019,11, 658. [C ossRe ] 40. Johns on, M.C.; Sco , C.J. An ibody conjuga ed nanopa icles as a no el o m o an ibody d ug conjuga e chemo he apy. D ug Disco . Today Technol. 2018,30, 63–69. [C ossRe ] 41. Agnello, L.; To o ella, S.; d’A genio, A.; Ca bone, C.; Camo ani, S.; Loca elli, E.; Ce chia, L. Op imizing cispla in deli e y o iple-nega i e b eas cance h ough no el EGFR ap ame -conjuga ed polyme ic nano ec o s. J. Exp. Clin. Cance Res. 2021 ,40, 239. [C ossRe ] [PubMed] 42. Ma suki, M.; Hoshi, T.; Yamamo o, Y.; Ikemo i-Kawada, M.; Minoshima, Y.; Funahashi, Y.; Ma sui, J. Len a inib inhibi s angiogenesis and umo ib oblas g ow h ac o signaling pa hways in human hepa ocellula ca cinoma models. Cance Med. 2018,7, 2641–2653. [C ossRe ] [PubMed] 43. Ren, X.; Zhang, Q.; Guo, W.; Wang, L.; Wu, T.; Zhang, W.; Kong, D. Cell-cycle and apop osis ela ed and p o eomics-based signaling pa hways o human hepa oma Huh-7 cells ea ed by h ee cu en ly used mul i-RTK inhibi o s. F on . Pha macol. 2022 , 13, 944893. [C ossRe ] [PubMed] 44. Duwa, R.; Bans ola, A.; Emami, F.; Jeong, J.H.; Lee, S.; Yook, S. Ce uximab conjuga ed emozolomide-loaded poly (lac ic-co-glycolic acid) nanopa icles o a ge ed nanomedicine in EGFR o e exp essing cance cells. J. D ug Deli . Sci. Technol. 2020 ,60, 101928. [C ossRe ] 45. Gue a-Cas ellano, A.; Má quez, I.; Pé ez-Mejías, G.; Díaz-Quin ana, A.; De La Rosa, M.A.; Díaz-Mo eno, I. Pos -T ansla ional Modi ica ions o Cy och ome c in Cell Li e and Disease. In . J. Mol. Sci. 2020,21, 8483. [C ossRe ] 46. A us, C.; Bouj ad, H.; Bouha ou , A.; B unelle, M.N.; Hoos, S.; Yus e, V.J.; Susin, S.A. AIF p omo es ch oma inolysis and caspase-independen p og ammed nec osis by in e ac ing wi h his one H2AX. EMBO J. 2010 ,29, 1585–1599. [C ossRe ] [PubMed] 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 .