Bioinspi ed o hogonal-shaped p o ein–biome al nanoc ys als enable o al
p o ein abso p ion
Ma ilde Du ´
an-Loba o
a,b,c,d
, Sulay To a
a,b,e
, Juan Cu˜
na o
a,b
, Rocío Ramos-Memb i e
,g
,
I ´
an Pe˜
nuelas
,g
, Ila ia Ma igo
h,i
, Fede ico Bene i
j
, Miguel Chenlo
k
, Cla a V. ´
Al a ez
k
,
Vashegyi Ildik´
o
l
, Rudol U banics
l,m
, J´
anos Szebeni
n,o,p
, Ma ía Jos´
e Alonso
a,b,c,*
a
Cen e o Resea ch in Molecula Medicine & Ch onic Diseases (CIMUS), Uni e si y o San iago de Compos ela, Campus Vida, 15782 San iago de Compos ela, Spain
b
Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS), Uni e si y o San iago de Compos ela, Campus Vida, 15782 San iago de Compos ela, Spain
c
Depa men o Pha macology, Pha maceu ics and Pha maceu ical Technology, School o Pha macy, Uni e si y o San iago de Compos ela, 15782 San iago de
Compos ela, Spain
d
Depa amen o de Fa macia y Tecnología Fa mac´
eu ica, Facul ad de Fa macia, Uni e sidad de Se illa, P o . Ga cía Gonz´
alez, 2, 41012 Se ille, Spain
e
CIBER Fisiopa ología de la Obesidad y Nu ici´
on (CIBERobn), Mad id, Spain
Radiopha macy Uni , Depa men o Nuclea Medicine, Clínica Uni e sidad de Na a a, Ins i u o de In es igaci´
on Sani a ia de Na a a (IdiSNA), A . Pío XII 36,
31008 Pamplona, Spain
g
T ansla ional Molecula Imaging Uni , Depa men o Nuclea Medicine, Clínica Uni e sidad de Na a a, Ins i u o de In es igaci´
on Sani a ia de Na a a (IdiSNA), A .
Pío XII 36, 31008 Pamplona, Spain
h
Depa men o Su ge y, Oncology and Gas oen e ology, Uni e si y o Pado a, Pado a, I aly
i
Vene o Ins i u e o Oncology IOV-IRCCS, Pado a, I aly
j
ECSIN-Eu opean Cen e o he Sus ainable Impac o Nano echnology, ECAMRICERT SRL, Pado a, I aly
k
Neoplasia & Endoc ine Di e en ia ion P0L5, Cen e o esea ch in Molecula Medicine and Ch onic Disease (CIMUS), A Ba celona s/n, 15782 San iago de
Compos ela, Spain
l
Se oScience L d, Budapes , Hunga y
m
Nanomedicine Resea ch and Educa ion Cen e , Depa men o Pa hophysiology, Semmelweis Uni e si y, Budapes , Hunga y
n
Nanomedicine Resea ch and Educa ion Cen e , Depa men o T ansla ional Medicine, Semmelweis Uni e si y, Budapes 1089, Hunga y
o
Depa men o Nanobio echnology and Regene a i e Medicine, Facul y o Heal h Sciences, Miskolc Uni e si y, Miskolc 2880, Hunga y
p
School o Chemical Enginee ing and T ansla ional Nanobioscience Resea ch Cen e , Sungkyunkwan Uni e si y, Suwon 16419, Republic o Ko ea
ARTICLE INFO
Keywo ds:
Pep ide deli e y
P o ein deli e y
O al deli e y
T ansmucosal
Biologicals
Nanocomplexes
ABSTRACT
Wi h he g owing numbe o ma ke ed biological d ugs, he de elopmen o echnological s a egies o hei o al
sys emic abso p ion, becomes inc easingly impo an . The ha sh gas oin es inal en i onmen and low pe me-
abili y o he in es inal epi helium, ep esen a huge challenge o hei sys emic deli e y. He ein, bioinspi ed in
he physiological insulin-Zn in e ac ion, he design o o hogonal-shaped p o ein-biome al hyb id nanoc ys als,
u he en eloped by a bilaye o unc ional bioma e ials, is epo ed. The nanoc ys als exhibi ed a size o 80 nm,
a neu al su ace cha ge and a high insulin loading. In i o s udies showed he capaci y o he nanocomplexes o
con ol he elease o he associa ed insulin, while p ese ing i s s abili y. In i o e alua ion showed sus ained
blood glucose educ ions in bo h heal hy and diabe ic a s (up o 40 % and 80 %, espec i ely), while ch onic
immuno oxici y s udies in mice indica ed no oxici y e ec . P elimina y e icacy s udies in heal hy awake pigs
ollowing o al capsule adminis a ion showed o e 20 % absolu e bioa ailabili y.
1. In oduc ion
P o ein/pep ide he apeu ics ha e a p ominen ole in cu en and
u u e heal hca e ma ke scena ios, mainly because hei
mac omolecula s uc u es make hem mo e speci ic and po en han
small molecules [1]. Howe e , hei s uc u al complexi y makes hem
ulne able in physiological en i onmen s and ine icien a o e coming
biological ba ie s o sys emic deli e y [1,2]. As a esul , pep ide
* Co esponding au ho a : Cen e o Resea ch in Molecula Medicine & Ch onic Diseases (CIMUS), Uni e si y o San iago de Compos ela, Campus Vida, 15782
San iago de Compos ela, Spain.
E-mail add ess: [email p o ec ed] (M.J. Alonso).
Con en s lis s a ailable a ScienceDi ec
Jou nal o Con olled Release
jou nal homepage: www.else ie .com/loca e/jcon el
h ps://doi.o g/10.1016/j.jcon el.2024.11.016
Recei ed 30 Ma ch 2024; Recei ed in e ised o m 13 Oc obe 2024; Accep ed 7 No embe 2024
Jou nal o Con olled Release 377 (2025) 17–36
A ailable online 17 No embe 2024
0168-3659/© 2024 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (
h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/ ).
he apeu ics a e cu en ly adminis e ed mainly ia injec ion [3]. which
limi s hei use in ch onic ea men s.
The o al ou e is by a he mos use ul and accep able modali y o
d ug adminis a ion [4]. Consequen ly, signi ican e o s ha e been
de o ed o o e coming each o he majo ba ie s iden i ied o o al
p o ein deli e y, namely, he ha sh gas oin es inal en i onmen , he
mucus laye and he unde lying epi helial ba ie [5]. The s a egies
de eloped hus a include he use o p o ease inhibi o s and/o
pe mea ion enhance s, he chemical modi ica ion o pep ides o uning
hei physicochemical p o iles [6], and a wide ange o polyme ic-, lipid-
and complex-based nanoca ie s [3,7]. Fu he mo e, echnologies p e-
iously applied in o he ields ha e been explo ed o o al- ou e appli-
ca ions, such as ionic liquids [8] and ion opho esis [9], while complex
mic oneedle capsule designs ha e eme ged [10–14]. Ne e heless,
success has been elusi e hus a ; he e has been ex ensi e p eclinical
esea ch bu cu en ly e y ew echnologies in ea ly clinical e alua ion
[5,7], and only wo o mula ions, based on pe mea ion enhance s, ha e
been ecen ly app o ed o comme cializa ion [15,16].
The ul ima e emaining obs acle is low o al bioa ailabili y along
wi h high in a- and in e subjec esponse a iabili y [7]. On he o he
hand, ansla ional aspec s, such as bioma e ial quali y and sa e y,
obus design pe o mance, con e sion in o a inal solid dosage o m,
p oduc ion echnology scalabili y and cos /bene i balance equen ly
emain unde a ed, hampe ing clinical ansla ion [7,17].
In o de o o e come bo h biological and echnological ba ie s, his
wo k ocused on designing a new gene a ion o o al p o ein nano-
medicines inspi ed by physiological phenomena. Speci ically, ou
inspi a ion de i ed om he obse a ion o na u al insulin-Zn com-
plexes in he panc ea ic s o age deposi s [18]. This ex ensi ely s udied
p o ein–biome al in e ac ion [19,20] has been widely exploi ed o
p olonging insulin elease ollowing pa en e al adminis a ion, leading
o he co-addi ion o Zn sal s o encapsula e insulin in mic o- and
nanoca ie s [21–24], whe e an excess o Zn dec eased he solubili y o
insulin [24]. Howe e , he po en ial o such an in e ac ion has ne e
been exploi ed as a nanoca ie in i sel , and mo e speci ically o he
o ma ion o ul asmall (below 100 nm), o hogonally shaped insulin-Zn
nanoc ys als, and hei use o o al insulin deli e y. Hence, he ein, we
epo a po en ial nanomedicine candida e consis ing o unable insulin-
Zn o hogonal nanocomplexes endowed wi h he capaci y o o e come
he biological ba ie s associa ed wi h he o al ou e. To enhance hei
s abili y and p omo e in e ac ion wi h en e ocy es, he nanoc ys als
we e coa ed wi h a bilaye o amphiphilic ma e ials, speci ically lau oyl
a gina e es e (LAE) and polye hylene glycol s ea a e (PEGs ). This
bilaye was designed o mimic he phospholipid a chi ec u e o a cell
memb ane while exposing a neu al hyd ophilic su ace.
The nanoca ie s we e e alua ed in e ms o hei key physico-
chemical p ope ies and colloidal s abili y, con olled elease and p o-
ec ion agains enzyma ic deg ada ion in biologically ele an media.
Addi ionally, he eeze-d ied o m o he nanoca ie s was e alua ed o
assess he p ese a ion o nanoca ie p ope ies and he bioac i i y o
he p ocessed p o ein. Fu he mo e, in i o e icacy s udies we e con-
duc ed in bo h heal hy and diabe ic a models and a 28-day ch onic
immuno oxici y s udy in mice was pe o med. In a inal s ep, an en e ic
capsule dosage o m con aining he eeze-d ied powde o he o mu-
la ion was adminis e ed o heal hy awake pigs and he insulin
bioa ailabili y was de e mined.
2. Resul s and discussion
2.1. Ra ional design and p oduc ion o en eloped insulin-Zn
nanocomplexes
The design was based on he hypo hesis ha he new nano-
o mula ion would p o ec insulin om in es inal enzymes, na iga e
h ough he mucus laye and acili a e he in e ac ion wi h he unde -
lying epi helium. The key quali y a ibu es o a a ge ed insulin
nanoca ie include a ep oduceable non-sphe ical nanoscale size, high
p o ein associa ion and loading, sus ained elease, colloidal s abili y in
in es inal luids, mucodi usi e cha ac e and he abili y o p omo e he
in e ac ion wi h he epi helium. Addi ionally, he echnological equi-
si es include bioma e ial sa e y, p oduc ion scalabili y and ep oduc-
ibili y, and he gene a ion o a inal solid dosage o m sui able o o al
adminis a ion.
Gi en he equi ed insulin dose and he dilu ion in he o ally
adminis e ed d ied o m, signi ican a en ion was ocused on achie ing
a high insulin loading (quan i y o insulin/quan i y o nanoca ie ). To
his aim, ou bioinspi a ion elied on he insulin-Zn complex na u ally
occu ing in he panc eas and in ol ed in physiological insulin me a-
bolism [25]. This in e ac ion has se ed as he basis o some sus ained-
elease o mula ions al eady on he ma ke [21,23,26], and Zn sal s and
insulin mix u es ha e been widely employed as encapsula ed ma e ials
in mic o- and nanoca ie s [21–23] o p o ein agg ega ion, hus a-
o ing insulin encapsula ion and delaying i s elease. Howe e , he
echnological app oach adop ed he e is adically di e en . Indeed, he
aim was o design and de elop hyb id o hogonal-shaped insulin-Zn
nanoc ys als s abilized by a bilaye o unc ional bioma e ials wi h
pene a ion enhancing p ope ies. Such con igu a ion was achie ed in a
i s s age by adop ing a bo om-up me hodology, ha allowed o p o-
duce s uc u ed insulin-Zn nanocomplexes wi h unable physicochem-
ical p ope ies con olled by selec ed p ocess condi ions. We iden i ied
he ollowing pa ame e s as c i ical o achie ing op imal nanosys em
p ope ies: i) he op imal insulin:Zn mola a io was 1:6, ensu ing he
comple e hexame ic complexa ion o he p o ein and maximized asso-
cia ion (Fig. 1A); ii) he op imal incuba ion media pH was 5.1, esul ing
in a low pa icle size (<100 nm) and polydispe sion index (PDI) o <0.3
(Fig. 1B, C, Table S1), which was a ibu ed o op imized p o ein ioni-
za ion and subsequen in e ac ion wi h Zn ca ions; and iii) s abiliza ion
o he complexa ion s ep a 4 ◦C, leading o a educed pa icle size and
mo e homogenous sys em c ys alliza ion (Table S2). The nano-
complexes p esen ed an ~80 nm size, a 0.3 PDI and a nega i e ze a
po en ial (ZP) (Fig. 1E). Thei small size was expec ed o a o in es inal
cell in e ac ions and mucodi usion [27], and he nega i ely cha ged
su ace was a ibu ed o nega i ely cha ged insulin esidues a he inal
pH o 6.8 o he nanocomplex suspension media [28]. This is consis en
wi h he hexame ic s uc u e o he insulin-Zn complex, whe e Zn ions
a e ound in an inne axial posi ion coo dina ed wi h his idine side
chains [29]. A ema kable associa ion e iciency o close o ~100 % wi h
a inal loading o o e 90 % was ob ained (Fig. 1E). Such excep ional
d ug loading is c ucial o achie ing he equi ed o al dose a e inal
packaging in o o al solid dosage o ms.
T ansmission elec on mic oscopy (TEM) imaging (Fig. 1F) in e -
es ingly e ealed a popula ion o non-sphe ical polygonal nano-
s uc u es wi h a signi ican ly smalle size o app oxima ely 50 nm and
lowe polydispe sion compa ed o dynamic ligh sca e ing (DLS) anal-
ysis. The o hogonal shape o he s uc u es was a ibu ed o he c ys al
na u e o he pu e insulin-Zn complex [30]. This shape was conside ed
o be he cause o he o e es ima ion o he pa icle size and poly-
dispe si y alues ob ained by DLS analysis, as his echnique is adap ed
o sphe ical pa icles [31]. Nanopa icle geome y has been ecen ly
iden i ied as a pa ame e wi h subs an ial impac on o al up ake and
anspo [32], in luencing he e en ion ime in he gas oin es inal
ac [33], mucus pe mea ion, cellula up ake, in acellula p ocessing
and ansmemb ane anspo [33–35]. O e all, he imp o ed pe o -
mance o non-sphe ical pa icles has been a ibu ed o hei la ge
con ac su ace a ea [32], and modi ied B ownian mo emen , o a ion
a ound he mucus ne wo ks and shea low [34,36].
2.2. Su ace en elopmen o he nanocomplexes wi h a cosu ac an
bilaye
Nex , he nanocomplexes we e en eloped by a hyd ophilic neu al
su ac an bilaye o p o ide colloidal s abili y, imp o ed mucodi usion
M. Du ´
an-Loba o e al.
Jou nal o Con olled Release 377 (2025) 17–36
18
Fig. 1. Bo om-up p oduc ion o en eloped nanocomplexes. A, pH alues adjus ed du ing he p oduc ion p ocess and esul ing physicochemical pa ame e s o he
o mula ions as a unc ion o he employed insulin:Zn a io. Se e al a ios [37] we e assayed o ensu e maximized p o ein hexame ic complexa ion, and a a io o 1:6
was inally selec ed as op imal (o e 90 % associa ion). B, pH alues in he p oduc ion p ocess, pa icle size and PDI measu ed by DLS o sc eened o mula ions as a
unc ion o he employed acid-base olume a ios (NaOH 0.1 ɴ: HCl 0.01 ɴ). C, G aphic ep esen a ion o he pa icle size and PDI o he o mula ions as a unc ion o
pH a he incuba ion s ep. A pH alue o 5.1 (NaOH:HCl olume a io 0.08) was selec ed o u he de elopmen , since i allowed us o main ain a small pa icle size
(<100 nm) along wi h an accep able PDI alue (0.3). D, ZP and PDI alues o he nanocomplexes upon addi ion o inc easing concen a ions o LAE-PEGs coa ing,
leading o neu al ZP alues and inc eased PDI. E, Physicochemical pa ame e s o o mula ions be o e and a e coa ing and upon inc easing ba ch size (10- and 100-
old) a e adap ing he p oduc ion p ocess, alida ing he scaling-up ial. F, TEM images o plain nanocomplexes. G, TEM images o en eloped nanocomplexes. All
measu emen s and e o ba s a e displayed as he mean alues ±SDs (n =3).
M. Du ´
an-Loba o e al.
Jou nal o Con olled Release 377 (2025) 17–36
19
[38], p o ec ion agains enzyma ic deg ada ion [39] and enhanced
epi helial pene a ion [40]. Fo his pu pose, a combina ion o neu al
and posi i ely cha ged amphiphilic molecules was selec ed, speci ically
PEGs , a widely employed FDA-app o ed excipien , and LAE, an
a ginine-based biologically de i ed gene ally- ega ded-as-sa e (GRAS)
excipien [41]. Bo h molecules we e expec ed o ac syne gis ically
based on hei epo ed capaci y o in e ac wi h cell memb anes and
al e hei pe meabili y [42]. B ie ly, PEGs and LAE we e sequen ially
added o a nanocomplex suspension a an 8:1 PEGs :LAE mass a io,
since a highe ela i e amoun o LAE compa ed o PEGs (lowe PEGs :
LAE mass a io) led o nanocomplex agg ega ion. By inc easing he
amoun o bo h PEGs and LAE added o he o mula ion, while main-
aining he op imized PEGs :LAE mass a io, he ZP o he o mula ion
inc eased o neu al alues (Fig. 1D) while he mean pa icle size was
main ained, hus indica ing he su ac an in e ac ion wi h he nano-
sys em su ace. In e es ingly, he PDI alues inc eased, pa icula ly
when he ZP alues a ained neu ali y (Fig. 1D), due o an excess o
su ac an molecules o ming micelles. TEM imaging con i med ha he
nanocomplex size and mo phology emained unal e ed a e he en el-
oping s ep (Fig. 1G). A concen a ion o 0.8 mg/mL LAE–6.4 mg/mL
PEGs in he inal nanocomplex suspension was selec ed on accoun o
he esul ing neu al ZP, indica ing he comple e co e age o he
nanocomplex su ace. The esul ing o mula ion was success ully eeze-
d ied (Fig. S1 and S2).
2.3. A chi ec u al o ganiza ion o he o hogonal c ys alline
nanocomplexes en eloped by a su ac an bilaye
F eeze-d ied samples o he en eloped nanocomplexes we e
analyzed by X- ay di ac ion (XRD), whe e he widening o he highes
in ensi y peaks in a di ac og am is ela ed o he size o he c ys alline
domains. O e all, wide peaks o bands a ibu able o nanoc ys alline
domains we e iden i ied o bo h ehalose and PVP-manni ol o mula-
ion powde s (Fig. 2B, C), while con ol samples p esen ed ei he no
peaks o na ow peaks indica i e o nonnanoc ys alline domains, al o-
ge he sugges ing he p esence o nanoc ys alli es in he o mula ion.
The cosu ac an en elope a chi ec u e, ini ially unde s ood as
depic ed in Fig. 3A based on expe imen al esul s, was u he in es i-
ga ed. Fo ins ance, when only LAE was added o he nanoc ys al sus-
pension, he nanopa icles immedia ely agg ega ed due o he
in e ac ion o posi i ely cha ged LAE molecules wi h he nega i ely
cha ged nanocomplexes. On he o he hand, when only PEGs was added
o he nanocomplexes, no change in he physicochemical p ope ies o
he nanocomplexes was obse ed. Howe e , when PEGs and LAE we e
Fig. 2. Cha ac e iza ion o he inne c ys alline nanocomplex s uc u e. X- ay analysis was pe o med o assess whe he he nanocomplexes p esen ed he known
c ys alline s uc u e [30] o he insulin-Zn hexame ic complex. A, TEM imaging o nanocomplexes showing inne s uc u al pa e ns. B, X- ay di ac og am o
nanocomplexes eeze-d ied wi h PVP-manni ol (pu ple line) and con ol sample o he eeze-d ied solu ion o PVP-manni ol and zinc (g een line) a he same
concen a ions as in he o mula ions. The di ac og am o he nanocomplexes (pu ple line) p esen ed se e al na ow peaks coinciden wi h hose o he zinc con ol
sample (g een line), which indica ed he nonnanoc ys alline domains a ibu ed o manni ol. Howe e , he o mula ion powde also p esen ed wide peaks
a ibu able o nanoc ys alline domains, which we e no p esen in he zinc con ol sample di ac og am, whe e highe na owe peaks appea ed ins ead (14 o
18◦2Th and 23 o 24◦2Th). C, X- ay di ac og am o nanocomplexes eeze-d ied wi h ehalose (pu ple) and con ol samples o eeze-d ied ehalose and zinc
(g een line). The absence o c ys alline phases o he con ol ehalose‑zinc sample (g een line) allowed mo e accu a e iden i ica ion o c ys alline domains o he
eeze-d ied o mula ion (pu ple line). The di ac og am o nanocomplexes eeze-d ied wi h ehalose (pu ple line) p esen ed se e al wide peaks o bands indica i e
o nanoc ys alline domains, a ibu ed o he nanocomplex co e s uc u e. (Fo in e p e a ion o he e e ences o colou in his igu e legend, he eade is e e ed o
he web e sion o his a icle.)
M. Du ´
an-Loba o e al.
Jou nal o Con olled Release 377 (2025) 17–36
20
adequa ely combined (8:1 mass a io) and added, LAE molecules sup-
posedly in e ac ed wi h he nanocomplexes, while hei agg ega ion was
p e en ed by he subsequen a achmen o PEGs molecules. In ac , he
o e all neu al ZP o he esul an en eloped nanocomplexes sugges ed
ha he hyd ophilic PEG po ions o PEGs we e o ien ed owa d he
ou e media. The in e ac ion o bo h su ac an s would hypo he ically
occu h ough hei hyd oca bon chains, cons i u ing a su ac an
bilaye (Fig. 3A).
To con i m his hypo hesis,
1
H NMR analysis (Fig. 3B, S4 and S5) and
wa e -ligand obse ed ia g adien spec oscopy (wa e LOGSY)
expe imen s (Fig. 3B and S6) we e ca ied ou on he en eloped nano-
complexes, whe e bulk sol en (H
2
O) p o ons we e selec i ely sa u a ed,
and his magne iza ion was ans e ed ia c oss- elaxa ion o ee
molecules in con ac wi h wa e media. As a esul , he esonances o
molecula egions in e ac ing wi h wa e appea ed wi h opposi e signs
o hose in e ac ing wi hin a s uc u e, such as a nanopa icle [43]. The
esul ing spec um (Fig. 3B) clea ly showed a 3.71 ppm in e ed peak
co esponding o he PEG egions o PEGs , con i ming hei ex e nal
loca ion on he nanocomplex su ace in con ac wi h he aqueous me-
dium and wi h highe mobili y compa ed o he s ea a e egion. On he
Fig. 3. Cha ac e iza ion o he su ac an bilaye en elope. A, Schema ic pic u e showing he hypo hesized con o ma ion o he bilaye o amphiphilic bioma e ials
based on expe imen al esul s. B,
1
H NMR and wa e LOGSY (wa e -ligand obse ed ia g adien spec oscopy) analysis o he en eloped nanocomplexes. De ailed
1
H
NMR signal assignmen and di usion-o de ed spec oscopy (DOSY) expe imen s dis ega ding he in luence o su ac an molecules no a ached o he nanocomplex
su ace a e a ailable in he SI sec ion. The wa e LOGSY spec um clea ly showed a 3.71 ppm in e ed peak co esponding o PEG egions, while he signal co -
esponding o bulk alipha ic chains om bo h su ac an s (1.27 ppm) emained nonin e ed. The obse ed changes in he signals we e p opo ional o he leng h o
he applied pulse ( om 3 o 1 ms, SI sec ion), hus con i ming ha hey we e due o he wa e LOGSY e ec . This obse a ion indica ed he ex e nal loca ion o he
PEG egions and in e nal loca ion o he alipha ic chains, suppo ing he hypo hesized en elope bilaye a chi ec u e.
M. Du ´
an-Loba o e al.
Jou nal o Con olled Release 377 (2025) 17–36
21
o he hand, he signal co esponding o bulk alipha ic chains om bo h
PEGs and LAE (1.27 ppm) emained nonin e ed, indica ing hei in-
e nal loca ion wi hin he s uc u e. Al oge he , hese esul s suppo ed
he hypo hesized en elope bilaye a chi ec u e, whe e PEG egions
emain o ien ed owa d he ex e nal aqueous media, while alipha ic
chains om bo h su ac an s a e loca ed inside he nanos uc u e.
2.4. In i o pe o mance o en eloped nanocomplexes and hei po en ial
o scalabili y and ansla ion
The high ionic o ce and complex composi ion o in es inal luids
ep esen one o he ba ie s o o al deli e y, whe e adequa ely uning
he nanopa icle su ace may d as ically de e mine he capaci y o he
ca ie s o main ain hei physicochemical p ope ies unde physiolog-
ical condi ions [44,45]. The e alua ion o ou nanocomplexes in bio-
ele an luids e ealed ha hei en eloping shell p o ided adequa e
colloidal s abili y [44] (Fig. 4A), while p o ec ion agains enzyma ic
deg ada ion (Fig. 4B) and sus ained elease (Fig. 5C) we e a ibu ed o
Zn because o i s enzyma ic inhibi o y [46] and insulin complexa ion
[24,47] p ope ies, espec i ely. Addi ionally, he ba ch size was
inc eased by 100- old (Fig. 1E), indica ing he success ul iden i ica ion
and con ol o key p ocess con ol pa ame e s. No ably, scaling-up po-
en ial cons i u es a highly impo an added alue ha cu en
nano echnology-based d ug deli e y app oaches a ely ha e [48,49].
In addi ion, gi en he pa icula i ies o he insulin-Zn in e ac ion, he
possibili y o ansla ing his echnology o o he biomac omolecules
was unce ain. Thus, we explo ed he nanocomplexa ion o an RNA
model molecule wi h biome als by adap ing he p oduc ion p ocedu e
and he biomolecule–biome al mass a ios. Nanocomplexes wi h unable
su ace cha ges coa ed wi h se e al polyme s we e ob ained (Table S5,
S6 and S7), o e ing possibili ies o he u he modula ion o nano-
ca ie p ope ies. While he p elimina y cha ac e o hese s udies is
no ed, he esul s sugges ha p oducing coa ed nanocomplexes o
biome als and biomac omolecules is easible, which opens up new a -
enues o o mula ion de elopmen based on his s a egy. Pa icula ly,
explo ing combina ions o biomac omolecules and biome als be ween
which speci ic physiological in e ac ions a e known o ake place could
be an app oach o in e es .
Fig. 4. In i o physicochemical cha ac e iza ion o he coa ed nanocomplexes. A, Colloidal s abili y o en eloped nanocomplexes as a unc ion o a e age pa icle
size and coun a e measu ed by DLS (mean alue ±S.D. (n =3)). The o mula ion p o ed o be s able o up o 4 h, as opposed o he noncoa ed nanocomplexes,
which immedia ely agg ega ed (da a no shown). B, P o ec ion o insulin agains panc ea ic deg ada ion as a unc ion o emaining nondeg aded insulin. Bo h coa ed
and noncoa ed o mula ions displayed simila p o iles, inc easing insulin
1/2
by 4- old wi h espec o ha o ee insulin (mean alues ±SDs (n =3)). C, Insulin
elease om coa ed nanocomplexes. A sus ained elease o e he incuba ion ime (up o 4 h) was displayed (mean alues ±SDs (n =3)). D, Insulin bioac i i y
e alua ion in pSynSRE-T-luc- ans ec ed cells as a unc ion o luci e ase ac i i y o e a ans ec ion con ol p omo e (be a-galac osidase). F esh solu ions o ee
insulin o inc eased concen a ions as a posi i e con ol induced dose– esponse luci e ase ac i i y in pSynSRE-T-luc- ans ec ed cells, while no s a is ically signi ican
e ec was obse ed in pSynSRE-Mu -T-luc- ans ec ed cells, bea ing ou -poin mu a ions in he insulin- esponsi e elemen , he e o e alida ing he assay. Recon-
s i u ed eeze-d ied coa ed nanocomplexes a insulin concen a ions equi alen o hose o he posi i e con ol esul ed in he speci ic ac i a ion o he insulin
ecep o , shown as del a luci e ase, compa ed o he con ol. The luci e ase ac i i y alues ob ained we e in he same ange as hose ob ained wi h esh insulin
solu ions, con i ming he p ese ed bioac i i y o insulin a e o mula ion p ocessing and eeze-d ying. Cell s udy esul s a e p esen ed as he means ±SDs, n =5
independen expe imen s wi h 6–8 eplica es pe condi ion in each (one-way ANOVA ollowed by Tukey’s mul iple compa ison es ; signi icance le els compa ed o
he con ol *p ≤0.05; ***p ≤0.001, ****p ≤0.001).
M. Du ´
an-Loba o e al.
Jou nal o Con olled Release 377 (2025) 17–36
22
An ul ima e cons ain speci ic o o al adminis a ion a ises om he
necessi y o gene a e a solid dosage o m as he end p oduc . This usu-
ally equi es he eeze-d ying o o mula ions p oduced in aqueous-
based media, which may comp omise he p ese a ion o he ini ial
physicochemical p ope ies o nanopa icles as well as he bioac i i y o
he loaded d ug [17,50]. To assess whe he he lyophilized nano-
complexes main ained hei key p ope ies, samples om eeze-d ied,
small and la ge ba ches we e e alua ed in i o, showing p ese ed in-
sulin bioac i i y on human INSR in human HepG2 hepa ocy es [51,52]
(Fig. 4D) and physicochemical p ope ies (Fig. S2) a e 2 mon hs o
s o age.
Nex , he eeze-d ied nanocomplexes we e loaded in o gela in cap-
sules along wi h a composi ion in ended o gene a e ca bon dioxide
(CO
2
) gas bubbles ha would p omo e a as esuspension o he nano-
complexes and hei p ojec ion owa d he in es inal wall, comp ising
a a ic acid (TA), sodium bica bona e (SBC) and Poloxame 199 (P188).
This acid-base eac ion has been employed in a simila app oach using
ci ic acid and SBC o p omo e able disin eg a ion [53,54]. I was also
employed o p omo e he in si u assembling and/o s abiliza ion o
nanos uc u es [55–57], whe e in es inal pe mea ion was ul ima ely
dependen upon he ac ion o pe mea ion enhance s such as ime hyl
chi osan (TMC) [54] o sodium dodecyl sulpha e (SDS) [55–57] o he
pe o mance o nanoca ie s [53] in his composi ion, TA and SBC we e
selec ed o gene a e he CO
2
bubbles, P188 was employed as su ac an
o dec ease su ace ension and he e o e inc ease he in ensi y o he
eac ion e ec [58], and in es inal pe mea ion was expec ed o be
a ained on accoun o he a ionally designed nanocomplexes. The
capsules we e u he coa ed wi h an en e ic polyme o p e en gas ic
deg ada ion, and he in eg i y o he coa ing and he igge ing o he gas
p oducing eac ion a e coa ing dissolu ion we e assessed in simula ed
gas ic and in es inal luids espec i ely. In he la e , he acid-base e-
ac ion was obse ed o ake place wi hin 2 min upon coa ing dissolu ion,
wi h an in ense gas p oduc ion (Fig. S7). The capsules we e s o ed a RT
p o ec ed om humidi y up o 1.5 mon hs be o e adminis a ion o pigs,
when hei con en homogenei y and d ug bioac i i y was p e iously
e alua ed by s.c. adminis a ion o heal hy a s.
2.5. In i o in e ac ion o en eloped nanocomplexes wi h he in es inal
wall
The nanoca ie s we e adiolabeled wi h echne ium-99 m (
99m
Tc)
ia linkage o he PEG chains on he nanoca ie en elope [59], ollowed
by o al adminis a ion o a s and subsequen single pho on emission
compu ed omog aphy-compu ed omog aphy (SPECT-CT) e alua ion
[60]. P io o he s udy, he s abili y and lack o elease o he adio-
iso ope in simula ed gas ic luid (SGF) we e assayed (Fig. S8 and S9).
The esul s (Fig. 5) showed ha , while he ee echne ium con ol
(sodium pe echne a e solu ion) emained localized in he s omach, he
Fig. 5. Biodis ibu ion o echne ium-99 m adiolabeled nanoca ie s in a s. A, % Radioac i i y de ec ed pe o gan a e o al adminis a ion o he adiolabeled
o mula ion. B, % Radioac i i y de ec ed pe o gan a e o al adminis a ion o [
99m
Tc]‑sodium pe echne a e (con ol). C, SPECT-CT images o he a s a e o al
adminis a ion o he adiolabeled o mula ion and ee
99m
Tc con ol; s =s omach; I =in es ine; c =cecum; cl =colon; = ec um. The adiolabeled ca ie s
emained mos ly in he small bowel (up o 2 h) and in he cecum (4 h, 8 h and 11 h) o he du a ion o he s udy, whe eas he ee echne ium con ol emained in he
s omach. The esul s a e p esen ed as he mean alues o n =4.
M. Du ´
an-Loba o e al.
Jou nal o Con olled Release 377 (2025) 17–36
23
adiolabeled ca ie s emained mos ly in he small bowel (up o 2 h) and
cecum (4 h, 8 h and 11 h) o he du a ion o he s udy. This p olonged
in es inal ansi p o ile is in ag eemen wi h simila expe imen s ca ied
ou wi h nanopa icula e o mula ions [59,61] and is suppo ed by he
lowe anspo eloci y in dis al segmen s o he gas oin es inal (GI)
ac [62] and he ac ha iso lu ane anes hesia is known o dec ease GI
mo ili y and p olong ansi imes due o muscle elaxa ion [63].
O e all, he
99m
Tc-labeled o mula ion signi ican ly in e ac ed wi h he
in es ine, ha ing been e ained o up o 26 h, in con as o he ech-
ne ium solu ion con ol, which emained mos ly in he s omach. This
enhanced in es inal in e ac ion o he o mula ion could be a ibu ed o
he nanoca ie ea u es, namely, i) hei small size o a o ing cellula
in e ac ions and mucodi usion [32]; ii) a PEGyla ed su ace o acili-
a ing mucodi usion [38]; and iii) he p esence o LAE, a su ac an wi h
a shown capaci y o dis up cellula memb anes [42], hypo he ically
po en ia ed by PEGs . Fo ins ance, PEG- a y acid es e s ha e shown
enhanced skin d ug pene a ion [64], while o he nonionic poly-
oxye hyla ed su ac an s ha e been widely s udied as opical ocula
pene a ion enhance s [65]. In e es ingly, no sys emic abso p ion o
99m
Tc was obse ed. This lack o abso p ion o he nanosys em was
expec ed based on p e ious li e a u e pe aining o nanopa icle ans-
po ac oss he in es inal wall. The key issue was whe he he enhanced
in e ac ion o he nanosys em wi h he in es inal wall may be ansla ed
in o g ea e insulin abso p ion. The absence o he sys emic abso p ion
o he nanosys em i sel may be conside ed a posi i e ea u e in e ms o
pha maceu ical pu poses, since i would seem o imply educed oxi-
cological conce ns [61].
2.6. Pha macological pe o mance o he en eloped nanocomplexes
The en eloped nanocomplex e icacy was i s e alua ed ollowing
subcu aneous (s.c.) adminis a ion o heal hy a s [44] (Fig. 6A).
Fig. 6. In i o e icacy e alua ion in small and la ge animal models. E icacy esul s a e displayed as % blood glucose alues wi h ega d o he baseline alues a
ime 0 h. Plasma insulin esul s a e displayed as plasma insulin concen a ions (ppb (ng/L)). A, Blood glucose a e s.c. adminis a ion o eshly p epa ed and eeze-
d ied econs i u ed o mula ion, s. insulin con ol o heal hy a s. B, Blood glucose a e s.c. adminis a ion o insulin ex ac ed om en e ic capsule powde a e 2
mon hs o s o age s. insulin con ol o heal hy a s. C, Blood glucose p o ile a e IJ adminis a ion o he o mula ion (n =5) s. an insulin solu ion (nega i e
con ol) (n =6) o heal hy a s. D, Blood glucose p o ile a e IJ adminis a ion o he o mula ion (n =9) s. an insulin solu ion (nega i e con ol) (n =8) o diabe ic
a s. E, Blood glucose p o ile a e o al adminis a ion o en e ic capsule o mula ion s. o al adminis a ion o PBS (nega i e con ol) and SC adminis a ion o an
insulin solu ion (posi i e con ol) (n =3) o awake domes ic pigs. Oscilla ions in le els om 6 h onwa d we e due o i. . adminis a ions o glucose in esponse o
hypoglycemic shock signs. F, Plasma insulin analysis by LC–MS esul ing om he adminis a ion o he en e ic capsule s. nega i e and posi i e con ol o awake
domes ic pigs. No plasma samples we e wi hd awn a e 6 h due o conce n abou he heal hy s a e o he animals. All esul s a e displayed as he mean alues ±
SEMs. A wo-way ANOVA ollowed by a Holm–Sidak mul iple compa ison es was applied; signi icance le els compa ed o he con ol *p ≤0.05; **p ≤0.01; ***p ≤
0.001, ****p ≤0.001).
M. Du ´
an-Loba o e al.
Jou nal o Con olled Release 377 (2025) 17–36
24
Subsequen ly, he o mula ion was assayed o i s e icacy in i o by
in es inal di ec injec ion [44,66–69], which a oids he a iabili y
associa ed wi h gas ic ansi imes, bo h in heal hy and diabe ic a s
[69]. The heal hy a model main ains he physiological insulin au o -
egula ion mechanisms [70] and a oids he high a iabili y associa ed
wi h STZ diaba iza ion [68,71]. As a coun e pa , only modes esponses
a e o be expec ed [44,68], and as ing pe iods need o be dec eased
[69]. Hence, he in ajejunal (IJ) adminis a ion o he coa ed nano-
complexes o heal hy a s (Fig. 6C) yielded an expec ed modes , bu
signi ican , 42 % dec ease in he ini ial blood glucose le els. Impo -
an ly, he e ec was main ained o up o 6 h. Simila esponses we e
epo ed o o he deli e y ca ie s [68,72,73]. This inding con i med
ha he insulin associa ed wi h he nanoca ie was abso bed in a su -
icien amoun o exe a p olonged blood glucose educ ion esponse
[68,70,74]. Hypo he ically, his could be due o he enhanced nano-
s uc u e in e ac ion wi h and pene a ion in o he in es inal epi helium,
as no ed o he adiolabeled o mula ion.
On he o he hand, β-cell de iciency in he diabe ic model allows low
amoun s o abso bed insulin o elici a ma kedly dec eased glucose
esponse [68], and hence, po en ial e ec s o an o al o mula ion would
ha e a highe chance o being iden i ied. Following di ec IJ injec ion o
he o mula ions, a ema kable blood glucose dec ease o up o 80 % was
ob ained (Fig. 6D), wi h clea di e ences ega ding he nega i e con ol
e en a he end o he measu emen pe iod (8 h). This high and p o-
longed esponse was compa able o o highe han hose epo ed in
e e ences in he ield [75–77]. As expec ed, he esul s showed a clea e
e ec in compa ison wi h hose o he heal hy a model, u he con-
i ming insulin in es inal abso p ion. Along wi h biodis ibu ion s udies,
he esul s indica ed ha he nanocomplexes enabled insulin abso p ion
wi hou nanoca ie ansloca ion.
2.7. P elimina y assessmen o he lack o ch onic immuno oxici y in mice
Ch onic exposu e o insulin ea men equi es he conside a ion o
Fig. 7. P elimina y ch onic immuno oxici y e alua ion o he o mula ion in mice. The esul s om FACS analysis o he immune in lamma o y s a us o lymphoid
o gans a e 28 days o ea men . A, Pe cen age o myeloid cells in spleens ollowing ch onic adminis a ion o o mula ion s. insulin o up o 28 days. Di e en
myeloid subpopula ions (in lamma o y monocy ic MDSCs (MO-MDSCs), g anulocy ic-MDSCs (PMN-MDSCs), mac ophages and dend i ic cells (DCs)) we e e alua ed
o each ea men g oup. B, E alua ion o DC ma u a ion a e o mula ion s. insulin adminis a ion in e ms o pe cen age. C, E alua ion o DC ma u a ion in e ms
o luo escence in ensi y o each ea men g oup. D, Pe cen age o lymphocy es a e ch onic adminis a ion o o mula ion s. insulin; pe cen age o T lymphocy es
CD3 o al, CD4 o CD8. The T-cell pe cen ages om each ea men g oup we e simila . E, Immunohis ochemis y o in es inal egions a e o mula ion adminis-
a ion wi h CD3 and CD68. In he Fig., colocaliza ion wi h DAPI is epo ed (magni ica ion 20×wi h d y objec i e). No inc eases in lymphocy e (CD3) o
mac ophage (CD68) signals we e obse ed a he le el o he small in es ine a e ch onic adminis a ion o insulin (da a no shown) o he nanoca ie s. Ba g aphs
ep esen he means ±SDs; n =5 mice pe g oup. *p ≤0.05; **p ≤0.01; ***p ≤0.001, by one-way ANOVA.
M. Du ´
an-Loba o e al.
Jou nal o Con olled Release 377 (2025) 17–36
25
s.c. adminis a ion o a s along wi h ee insulin o compa ison [44].
To his aim, male Sp ague Dawley a s (250 g) we e as ed o 4 h be o e
he s udy wi h ee access o wa e . A solu ion o ee insulin, eshly
p epa ed o mula ion and p e iously eeze-d ied, econs i u ed
o mula ion we e adminis e ed a a dose o 1 IU/kg. Be o e adminis-
a ion (0 h), blood samples we e wi hd awn om he ail ein, and
ini ial blood glucose le els we e measu ed wi h a hand-held glucome e
(Glucoca d™ G +me e , A k ay Fac o y, Japan) and moni o ed e e y
hou he ea e .
4.2.15. In i o e icacy s udies in pigs
In i o e icacy s udies in pigs we e conduc ed on 12- o 14-week-old
neu e ed male domes ic pigs (Sus sc o a domes icus) (15–20 kg) om he
Resea ch Ins i u e o Animal B eeding and Nu i ion, He ceghalom,
Hunga y. They we e housed in indi idual caging (1 m ×2.5 m), ed a
s anda d powde die o domes ic pigs wice a day wi h access o wa e
ad libi um, and allowed o acclima ize o 1 week p io o he s udy. The
s udies we e designed in acco dance wi h accep ed pha macological
p inciples o mee he equi emen s o he p inciples o Hunga ian Ac
1998: XXVIII egula ing animal p o ec ion (la es modi ied by Ac 2011
CLVIII) and Go e nmen Dec ee 40/2013 on animal expe imen s; EEC
Di ec i e 2004/27/EC o he Eu opean Pa liamen and o he Council o
Ma ch 31, 2004 amending Di ec i e 2001/83/EC on he Communi y
code ela ing o medical p oduc s o human use (O icial Jou nal L-136,
30/04/2004, pp. 34-57). Animal handling and ca e we e conduc ed
acco ding o he Guide o he Ca e and Use o Labo a o y Animals, NRC,
2011 and Di ec i e 2010/63/EU (Eu opean Pa liamen and Council,
ook ull e ec on 1 Janua y 2013). Special pe mission o animal
s udies unde numbe PEI/001/3948-6/2014 was issued by he Pes
Coun y Go e nmen O ice o Food Sa e y and Animal Heal h Di ec-
o a e. All p ocedu es ca ied ou on animals we e app o ed by he local
e hical commi ee o Semmelweis Uni e si y.
4.2.15.1. Su gical p ocedu e. On he day o ins umen a ion, animals
we e p eanes he ized in amuscula ly wi h Calypsol/Dexdomi o (2–3/
0.2-0.3 mL, based on body weigh ) injec ions in he s alls o a oid s ess
and we e anspo ed o he ope a ing oom seda ed. The anes hesia was
main ained using iso lu ane inhala ion na cosis (2–3 %) wi h oxygen
h ough a muzzle mask. Animals we e allowed o b ea he spon ane-
ously. Thei espi a ion was moni o ed using a pulse-oxime e ( ixed on
he ail), measu ing he blood oxygen sa u a ion (SpO2), and hei
empe a u e was measu ed ec ally (bo h pa ame e s we e moni o ed by
InnoCa e-T Anes hesia Moni o - Innomed Medical Co. Hunga y). A
capnog aph was connec ed o he muzzle mask o moni o e CO2 (% o
end- idal CO2) and he espi a o y a e (CAP10 Medlab, Medlab Medi-
zinische Diagnosege a e GmbH, Ka ls uhe, Ge many) du ing he
ope a ion.
The pigs we e ins umen ed wi h a cen al enous single-lumen
ca he e se (BB aun Ce o ix Mono V330, 5 F , 30 cm, 16G, Ca .
4,160,290 N) in oduced in o he ena ca a c anialis h ough he ena
maxilla is ex e na
➔
ena jugula is ex e na. The ca he e s’ ails wi h
sampling po s we e led ou subcu aneously h ough he back, ollowed
by ixa ion a he exi si es wi h su u es. All incision a eas we e sha ed
and disin ec ed by libe al applica ion o po idone‑iodine 10 % p io o
he ope a ion and closed a e he ixa ion o cannulas. The cannulas
we e washed daily wi h hepa in-saline du ing he ecupe a ion pe iod.
Following he ope a ion, he pigs we e anspo ed back o he s alls and
obse ed un il spon aneous awakening. P e en i e an ibio ic ea men
(Sy aquinol a a dose o 2.5 mg/kg body weigh ) and analgesia ( heu-
mocam a a dose o 0.4 mg/kg body weigh ) we e applied.
4.2.15.2. Fo mula ion adminis a ion and sampling. P io o o mula ion
adminis a ion, he pigs we e as ed o e nigh p io o pe os adminis-
a ion o he en e ic capsules wi h a plunge applica o . Be o e and
ollowing adminis a ion, p e- (0’, con ol) and pos ea men whole
blood samples we e collec ed a p ede e mined in e als o blood cell
and blood glucose analysis, and K3EDTA plasma samples we e collec ed
o insulin analysis. Con ulsions and hypoglycemic shock signs 6 h a e
adminis a ion in esponse o he unexpec edly in ense e ec o he
o mula ions we e ea ed by i. . injec ions o 10 % w/ glucose. The
animals we e u he moni o ed un il hei glycemic p o iles s abilized.
K3EDTA samples we e cen i uged immedia ely a e collec ion
(1500g, 10 min a 4 ◦C), aliquo ed, ozen a −20 ◦C and subsequen ly
ans e ed o −80 ◦C o s o age un il analysis. Whole blood samples
we e analyzed immedia ely by a Dia on Ve hema ology analyze
(Dia on MI PLC, Budapes , Hunga y) o he ollowing pa ame e s:
whi e blood cells (WBC), ed blood cells (RBC), hemoglobin (Hgb),
pla ele s (PLT), lymphocy es (Ly) and g anulocy es (G ). A e comple-
ion o he s udy, he pigs we e eu hanized by i. . adminis a ion o
Eu hasol (1 mL/10 kg body weigh ) ollowed by i. . injec ion o cc. KCl
solu ion (15 mL/10 kg body weigh ). Eu hanasia was con i med be o e
disposing he ca cass by obse ing ha he e was no espi a o y
mo emen o a leas 3 min and ha he hea bea had ceased.
4.2.15.3. Pha macokine ic e alua ion by LC–MS. The insulin con en in
he plasma samples ob ained om he pig s udies was analyzed by liquid
ch oma og aphy/ andem mass spec ome y (LC/MS) ollowing a sam-
ple ex ac ion and analysis me hodology desc ibed elsewhe e [100]
wi h some modi ica ions. S ock solu ions o bo ine insulin (in e nal
s anda d, IS) and human insulin o he cons uc ion o calib a ion
cu es we e ini ially p epa ed in HCl 0.01 N a 5 mg/mL and subse-
quen ly dilu ed in a eshly p epa ed p emixed sol en o ul apu e
wa e /me hanol (80/20) wi h 0.1 % / ace ic acid. Plasma samples o
250
μ
L we e added o 25
μ
L o 500 ppb IS s ock solu ion; 0–25
μ
L human
insulin s ock solu ions (25 ppb and 100 ppb) we e used when applicable
o he cons uc ion o calib a ion cu es; and up o 100
μ
L o he said
sol en was used o a cons an inal olume o 350
μ
L in all samples.
Nex , 750
μ
L o an ace oni ile–me hanol mix u e (1:1 / ) was added o
each sample as a p ecipi an . The samples we e o exed and main ained
unde s i ing in an incuba o (Heidolph Ins umen s GmbH & Co. KG,
Schwabach, Ge many) o 10 min (37 ◦C, 600 pm) and hen cen i uged
(21,000 c , 15 ◦C, 10 min, Eppendo Cen i uge 5430 R, Eppendo
Ib´
e ica S.L.U., Mad id, Spain). The ob ained supe na an was il e ed
h ough 0.22
μ
m PVDF hyd ophilic il e s (4 mm, SLGVR04NL, Me ck
Millipo e, Ge many) and acuumed a 37 ◦C un il comple ely e apo-
a ed wi h he aid o a SpeedVac. Finally, he d y samples we e econ-
s i u ed wi h 90
μ
L o a eshly p epa ed p emixed sol en o ul apu e
wa e /me hanol (80/20) wi h 0.1 % / ace ic acid and subsequen ly
analyzed. All sol en s employed we e o LC/MS g ade wi h he excep-
ion o ace ic acid, which was USP, pH. Eu . eagen g ade, and low-
binding ma e ials and ecipien s we e used a all imes.
The UPLC sys em consis ed o an Acqui y UPLC® H-class sys em
(Wa e s Co p, Mil o d, USA) and a column compa men (Acqui y
UPLC® CSH™ C18 (2.1 ×50 mm, 1.7
μ
m; Pa numbe 186005296,
Se ial no. 01463731715175, Cieny ech, San iago de Compos ela,
Spain). The expe imen al analy ical condi ions we e as ollows: he
mobile phase consis ed o 0.1 % o mic acid aqueous solu ion (A) and
0.1 % o mic acid ace oni ile solu ion (B). A g adien p og am was used
as ollows: 80 % o 63 % A om 0 o 3.8 min; 63 % o 2 % A om 3.8 o
3.9 min; 2 % A main ained om 3.9 o 4.9 min; 2 % o 80 % A om 5.9 o
5 min, and kep cons an up o 7 min o allow he sys em o equilib a e.
The o al un ime was 7 min. The column empe a u e was main ained
a 40 ◦C, and he au osample was he mos a ized a 10 ◦C. The injec ed
olume was 50
μ
L. Unde hese condi ions, human insulin was elu ed a
3.34 ±0.02 min, and bo ine insulin was employed as an in e nal
s anda d a 3.19 ±0.02 min. The UPLC sys em was coupled o a Xe o®
T iple Quad upole De ec o (TQD) (Wa e s Co p, Mil o d, USA) wi h an
elec osp ay ioniza ion (ESI) in e ace. A empe a u e o 525 ◦C was
selec ed as he sou ce empe a u e, and 60 ◦C was selec ed as he des-
ol a ion empe a u e. The capilla y ol age was 2.0 kV, and he cone
M. Du ´
an-Loba o e al.
Jou nal o Con olled Release 377 (2025) 17–36
32
ol age and collision ene gy we e se a 50 V and 3 V, espec i ely.
Ni ogen was used o desol a ion and as he cone gas a low a es o
1000 L/h and 80 L/h, espec i ely. A gon was used as he collision gas.
Mass spec ome ic de ec ion was ope a ed in posi i e mode and se up
o mul iple eac ion moni o ing (MRM) o moni o he human insulin
ansi ions o m/z 969.0 >> 1133.0 (cone ol age 50 V, collision ene gy
25 V); 1162.6 >> 143.2 (cone ol age 60 V, collision ene gy 40 V);
1162.6 >> 219.0 (cone ol age 60 V, collision ene gy 50 V); 1162.6 >>
226.0 (cone ol age 60 V, collision ene gy 40 V), and bo ine insulin
ansi ions o m/z 956.5 >> 1114.8 (cone ol age 50 V, collision ene gy
18 V); 956.6 >> 315.2 (cone ol age 50 V, collision ene gy 50 V); 956.6
>> 1121.2 (cone ol age 50 V, collision ene gy 18 V); and 1147.5 >>
315.5 (cone ol age 70 V, collision ene gy 52 V). Calib a ion cu es
we e cons uc ed o e a ange o 0.25–5 ppb, wi h an LOQ o 0.5 ppb.
Da a acquisi ion and analysis we e pe o med using Ta ge Lynx 4.1
so wa e (Wa e s Co p., Mil o d, USA).
4.2.16. P oduc ion and physicochemical cha ac e iza ion o
biome al– RNA nanocomplexes
Nanocomplexes o RNA-Zn we e p epa ed by adding a 20 mg/mL
zinc ace a e dihyd a e aqueous solu ion o e 500
μ
L o a 0.1 mg/mL
RNA solu ion in HCl 0.01 ɴ o a ain a cha ge a io (Zn mol–nucleo ide)
o ei he 5:1 o 10:1. The mix u e was main ained unde magne ic
s i ing a 300 pm while 0.1 ɴ NaOH was added o adjus he pH o 7.4.
Nex , he mix u e was incuba ed a 4 ◦C o 3 h. Subsequen ly, he
mix u e was allowed o empe a RT o 4 min, o exed o 10 s and
hen cha ac e ized in e ms o pa icle size and ZP.
Rega ding he RNA–Fe nanocomplex p oduc ion, wo ypes o
nanocomplexes we e p oduced, namely, Type I nanocomplexes, exhib-
i ing a nega i e su ace cha ge and abili y o be coa ed wi h a posi i ely
cha ged ma e ial, and Type II nanocomplexes, exhibi ing a posi i e
su ace cha ge and abili y o be coa ed wi h a nega i ely cha ged ma-
e ial. To p oduce Type I nanocomplexes, a 0.1 mg/mL RNA solu ion in
RNAse- ee wa e and a 0.246 mg/mL e ic chlo ide hexahyd a e so-
lu ion in ace a e bu e (100 mᴍ) (pH 6) we e p epa ed. Nex , he e ic
chlo ide hexahyd a e solu ion was added d opwise o e he RNA so-
lu ion unde s i ing (500 pm) a a 1:1 / a io, and he mix u e was
s i ed o 10 min. To coa he esul ing nanocomplexes, he ob ained
suspension was immedia ely added d opwise o e a 0.1 mg/mL solu ion
o he selec ed posi i ely cha ged polyme (chi osan o polya ginine
(PARG)) unde s i ing (500 pm) a a 1:1 / a io, and he mix u e was
s i ed o 10 min. To p oduce Type II nanocomplexes, a 0.1 mg/mL
RNA solu ion in RNAse- ee wa e and a 2.216 mg/mL e ic chlo ide
hexahyd a e solu ion in ace a e bu e (100 mM) (pH 6) we e p epa ed.
Nex , he RNA solu ion was added d opwise o e he e ic chlo ide
hexahyd a e solu ion unde s i ing (500 pm) a a 1:1 / a io, and he
mix u e was s i ed o 10 min. To coa he esul ing nanocomplexes, he
ob ained suspension was immedia ely added d opwise o e a 2 mg/mL
solu ion o he selec ed nega i ely cha ged polyme ((HA), CS, PEG(5 k)-
PGA(10) o PSA) unde s i ing (500 pm) a a 1:1 / a io, and he
mix u e was s i ed o 10 min.
The pa icle size dis ibu ion and PDI we e de e mined by DLS, and
he ZP was de e mined om he elec opho e ic mobili y alues ob-
ained by LDA using Mal e n Ze asize equipmen (NanoZS ZEN 3600,
Mal e n Ins umen s, Wo ces e shi e, UK) equipped wi h a ed lase
ligh beam (λ =632.8 nm). The o mula ions we e di ec ly measu ed
wi hou dilu ion a 25 ◦C wi h a leas h ee di e en ba ches and ip-
lica e analysis o each ba ch.
4.2.17. S a is ics
All expe imen s we e pe o med a leas in iplica e, and da a a e
p esen ed as he means ±s anda d de ia ions (SDs) in physicochemical
s udies and he s anda d e o s o he mean (SEMs) o in i o s udies.
S a is ical analysis was ca ied ou using one-way analysis o a iance
(ANOVA) wi h a mul iple compa isons es (G aphPad P ism, G aphPad
so wa e Inc., CA, USA). All o he analyses we e pe o med using one-
way analysis o a iance (ANOVA). The le el o signi icance was se a
p obabili ies o *p <0.05, **p <0.01, ***p <0.001 and **** p <
0.0001.
Funding s a emen
This wo k was suppo ed by he Eu opean TRANS-INT Conso ium,
which ecei ed unding om he Eu opean Union’s Se en h F amewo k
P og amme o esea ch, echnological de elopmen and demons a ion
unde g an ag eemen NO. 281035. M. Du ´
an-Loba o acknowledges a
pos doc o al ellowship (Con a o de Acceso al Sis ema Espa˜
nol de
Ciencia, Tecnología e Inno aci´
on (g an numbe USE-19533-Y)) g an ed
by “VI Plan P opio” om he Uni e si y o Se ille.
CRediT au ho ship con ibu ion s a emen
Ma ilde Du ´
an-Loba o: W i ing – e iew & edi ing, W i ing –
o iginal d a , Visualiza ion, Valida ion, Me hodology, In es iga ion,
Fo mal analysis, Da a cu a ion, Concep ualiza ion. Sulay To a :
W i ing – e iew & edi ing, Supe ision, Resou ces, P ojec adminis-
a ion, Me hodology, In es iga ion, Funding acquisi ion, Fo mal anal-
ysis, Concep ualiza ion. Juan Cu˜
na o: Valida ion, In es iga ion,
Fo mal analysis, Da a cu a ion. Rocío Ramos-Memb i e: Valida ion,
In es iga ion, Fo mal analysis, Da a cu a ion. I ´
an Pe˜
nuelas: W i ing –
e iew & edi ing, Supe ision, Resou ces, Me hodology, In es iga ion,
Funding acquisi ion, Concep ualiza ion. Ila ia Ma igo: W i ing – e-
iew & edi ing, Valida ion, Supe ision, Resou ces, P ojec adminis-
a ion, Me hodology, In es iga ion, Funding acquisi ion, Fo mal
analysis, Da a cu a ion, Concep ualiza ion. Fede ico Bene i: W i ing –
e iew & edi ing, Valida ion, Resou ces, Me hodology, In es iga ion,
Funding acquisi ion, Fo mal analysis, Da a cu a ion, Concep ualiza ion.
Miguel Chenlo: Valida ion, Supe ision, Resou ces, Me hodology,
Fo mal analysis, Da a cu a ion. Cla a V. ´
Al a ez: W i ing – e iew &
edi ing, Supe ision, Resou ces, Me hodology, Funding acquisi ion,
Concep ualiza ion. Vashegyi Ildik´
o: Valida ion, In es iga ion, Fo mal
analysis, Da a cu a ion, Concep ualiza ion. Rudol U banics: Valida-
ion, Supe ision, Me hodology, In es iga ion, Fo mal analysis, Da a
cu a ion, Concep ualiza ion. J´
anos Szebeni: W i ing – e iew & edi -
ing, Supe ision, Resou ces, Funding acquisi ion, Concep ualiza ion.
Ma ía Jos´
e Alonso: W i ing – e iew & edi ing, Supe ision, Resou ces,
Funding acquisi ion, Concep ualiza ion.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Da a a ailabili y
Da a will be made a ailable on eques .
Acknowledgmen s
The au ho s a e especially g a e ul o D . ´
Al a o An elo o his
assis ance wi h LC–MS me hod de elopmen and analysis, D . Manuel
Ma ín-Pas o (Nuclea Magne ic Resonance Uni , RIADT, Uni e sidade
de San iago de Compos ela) o his assis ance wi h NMR expe imen al
design and in e p e a ion, D . B uno Dacu˜
na Ma i˜
no o his help wi h Rx
expe imen al design and in e p e a ion (X- ay Uni , C ys alline Powde
Sec ion, RIAIDT, Uni e sidade de San iago de Compos ela, Ma ia
Sua ez-Fa i˜
na o he echnical help wi h cell-based bioac i i y assays,
and Sano i o p o iding he human insulin pep ide as well as assis ance
wi h pep ide handling and analysis.
M. Du ´
an-Loba o e al.
Jou nal o Con olled Release 377 (2025) 17–36
33
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j.jcon el.2024.11.016.
Re e ences
[1] A.N. Zelikin, C. Eh ha d , A.M. Healy, Ma e ials and me hods o deli e y o
biological d ugs, Na . Chem. 8 (2016) 997–1007, h ps://doi.o g/10.1038/
nchem.2629.
[2] J. By ne, H.W. Huang, J.C. McRae, S. Babaee, A. Sol ani, S.L. Becke , G. T a e so,
De ices o d ug deli e y in he gas oin es inal ac : a e iew o sys ems
physically in e ac ing wi h he mucosa o enhanced deli e y, Ad . D ug Deli .
Re . 177 (2021) 113926, h ps://doi.o g/10.1016/j.add .2021.113926.
[3] T.D. B own, K.A. Whi ehead, S. Mi ago i, Ma e ials o o al deli e y o p o eins
and pep ides, Na . Re . Ma e . 5 (2020) 127–148, h ps://doi.o g/10.1038/
s41578-019-0156-6.
[4] A.C. Anselmo, Y. Goka n, S. Mi ago i, Non-in asi e deli e y s a egies o
biologics, Na . Re . D ug Disco . 18 (2018) 19–40, h ps://doi.o g/10.1038/
n d.2018.183.
[5] M. Du ´
an-Loba o, A.M. L´
opez-Es ´
e ez, A.S. Co dei o, T.G. Dacoba, J. C ecen e-
Campo, D. To es, M.J. Alonso, Nano echnologies o he deli e y o biologicals:
his o ical pe spec i e and cu en landscape, Ad . D ug Deli . Re . 176 (2021),
h ps://doi.o g/10.1016/j.add .2021.113899.
[6] D.J. B ayden, T.A. Hill, D.P. Fai lie, S. Mahe , R.J. M sny, Sys emic deli e y o
pep ides by he o al ou e: o mula ion and medicinal chemis y app oaches, Ad .
D ug Deli . Re . (2020), h ps://doi.o g/10.1016/j.add .2020.05.007.
[7] M. Du ´
an-Loba o, Z. Niu, M.J. Alonso, O al deli e y o biologics o p ecision
medicine, Ad . Ma e . 32 (2019), h ps://doi.o g/10.1002/adma.201901935.
[8] A. Bane jee, K. Ibsen, T. B own, R. Chen, C. Aga emo , S. Mi ago i, Ionic liquids
o o al insulin deli e y, P oc. Na l. Acad. Sci. 115 (2018) 7296–7301, h ps://
doi.o g/10.1073/pnas.1722338115.
[9] A. Bane jee, R. Chen, S. A a in, S. Mi ago i, In es inal ion opho esis om
mucoadhesi e pa ches: a s a egy o o al deli e y, J. Con ol. Release 297 (2019)
71–78, h ps://doi.o g/10.1016/j.jcon el.2019.01.037.
[10] A. Ab amson, E. Ca a el-Sal ado , M. Khang, D. Dellal, D. Sil e s ein, Y. Gao, M.
R. F ede iksen, A. Vegge, F. Hubalek, J.J. Wa e , A.V. F iede ichses, J. Fels, R.
K. Ki k, C. Cle eland, J. Collins, S. Tamang, A. Haywa d, T. Landh, S.T. Buckley,
N. Roxhed, U. Rahbek, R. Lange , G. T a e so, An inges ible sel -o ien ing
applica o o o al deli e y o mac omolecules, Sci. (Unde Re .) 363 (2019)
611–615, h ps://doi.o g/10.1126/SCIENCE.AAU2277.
[11] A. Ab amson, E. Ca a el-Sal ado , V. Soa es, D. Minahan, R.Y. Tian, X. Lu,
D. Dellal, Y. Gao, S. Kim, J. Waine , J. Collins, S. Tamang, A. Haywa d,
T. Yoshi ake, H.C. Lee, J. Fujimo o, J. Fels, M.R. F ede iksen, U. Rahbek,
N. Roxhed, R. Lange , G. T a e so, A luminal un olding mic oneedle injec o o
o al deli e y o mac omolecules, Na . Med. 25 (2019) 1512–1518, h ps://doi.
o g/10.1038/s41591-019-0598-9.
[12] A. Ab amson, M.R. F ede iksen, A. Vegge, B. Jensen, M. Poulsen, B. Mou idsen,
M.O. Jespe sen, R.K. Ki k, J. Windum, F. Hub´
alek, J.J. Wa e , J. Fels, S.
B. Gunna sson, A. Boh , E.M. S aa up, M.W.H. Ley, X. Lu, J. Waine , J. Collins,
S. Tamang, K. Ishida, A. Haywa d, P. He skind, S.T. Buckley, N. Roxhed,
R. Lange , U. Rahbek, G. T a e so, O al deli e y o sys emic monoclonal
an ibodies, pep ides and small molecules using gas ic au o-injec o s, Na .
Bio echnol. 40 (2022) 103–109, h ps://doi.o g/10.1038/s41587-021-01024-0.
[13] M. Hashim, R. Ko upolu, B. Syed, K. Ho len, S. Be aki, P. Ka amchedu, A.
K. Dhalla, R. Ru y, M. Im an, Jejunal wall deli e y o insulin ia an inges ible
capsule in anes he ized swine—a pha macokine ic and pha macodynamic s udy,
Pha macol. Res. Pe spec . 7 (2019) 1–6, h ps://doi.o g/10.1002/p p2.522.
[14] W. Chen, J. Waine , S.W. Ryoo, X. Qi, R. Chang, J. Li, S.H. Lee, S. Min,
A. Wen wo h, J.E. Collins, S. Tamang, K. Ishida, A. Haywa d, R. Lange ,
G. T a e so, Dynamic omnidi ec ional adhesi e mic oneedle sys em o o al
mac omolecula d ug deli e y, Sci. Ad . 8 (2022) 1–11, h ps://doi.o g/
10.1126/sciad .abk1792.
[15] S.T. Buckley, T.A. Bækdal, A. Vegge, S.J. Maa bje g, C. Pyke, J. Ahn el -Rønne, K.
G. Madsen, S.G. Sch´
eele, T. Alanen alo, R.K. Ki k, B.L. Pede sen, R.
B. Skyggebje g, A.J. Benie, H.M. S auss, P.O. Wahlund, S. Bje egaa d, E. Fa kas,
C. Feke e, F.L. Sønde gaa d, J. Bo egaa d, M.L. Ha o -Nielsen, L.B. Knudsen,
T anscellula s omach abso p ion o a de i a ized glucagon-like pep ide-1
ecep o agonis , Sci. T ansl. Med. 10 (2018), h ps://doi.o g/10.1126/
sci anslmed.aa 7047.
[16] D.J. B ayden, S. Mahe , T ansien pe mea ion enhance ® (TPE®) echnology o
o al deli e y o oc eo ide: a echnological e alua ion, Expe Opin. D ug Deli .
18 (2021) 1501–1512, h ps://doi.o g/10.1080/17425247.2021.1942838.
[17] E. Ca a el-Sal ado , A. Ab amson, R. Lange , G. T a e so, O al deli e y o
biologics using d ug-de ice combina ions, Cu . Opin. Pha macol. 36 (2017)
8–13, h ps://doi.o g/10.1016/j.coph.2017.07.003.
[18] T.V. O’Hallo an, M. Kebede, S.J. Philips, A.D. A ie, Zinc, insulin, and he li e : a
m´
enage `
a ois, J. Clin. In es . 123 (2013) 4136–4139, h ps://doi.o g/10.1172/
JCI72325.
[19] J.J. Abel, C ys alline Insulin, P oc. Na l. Acad. Sci. USA 12 (1926) 132–136,
h ps://doi.o g/10.1073/pnas.12.2.132.
[20] K. Hallas-Mølle , K. Pe e sen, J. Schlich k ull, C ys alline and amo phous insulin-
zinc compounds wi h p olonged ac ion, Science 116 (1952) 394–398, h ps://doi.
o g/10.1126/science.116.3015.394.
[21] G.P. Ca ino, J.S. Jacob, E. Ma hiowi z, Nanosphe e based o al insulin deli e y,
J. Con ol. Release 65 (2000) 261–269, h ps://doi.o g/10.1016/S0168-3659
(99)00247-3.
[22] X. Han, Y. Lu, J. Xie, E. Zhang, H. Zhu, H. Du, K. Wang, B. Song, C. Yang, Y. Shi,
Z. Cao, Zwi e ionic micelles e icien ly deli e o al insulin wi hou opening igh
junc ions, Na . Nano echnol. 15 (2020) 605–614, h ps://doi.o g/10.1038/
s41565-020-0693-6.
[23] D. H is o , F. McCa ney, J. Bei ne, E. Mahon, S. Reid, S. Bha acha jee,
G. Pena ie , U. We ne , D. Bazile, D.J. B ayden, Silica-coa ed nanopa icles wi h a
Co e o zinc, l -a ginine, and a pep ide designed o O al deli e y, ACS Appl.
Ma e . In e aces 12 (2020) 1257–1269, h ps://doi.o g/10.1021/
acsami.9b16104.
[24] M.F. Dunn, Zinc-ligand in e ac ions modula e assembly and s abili y o he
insulin hexame - a e iew, in, BioMe als (2005) 295–303, h ps://doi.o g/
10.1007/s10534-005-3685-y.
[25] Y.V. Li, Zinc and insulin in panc ea ic be a-cells, Endoc ine 45 (2014) 178–189,
h ps://doi.o g/10.1007/s12020-013-0032-x.
[26] X. Han, Y. Lu, J. Xie, E. Zhang, H. Zhu, H. Du, K. Wang, B. Song, C. Yang, Y. Shi,
Z. Cao, Zwi e ionic micelles e icien ly deli e o al insulin wi hou opening igh
junc ions, Na . Nano echnol. 15 (2020) 605–614, h ps://doi.o g/10.1038/
s41565-020-0693-6.
[27] A. Bane jee, J. Qi, R. Gogoi, J. Wong, S. Mi ago i, Role o nanopa icle size,
shape and su ace chemis y in o al d ug deli e y, J. Con ol. Release 238 (2016)
176–185, h ps://doi.o g/10.1016/j.jcon el.2016.07.051.
[28] K. Nadendla, S.H. F iedman, Ligh Con ol o P o ein Solubili y Th ough
Isoelec ic Poin Modula ion, 2017, h ps://doi.o g/10.1021/jacs.7b08465.
[29] M. Weiss, Design o ul a-s able insulin analogues o he de eloping wo ld,
J. Heal h Special ies 1 (2013) 59, h ps://doi.o g/10.4103/1658-600X.114683.
[30] T.L. Blundell, J.F. Cu ield, S.M. Cu ield, E.J. Dodson, G.G. Dodson, D.
C. Hodgkin, D.A. Me cola, M. Vijayan, A omic posi ions in hombohed al 2-zinc
insulin c ys als, Na u e 231 (1971) 506–511, h ps://doi.o g/10.1038/
231506a0.
[31] P.J. Wya , Measu emen o special nanopa icle s uc u es by ligh sca e ing,
Anal. Chem. 86 (2014) 7171–7183, h ps://doi.o g/10.1021/ac500185w.
[32] A. Bane jee, J. Qi, R. Gogoi, J. Wong, S. Mi ago i, Role o nanopa icle size,
shape and su ace chemis y in o al d ug deli e y, J. Con ol. Release 238 (2016)
176–185, h ps://doi.o g/10.1016/j.jcon el.2016.07.051.
[33] D. Li, J. Zhuang, H. He, S. Jiang, A. Bane jee, Y. Lu, W. Wu, S. Mi ago i, L. Gan,
J. Qi, In luence o pa icle geome y on gas oin es inal ansi and abso p ion
ollowing O al adminis a ion, ACS Appl. Ma e . In e aces 9 (2017)
42492–42502, h ps://doi.o g/10.1021/acsami.7b11821.
[34] M. Guo, M. Wei, W. Li, M. Guo, C. Guo, M. Ma, Y. Wang, Z. Yang, M. Li, Q. Fu,
L. Yang, Z. He, Impac s o pa icle shapes on he o al deli e y o d ug
nanoc ys als: mucus pe mea ion, ansepi helial anspo and bioa ailabili y,
J. Con ol. Release 307 (2019) 64–75, h ps://doi.o g/10.1016/j.
jcon el.2019.06.015.
[35] R. Gup a, Y. Badhe, S. Mi ago i, B. Rai, Pe mea ion o nanopa icles ac oss he
in es inal lipid memb ane: dependence on shape and su ace chemis y s udied
h ough molecula simula ions, Nanoscale 12 (2020) 6318–6333, h ps://doi.
o g/10.1039/c9n 09947 .
[36] C. Bao, B. Liu, B. Li, J. Chai, L. Zhang, L. Jiao, D. Li, Z. Yu, F. Ren, X. Shi, Y. Li,
Enhanced anspo o shape and igidi y- uned
α
-Lac albumin nano ubes ac oss
in es inal mucus and cellula ba ie s, Nano Le . 20 (2020) 1352–1361, h ps://
doi.o g/10.1021/acs.nanole .9b04841.
[37] C. Manoha an, J. Singh, Insulin loaded PLGA mic osphe es: e ec o zinc sal s on
encapsula ion, elease, and s abili y, J. Pha m. Sci. 98 (2009) 529–542, h ps://
doi.o g/10.1002/jps.21445.
[38] Q. Xu, L.M. Ensign, N.J. Boylan, A. Sch¨
on, X. Gong, J.C. Yang, N.W. Lamb, S. Cai,
T. Yu, E. F ei e, J. Hanes, Impac o su ace polye hylene glycol (PEG) densi y on
biodeg adable nanopa icle anspo in mucus ex i o and dis ibu ion in i o,
ACS Nano 9 (2015) 9217–9227, h ps://doi.o g/10.1021/acsnano.5b03876.
[39] A. Vila, A. S´
anchez, M. Tobío, P. Cal o, M.J. Alonso, Design o biodeg adable
pa icles o p o ein deli e y, J. Con ol. Release 78 (2002) 15–24, h ps://doi.
o g/10.1016/S0168-3659(01)00486-2.
[40] S. Mahe , R.J. M sny, D.J. B ayden, In es inal pe mea ion enhance s o o al
pep ide deli e y, Ad . D ug Deli . Re . 106 (2016) 277–319, h ps://doi.o g/
10.1016/j.add .2016.06.005.
[41] K. Chi, J.M. Ca chma k, C ys alline nanocellulose/lau ic a gina e complexes,
Ca bohyd . Polym. 175 (2017) 320–329, h ps://doi.o g/10.1016/j.
ca bpol.2017.08.005.
[42] X. Yang, R. Rai, C.N. Huu, N. Ni in, Syne gis ic an imic obial ac i i y by ligh o
he mal ea men and lau ic a gina e: memb ane damage and oxida i e s ess,
Appl. En i on. Mic obiol. 85 (2019) 1–14, h ps://doi.o g/10.1128/AEM.01033-
19.
[43] A. Pensado, M. Ma ín-Pas o , G.K. Zo zi, E.S. Ca alho, A. Sanchez, S uc u al
analysis o nanosys ems: solid So bi an es e s nanopa icles (SSN) as a case s udy,
Eu . J. Pha m. Biopha m. 104 (2016) 189–199, h ps://doi.o g/10.1016/j.
ejpb.2016.05.002.
[44] Z. Niu, E. Tedesco, F. Bene i, A. Mabondzo, I.M.I.M. Mon agne , I. Ma igo,
D. Gonzalez-Touceda, S. To a , C. Di´
eguez, M.J.M.J. San ande -O ega, M.J.M.
J. Alonso, Ra ional design o polya ginine nanocapsules in ended o help pep ides
o e coming in es inal ba ie s, J. Con ol. Release 263 (2017) 4–17, h ps://doi.
o g/10.1016/j.jcon el.2017.02.024.
[45] L.N. Thwala, D.P. Delgado, K. Leone, I. Ma igo, F. Bene i, M. Chenlo, C.
V. Al a ez, S. To a , C. Dieguez, N.S. Csaba, M.J. Alonso, P o amine
M. Du ´
an-Loba o e al.
Jou nal o Con olled Release 377 (2025) 17–36
34
nanocapsules as ca ie s o o al pep ide deli e y, J. Con ol. Release 291 (2018)
157–168, h ps://doi.o g/10.1016/j.jcon el.2018.10.022.
[46] T.V. O’Hallo an, M. Kebede, S.J. Philips, A.D. A ie, Zinc, insulin, and he li e : a
m´
enage `
a ois, J. Clin. In es . 123 (2013) 4136–4139, h ps://doi.o g/10.1172/
JCI72325.
[47] R. Pohl, R. Hause , M. Li, E. De Souza, R. Felds ein, R. Seibe , K. Ozhan,
N. Kashyap, S. S eine , Ul a- apid abso p ion o ecombinan human insulin
induced by zinc chela ion and su ace cha ge masking, J. Diabe es Sci. Technol. 6
(2012), h ps://doi.o g/10.1177/193229681200600404.
[48] A.C. Anselmo, S. Mi ago i, Nanopa icles in he clinic, Bioeng. T ansl. Med. 1
(2016) 10–29, h ps://doi.o g/10.1002/b m2.10003.
[49] S. Hassan, G. P akash, A. Bal Oz u k, S. Saghazadeh, M. Fa han Sohail, J. Seo,
M. Remzi Dokmeci, Y.S. Zhang, A. Khademhosseini, E olu ion and clinical
ansla ion o d ug deli e y nanoma e ials, Nano Today 15 (2017) 91–106,
h ps://doi.o g/10.1016/j.nan od.2017.06.008.
[50] M. Du ´
an-Loba o, Z. Niu, M.J. Alonso, O al deli e y o biologics o p ecision
medicine, Ad . Ma e . 32 (2019), h ps://doi.o g/10.1002/adma.201901935.
[51] E. P esas, F. McCa ney, E. Sul an, C. Hunge , S. Nellen, C.V. Al a ez, U. We ne ,
D. Bazile, D.J. B ayden, C.M. O’D iscoll, Physicochemical, pha macokine ic and
pha macodynamic analyses o amphiphilic cyclodex in-based nanopa icles
designed o enhance in es inal deli e y o insulin, J. Con ol. Release 286 (2018)
402–414, h ps://doi.o g/10.1016/j.jcon el.2018.07.045.
[52] J. Heade, F. McCa ney, M. Chenlo, O.M. Ma o, M. Se e ic, R. Ken , S.B. Bleiel,
C.V. Al a ez, B.T. G i in, D.J. B ayden, Syn hesis and in i o e alua ion o
insulin-loaded whey beads as an o al pep ide deli e y sys em, Pha maceu ics 13
(2021), h ps://doi.o g/10.3390/pha maceu ics13050656.
[53] Y. Zhou, Z. Chen, D. Zhao, D. Li, C. He, X. Chen, A pH- igge ed sel -unpacking
capsule con aining Zwi e ionic hyd ogel-coa ed MOF nanopa icles o e icien
O al Exendin-4 deli e y, Ad . Ma e . 33 (2021) 1–10, h ps://doi.o g/10.1002/
adma.202102044.
[54] A.M.M. Sadeghi, M.R. A adi, S. Ej emaimeh , S. Abashzadeh, A. Pa oaza ,
F. Do koosh, M. Faghihi, M. Ra iee-Teh ani, H.E. Junginge , De elopmen o a
gas empowe ed d ug deli e y sys em o pep ide deli e y in he small in es ine,
J. Con ol. Release 134 (2009) 11–17, h ps://doi.o g/10.1016/j.
jcon el.2008.10.012.
[55] E.Y. Chuang, K.J. Lin, P.Y. Lin, H.L. Chen, S.P. Wey, F.L. Mi, H.C. Hsiao, C.
T. Chen, H.W. Sung, Sel -assembling bubble ca ie s o o al p o ein deli e y,
Bioma e ials 64 (2015) 115–124, h ps://doi.o g/10.1016/j.
bioma e ials.2015.06.035.
[56] P.Y. Lin, E.Y. Chuang, Y.H. Chiu, H.L. Chen, K.J. Lin, J.H. Juang, C.H. Chiang, F.
L. Mi, H.W. Sung, Sa e y and e icacy o sel -assembling bubble ca ie s s abilized
wi h sodium dodecyl sul a e o o al deli e y o he apeu ic p o eins, J. Con ol.
Release 259 (2017) 168–175, h ps://doi.o g/10.1016/j.jcon el.2016.12.018.
[57] A. Ka ole, S. Pa ez, R.S. Thaku , S.L. Muda a h, E e escen based nano-gas
ca ie enhanced he bioa ailabili y o poo ly aqueous soluble d ug: a
comp ehensi e mechanis ic unde s anding, J. D ug Deli . Sci. Technol. 69 (2022)
103167, h ps://doi.o g/10.1016/j.jdds .2022.103167.
[58] T.S. Co ey, Die coke and Men os: wha is eally behind his physical eac ion?
Am. J. Phys. 76 (2008) 551–557, h ps://doi.o g/10.1119/1.2888546.
[59] Z. Niu, E. Sama idou, E. Jaumain, J. Co¨
ene, G. Ullio, N. Sh es ha, J. Ga cia,
M. Du ´
an-Loba o, S. To a , M.J. San ande -O ega, M.V. Lozano, M.M. A oyo-
Jimenez, R. Ramos-Memb i e, I. Pe˜
nuelas, A. Mabondzo, V. P ´
ea , M. Teixid´
o,
E. Gi al , M.J. Alonso, PEG-PGA en eloped oc aa ginine-pep ide nanocomplexes:
an o al pep ide deli e y s a egy, J. Con ol. Release 276 (2018) 125–139,
h ps://doi.o g/10.1016/j.jcon el.2018.03.004.
[60] X. Yu, D. Gao, L. Gao, J. Lai, C. Zhang, Y. Zhao, L. Zhong, B. Jia, F. Wang, X. Chen,
Z. Liu, Inhibi ing me as asis and p e en ing umo elapse by igge ing hos
immuni y wi h umo - a ge ed pho odynamic he apy using pho osensi ize -
loaded unc ional nanog aphenes, ACS Nano 11 (2017) 10147–10158, h ps://
doi.o g/10.1021/acsnano.7b04736.
[61] P. A eses, M.T. Agüe os, G. Quincoces, M. Collan es, J.A. Rich e , L. L´
opez-
S´
anchez, M. S´
anchez-Ma ínez, J.M. I ache, I. Pe˜
nuelas, Molecula imaging
echniques o s udy he biodis ibu ion o o ally adminis e ed 99m Tc-labelled
nai e and ligand- agged nanopa icles, Mol. Imaging Biol. 13 (2011) 1215–1223,
h ps://doi.o g/10.1007/s11307-010-0456-0.
[62] J.M. DeSesso, C.F. Jacobson, Ana omical and physiological pa ame e s a ec ing
gas oin es inal abso p ion in humans and a s, Food Chem. Toxicol. 39 (2001)
209–228.
[63] M.C. To jman, J.I. Joseph, C. Munsick, M. Mo ishi a, Z. G unwald, E ec s o
iso lu ane on gas oin es inal mo ili y a e b ie exposu e in a s, In . J. Pha m.
294 (2005) 65–71, h ps://doi.o g/10.1016/j.ijpha m.2004.12.028.
[64] N. D agice ic, H.I. Maibach, Nina D agice ic, Howa d I. Maibach (Eds.),
Pe cu aneous Pene a ion Enhance s Chemical Me hods in Pene a ion
Enhancemen _ Modi ica ion o he S a um Co neum (2015), Sp inge -Ve lag,
2015. Be lin Heid.pd .
[65] J. Jiao, Polyoxye hyla ed nonionic su ac an s and hei applica ions in opical
ocula d ug deli e y, Ad . D ug Deli . Re . 60 (2008) 1663–1673, h ps://doi.
o g/10.1016/j.add .2008.09.002.
[66] M. Mo ishi a, T. Go o, N.A. Peppas, J.I. Joseph, M.C. To jman, C. Munsick,
K. Nakamu a, T. Yamaga a, K. Takayama, A.M. Lowman, Mucosal insulin deli e y
sys ems based on complexa ion polyme hyd ogels: e ec o pa icle size on
insulin en e al abso p ion, J. Con ol. Release 97 (2004) 115–124, h ps://doi.
o g/10.1016/j.jcon el.2004.03.008.
[67] M. Mo ishi a, N. Kamei, J. Eha a, K. Isowa, K. Takayama, A no el app oach using
unc ional pep ides o e icien in es inal abso p ion o insulin, J. Con ol.
Release 118 (2007) 177–184, h ps://doi.o g/10.1016/j.jcon el.2006.12.022.
[68] L.N. Thwala, A. Beloqui, N.S. Csaba, D. Gonz´
alez-Touceda, S. To a , C. Dieguez,
M.J. Alonso, V. P ´
ea , V.P. Lungile Nomcebo Thwala, Ana Beloqui, Noemi
S e ania Csaba, Da id Gonz´
alez-Touceda, Sulay To a , Ca los Dieguez, Ma ia
Jose Alonso, The in e ac ion o p o amine nanocapsules wi h he in es inal
epi helium: a mechanis ic app oach, J. Con ol. Release 243 (2016) 109–120,
h ps://doi.o g/10.1016/j.jcon el.2016.10.002.
[69] I. San alices, C. V´
azquez-V´
azquez, M.J. San ande -O ega, V. Lozano, F. A aújo,
B. Sa men o, N. Sh es ha, V. P ´
ea , M. Chenlo, C.V. Al a ez, F. Bene i,
J. Cu˜
na o, S. To a , D. To es, M.J. Alonso, A nanoemulsion/micelles mixed
nanosys em o he o al adminis a ion o hyd ophobically modi ied insulin, D ug
Deli . T ansl. Res. 11 (2021) 524–545, h ps://doi.o g/10.1007/s13346-021-
00920-x.
[70] C. Damge, C. Michel, M. Ap ahamian, P. Cou eu , New app oach o o al
adminis a ion o insulin wi h polyalkylcyanoac yla e nanocapsules as d ug
ca ie , Diabe es 37 (1988) 246–251, h ps://doi.o g/10.2337/diab.37.2.246.
[71] B.L. Fu man, S ep ozo ocin-induced diabe ic models in mice and a s, Cu .
P o oc. Pha macol. 70 (2015) 5.47.1–5.47.20, h ps://doi.o g/10.1002/
0471141755.ph0547s70.
[72] A. Vieho , L. Ja o , A. B´
eduneau, Y. Pelleque , A. Lamp ech , O al insulin deli e y
in a s by nanopa icles p epa ed wi h non- oxic sol en s, In . J. Pha m. 443
(2013) 169–174, h ps://doi.o g/10.1016/j.ijpha m.2013.01.017.
[73] A. Makhlo , Y. Tozuka, H. Takeuchi, Design and e alua ion o no el pH-sensi i e
chi osan nanopa icles o o al insulin deli e y, Eu . J. Pha m. Sci. 42 (2011)
445–451, h ps://doi.o g/10.1016/j.ejps.2010.12.007.
[74] C. Damg´
e, C. Michel, M. Ap ahamian, P. Cou eu , J.P. De issague ,
Nanocapsules as ca ie s o o al pep ide deli e y, J. Con ol. Release 13 (1990)
233–239, h ps://doi.o g/10.1016/0168-3659(90)90013-J.
[75] E.M. P idgen, F. Alexis, T.T. Kuo, E. Le y-Nissenbaum, R. Ka nik, R.S. Blumbe g,
R. Lange , O.C. Fa okhzad, T ansepi helial anspo o c- a ge ed nanopa icles
by he neona al c ecep o o o al deli e y, Sci. T ansl. Med. 5 (2013), h ps://
doi.o g/10.1126/sci anslmed.3007049.
[76] W. Shan, X. Zhu, M. Liu, L. Li, J. Zhong, W. Sun, Z. Zhang, Y. Huang, O e coming
he di usion ba ie o mucus and abso p ion ba ie o epi helium by sel -
assembled nanopa icles o o al deli e y o insulin, ACS Nano 9 (2015)
2345–2356, h ps://doi.o g/10.1021/acsnano.5b00028.
[77] M. Mo ishi a, T. Go o, K. Nakamu a, A.M. Lowman, K. Takayama, N.A. Peppas,
No el o al insulin deli e y sys ems based on complexa ion polyme hyd ogels:
single and mul iple adminis a ion s udies in ype 1 and 2 diabe ic a s,
J. Con ol. Release 110 (2006) 587–594, h ps://doi.o g/10.1016/j.
jcon el.2005.10.029.
[78] R.L. Jump, A.D. Le ine, Mechanisms o na u al ole ance in he in es ine:
implica ions o in lamma o y bowel disease, In lamm. Bowel Dis. 10 (2004)
462–478, h ps://doi.o g/10.1097/00054725-200407000-00023.
[79] R.V. O e gaa d, A. Na a ia, S.H. Ingwe sen, T.A. Bækdal, R.J. Kildemoes,
Clinical pha macokine ics o O al Semaglu ide: analyses o da a om clinical
pha macology ials, Clin. Pha macokine . 60 (2021) 1335–1348, h ps://doi.
o g/10.1007/s40262-021-01025-x.
[80] P. Tyagi, R. T i edi, S. Pecheno , C. Pa el, J. Re ell, S. Wills, Y. Huang, A.
I. Rosenbaum, J.A. Sub amony, Ta ge ed o al pep ide deli e y using mul i-uni
pa icula es: d ug and pe mea ion enhance laye ing app oach, J. Con ol.
Release 338 (2021) 784–791, h ps://doi.o g/10.1016/j.jcon el.2021.09.002.
[81] S. Bonengel, M. Jelkmann, M. Abdulka im, M. Gumble on, V. Reins adle ,
H. Obe ache , F. P ü e , A. Be nkop-Schnü ch, Impac o di e en hyd ophobic
ion pai s o oc eo ide on i s o al bioa ailabili y in pigs, J. Con ol. Release 273
(2018) 21–29, h ps://doi.o g/10.1016/j.jcon el.2018.01.012.
[82] S. Be g, J. K ause, A. Bj¨
o kbom, K. Wal e , S. Ha un, A. G an eld , D. Janz´
en, S.
F. Nunes, M. An onsson, N. Van Zuydam, S. Sk ic, A. Huge h, W. Wei schies,
N. Da ies, B. Ab ahamsson, C.A.S. Be gs ¨
om, In i o and in i o e alua ion o
3D p in ed capsules wi h p essu e igge ed elease mechanism o O al pep ide
deli e y, J. Pha m. Sci. 110 (2021) 228–238, h ps://doi.o g/10.1016/j.
xphs.2020.10.066.
[83] F.A. Do koosh, J.C. Ve hoe , J.H.M. Ve heijden, M. Ra iee-Teh ani, G. Bo cha d,
H.E. Junginge , Pe o al abso p ion o oc eo ide in pigs o mula ed in deli e y
sys ems on he basis o supe po ous hyd ogel polyme s, Pha m. Res. 19 (2002)
1532–1536, h ps://doi.o g/10.1023/A:1020416918624.
[84] J. Wang, V. Yada , A.L. Sma , S. Taji i, A.W. Basi , Towa d o al deli e y o
biopha maceu icals: an assessmen o he gas oin es inal s abili y o 17 pep ide
d ugs, Mol. Pha m. 12 (2015) 966–973, h ps://doi.o g/10.1021/mp500809 .
[85] A. Klepach, H. T an, F. Ahmad Mohammed, M.E.H. ElSayed, Cha ac e iza ion and
impac o pep ide physicochemical p ope ies on o al and subcu aneous deli e y,
Ad . D ug Deli . Re . 186 (2022) 114322, h ps://doi.o g/10.1016/j.
add .2022.114322.
[86] F.A. Do koosh, J.C. Ve hoe , G. Bo cha d, M. Ra iee-Teh ani, J.H.M. Ve heijden,
H.E. Junginge , In es inal abso p ion o human insulin in pigs using deli e y
sys ems based on supe po ous hyd ogel polyme s, In . J. Pha m. 247 (2002)
47–55, h ps://doi.o g/10.1016/S0378-5173(02)00361-7.
[87] A.K. Dhalla, Z. Al-Shamsie, S. Be aki, A. Dasa i, L.C. Fung, L. Fusa o, A. Ga apa y,
B. Gu ie ez, D. G a a, M. Hashim, K. Ho len, P. Ka amchedu, R. Ko upolu,
E. Liang, C. Ong, Z. Owyang, V. Salgo a, S. Sha ma, B. Syed, M. Syed, A.T. Vo,
R. Abdul-Wahab, A. Wasi, A. Yamaguchi, S. Yen, M. Im an, A obo ic pill o o al
deli e y o bio he apeu ics: sa e y, ole abili y, and pe o mance in heal hy
subjec s, D ug Deli . T ansl. Res. 12 (2022) 294–305, h ps://doi.o g/10.1007/
s13346-021-00938-1.
[88] Y.I.I.O. Yamaga a, Sus ained-Release P epa a ion Con aining a Me al Sal o a
Pep ide, EP1002529A1, 2000.
M. Du ´
an-Loba o e al.
Jou nal o Con olled Release 377 (2025) 17–36
35
[89] C. Yoshizane, A. Mizo e, M. Yamada, N. A ai, S. A ai, K. Ma u a, H. Mi suzumi,
T. A iyasu, S. Ushio, S. Fukuda, Glycemic, insulinemic and inc e in esponses
a e o al ehalose inges ion in heal hy subjec s, Nu . J. 16 (2017) 9, h ps://doi.
o g/10.1186/s12937-017-0233-x.
[90] H.A. Me chan , E.L. McConnell, F. Liu, C. Ramaswamy, R.P. Kulka ni, A.W. Basi ,
S. Mu dan, Assessmen o gas oin es inal pH, luid and lymphoid issue in he
guinea pig, abbi and pig, and implica ions o hei use in d ug de elopmen ,
Eu . J. Pha m. Sci. 42 (2011) 3–10, h ps://doi.o g/10.1016/j.ejps.2010.09.019.
[91] G. Zheng, A.M. To es, W.S. P ice, Wa e Con ol: sel -di usion based sol en
signal supp ession enhanced by selec i e in e sion, Magn. Reson. Chem. 55
(2017) 447–451, h ps://doi.o g/10.1002/m c.4420.
[92] D. Sinnae e, Simul aneous sol en and J-modula ion supp ession in PGSTE-based
di usion expe imen s, J. Magn. Reson. 245 (2014), h ps://doi.o g/10.1016/j.
jm .2014.05.007.
[93] C. Dal i , G.P. Foglia o, A. S ewa , M. Ve onesi, B. S ockman, Wa e LOGSY as a
me hod o p ima y NMR sc eening: p ac ical aspec s and ange o applicabili y,
J. Biomol. NMR 21 (2001) 349–359, h ps://doi.o g/10.1023/A:
1013302231549.
[94] E.L. McConnell, A.W. Basi , S. Mu dan, Measu emen s o a and mouse
gas oin es inal pH, luid and lymphoid issue, and implica ions o in- i o
expe imen s, J. Pha m. Pha macol. 60 (2010) 63–70, h ps://doi.o g/10.1211/
jpp.60.1.0008.
[95] I.R. Ha is, H. H¨
oppne , W. Sie ken, K.-P. Wi e n, A.M. Fa ell, Regula ion o
HMG-CoA syn hase and HMG-CoA educ ase by insulin and epide mal g ow h
ac o in HaCaT ke a inocy es, J. In es . De ma ol. 114 (2000) 83–87, h ps://
doi.o g/10.1046/j.1523-1747.2000.00822.x.
[96] A.R. Ga cia-Rendueles, J.S. Rod igues, M.E.R. Ga cia-Rendueles, M. Sua ez-
Fa i˜
na, S. Pe ez-Rome o, F. Ba ei o, I. Be nabeu, J. Rod iguez-Ga cia,
L. Fugazzola, T. Sakai, F. Liu, J. Cameselle-Teijei o, S.B. B a o, C.V. Al a ez,
Rewi ing o he apop o ic TGF-β-SMAD/NFκB pa hway h ough an oncogenic
unc ion o p27 in human papilla y hy oid cance , Oncogene 36 (2017) 652–666,
h ps://doi.o g/10.1038/onc.2016.233.
[97] Z. Zeng, D. Qi, L. Yang, J. Liu, Y. Tang, H. Chen, X. Feng, S imuli- esponsi e sel -
assembled dend ime s o o al p o ein deli e y, J. Con ol. Release 315 (2019)
206–213, h ps://doi.o g/10.1016/j.jcon el.2019.10.049.
[98] B. Chassaing, J.D. Ai ken, M. Malleshappa, M. Vijay-Kuma , Dex an sul a e
sodium (DSS)-induced coli is in mice, Cu . P o oc. Immunol. (2014), h ps://doi.
o g/10.1002/0471142735.im1525s104.
[99] I. Ma igo, S. Zilio, G. Desan is, B. Mlecnik, A.H.R. Agnellini, S. Ugel, M.S. Sasso, J.
E. Qualls, F. K a och ill, P. Zano ello, B. Molon, C.H. Ries, V. Runza, S. Ho es, A.
M. Bilocq, G. Bindea, E.M.C. Mazza, S. Biccia o, J. Galon, P.J. Mu ay, V. B on e,
T cell Cance he apy equi es CD40-CD40L ac i a ion o umo nec osis ac o
and inducible ni ic-oxide-syn hase-p oducing dend i ic cells, Cance Cell 30
(2016) 377–390, h ps://doi.o g/10.1016/j.ccell.2016.08.004.
[100] Z. Wei, E. Ting, Q.M. Yap, Z. Sun, J. Xing, Z. Zhan, A. De elopmen , S. Cen e,
M. Sciences, De elopmen o 2D-LC/MS/MS bioanaly ical me hod o
quan i a i e de e mina ion o insulin gla gine in human plasma, ASMS MP 311
(2016) 1–7.
M. Du ´
an-Loba o e al.
Jou nal o Con olled Release 377 (2025) 17–36
36
