Enginee ing nanoscale glyco-zeoli ic-imidazola e amewo ks: Insigh s in o
he mechanism o o ma ion
Rocío Rod íguez-Ma ín
a,1
, Sal ado R.G. Bales a
b,1
, Said Hamad
b
,
Elena M. S´
anchez-Fe n´
andez
a,**
, Ca olina Ca illo-Ca i´
on
c,*
a
Depa men o O ganic Chemis y, Facul y o Chemis y, Uni e si y o Se illa, C/ P o eso Ga cía Gonz´
alez 1, 41012, Se illa, Spain
b
Depa men o Physical, Chemical, and Na u al Sys ems, Uni e si y Pablo de Ola ide, C a. U e a km 1, 41013, Se illa, Spain
c
Ins i u e o Chemical Resea ch (IIQ), CSIC-Uni e si y o Se ille, A da. Am´
e ico Vespucio 49, 41092, Se illa, Spain
ARTICLE INFO
Keywo ds:
Zeoli ic-imidazola e amewo ks
Glycomime ics
Glycolipids
De no o encapsula ion
Elec onic s uc u e calcula ions
ABSTRACT
The e icien encapsula ion o la ge ca bohyd a es in o po ous me al-o ganic amewo ks (MOFs), and no simply
a ached o he MOF’s su ace, is s ill challenging and unde explo ed. In his wo k we ha e in es iga ed he scope
o an op imized syn he ic p ocedu e ollowing a biomime ic mine aliza ion s a egy o he encapsula ion o a
a ie y o he apeu ic glycolipids wi hin a Zeoli ic-Imidazola e F amewo k-8 nanos uc u e (GlycoZIFs). In all
cases, ega dless o he glycosidic linkage na u e o he glycolipid, we ob ained uni o m, c ys alline and
ep oducible GlycoZIFs nanopa icles by using he same op imized expe imen al condi ions, which demons a e
he e sa ili y o ou app oach. Ou expe imen al da a e ealed ha he o ma ion o glyco-micelles, by aking
ad an age o he su ac an -like cha ac e o hese glycolipids, is key o p omo e he nuclea ion o ZIF-8 a ound,
allowing hus a p ecise con ol o he spa ial loca ion and amoun o glycod ug encapsula ed in each ZIF-pa icle.
In u n, he elec onic s uc u e calcula ions showed ha he e is a s ong in e ac ion be ween he hyd oxyl
g oups in posi ions C3 and C4 o he glycone co e o he glycolipid and he Zn a oms on he ZIF-8 su ace,
sugges ing ha hose a ou able glyco-ZIF in e ac ions also played an impo an ole o induce he ZIF-8
nuclea ion. Expe imen al con ol da a and compu a ional s udies ob ained wi h a p o ec ed glycolipid
ea u ing O-ace yl g oups suppo ed ha conclusion.
1. In oduc ion
The implemen a ion o e icien syn he ic s a egies aimed a
designing glycomime ic d ugs making use o a chemically e sa ile
amewo k e med sp
2
-iminosuga , has gi en ise o an ex ensi e
collec ion o me abolically s able sp
2
-iminoglycolipids (sp
2
-IGLs) wi h
immunomodula o y capabili ies. They p esen ema kable ac i i y
agains pa hological complica ions de i ed om in lamma o y e en s (i.
e., diabe ic e inopa hy and neph opa hy, as hma, and acu e in lam-
ma ion), p oli e a ion o umo cells o e en in ec ions caused by pa -
asi es [1]. In he con ex o in lamma ion-associa ed diseases, an
ex emely complex egula o y ne wo k akes place du ing he in lam-
ma o y p ocess, in ol ing wo ypes o cellula esponses, p oin-
lamma o y and an iin lamma o y, which a e ac i a ed by comple ely
di e en signals. An imbalance o bo h esponses leads o dys egula ion
o he immune sys em, igge ing he in lamma o y esponse. Se e al
wo ks ha e poin ed ou he ole o some sp
2
-IGLs as modula o s o he
immune esponse, esul ing in a dec ease o he exp ession o p oin-
lamma o y cy okines along wi h he concomi an inc ease o an iin-
lamma o y ma ke s [1]. In he con ex o cance , sp
2
-IGLs demons a ed
o educe bo h he p oli e a ion and he mig a ion o malignan cells
suppo ing hei abili ies as an imi o ic, p oapop o ic, and an i-
me as a ic agen s. Likewise, he g ow h inhibi ion o in acellula o ms
o he pa asi e Leishmania dono ani by some sp
2
-IGLs aised hei po-
en ial as an ileishmanial agen s.
The p i ileged s uc u e o he sp
2
-iminosuga s, ca bohyd a e-based
analogues ea u ing a pseudoamide- ype unc ionali y wi h sp
2
-hyb id-
a ion, makes i a sui able sca old o pe o m N-, C-, O-, S-, Se-glyco-
sida ion eac ions by di e en and e icien syn he ic me hodologies [2,
3]. In ecen yea s, b owsing he aglycone space o hese amphiphilic
* Co esponding au ho .
** Co esponding au ho .
E-mail add esses: [email p o ec ed] (E.M. S´
anchez-Fe n´
andez), [email p o ec ed] (C. Ca illo-Ca i´
on).
1
Equal con ibu ion.
Con en s lis s a ailable a ScienceDi ec
Ma e ials Today Chemis y
jou nal homepage: www.jou nals.else ie .com/ma e ials- oday-chemis y/
h ps://doi.o g/10.1016/j.m chem.2025.102546
Recei ed 24 Sep embe 2024; Recei ed in e ised o m 30 Decembe 2024; Accep ed 22 Janua y 2025
Ma e ials Today Chemis y 44 (2025) 102546
A ailable online 31 Janua y 2025
2468-5194/© 2025 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY-NC license ( h p://c ea i ecommons.o g/licenses/by-
nc/4.0/ ).
molecules by nume ous s uc u e-biological ac i i y ela ionship s udies
ha e allowed us o selec some sp
2
-IGL-based immunomodula o y gly-
cod ugs bea ing a hyd ophobic ail, speci ically a dodecyl lipid chain
(C
12
H
25
) a ached o di e en unna u al glycosidic connec o s (sul ox-
ides, sul ones, selenou eas, hiou eas, among o he s) [4,5]. The la ge
numbe o pa hological p ocesses in which sp
2
-IGLs can in e ene,
p o iding signi ican pha macological bene i s, makes hem an
ou s anding g oup o s able chemical and s uc u al ca bohyd a e mi-
me ics wi h g ea added alue. Two ep esen a i e examples o his
amily o sp
2
-glycolipids selec ed by hei ema kable he apeu ic
p ope ies in in lamma o y in i o models a e depic ed in Scheme 1A
(compounds 1 [6] and 2 [7]). The body o esul s achie ed om
di e en p eclinical models spanning om in i o, ex i o and in i o
assays, ce ainly e lec s he bene i s igge ed by a a ie y o sp
2
-IGLs
agains dele e ious e ec s p o oked by ad e se in lamma o y p ocesses
[1,8,9].
Howe e , a cons aining ac o o hese glycod ugs, and also
equen ly ound in o he amphiphilic pha macological agen s, is hei
low solubili y in aqueous solu ions and biological luids, es ic ing hei
in i o sys emic bioa ailabili y. Inc easing d ug doses ha o se he
poo biodis ibu ion is no conside ed a sui able op ion due o he po-
en ial unwan ed e ec s, hence d ug solubili y enhancemen has
become a challenging ask o he scien i ic communi y o e he yea s
[10]. The use o nanopa icle (NP)-based echnology ha enables mo e
e icien deli e y o he bioac i e loaded compound is well ecognized as
an appealing he apeu ic app oach. Among he di e se ypes o NPs used
as nanoca ie s (lipid-based NPs, polyme ics, lipid-polyme hyb ids,
ino ganics), me al-o ganic amewo ks (MOFs)-based d ug deli e y
sys ems (DDSs) ha e ecen ly achie ed p ominen accomplishmen s in
he biomedicine ield when p epa ed on a nanome ic scale (nanoMOFs)
[11–13]. The unique ea u es o some amilies o nanoMOFs (e.g., high
d ug loading capaci y, good biocompa ibili y, unable po osi y and
chemical composi ion, con olled and unable bios abili y o minimize
hei in i o accumula ion) le hem o be conside ed one o he mos
p omising candida es o de eloping in acellula DDSs [13].
Al hough a g ea numbe o small he apeu ic molecules has been
succes ully loaded in o MOFs o acili a e cell-up ake and a ain
imp o ed he apeu ic p ope ies [14], he encapsula ion o la ge bio-
molecules (e.g., p o eins, nucleic acids, ca bohyd a es) in a p ecise and
con olled way is no an easy endea ou . Symme y de ec and labile
me al-ligand coo dina ion a e he main obs acles o syn hesis o
well-o de ed c ys alline ma e ials inco po a ing biomolecules. Signi i-
can ad ances ha e been achie ed in he las yea s o he encapsula ion
o p o eins and nucleic acids [14–17]; howe e , he encapsula ion o
ca bohyd a es has been spa sely epo ed so a . To he bes o ou
knowledge, only Falca o and cowo ke s ha e inco po a ed a a ie y o
glycosaminoglycan (GAG)-based d ugs in me al-azola e amewo ks
[18]. They obse ed ha a e encapsula ing he GAGs, he c ys allini y
and con ol on he pa icle size o he esul ing biocomposi es we e
se iously comp omised, which may impose signi ican limi a ions in
hei u he he apeu ic applica ions. Using a di e en syn he ic s a -
egy, ou g oup has ecen ly epo ed he con olled encapsula ion o a
glycolipid wi hin ZIF-8, speci ically a selenium-based sp
2
-IGL (com-
pound 1, Scheme 1A), wi h a p ecise con ol on he spa ial dis ibu ion
o he glycod ug [19]. Ou me hod o he p epa a ion o 1@ZIF nano-
pa icles was ca ied ou unde mild condi ions a oom empe a u e
(RT) and in wa e , p e en ing hus he po en ial deg ada ion o inac i-
a ion o he bioac i e glycolipid mime ics du ing hei encapsula ion,
and p o ec ing hem as hey a el o a ge cells. In addi ion, by
exploi ing he pH-dependen deg ada ion o he ZIF-8 s uc u e, we
a ained an e icien in acellula deli e y o he glycod ug unde he
acidic condi ions inside endosomes upon up ake. This allowed us o
demons a e, o he i s ime, he syne gis ic an ioxidan e ec o
1@ZIF nanopa icles in human endo helial cells. No ably, 1@ZIF was
also able o e e se, o a ce ain le el, he oxida i e s ess induced in
cells, which was bene icial o p ese ing c i ical endo helial unc ions
such as angiogenesis and cell mig a ion [19].
Encou aged by his ecen achie emen , he ein we se ou o explo e
whe he he syn he ic me hodology applied o he encapsula ion o he
selenium-con aining glycomime ic 1 in ZIF-8 was ex ensible o o he
iminosuga -based d ugs, aimed o in es iga e he e sa ili y o ou
op imized app oach o he syn hesis o a collec ion o di e se GlycoZIFs
wi h no ewo hy he apeu ic applica ions. The selec ed sp
2
-IGLs (com-
pounds 1–5, Scheme 1A) ea u e di e en glycosidic linkage- ypes (i.e.,
selenou ea, sul u a om and sul one unc ionali y), and also p esen
di e ences in hei glycone moie y, speci ically he lack o hyd oxyl
g oup in C2 posi ion in compounds 3 and 4 ( e med 2-deoxy-sp
2
-IGLs),
and he blocking o hyd oxyl g oups in C3/C4 posi ions by O-ace yl
g oups (compound 5). Wi h his se o glycolipids, we will be able o
e alua e he in luence o bo h he aglycone na u e and he saccha ide
esidue on he physico-chemical p ope ies o he syn hesized GlycoZIFs.
Combining expe imen al and compu a ional s udies, we plan o in es-
iga e he encapsula ion mechanism o he glycolipid in o he ZIF-
s uc u e and o de ine he key s uc u al ea u es equi ed o he suc-
cess ul o ma ion o he GlycoZIFs, which will allow us o p edic , in he
nex u u e, he possible ex ension o he me hod o o he amilies o
ca bohyd a e-based d ugs.
Scheme 1. (A) Gene al ep esen a ion o he sp
2
-iminosuga -based glycod ugs, and speci ic chemical s uc u es o he glycomime ics s udied: sp
2
-IGLs (1, 2), 2-
deoxy-sp
2
-IGLs (3, 4) and di-O-ace yla ed-2-deoxy-sp
2
-IGL (5). (B) Schema ic ep esen a ion o he one-po syn hesis o GlycoZIFs nanopa icles, indica ing he main
objec i es planned in his wo k.
R. Rod íguez-Ma ín e al.
Ma e ials Today Chemis y 44 (2025) 102546
2
2. Ma e ials and me hods
2.1. Syn he ic p ocedu e o he p epa a ion o glycod ugs
Full s uc u al cha ac e iza ion o new 2-deoxy-sp
2
-IGLs is desc ibed
in de ail in he Suppo ing in o ma ion (Scheme S1, Figs. S1–S3). The
ollowing compounds: (1S)-(N
′
-dodecylselenou eido)-5N,6O-oxome hy-
lidenenoji imycin (1) [6], (1R)-1-dodecylsul onyl-5N,6O-oxome hyli-
denenoji imycin (2) [20], 3,4-di-O-ace yl-5N,6O-(oxome hylidene)
noji imycin iminoglycal [21] and (1R)-3,4-di-O-ace yl-2-deox-
y-1-S-dodecyl-5N,6O-oxome hylidene-1- hionoji imycin (5) [22], we e
p epa ed acco ding o p e iously epo ed p ocedu es. The wo no el
2-deoxy-
α
-glycomime ics, 3 and 4, we e success ully syn hesized
ollowing he op imized syn he ic p o ocol epo ed by ou g oup o he
s e eoselec i e syn hesis o iminosuga 2-deoxy- hioglycosides om he
bicyclic sp
2
-iminoglycal ca bama e p omo ed by ce ium(IV) ammonium
ni a e in he p esence o dodecane-1- hiol [22]. Nex , oxida ion o he
sul u a om o he esul ing di-O-ace yla ed-2-deoxy-
α
-S-glycoside (5)
employing an excess o me a-chlo ope benzoic acid (MCPBA) p o ided
he
α
-glycosylsul onyl de i a i e. Subsequen dep o ec ion eac ions
unde basic condi ions o he oxidized sp
2
-IGL and i s p ecu so hio-
e he led o he a ge ully unp o ec ed compounds 3 and 4 in excellen
yields (see ESM o de ails, Scheme S1). The chemical s uc u es o he
as-syn hesized 2-deoxy-sp
2
-IGLs we e con i med by nuclea magne ic
esonance (NMR), mass spec ome y (MS) and elemen al analysis (see
ESM). As expec ed, analysis o he coupling cons an s (J
1,2a
~ 5–7 Hz)
e ealed he exclusi e o ma ion o he
α
-anome s in bo h cases due o
he p onounced anome ic e ec p esen in his amily o compounds [1].
2.2. Syn hesis o GlycoZIFs
The co esponding GlycoZIFs we e p epa ed ollowing he expe i-
men al p ocedu e epo ed ecen ly by ou g oup [19]. B ie ly, 3 mL o
an aqueous solu ion o zinc ni a e (Zn(NO
3
)
2
⋅6H
2
O; 25 mM) was added
o e 3 mL o an aqueous solu ion o 2-me hylimidazol (HmIM; 1.3 M) a
RT unde con inuous s i ing (350 pm). Nex , 0.6 mL o a me hanolic
solu ion o he co esponding sp
2
-IGL (1–4; 10 mM) was added d opwise
unde s i ing. The esul ing mix u e was s i ed o 2 min and le o
s and o 2 h. The appea ance o a homogeneous u bidi y o e ime is
indica i e o he o ma ion o he GlycoZIFs pa icles. A e 2 h, he
pa icles we e collec ed by cen i uga ion (12,000 c , 15 min) and
washed h ee imes wi h me hanol (MeOH). Finally, he pa icles we e
edispe sed in MeOH a a concen a ion o 10 mg/mL and s o ed in he
idge (4 ◦C) un il use. The as-p epa ed GlycoZIFs we e named 1@ZIF,
2@ZIF, 3@ZIF, 4@ZIF. The same expe imen al condi ions we e
employed using he p o ec ed sp
2
-IGL (1R)-3,4-di-O-ace yl-2-deox-
y-1-S-dodecyl-5N,6O-oxome hylidene-1- hionoji imycin (5) o a o d
5@ZIF.
2.3. Syn hesis o ZIF-8 pa icles as con ols
Two ypes o ZIF-8 con ol pa icles we e syn hesized, ZIF-8 con ol 1
(ZIF-8/Cn 1) and ZIF-8 con ol 2 (ZIF-8/Cn 2), ollowing he same
expe imen al p ocedu e desc ibed abo e bu eplacing he sp
2
-IGL so-
lu ion by he addi ion o 0.6 mL o MeOH in he case o ZIF-8/Cn 1 o 3
mL o an aqueous solu ion o ce yl ime hylammonium b omide (CTAB;
2.0 ×10
−3
M) in he case o ZIF-8/Cn 2. In bo h cases, he pu i ica ion
s ep was iden ical as ha pe o med wi h he co esponding GlycoZIFs.
ZIF-8/Cn 1 and ZIF-8/Cn 2 pa icles we e inally edispe sed in MeOH
a a concen a ion o 10 mg/mL and s o ed in he idge (4 ◦C) un il use.
2.4. Gene al in o ma ion o cha ac e iza ion o GlycoZIFs
T ansi ion Elec on Mic oscopy (TEM) images we e acqui ed using a
JEOL TEM 1400 ope a ed a 200 kV. Samples we e p epa ed by d ying a
dilu ed dispe sion o he pa icles on 200 mesh coppe g ids coa ed wi h
Fo m a /ca bon ilm.
Measu emen s o dynamic ligh sca e ing (DLS) and ze a-po en ial
(ζ-po en ial) we e pe o med using a Mal e n Ze asize Nano ZSP
equipped wi h a 10 mW He–Ne lase ope a ing a a wa eleng h o 633
nm and ixed sca e ing angle o 173◦. Fo DLS analysis, dilu ed samples
we e loaded in o a qua z cu e e and h ee measu emen s, each con-
sis ing o wel e da a uns, we e aken a RT a e an equilib a ion s ep
o 120 s. The ζ-po en ial o he nanopa icles dispe sed in Milli-Q wa e
was measu ed wi h lase Dopple anemome y (LDA) by using he same
Mal e n Ze asize Nano ZSP ins umen .
High-pe o mance liquid ch oma og aphy-mass spec ome y
(HPLC-MS) analysis we e done using a Wa e s Alliance 2695 HPLC
coupled o an ESI-ion ap mass spec ome e ins umen (B uke
AmaZon). Samples we e analyzed using 0.1 % o mic acid elu ing g a-
dien s a a low a e o 0.3 mL/min. Spec a we e egis e ed in bo h
posi i e and nega i e modes in he m/z 100–2000 ange.
Powde X-Ray Di ac ion (PXRD) was pe o med using a B uke D8-
Ad ance Di ac ome e . X- ay adia ion o Cu K
α
was used, and he
measu emen ange was om 5◦ o 70◦(2θ) wi h a s ep o 0.02◦(2θ).
1
H NMR spec a o he GlycoZIFs we e eco ded in CD
3
OD using a
400 MHz B uke A ance III HD spec ome e .
N
2
so p ion iso he ms (77 K) o powde samples we e ca ied ou in a
Mic ome i ics T is a II 3020 sys em. Be o e analysis, samples we e
degassed unde acuum o 18 h a 120 ◦C. The appa en su ace a eas
we e calcula ed om he Ba e –Emme –Telle (BET) me hod in he
p essu e in e al P/P
o
=0.01–0.3 (being P
o
he sa u a ion p essu e).
Po e olume and ex e nal su ace a ea we e calcula ed by he -plo
me hod. The po e size dis ibu ions o mesopo es we e calcula ed om
he deso p ion b anch o he iso he m using he Ba e –Joyne –Halenda
(BJH) me hod.
The mog a ime ic Analysis (TGA) o powde samples was pe -
o med using a The mal Ad an age SDT-600 ins umen wi h a gene al
hea ing p o ile om 30 o 650 ◦C and using a hea ing a e o 5 ◦C/min
unde ai in a low o 100 mL/min.
2.5. De e mina ion o c i ical micelle concen a ion (CMC) o glycod ugs
The CMC o he s udied sp
2
-IGL compounds was de e mined by DLS.
Measu emen s we e ca ied ou in a glass cu e e a 25 ◦C and epea ed
h ee imes. A se ies o solu ions anging om 0.02 o 1.0 mM was
p epa ed in Milli-Q wa e om a me hanolic s ock solu ion o sp
2
-IGL
(10 mM). The in ensi y alues o sca e ed ligh as a unc ion o con-
cen a ion o he glycomime ics we e depic ed (see ESM o de ails).
The sca e ing in ensi ies de ec ed o sp
2
-IGL concen a ions below
CMC ga e an app oxima ely cons an alue co esponding o ha o
wa e . The in ensi y s a ed o show a linea inc ease wi h concen a ion
a he CMC, since he numbe o micelles inc eased in he solu ion. The
in e sec ion o bes i lines d awn h ough he da a poin s co esponds
o he CMC alue. Fu he e idence o micelle o ma ion came om he
co ela ion unc ion cu es, whe e in e cep s o co ela ion unc ions
became much highe (>0.7) a e micelles o ma ion.
2.6. Compu a ional me hods
Tigh -Binding Densi y Func ional Theo y calcula ions we e ca ied
ou using he DFTB+( . 24.1) code [23], wi hin he GFN2-xTB Hamil-
onian [24], which is he i s pa ame ized igh -binding me hod o
include elec os a ic in e ac ions and exchange-co ela ion e ec s up o
second o de in he mul ipole expansion, as well as he D4 G imme
dispe sion model [25]. As a model o he ZIF-8 su ace we used he
(100) su ace, which is he mos s able. The su ace dipole was emo ed
by ans e ing wo imidazola e a oms om he op o he bo om o he
model su ace. The plana models o he micelles we e c ea ed using he
Packmol code [26].
Fo all sys ems s udied, in o de o accommoda e he eac i e unc-
ional g oups o he glycolipid molecules nea he eac ion si es, we i s
R. Rod íguez-Ma ín e al.
Ma e ials Today Chemis y 44 (2025) 102546
3
ca ied ou es ained Molecula Dynamics (MD) simula ions, using he
Plumed code [27], in oducing linea and ha monic es ain s on he
dis ance be ween he po en ially eac i e O a oms ( hose in he OH
g oups in sys em 1 and in he O-ace yl g oups in sys em 2). The
es aining ene gy (δE) is gi en by: δE=∑ijk
2(dij −a)2+m(dij −a),
whe e k=25 kJ/(mol⋅
˚
A2), m=25 kJ/(mol ⋅
˚
A), a=2.7˚
A, and d is he
dis ance be ween he eac i e O a om and he supe icial Zn a om. In
o de o acili a e molecula eassembling, he empe a u e was se o
450 K. The p essu e was 1 ba . In all MD simula ions he imes eps we e
se o 0.5 s, and Nose-Hoo e ba os a s (wi h a 10 s ime cons an ) and
he mos a s (wi h a 100 s ime cons an ) we e employed o ca y ou he
NPT MD simula ions. The simula ions we e un o 10 ps. The las
con igu a ions we e ene gy-minimised wi h RFO algo i hms. Once he
eac i e unc ional g oups we e close o he eac ion si es, we emo ed
he es ain s and pe o med NPT MD simula ions, o 100 ps, s a ing
om he ene gy-minimised s uc u es, a a p essu e o 1 ba and em-
pe a u e o 298 K.
3. Resul s and discussion
3.1. Syn hesis o ZIF-based nanos uc u es con aining glycod ugs
(GlycoZIFs)
Being ully awa e ha solubili y issues in in i o sys ems o he
a o emen ioned amphiphilic sp
2
-glycod ugs may diminish hei
ex ao dina y in insic he apeu ic ac i i y owa ds di e en pa hol-
ogies, we conside in his wo k he possibili y o expanding he ZIF-8-
based nano echnological app oach epo ed o compound 1. Ex end-
ing he me hodology o o he sp
2
-IGLs would allow us o imp o e he
in acellula deli e y o each o hese glycod ugs, and he eby maximize
hei he apeu ic bene i s. To explo e he scope o he encapsula ion
app oach, we selec ed ou sp
2
-IGLs as a ge candida es (1–4, Scheme
1A), being 1 he compound al eady used in ou p e ious wo k [19].
Since addi ional cha ac e iza ion o 1@ZIF ha e been pe o med in his
wo k, we ha e included i h oughou he manusc ip . F om he chem-
ical poin o iew, he
α
-glycomime ics examined in his s udy showcase
no able di e ences in hei s uc u al ea u es no only ega ding he
α
-glycosidic connec o - ype (S, SO
2
o SeC(NH)
2
), bu also in he glycone
co e due o he eplacemen o he hyd oxyl g oup loca ed in C2 posi ion
by a hyd ogen a om in he pipe idine esidue, compounds 1 and 2 s
2-deoxy-de i a i es 3 and 4 (Scheme 1A).
Once we p epa ed he selec ed sp
2
-IGLs, he co esponding Glyco-
ZIFs we e success ully syn hesized by ollowing a de no o o biomime ic
mine aliza ion encapsula ion app oach as p e iously epo ed o com-
pound 1 [19]; see he de ailed expe imen al p ocedu e in sec ion 2.2.
The as-p epa ed GlycoZIFs we e e med 1@ZIF, 2@ZIF, 3@ZIF and
4@ZIF, which s and o he pa icles con aining he sp
2
-IGLs 1–4. We did
no obse e isual changes in he encapsula ion p ocess wi h he
di e en glycolipids, de ec ing in all cases he g adual inc ease in
u bidi y o e ime as he GlycoZIF c ys als g ew. Fu he mo e, HPLC
analyses o he supe na an s a e pu i ica ion o GlycoZIFs con i med
he e icien encapsula ion o he glycod ugs in all cases, ob aining e y
simila alues o encapsula ion e iciencies (96–98 %) and loading ca-
paci ies (3–4 w %). Also, he e med ZIF-8 con ol 1 (ZIF-8/Cn 1) and
ZIF-8 con ol 2 (ZIF-8/Cn 2) we e p epa ed in absence o glycod ug o
compa ison. Fo ZIF-8/Cn 1, he syn hesis was pe o med employing he
same op imized expe imen al condi ions bu eplacing he co espond-
ing sp
2
-IGL by me hanol, o keep he amoun o me hanol cons an in he
inal eac ion mix u e. No e ha he glycod ug s ock solu ion was p e-
pa ed in me hanol o solubili y easons. In his case, la ge pa icles
we e ob ained due o he absence o he amphiphilic glycolipid ha ac s
as a modula o agen du ing he c ys al o ma ion. Howe e , he size o
he pa icles is decisi e in hei biological pe o mance, since i a ec s
he cellula up ake p ocess and in acellula s abili y, so i is desi able o
ha e con ol pa icles (wi hou loaded d ug) wi h a size simila o ha o
he he apeu ic nanosys em. In his line, ZIF-8/Cn 2 was also p epa ed,
whe e he sp
2
-IGL was eplaced by he su ac an CTAB as a size con-
olling agen , allowing he pa icle size o be con olled (<100 nm) so
ha i was simila o ha o GlycoZIFs. Nex , he esul ing GlycoZIFs and
con ol pa icles we e analyzed o de e mine hei physico-chemical
p ope ies using di e en echniques: DLS, TEM, PXRD, NMR, N
2
physiso p ion and TGA.
3.2. Mo phological and s uc u al cha ac e iza ion o GlycoZIFs
The hyd odynamic diame e (d
h
) o he GlycoZIFs pa icles, 1@ZIF,
2@ZIF, 3@ZIF and 4@ZIF, dispe sed ei he in MeOH (Fig. 1A) o Milli-Q
wa e (Fig. 1B) was measu ed by DLS analysis. I is wo hwhile no ing
ha he edispe sion o he GlycoZIFs in wa e did no signi ican ly
modi y he hyd odynamic size. The esul s (Table S1, ESM) e ealed ha
all GlycoZIFs, ega dless o bo h hei s uc u al di e ences and he
sol en employed, p esen ed e y simila hyd odynamic sizes (ca. 80
nm), and impo an ly, a he low polydispe si y index (PDI), which was
indica i e o a homogenous popula ion o pa icles. Compa ison o in-
ensi y, olume and numbe -weigh ed size dis ibu ions ob ained o he
di e en GlycoZIFs pa icles showed small di e ences as expec ed due
o he na ow size dis ibu ions (Fig. S4). The su ace cha ge o hese
GlycoZIFs dispe sed in wa e and in a phospha e bu e (PB) solu ion
(0.01 M, pH =7.4) was also s udied by ζ-po en ial measu emen s
(Fig. 1C). The alues o ζ-po en ial o he GlycoZIFs dispe sed in wa e
we e ca. 10 mV, while GlycoZIFs dispe sed in a PB solu ion (0.01 M, pH
=7.4) exhibi ed nega i e cha ge, ca. −30 mV, likely due o he coo -
dina ion o he HPO
4
2−
o he Zn
2+
in he GlycoZIF su ace. No signi ican
changes in he su ace cha ge o GlycoZIFs compa ed o he con ol
nanosized ZIF-8 pa icles (ZIF-8/Cn 2) in any o he es ed media
con i med ha glycod ug molecules we e indeed encapsula ed wi hin
he ZIF-8 s uc u e and no adso bed on he su ace o he pa icles.
In o de o check possible di e ences in he mo phology (shape and
size) and he homogenei y o he GlycoZIF nanopa icles depending on
he sp
2
-IGL encapsula ed, he pa icles we e examined unde he mi-
c oscope. Rep esen a i e TEM images o 1@ZIF, 2@ZIF, 3@ZIF, 4@ZIF
and he con ols (ZIF-8/Cn 1 and ZIF-8/Cn 2) a e shown in Fig. 2. These
esul s e ealed ha he sp
2
-IGLs molecules we e inco po a ed o ming
micelles which a e loca ed oughly in he cen e o he s uc u e. This
ac seems o indica e ha , unde he op imized expe imen al condi-
ions, he micelles p esen in he medium du ing he syn hesis (i.e.,
aqueous mix u e con aining he p ecu so s) ac as seeds o he subse-
quen o ma ion o a ZIF-8 shell a ound hem. No ably, small di e ences
in he s uc u al ea u es o he sp
2
-IGLs led o some changes in he inal
mo phology o he GlycoZIFs pa icles. In his ega d, some GlycoZIFs
had a cubic mo phology wi h ounded co ne s (1@ZIF, 2@ZIF), while
o he s p esen ed a quasi-sphe ical shape (3@ZIF, 4@ZIF). Con ol ZIF-
8/Cn 1 pa icles showed he ypical dodecahed al shape, while he
con ol ZIF-8/Cn 2 p esen ed a cubic shape, as expec ed when using
CTAB as su ac an agen [19]. This inding is no su p ising since p e-
iously desc ibed esul s ha e al eady shown ha di e en mo phol-
ogies can be achie ed by using di e en modula o s agen s (su ac an s
o o ganic amines) o by a ying he concen a ion o hese modula o s
in he mo he solu ion [28].
Nex , he c ys allini y o he GlycoZIFs was analyzed by PXRD
(Fig. 3), con i ming he cha ac e is ic ZIF-8 single-phase sodali e o-
pology c ys allini y o all he GlycoZIFs ega dless o he s uc u al
di e ences o he encapsula ed sp
2
-IGL. As obse ed, all he di ac ion
peaks i well wi h he simula ed ZIF-8 and wi h bo h ZIF-8 con ols,
which e eal ha he encapsula ion o sp
2
-IGL micelles wi hin he
s uc u e did no a ec signi ican ly he c ys allini y o he ZIF-8 shell
g own a ound hem. Howe e , a sligh b oadening o some di ac ion
peaks in all GlycoZIFs was also e iden . This ac could be ela ed o
some s uc u al de ec s wi hin he nanos uc u e as esul o he inco -
po a ion o he glycomime ics, as well as due o he smalle nanopa icle
size o he GlycoZIFs (d
h
~ 75–89 nm) compa ed o ZIF-8/Cn 1 (d
h
~
R. Rod íguez-Ma ín e al.
Ma e ials Today Chemis y 44 (2025) 102546
4
509–515 nm) and ZIF-8/Cn 2 (d
h
~ 103–110 nm) (Table S1).
Likewise, i espec i e o ei he he unc ionali y a he glycosidic
linkage o he p esence/absence o he hyd oxyl g oup loca ed in C2 o
he glycone moie y,
1
H NMR analyses allowed us o co obo a e ha
hese
α
-glycomime ics we e no adso bed on he su ace o he
nanopa icles bu encapsula ed inside he ZIF-8 amewo k, as p e i-
ously epo ed o he hyb id 1@ZIF [19]. To his end,
1
H NMR spec a
o compounds 2, 3 and 4, dissol ed in CD
3
OD, and hei co esponding
GlycoZIFs (2@ZIF, 3@ZIF and 4@ZIF) dispe sed in CD
3
OD, we e
eco ded a 400 MHz and compa ed o hose
1
H NMR spec a ob ained
Fig. 1. DLS numbe dis ibu ions o d
h
o GlycoZIFs and con ol ZIF-8 pa icles as dispe sed in (A) MeOH o (B) Milli-Q wa e . (C) ζ-po en ial o GlycoZIFs and
con ol pa icles dispe sed in ei he wa e o PB (0.01 M, pH =7.4).
R. Rod íguez-Ma ín e al.
Ma e ials Today Chemis y 44 (2025) 102546
5

a e dissol ing he GlycoZIF-based nanos uc u es unde he p esence
o dilu ed sul u ic acid. The same p o ocol was ollowed wi h
ZIF-8/Cn 1. Be o e he acid dissolu ion o he pa icles,
1
H NMR spec a
o all he GlycoZIFs showed wo signals co esponding o he p o ons o
HmIM and no signals a ibu able o he p o ons o ei he he glycone
co e o he alipha ic linea chain. This esul ag ees wi h he inco po-
a ion o hese glycomime ics wi hin he igid and solid ZIF-8 shell,
being hei esonance p e en ed when a magne ic ield is applied.
Howe e , when 2@ZIF, 3@ZIF and 4@ZIF nanos uc u es we e dis-
sol ed, due o he b eaking o Zn–N bonds unde acid expe imen al
condi ions, ep esen a i e signals o p o ons belonging o he eleased
sp
2
-IGLs we e iden i ied. As a ep esen a i e example,
1
H NMR analyses
o 2@ZIF a e depic ed in Fig. 4.
1
H NMR spec a o 3@ZIF, 4@ZIF and
ZIF-8/Cn 1 a e shown in he ESM (Figs. S5–S8). These esul s o all he
sp
2
-IGLs unde s udy clea ly e eal hei success ul encapsula ion in o
he ZIF-8 c ys alline s uc u e.
To e alua e he e ec on he po osi y o ZIF-8 a e he encapsula ion
o 1, 2, 3 and 4, N
2
so p ion iso he ms o he GlycoZIFs we e measu ed
(Fig. 5). Whe eas ZIF-8/Cn 1 and ZIF-8/Cn 2 pa icles p esen ed a
e e sible ype I iso he m, ypical o mic opo ous ma e ials, GlycoZIFs
displayed ype I/ ype IV iso he ms wi h a hys e esis loop a high ela i e
p essu es, sugges ing he p esence o mesopo ous. The B u-
naue −Emme −Telle (BET) appa en su ace a ea (S
BET
) dec eased
no ably in GlycoZIFs (Table S2), which is a ibu ed o he inco po a ion
o sp
2
-IGLs wi hin he s uc u e. Anyway, all he GlycoZIFs showed e y
simila ex u al p ope ies (mic opo e a ea (S
mic o
), ex e nal su ace a ea
(S
ex
), mic opo e olume (V
mic o
) and mesopo e olume (V
meso
), see
Table S2 o de ails), con i ming again he simila beha iou o he
di e en s udied glycolipids. The signi ican dec ease in he mic opo e
a ea accompanied wi h he no able inc ease o ex e nal su ace a ea may
be a ibu ed o he p esence o glyco-micelles inside he amewo k.
No e ha he inclusion o d ugs as indi idual compounds (no o ming
assemblies o micelles) does no usually lead o such a la ge inc ease in
he ex e nal su ace a ea. BJH po e size dis ibu ions cu e o GlycoZIFs
(Fig. S9 and Table S2) showed ha mesopo ous we e p esen wi hin he
s uc u e. The e o e, hese esul s seem o indica e ha he sp
2
-IGLs
we e encapsula ed in o ZIF-8 in he o m o micelles, consis en wi h
TEM obse a ions, and he eby p oduced some mesopo osi y while
p ese ing he mic opo osi y o ZIF-s uc u e o a la ge ex en .
Nex , he he mal beha iou o he GlycoZIFs was also e alua ed
(Fig. S10). The TGA cu e o con ol ZIF-8/Cn 1 pa icles ma ched well
wi h he epo ed li e a u e [29], wi h a sha p weigh loss a a ound
440 ◦C ha co esponds o he ans o ma ion om ZIF-8 in o ZnO.
No ably, he encapsula ion o sp
2
-IGLs led o changes in TGA p o iles o
he co esponding GlycoZIFs, showing an ea ly mass loss a ~300 ◦C,
which could be a ibu ed o he decomposi ion o sp
2
-IGLs molecules
encapsula ed wi hin he ZIF-s uc u e.
Finally, he elease o glycod ugs om he co esponding GlycoZIFs
was s udied unde wo biological condi ions, i.e., ex acellula and
in acellula en i onmen s, which di e in he pH o he su ounding
media. To do his, he GlycoZIFs we e incuba ed o 24 h in ei he T is
bu e solu ion a pH =7.4 (emula ing ex acellula condi ions) o in
ace a e bu e solu ion pH =4.5 (emula ing condi ions inside he
endosomes), and we quan i ied he amoun o glycod ug (1, 2, 3 o 4)
Fig. 2. Rep esen a i e TEM images o GlycoZIFs: 1@ZIF, 2@ZIF, 3@ZIF, 4@ZIF, and con ol pa icles: ZIF-8/Cn 1 and ZIF-8/Cn 2. No e ha in some cases o mo e
clea ly obse e he micelle inside he pa icle i is necessa y o ake he image ou o ocus.
R. Rod íguez-Ma ín e al.
Ma e ials Today Chemis y 44 (2025) 102546
6
deli e ed o he medium by HPLC. As shown in Fig. S11, he e was a
success ul elease a pH =4.5 o all GlycoZIFs, eaching be ween 74 and
79 % a e 24 h, while he amoun o glycod ug eleased was minimal
(<10 %) a pH =7.4. These esul s we e expec ed due o he pH-
dependan s abili y o he ZIF-8, as he Zn–N coo dina ion bonds a e
b oken a acidic pH, esul ing in he dissolu ion o he pa icles and
subsequen elease o he encapsula ed ca go. Taking ad an age o his
pH- esponsi eness o he GlycoZIFs, we could achie e a con olled
in acellula deli e y o he a ge glycod ugs, sol ing hei solubili y
limi a ions and inc easing he amoun o glycod ug eaching he in e io
o cells, which is an icipa ed o esul in be e he apeu ic e iciency.
3.3. Mechanis ic hypo hesis o he o ma ion o GlycoZIFs
All he abo e cha ac e iza ion esul s oge he demons a e ha he
s udied sp
2
-IGLs 1–4 p esen an analogous beha iou wi h ega d o
hei inco po a ion in o he ZIF-8 s uc u e du ing c ys al o ma ion,
also gi ing ise o GlycoZIFs wi h e y simila physical-chemical p op-
e ies. On his basis, we could in e ed ha he common sp
2
-iminosuga
pola head and he hyd ophobic alkyl chain a e key s uc u al mo i s
ha allow he success ul p epa a ion o GlycoZIFs’ s uc u es. No ably,
he na u e o he
α
-glycosidic connec o o he lack o he –OH g oup
loca ed in C2 posi ion o he glycone co e we e ound o be i ele an o
he GlycoZIFs o ma ion. Based on he expe imen al esul s achie ed so
a , we aimed o disclose he molecula mechanism in ol ed in he
nuclea ion and g ow h s ages du ing he o ma ion o GlycoZIFs pa i-
cles. We obse ed ha he concen a ion o he glycolipid was a key
ac o o achie ing hei p ope encapsula ion [19], which seems o be
associa ed o he p esence/absence o glycolipid micelles in he eac ion
mix u e. Low glycolipid concen a ions (<0.2 mM) did no lead o he
o ma ion o GlycoZIFs, bu ZIF-8 pa icles we e ob ained in which,
cu iously, changes in size and mo phology we e obse ed when
compa ed wi h he con ol ZIF-8 sample (ZIF-8/Cn 1, p epa ed in he
absence o glycolipid). This inding indica ed ha glycolipid is capable
Fig. 3. PXRD pa e ns o 1@ZIF, 2@ZIF, 3@ZIF, 4@ZIF and con ol pa icles (ZIF-8/Cn 1, ZIF-8/Cn 2). Magni ica ions o he 2θ ange o clea ly isualize some
b oadened peaks in he GlycoZIFs pa e ns a e p esen ed as inse s. Simula ion o ZIF-8 (C ys allog aphy Open Da abase: 7111970) wi h hkl planes co esponding o
each peak is also shown.
R. Rod íguez-Ma ín e al.
Ma e ials Today Chemis y 44 (2025) 102546
7
o ac ing as a size/shape-con olling agen , jus as epo ed o o he
su ac an molecules such as CTAB. In con as , when he glycolipid was
added o he eac ion media a a inal concen a ion o 0.9 mM, a which
he compound is mos ly as micelles, we ob ained GlycoZIFs pa icles as
hose p esen ed along he wo k, inco po a ing he glycolipid wi h a
good encapsula ion e iciency and showing he p esence o one
glyco-micelle pe ZIF-pa icle as obse ed unde he mic ocoscope. A
such concen a ion, he glycolipids would be mos ly as micelles in he
media, H
2
O:MeOH (10:1), acco ding o CMC es ima ed om DLS mea-
su emen s (discussed la e ). These esul s poin ou ha he
glyco-micelles igge he spon aneous o ma ion o ZIF-8 shell, likely
ac ing as nuclea ion seeds in a simila way o wha was epo ed wi h
ino ganic nanopa icles (e.g., Au NPs and Pd NPs) in he o ma ion o
co e-shell NP/ZIF-8 composi es [30,31]. The e o e, we hypo hesize ha
he mechanism begins wi h he as o ma ion o micelles when he
glycolipid is added o he eac ion mix u e con aining he ZIF-8
Fig. 4.
1
H NMR spec a (400 MHz, CD
3
OD) o : (a) 2@ZIF, (b) dissol ed 2@ZIF, (c) ee sp
2
-IGL 2 bea ing a sul onyl g oup as glycosidic linkage.
R. Rod íguez-Ma ín e al.
Ma e ials Today Chemis y 44 (2025) 102546
8
p ecu so s. As depic ed in Fig. 6, Zn
+2
ions a e concen a ed o he
glyco-micelle su ace due o Zn–OH coo dina ing bonds, p oducing a
local supe sa u a ion ha a o s ZIF-8 c ys al g ow h a ound hose
micelles. As a esul , GlycoZIFs c ys alline nanopa icles a e o med in
jus a ew minu es, isually obse able by he appea ance o sligh
u bidi y ins an ly a e he addi ion o any o he glycolipids, whe eas
he pa icles size inc eases u he wi h ime un il eaching he
maximum size a e 2 h, acco ding o p e ious expe imen al s udies
[19].
To ob ain some expe imen al e idence ha suppo s he p oposed
mechanis ic hypo hesis, we conduc ed he ollowing di e en expe i-
men s. Fi s , o de e mine i sp
2
-IGLs we e able o o m micelles as
p oposed in he eac ion media, we add essed he de e mina ion o CMC
alues o 1, 2, 3 and 4 using a me hod based on DLS measu emen s (see
ESM, Table S3). Acco ding o he esul s, we could hus con i m ha a
he concen a ions used in he syn hesis o he GlycoZIFs, sp
2
-IGLs
Fig. 5. N
2
adso p ion ( illed ci cles) and deso p ion (emp y ci cles) iso he ms o he as-p epa ed GlycoZIFs and con ol ZIF-8 pa icles.
Fig. 6. P oposed mechanism o he o ma ion o GlycoZIFs pa icles, showing he ole o he glyco-micelles as nuclea ion seeds o p omo e he g ow h o he ZIF-8
shell a ound.
R. Rod íguez-Ma ín e al.
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9