High-yield halide-assisted synthesis of metal–organic framework UiO-based nanocarriers

Nanoscale
PAPER
Ci e his: DOI: 10.1039/d1n 08305h
Recei ed 17 h Decembe 2021,
Accep ed 15 h Ma ch 2022
DOI: 10.1039/d1n 08305h
sc.li/nanoscale
High-yield halide-assis ed syn hesis o me al–
o ganic amewo k UiO-based nanoca ie s†
Manuel Ceballos,
a
Manuela Ced ún-Mo ales,
a
Manuel Rod íguez-Pé ez,
b
Samuel Funes-He nando,
b
José Manuel Vila-Funguei iño,
b
Giulia Zampini,
b
Ma ia F. Na a o Poupa d,
b
Es e Polo,
c
Pablo del Pino *
a
and
Bea iz Pelaz *
d
The syn hesis o nanosized me al–o ganic amewo ks (NMOFs) is equisi e o hei applica ion as injec -
able d ug deli e y sys ems (DDSs) and o he bio ele an pu poses. He ein, we ha e c i ically examined he
ole o diffe en syn he ic pa ame e s leading o he p oduc ion o UiO-66 c ys als smalle han 100 nm.
O no e, we demons a e he co-modula o ole con e ed by halide ions, no only o p oduce NMOFs
wi h p ecise mo phology and size, bu also o significan ly imp o e he eac ion yield. The esul ing
NMOFs a e highly c ys alline and exhibi sus ained colloidal s abili y in diffe en biologically ele an
media. As a p oo o concep , hese NMOFs we e loaded wi h Rhodamine 6G (R6G), which emained
apped in mos common biologically ele an media. When incuba ed wi h li ing mammalian cells, he
R6G-loaded NMOFs we e efficien ly in e nalized and did no impai cell iabili y e en a ela i ely high
doses.
In oduc ion
Me al–o ganic amewo ks (MOFs) a e a p oli ic amily o
po ous ma e ials buil using 3D ex ended ne wo ks comp ising
me allic cen es coo dina ed o ligands. These ma e ials ha e
aised conside able in e es due o hei unique physico-
chemical p ope ies, pa icula ly mo i a ed by he p esence o
o de ed oids wi h de ined sizes and chemical en i onmen s
as designed by hei cons i uen s.
1,2
These ma e ials ha e been
success ully applied wi h mani old pu poses such as gas
s o age/sepa a ion, d ug deli e y, ca alysis, chemical sensing,
and wa e ea men .
1,3,4
Fo he applica ion o MOFs in he li e sciences, ine
con ol o e size and mo phology is desi ed gene ally.
P oducing pa icles wi h dimensions below 100 nm is sough
o a ou he in e ac ion be ween cells and pa icles, as well as
o a oid ascula blockage when adminis e ed in a enously.
5,6
Many s udies in he ield o nanomedicine ha e p o ed ha
sizes a ound 50 nm a e op imal o a ou he in e naliza ion o
nanopa icles (NPs) by cells.
7
Likewise, p oducing small, col-
loidally s able, and homogenous nanoMOFs (NMOFs) is
desi ed o his ype o bioapplica ion.
Reduced size no only has a posi i e impac on hei po en ial
in e ac ion wi h li ing en i ies bu also p o ides in e es ing p o-
pe ies compa ed wi h hei bulk coun e pa s, such as accele -
a ed adso p ion/deso p ion kine ics and highe accessibili y o
he in e nal ac i e si es.
1
All in all, du ing ecen yea s he p o-
duc ion o NMOFs has expe ienced an exponen ial e olu ion.
1,2,8
UiO-66 is among he mos s udied MOFs due o i s high
s abili y, bo h he mal and hyd oly ic. The chemical o mula
o UiO-66 is [Z
6
O
4
(OH)
4
L
6
]
n
, which leads o he o ma ion o a
po ous c ys alline ex ended ne wo k.
9
I is no ewo hy ha
UiO-66 is commonly used o enginee ing de ec s in i s s uc-
u e as i can bea a high numbe o de ec s while keeping i s
s uc u e.
9–11
The sol o he mal syn hesis o he UiO-66 amily, assis ed
by diffe en modula o s, has been ex ensi ely s udied, includ-
ing ca boxyla ed modula o s such as o mic acid (FA), ace ic
acid (AA), i luo oace ic acid (TFA), and benzoic acid (BA), and
non-ca boxyla ed modula o s such as wa e and hyd ochlo ic
acid.
12
The g ow h, kine ics, size and shape o he o med pa -
icles a e highly in luenced by he Z p ecu so s, i.e., Z Cl
4
o
†Elec onic supplemen a y in o ma ion (ESI) a ailable. See DOI: h ps://doi.o g/
10.1039/d1n 08305h
a
Cen o Singula de In es igación en Química Biolóxica e Ma e iais Molecula es
(CiQUS), Depa amen o de Física de Pa ículas, Uni e sidade de San iago de
Compos ela, 15782 San iago de Compos ela, Spain. E-mail: [email p o ec ed]
b
Cen o Singula de In es igación en Química Biolóxica e Ma e iais Molecula es
(CiQUS), Uni e sidade de San iago de Compos ela, 15782 San iago de Compos ela,
Spain
c
Cen o Singula de In es igación en Química Biolóxica e Ma e iais Molecula es
(CiQUS), Depa amen o de Bioquímica, Uni e sidade de San iago de Compos ela,
15782 San iago de Compos ela, Spain
d
Cen o Singula de In es igación en Química Biolóxica e Ma e iais Molecula es
(CiQUS), Depa amen o de Química Ino gánica, Uni e sidade de San iago de
Compos ela, 15782 San iago de Compos ela, Spain. E-mail: bea iz.pela[email p o ec ed]
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Z OCl
2
, because o he diffe en kinds o Z -clus e s ha can
be o med. The use o Z OCl
2
leads o as e c ys alliza ion as
he hyd olysis p oduc is he Z e ame Z
4
(OH)
8
(H
2
O)
16
ins ead o he hexanuclea clus e [Z
6
O
4
(OH)
4
]
12+
.
13,14
In o de o con ol he MOF size, se e al ele an chemical
equilib ia du ing he c ys al g ow h p ocess ha e o be aken
in o accoun , including he linke dep o ona ion, he modu-
la o dep o ona ion, he linke complexa ion, and he e min-
a ion.
15
These equilib ia a e achie ed while he c ys als g ow
ollowing he LaMe model.
16
Expe imen ally se e al key pa a-
me e s ha e been iden i ied, such as he modula o iden i y
and concen a ion, he equi alen s o linke and modula o
and he me al–ligand bond in e ac ions.
15,17
Fo example, Beh ens e al.
18
p epa ed ca. 200 nm oc a-
hed al UiO-66 pa icles using a monoca boxyla e agen (BA) a
high equi alen s a io, as a compe i o o he linke . They
dec eased he equi alen s o BA o educe he size o he
MOFs. On he o he hand, when he syn hesis was pe o med
using a high numbe o equi alen s o BA, he pa icles p e-
sen ed an i egula shape and size o se e al mic ons. Simila
obse a ions ha e ound ha UiO-66 using lowe modula o
equi alen s in he case o AA, FA, TFA, and dichlo oace ic
acid (DCA). In hese cases, a e y low modula o equi alen s
(<50 eq.), UiO-66 NMOFs unde 100 nm ha e been p oduced.
15,19
On ano he no e, i is well known ha acidic species, such
as hyd ochlo ic acid, inhibi he linke dep o ona ion, inc eas-
ing he eac ion yield.
20
Ye hese species can also hyd olyse
N,N-dime hyl o mamide (DMF), ypically used as a sol en
du ing hese syn he ic me hods,
21
o ming FA in he media
ha can also ac as a modula o .
The effec o he p ecu so and modula o on he c ys alli-
ni y, s uc u al de ec s and po osi y has been widely in es i-
ga ed. Howe e , hei impac on he p oduc ion o UiO-66
NMOFs, pa icula ly amina ed-de i a e NMOFs, emains
unclea . Se e al a emp s o p oduce UiO-66-NH
2
NMOFs wi h
educed size ha e led o NMOFs a ound 100 nm by changing
he modula o and he eagen a ios.
22
The smalles NMOFs
p oduced ollowing hese s a egies used a mix u e o BA and
AA in a a io o 20 : 3.75 wi h espec o Z Cl
4
and 2-amino e -
eph halic acid (NH
2
–BDC), in a sol o he mal syn hesis kep a
120 °C du ing 24 hou s.
23
In he p esen wo k, we epo he effec o se e al syn he ic
pa ame e s in o de o p oduce UiO-66-NH
2
NMOFs wi h sizes
below 100 nm, aiming o hei applica ion as a d ug deli e y
sys em (DDS). Speci ically, we ha e explo ed he in luence o
he Z p ecu so and he modula o , and he ole o halides as
co-modula o s (Scheme 1), which was also con i med. The
esul ing UiO-66-NH
2
NMOFs (see Table 1) we e colloidally
s able in biologically ele an media and we e efficien ly in e -
nalized by li ing cells wi hou impai ing cell iabili y. As a
p oo o concep , hey we e loaded wi h Rhodamine 6G (R6G),
a po en mi ochond ial p obe.
24,25
The loading a e and he
Bea iz Pelaz
D Bea iz Pelaz is cu en ly an
ERC-S G g an ee, Ramón y Cajal
esea che and P incipal
In es iga o a he Cen e o
Chemical Biology and Molecula
Ma e ials (CiQUS, Uni e si y o
San iago de Compos ela). She
ecei ed he Ph.D. om he
Uni e si y o Za agoza (2012).
Then she was a pos doc o al
ellow a he Philipp-Uni e si ä
Ma bu g. Since 2017, she has co-
led he BioNanoTools (h ps://
www.usc.es/ciqus/es/g upos/
bionano ools) g oup in CiQUS. The esea ch o he g oup is
ocused in he de elopmen o emo ely con olled sma
ma e ials, and he s udy o hei in e ac ion wi h li ing en i ies.
Table 1 Summa y able o samples p epa ed
Name Z
p ecu so Ligand Co-
modula o Size
(nm)
UiO-66-
NH
2
(Z OCl
2
)Z OCl
2
NH
2
–
BDC
—225 ± 37
UiO-66-NH
2
Z O(NO
3
)
2
NH
2
–
BDC
—>1000
UiO-66-NH
2
:Cl
−
Z O(NO
3
)
2
NH
2
–
BDC Cl
−
94 ± 10
UiO-66-NH
2
:B
−
Z O(NO
3
)
2
NH
2
–
BDC B
−
81 ± 9
UiO-66-NH
2
:I
−
Z O(NO
3
)
2
NH
2
–
BDC I
−
109 ± 14
UiO-66(Z OCl
2
) Z OCl
2
BDC —186 ± 13
UiO-66 Z O(NO
3
)
2
BDC —>1000
UiO-66:Cl
−
Z O(NO
3
)
2
BDC Cl
−
174 ± 13
Scheme 1 Summa y o he s udied pa ame e s and hei effec du ing
he syn hesis o UiO-66-NH
2
.
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s abili y o R6G we e e alua ed be o e s udying he in e naliz-
a ion a e o he NMOFs.
Resul s and discussion
In luence o zi conium p ecu so
In o de o p oduce UiO-66-NH
2
NMOFs, as a s a ing poin ,
Z OCl
2
,NH
2
–BDC and AA we e used as he Z sou ce, ligand
and modula o , espec i ely, using a mola a io o 1 : 2.5 : 995,
he ein kep cons an unless o he wise speci ied (see he
Expe imen al sec ion). The i s pa ame e ha we a ied was
he Z sou ce, i.e., Z O(NO
3
)
2
ins ead o Z OCl
2
. While he Z
clus e (i.e. e ame ) p oduced by Z OCl
2
a ou s he kine ic
g ow h o he MOF, he impac o Z O(NO
3
)
2
(which o ms as a
clus e he ex ended oligome Z
8
O
28
(NO
3
)
8
)
26
has been p e-
iously s udied in e ms o he p oduc c ys allini y, ega dless
o how i in luences he size and shape o he MOFs.
27,28
We
obse ed ha using Z OCl
2
leads o oc ahed al c ys als wi h a
well-de ined shape and ace s bu a b oad size dis ibu ion,
anging om 200 o 400 nm (see Fig. 1a and Fig. S1 and S2 o
he ESI†). In he case o Z O(NO
3
)
2
, he o ma ion o winned
pa icles can be ela ed o he clus e o med, as shown in
Fig. 1b (see Fig. S1 and S2†). As he Z s oichiome y o he
clus e o med wi h oxyni a e p ecu so is double ha in he
case o he oxychlo ide one, we hypo hesized ha du ing MOF
c ys al g ow h se e al ace s g ow simul aneously, leading o
he o ma ion o oc ahed al pa icles.
In luence o he modula o a e
Aiming o p e en he o ma ion o winned pa icles, while
using he Z O(NO
3
)
2
p ecu so , he modula o equi alen s
we e a ied a ound ou s anda d condi ions (i.e., 995 equi a-
len s o AA, 1.8 mL), using he ollowing olumes (equi alen s)
o AA: 1.4, 1.6, 1.8, 2.0 and 2.2 mL (775, 885, 995, 1105 and
1215 equi alen s o AA, espec i ely). As shown in Fig. S3,† he
a ia ion o modula o equi alen s in his ange appea s unin-
luen ial wi h espec o he size o mo phology o he NMOFs
o med. In pa icula , in all cases we ob ained winned
pa icles.
In luence o he chlo ide as a co-modula o
The esul s om he in luence o he wo i s pa ame e s led
us o conclude ha he chlo ine ions p esen in he Z OCl
2
p ecu so we e in ol ed in he c ys al g ow h o he pa icles.
Thus, he effec o chlo ide as a co-modula o was in es iga ed
by adding i ex e nally du ing he syn hesis while using he
p ecu so Z O(NO
3
)
2
. We expec ed o p oduce pa icles wi h a
de ined mo phology (i.e., oc ahed al) as occu ed when using
Z OCl
2
. To con i m his hypo hesis, we selec ed wo diffe en
chlo ine sou ces, i.e., hyd ochlo ic acid (Fig. 1c) and sodium
chlo ide (Fig. 1d), a a ixed a io o 1 : 17 Z : Cl
−
, leading o
disc e e nanoc ys als wi h a size less han 100 nm.
HCl has p e iously been iden i ied as esponsible o
imp o ing he eac ion yield and educing eac ion imes o
he syn hesis o UiO-66 and UiO-67.
13,20
Goes en e al.
29
a gued ha his ac was su p ising when Z Cl
4
was used as a
p ecu so , conside ing ha upon he p ecu so hyd olysis
mo e HCl is p oduced in he media du ing he MOF g ow h.
One migh hink ha HCl neu aliza ion would ha e been a
pa ame e o imp o e he speed and he syn hesis yield
ins ead o he opposi e.
29
In his di ec ion, Mu e al.
30
ela ed
he ypically small UiO-66 c ys al size (a ound 200 nm) o he
pa ial ligand dep o ona ion as a esul o he inc emen o
p o ons in he solu ion du ing syn hesis. They used FA,
hypo hesizing ha i was esponsible o he pa ial p o ona-
ion o BDC on he MOF su ace, leading o he gene a ion o
disc e e pa icles. When a dep o ona ing agen such as i-
e hylamine (TEA) was added, winned pa icles we e o med
ins ead o oc ahed al ones.
30
As discussed, in ou case, he use
o he same amoun o Cl
−
ions om diffe en sou ces led us
o he same esul , which was oc ahed al NMOFs o less han
100 nm. This esul demons a es ha he pH effec is no a
c i ical ac o he e, con i ming ha chlo ine ions play an
ac i e ole, p e en ing he gene a ion o winned pa icles, and
mo e impo an ly, allowing us o ge disc e e pa icles wi h
sizes below 100 nm.
In luence o diffe en halides as co-modula o s o p oduce
UiO-66-NH
2
NMOFs
Some epo s ha e in es iga ed he ole o diffe en dihalides
o halides such as iodine and luo ide.
31,32
In he case o
iodine, i s eac ion wi h hiol ligands o o m UiO-66-SH has
been s udied. Iodine can o m an RS–I bond. Howe e , his
eac ion p e en s he MOF oxida ion, he eby inc easing i s
s abili y.
31
Fo luo ide ions, i has been epo ed ha i is
capable o s ongly in e ac ing wi h he me al si e,
32
ac ing as
blocking agen s o ca boxyla ed ligands and b eaking he
Z
8
O
28
(NO
3
)
8
oligome o med by he oxyni a e p ecu so .
26
The anion effec in Z –MOF has al eady been in es iga ed
by Reinsch e al.
33
They epo ed ha sulpha e and dich oma e
anions coo dina e o he me al posi ions in he Z clus e ,
lea ing a ailable jus eigh o he wel e posi ions o he
Fig. 1 Rep esen a i e mic og aphs o he UiO-66-NH
2
pa icles
ob ained using (a) Z OCl
2
o (b) Z O(NO
3
)
2
as Z p ecu so ; o using he
combina ion o (c) Z O(NO
3
)
2
and HCl; and (d) Z O(NO
3
)
2
and NaCl.
Scale ba s co espond o 500 nm.
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linke coo dina ion. This led o he o ma ion o UiO-66 wi h
an incomple e numbe o ligands, which is e y equen .
The e o e, we decided o sys ema ically s udy he ole o he
diffe en halides (X
−
) in he syn hesis o UiO-66-NH
2
NMOFs.
Fo ha , we es ablished a common syn he ic condi ion using
Z O(NO
3
)
2
as a Z sou ce and he co esponding sodium
halide (NaX) sal s, which a e, sodium luo ide, sodium chlo -
ide, sodium b omide, and sodium iodide, while ixing he
a io Z : X
−
. As p e iously discussed, he Z oxychlo ide
(Fig. 2a) and he oxyni a e p ecu so s (Fig. 2b) led o oc ahe-
d a wi h b oad size dis ibu ion (200–400 nm) and la ge
winned mic opa icles, espec i ely; hese samples will be
e e ed o as UiO-66-NH
2
(Z OCl
2
) and UiO-66-NH
2
, espec -
i ely (see Table 1).
Keeping cons an he mola a io o halide used du ing he
UiO-66-NH
2
NMOF syn hesis, he p e iously obse ed mo -
phology (oc ahed al and no - winned pa icles) and size ange
(i.e.,80–90 nm) obse ed using diffe en chlo ide sou ces
(Fig. 1c and d) we e main ained in he case o chlo ide,
b omide, and iodide ions (Fig. 2c, d and e, espec i ely; Fig. S4
and S5† o addi ional SEM and TEM images; Fig. S6†shows
he aspec o he solu ions in me hanol). The halide-assis ed
NMOFs will be deno ed as UiO-66-NH
2
:Cl
−
; UiO-66-NH
2
:B
−
and UiO-66-NH
2
:I
−
o he samples assis ed by chlo ine,
b omine and iodine, espec i ely; hey showed a e age sizes o
94 ± 10 nm, 81 ± 9 nm, and 109 ± 14 nm (Table 1 and
Fig. S7†), espec i ely, as measu ed om SEM mic og aphs.
We asc ibe he sligh ly la ge size o UiO-66-NH
2
:I
−
o he
pa ial oxida ion o iodide in he p esence o NO
3
−
ions
(E°
Cl2=Cl¼1:396V, E°
B 2=B 
¼1:098V, E°
I2=I
¼0:0620V, and
E°
NO3=NO2
¼0:0940V), dec easing he amoun o his co-
modula o and inc easing he size o he NMOFs.
Fo comple eness, we also ied NaF as a co-modula o ,
which howe e p e en ed he o ma ion o pa icles. We
Fig. 2 TEM images o (a) UiO-66-NH
2
(Z OCl
2
) (black), (b) UiO-66-NH
2
(o ange), (c) UiO-66-NH
2
:Cl
−
( ed), (d) UiO-66-NH
2
:B
−
(g een), and (e)
UiO-66-NH
2
:I
−
(blue), HRTEM o ( ) UiO-66-NH
2
:Cl
−
and (g) o UiO-66-NH
2
:B
−
and (h–j) STEM-EDX images o UiO-66-NH
2
:Cl
−
(h) wi h Z (i) and
Cl
−
( j) mapping, and (k–m) STEM-EDX images o UiO-66-NH
2
:B
−
(k) STEM, Z (l) and B
−
(m) mapping. Scale ba s co espond o 200 nm excep o
he HRTEM images, in which hey co espond o 10 nm.
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hypo hesize ha luo ine ions coo dina e o he zi conium
clus e , p e en ing u he c ys al g ow h. This hypo hesis was
con i med by measu ing he UV– is spec a o he o med clus-
e s using all he halides (Fig. S8†). Mo eo e , he o ma ion o
a p ecipi a e was obse ed in he case o he luo ide ions,
leading o high sca e ing in a b oad spec al ange as a esul
o he poo solubili y o Z F
4
o med in he solu ion (Fig. S9†).
The esul s ob ained wi h iodide and b omide con i med ou
hypo hesis abou he c i ical ole o halide ions in he g ow h
o he UiO-66-NH
2
NMOFs.
High- esolu ion ansmission elec on mic oscopy
(HRTEM) showed highly c ys alline NMOFs; in he case o
UiO-66-NH
2
:Cl
−
and UiO-66-NH
2
:B
−
, he as Fou ie ans-
o m (FFT) analysis o he co esponding TEM images o
single NMOFs showed he cha ac e is ic {200} index o he a
ace-cen e ed-cubic s uc u e o he Fm3
ˉ
mspace g oup (inse s
in Fig. 2 and g). The d-spacing, in his case, co esponds o
ca. 10.78 Å and ca. 10.000 Å nm, espec i ely. These d-spacings
a e in good ag eemen wi h he heo e ical d-spacing (200) o
UiO-66, 10.373 Å, and as a consequence wi h one hal o he
la ice pa ame e o he UiO-66 uni cell (Fig. S10 and
Table S1†). EDX expe imen s con i med, in all he NMOFs, a
homogeneous Z and N dis ibu ion (da a no shown). In he
case o he halides, Cl
−
and B
−
ions we e de ec ed (Fig. 2j and
m, espec i ely) dis ibu ed in all he c ys als. Howe e , he I
−
was no p esen in a de ec able amoun . We hypo hesized hey
we e washed ou du ing he washing s eps o he NMOFs as
hei coo dina ion abili y was educed compa ed wi h ha o
Cl
−
o B
−
.
Wide-angle X- ay sca e ing (WAXS) measu emen s we e pe -
o med in capilla y mode, he main e lec ions o which we e
in ag eemen wi h p e iously epo ed powde X- ay diff ac ion
pa e ns,
34
and which e ealed he p esence o b oad peaks a
low 2θ alues, in bo h he samples con aining halides and he
sample syn hesized wi h Z OCl
2
(Fig. 3a). To co obo a e his
b oad peak a ound 4° 2θ, small-angle X- ay sca e ing (SAXS)
measu emen s we e pe o med (Fig. 3b); hese b oad peaks
ha e been a ibu ed o he p esence o o de ed missing
clus e de ec s.
27,35
The in ensi ies o diff ac ed peaks we e
educed, pe o ming a uni -cell e inemen wi h Pawley’s
me hod on he diff ac og am in Fig. 3a (Fig. S11†), compa ing
each sample wi h Pawley’s model and he esidual, which was
less han 1% in all he samples. Wi h his me hod, he in ensi-
ies and backg ound we e co ec ed and he cell pa ame e s o
each NMOF we e calcula ed and compa ed wi h hose o a
UiO-66-NH
2
cubic cell (Table S2†). The e ined cell pa ame e s
a e in ag eemen wi h he e e ence used o ix he peak
posi ion.
35,36
Fou ie - ans o med in a ed spec a (FTIR) o he samples
we e compa ed wi h ha o pu e NH
2
–BDC as a e e ence
(Fig. 4a–c). The C O ib a ion a 1671 cm
−1
was shi ed o all
he expe imen s o 1558 cm
−1
o he samples syn hesized
wi h Z O(NO
3
)
2
, and o 1555 cm
−1
o UiO-66-NH
2
(Z OCl
2
). I
is well known ha he ib a ions o he bands o UiO-66-
NH
2
(Z OCl
2
) a e less in ense han hose o UiO-66-NH
2
:X
−
(X
−
:Cl
−
,B
−
and I
−
), in pa icula compa ed wi h UiO-66-NH
2
:
Cl
−
; his e idence co obo a es he be e complexa ion o he
ligand wi h he Z clus e in his sample compa ed wi h he
o he s, due o he ca boxyla e anion ha ing wo s ongly
coupled C–O bonds wi h a Z clus e .
37,38
The ib a ions a
3505 cm
−1
and 3390 cm
−1
co esponding o he asymme ic
and symme ic s e ching o N–H bond we e shi ed o
3465 cm
−1
and 3350 cm
−1
because he complexa ion changed
he chemical en i onmen in he MOF o ma ion.
39,40
Raman spec a (see Fig. S12†) o he diffe en UiO-66-NH
2
NMOFs samples showed, in all cases, C C s e ching o a o-
ma ic ings cen e ed a 1627 cm
−1
,O–C–O symme ic s e ch-
ing in-phase a 1425–1447 cm
−1
and C–O s e ching a
1270 cm
−1
ela ed o he o ganic linke ;
41
and o he me al–
ligand coo dina ion signa u es, peaks ela ed o Z –O we e
obse ed a 247, 365 and 620 cm
−1
.
42
The UV– is spec a o UiO-66-NH
2
:X
−
(X
−
:Cl
−
,B
−
and I
−
)
(Fig. 4d) show h ee sha p bands, one a 240 nm, a ansi ion
ha can be a ibu ed o an elec onic ansi ion be ween he
halide and he Z –O clus e ,
43
and wo a 270 and 370 nm
co esponding o he n–π* ansi ion o he amine g oup and
he π–π* ansi ion o he NH
2
–BDC and Z –O clus e s,
44
espec i ely. Fo UiO-66-NH
2
(Z OCl
2
) he bands shi ed sligh ly
o 246, 281 and 392 nm, espec i ely, due o he bigge pa icle
size. In e es ingly, in he case o UiO-66-NH
2
, wi hou halide
jus wo bands appea ed a 306 and 415 nm, ed shi ed due o
he size o he pa icles. In his case, he band a ibu ed o
he halide a sho e wa eleng hs was no p esen .
The mal g a ime ic analysis (TGA) o he samples (Fig. 5a)
shows he he mal s abili y o he MOFs. All o hem we e
s able up o 350 °C, and only UiO-66-NH
2
s a ed o decom-
Fig. 3 C ys allog aphic cha ac e iza ion o UiO-66-NH
2
NMOFs:
UiO-66-NH
2
(Z OCl
2
) (black), UiO-66-NH
2
(o ange), UiO-66-NH
2
:Cl
−
( ed), UiO-66-NH
2
:B
−
(g een) and UiO-66-NH
2
:I
−
(blue). (a) WAXS ana-
lysis measu ed a a sample- o-de ec o dis ance o 288 mm and (b)
SAXS analysis measu ed a a sample- o-de ec o dis ance o 1343 mm in
capilla y mode.
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pose be o e ha empe a u e. Also, i is in e es ing o obse e
ha he samples ha migh be ligand de ec i e a e UiO-66-
NH
2
and UiO-66-NH
2
:I
−
(see he ESI†), as indica ed by hei
lowe a e compa ed wi h he Z clus e .
45
This is sus ained by
ou obse a ions o an ac i e ole o chlo ide and b omide ions
as a pa o he c ys alline s uc u e o he NMOFs, as iodide
ions we e no obse ed in ou TEM s udies.
N
2
so p ion measu emen s a 77 K show ype I iso he ms
(Fig. 5b), cha ac e is ic o mic opo ous ma e ials. The
B unaue –Emme –Telle -speci ic su ace a ea (S
BET
) o UiO66-
NH
2
(Z OCl
2
) is 865.8 m
2
g
−1
, whe eas he a eas o UiO66-NH
2
:
X
−
(X
−
:Cl
−
,B
−
,I
−
) a e i ually he same, wi h alues o
1017.7, 1027.9 and 1040.0 m
2
g
−1
, espec i ely (Table 2). These
indings a e in good ag eemen wi h esul s epo ed
elsewhe e.
13,46,47
The lowe BET su ace a ea o UiO66-NH
2
(Z OCl
2
) can be
asc ibed o he highe dimension o he pa icles wi h espec
o ha o UiO-66-NH
2
:X
−
(X
−
:Cl
−
,B
−
,I
−
). Fu he mo e, he
halide samples p esen e y simila alues o S
BET
, indica ing
he high ep oducibili y o he syn he ic s a egy in e ms o
he p ecise mo phology and size, despi e he change in he
halide ion.
Induc i ely coupled plasma op ical emission spec oscopy
(ICP-OES) measu emen s we e pe o med o all he samples
(Table S3†). These measu emen s allowed us o de e mine
bo h he mola concen a ion and he numbe o NMOF pa -
icles pe millili e . This las calcula ion was done conside ing
hei dimensions as ob ained by SEM measu emen s (see
sec ion S2 om he ESI, Table S4†),
48
and on he o he hand
o de e mine he zi conium concen a ion (mg mL
−1
).
Addi ionally, hese measu emen s allow us o e alua e he
eac ion yields (Table S3†). An inc ease in he eac ion yield
was obse ed o all he syn heses in which a halide was
added. In pa icula , he yield o he eac ion inc eased om
10% and 34% o UiO-66-NH
2
(Z OCl
2
) and UiO-66-NH
2
,
espec i ely, o ca. 77%, 52% and 45% o UiO-66-NH
2
:Cl
−
,
UiO-66-NH
2
:B
−
and UiO-66-NH
2
:I
−
, espec i ely. The
yield inc emen was especially signi ican in he case o he
chlo ide ions, leading o mo e han wice he amoun o MOF
han in he equi alen syn hesis pe o med wi hou his
halide.
Nanopa icle acking analysis (NTA) and dynamic ligh
sca e ing (DLS) expe imen s we e ca ied ou o e alua e he
effec i e hyd odynamic diame e o he NMOFs (Fig. 6a and b).
NTA showed ha UiO-66-NH
2
(Z OCl
2
) has an effec i e dia-
me e o ca. 200 nm, while o UiO-66-NH
2
:X
−
(X
−
:Cl
−
,B
−
,I
−
)
he effec i e diame e s we e below 140 nm (Fig. 6a and
Table S5†). These da a we e in good ag eemen wi h he DLS
measu emen s ca ied ou whe e he hyd odynamic diame e
was abo e 200 nm o 66-NH
2
(Z OCl
2
) and a ound 150 nm o
he samples syn hesized wi h halides (see Fig. 6b, Fig. S13,
and Table S5†). Fo UiO-66-NH
2
he NTA measu emen s could
no be ca ied ou due o i s poo colloidal s abili y and DLS
showed hyd odynamic diame e s abo e 700 nm.
ζ-Po en ial measu emen showed alues abo e 5 mV o all
he NMOFs (see Fig. 6c, and Table S5†). This is explained by
he p esence o he amine g oup om NH
2
–BDC as a pendan
g oup.
To comple e he cha ac e iza ion o he UiO-66-NH
2
NMOFs, he colloidal s abili y in diffe en media a diffe en
imes was s udied. The selec ed media we e MilliQ wa e , lyso-
somal medium (phagolysosomal simulan luid (PSF), 0.02 M
and pH 5), comple e cell media (Dulbecco’s Modi ied Eagle
Medium (DMEM) supplemen ed wi h 10% oe al bo ine
se um (FBS)) and phospha e-buffe ed saline (PBS, 0.1 M, pH
7.4) (Fig. 6d, and Table S6†). UiO-66-NH
2
(Z OCl
2
) NMOFs
showed good s abili y in wa e , PSF and DMEM, while in PBS
an inc emen in he effec i e diame e was obse ed a e 1 h,
indica ing ha he NMOFs we e agg ega ing. In he case o
UiO-66-NH
2
:X
−
(X
−
:Cl
−
,B
−
,I
−
), in PFS an inc emen in size
Fig. 4 (a–c) FTIR analysis and (d) UV– is spec oscopy o UiO-66-NH
2
NMOFs: UiO-66-NH
2
(Z OCl
2
) (black), UiO-66-NH
2
(o ange), UiO-66-NH
2
:
Cl
−
( ed), UiO-66-NH
2
:B
−
(g een) and UiO-66-NH
2
:I
−
(blue).
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was obse ed wi h espec o he samples in wa e ; howe e ,
hey emained s able along he measu ed ime. Fo DMEM all
samples showed an inc emen in size consis en wi h he
p o ein co ona o ma ion;
49
inally in PBS he samples showed
a simila endency o UiO-66-NH
2
(Z OCl
2
) bu wi h much
lowe agg ega ion a es.
These da a show ha ou NMOFs ha e consis en colloidal
s abili y despi e lacking induced de ec s by he use o a de ec -
induce modula o du ing he syn hesis
19,50
o being modi ied
wi h an addi ional ex e nal su ace coa ing.
Chlo ine ions as co-modula o s o he UiO-66 NMOF
syn hesis
Fo comple eness, we s udied he syn hesis o UiO-66 NMOFs
using he same op imized condi ions as used o he syn hesis
o UiO-66-NH
2
NMOF. Tha is, using bo h Z p ecu so s
(Z OCl
2
and Z (NO
3
)
2
) and chlo ine ions (NaCl) in he same
op imized a io (see Table 1).
In e es ingly, we ob ained indi idual oc ahed al pa icles
smalle han 200 nm when chlo ide ions we e p esen on he
syn hesis, as pa o he p ecu so o added as a sal (UiO-66
Z OCl
2
and UiO-66:Cl
−
, Fig. 7a and b). In he absence o chlo -
ide ions, he pa icles ob ained we e bigge han 800 nm,
o ming mul i winned pa icles (Fig. 7c). This esul con i ms
he ole o he chlo ide ions in he gene a ion o small UiO-66
pa icles. We u he pe o med TEM (Fig. S14 and S15†) and
STEM-EDX expe imen s o UiO-66:Cl
−
NMOFs, and chlo ide
ions we e de ec ed, showing he same homogeneous dis i-
bu ion in he MOFs (Fig. 7d) as obse ed o he UiO-66-NH
2
:X
−
(X
−
:Cl
−
,B
−
) NMOFs. As p e iously done wi h UiO-66-NH
2
,
we cha ac e ized he UiO-66 p oduced using ICP-OES, UV– is
spec oscopy, DLS, ζ-po en ial and N
2
so p ion (see Fig. S16–S18
andTablesS7andS8†). UiO-66-NH
2
yielded bigge hyd odyn-
amic diame e s and a ζ-po en ial close o neu ali y. In he
case o UiO-66 (Z OCl
2
) and UiO-66:Cl
−
hey showed posi i e
po en ial alues. As expec ed, he samples p esen ed highe
alues o S
BET
wi h espec o he ela i e amina ed samples,
wi h alues o 1160.2 and 1231.1 m
2
g
−1
o UiO-66 (Z OCl
2
)
and UiO-66:Cl
−
, espec i ely (Table 1 and Fig. S19†).
Fig. 5 (a) TGA analysis and (b) BET iso he ms o UiO-66-NH
2
NMOFs:
UiO-66-NH
2
(Z OCl
2
) (black), UiO-66-NH
2
(o ange), UiO-66-NH
2
:Cl
−
( ed), UiO-66-NH
2
:B
−
(g een) and UiO-66-NH
2
:I
−
(blue).
Table 2 BET specific su ace a ea (S
BET
) o UiO-66 samples
Sample S
BET
(m
2
g
−1
)
UiO-66-NH
2
(Z OCl
2
) 865.8 ± 18.2
UiO-66-NH
2
:Cl
−
1017.7 ± 12.0
UiO-66-NH
2
:B
−
1027.9 ± 13.5
UiO-66-NH
2
:I
−
1040.0 ± 10.8
UiO-66 (Z OCl
2
) 1160.2 ± 13.7
UiO-66:Cl
−
1231.1 ± 13.6
Fig. 6 Effec i e diame e (hyd odynamic diame e ) o UiO-66-NH
2
NMOFs analyzed by (a) NTA and (b) DLS. (c) ζ-Po en ial measu emen s.
(d) Summa y o he s abili y es s in wa e and lysosomal medium. Black
line co esponds o UiO-66-NH
2
(Z OCl
2
), ed o UiO-66-NH
2
:Cl
−
,
g een o UiO-66-NH
2
:B
−
and blue o UiO-66-NH
2
:I
−
.
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UiO-66 NMOFs as DDSs
Loading o Rhodamine 6G. The UiO-66 MOF amily has
been ex ensi ely used as DDSs because o hei good s abili y,
he low oxici y o hei componen s, and hei la ge po osi y.
Acco ding o i s c ys al da a, UiO-66 has a cage size o 0.75 and
1.2 nm wi h a po e ape u e o 0.6 nm, and diffe en molecules
ha e been encapsula ed, such as calcein,
51
dichlo oace a e,
52,53
5- luo ou acil,
52
doxo ubicin,
3,54
5-aminole ulinic acid,
55
e c.
As a model molecule, we selec ed Rhodamine 6G (R6G,
Table S9 and Fig. S20†) o e alua e he loading abili ies o he
syn hesized NMOFs. To load he NMOFs, a pos -loading s a -
egy was applied by simply mixing he NMOFs wi h a solu ion
o R6G in a a io o 1 : 2 Z : R6G (w : w) (see Table S10†). A e
ho ough washing o he NMOFs, we e alua ed he loading
a e o UiO-66-NH
2
NMOFs, inding loading a es om 27 o
31%, co esponding o a a io o ca. 0.6 mg R6G pe mg Z and
a loading a e o R6G pe NMOF on he o de o 10
5
pe NMOF
(see sec ions S5–S7 o he ESI, Fig. S21 and Table S11†). The
loading o R6G inc eased he hyd odynamic size o he NMOFs
while hei ζ-po en ials we e kep posi i e excep o
NH
2
(Z OCl
2
)@R6G, he po en ial o which was nea ly 0 mV
(see Fig. S22–S25 and Table S12†). In e es ingly, we ound
simila UiO-loading a es o UiO-66 NMOFs (ca. 30% o
loading), wi h a a io o ca. 0.6 mg R6G pe mg Z , co es-
ponding o 6–7×10
5
molecules o R6G pe NMOF (see Tables
S13 and S14, and Fig. S26–S28†). The obse ed loading a io
co esponds o ca.17–20% w/w o all he syn hesized NMOFs,
UiO-66-NH
2
and UiO-66.
Despi e wo king wi h highly c ys alline s uc u es, his
pos -loading efficiency is highe han ha p e iously epo ed
wi h 5- luo ou acil, in which loadings o abou 2%
9
o 5%
52
w/w (in he case o UiO-66-NO
2
) we e achie ed using diffe en
syn he ic app oaches and wo king wi h highly de ec i e
UiO-66. An ex emely high loading o 1 mg doxo ubicin pe mg
MOF was epo ed by Chen e al.
54
and ano he high loading
a io highe han 25% w/w has been epo ed, bu as a conse-
quence o he inco po a ion o he d ugs as a pa o he
UiO-66 s uc u e.
9
On he o he hand, he numbe o mole-
cules pe NMOF was o he same o de o magni ude as he
encapsula ion o Hoechs o p o- luo opho es in ZIF-8-based
nanocomposi es p e iously epo ed by us.
56,57
The aim was o use hese NMOFs as DDSs o anspo he
R6G o he in acellula domain o cells. I is well known ha
mos nanoma e ials a e apped in lysosomal compa men s
whe e hey a e u he deg aded.
58,59
NMOFs a e no an
excep ion.
56,60,61
We s udied he s abili y o he loading o R6G
in diffe en media o diffe en imes up o 24 h. We used he
same media as p e iously selec ed o colloidal s abili y
s udies excep o PBS, ha is, wa e , comple e DMEM, and
PSF. The esul s p o ed ha o UiO-66-NH
2
NMOFs, he R6G
encapsula ed was s able in MilliQ wa e wi h no signi ican
elease in any o he samples (see Table S15, and Fig. S29†).
Howe e , he ime-dependen elease o R6G showed a clea
inc ease in he cases o UiO-66-NH
2
:Cl
−
and UiO-66-NH
2
:B
−
,
wi h 1 and 1.8% elease a 24 h, espec i ely, while o he es
he R6G elease emained below 0.7%. The same end was
obse ed in bo h PSF and DMEM. Fo PSF, bo h samples
showed a elease o ca. 3% a 24 h, while o DMEM a highe
elease was obse ed close o 4% and 4.5% o UiO-66-NH
2
:Cl
−
and UiO-66-NH
2
:B
−
, espec i ely.
In he case o UiO-66 he eleased R6G was highe in all he
cases and in all he media, in pa icula o UiO-66:Cl
−
in PSF
and DMEM a 24 h eleases o ca. 14% and 4% we e obse ed
(see Table S16†). These esul s show ha he p esence o he
amine g oup is unin luen ial conce ning he loading a e o
he NMOFs, ye i s abilizes RG6 wi hin he NMOFs.
Addi ionally, i demons a es ha he encapsula ion s abili y
in ou NMOFs is e y high.
I we compa e he ca go elease om o he UiO-66 nano-
sys ems (please no e ha we a e ypically e e ing o UiO-66
no o UiO-66-NH
2
NMOFs as, o he la e , li e a u e examples
a e sca ce), we ind ha he elease o doxo ubicin a e 24 h a
37 °C was highe han 20% a pH 5, 6.8 and 7.4, being e y
close o he 25% a pH 5,
54
while he elease o encapsula ed
calcein om UiO-66 MOFs has been epo ed o occu a
app oxima ely 80% mass elease a e 5 h in solu ion.
51
Cell s udies
Nex , we e alua ed he in e ac ion o UiO-66-NH
2
NMOFs wi h
cells in 2D cell cul u es o A549 adenoca cinoma cells. We
assessed he biocompa ibili y using he esazu in es , which
con i med he co ec me abolic a e o mi ochond ia in cell
cul u es. We used concen a ions o up o 100 μgZ mL
−1
(which co esponds o ca. 320 μgmL
−1
o 0.82, 2.1, 0.93 and
1.97 nM o UiO-66-NH
2
, UiO-66-NH
2
:Cl
−
, UiO-66-NH
2
:B
−
, and
UiO-66-NH
2
:I
−
, espec i ely), which showed no signi ican ox-
ici y o any sample a e 24 h o incuba ion (see Fig. S30†).
The same esul was ob ained when es ing he UiO-66 NMOFs
Fig. 7 Rep esen a i e TEM images o (a) UiO-66 (Z OCl
2
) (b) UiO-66 (c)
UiO-66:Cl
−
and, (d) EDX elemen al mapping o ca bon, zi conium and
chlo ide om a UiO-66:Cl
−
NMOF. Scale ba s co espond o 200 nm o
TEM and o 100 nm o STEM-EDX.
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(see Fig. S31†) in he same concen a ion ange. These esul s
a e in good ag eemen wi h p e ious oxici y es s epo ed
wi h he UiO-66 amily pa icles.
55
As men ioned abo e, he
biocompa ibili y o his MOF amily is e y high, being one o
he easons he educed oxici y o i s componen s, o
example, Z is one o he a e ansi ion me als ha a e na u-
ally p esen in he human body (ca. 300 mg o Z ).
51
A e con i ming he low oxici y o ou NMOFs, we s udied
he up ake o UiO-66-NH
2
NMOFs using luo escence
mic oscopy and low cy ome y (Fig. 8, Fig. S32 and
Table S17†). To do his, we used he R6G-loaded NMOFs. R6G
was selec ed as ca go o wo easons, (i) o p o ide he
NMOFs wi h luo escence o ack hem in acellula ly and (ii)
due o i s abili y o a ge unc ional mi ochond ia.
24,25
Con ocal mic oscopy imaging expe imen s showed he in a-
cellula loca ion o he NMOF in he pe inuclea egion, as
expec ed (Fig. 8a).
56,60
This in acellula localiza ion indica es
ha R6G emains inside he NMOFs, as mi ochond ia s aining
is no isible as a esul o in acellula R6G leakage. Also,
despi e p esen ing a posi i e ζ-po en ial, he loaded R6G is no
capable o ec o izing he NMOFs o he mi ochond ia ei he .
To s udy he cellula up ake by low cy ome y, 25 pM o
UiO-66-NH
2
(Z OCl
2
) and UiO-66-NH
2
:X
−
(X
−
:Cl
−
,B
−
,I
−
)
we eincuba ed o 6hincomple ecellmediaa 37°C,5%
CO
2
. We ound ha he cellula up ake was highe o
UiO-66-NH
2
(Z OCl
2
) compa ed wi h UiO-66-NH
2
:X
−
(X
−
:Cl
−
,
B
−
,I
−
), which p esen ed simila in e naliza ion a es (see
Fig. 8b and c). This diffe ence migh be ela ed o he con e -
sion o he concen a ion o NMOF om mass o mola con-
cen a ion, as UiO-66-NH
2
(Z OCl
2
) is he sample wi h a less
de ined mo phology and b oade size dis ibu ion. Howe e ,
his diffe ence in he up ake was signi ican ly educed when
he expe imen was pe o med using a cons an amoun o
Z (2.5 µg mL
−1
, which co esponds o 8 µg mL
−1
o NMOF).
In his case, he in e naliza ion a e was e y simila in all
cases. We only obse ed educed up ake o UiO-66-NH
2
:Cl
−
NMOFs (see Fig. 8c). These up ake a es a e expec ed based
on he physicochemical pa ame e s s udied du ing he
cha ac e iza ion o he NMOFs, i.e. hei hyd odynamic dia-
me e s and hei ζ-po en ial alues.
62
These esul s a e in
good ag eemen wi h he esul s ob ained by luo escence
mic oscopy analysis and e idence he c i ical impac o pe -
Fig. 8 (a) Rep esen a i e mic oscopy con ocal images o A549 cells incuba ed o 6 h wi h 25 pM o diffe en UiO-66-NH
2
@R6G and UiO-66-NH
2
:
X
−
@R6G NMOFs. Scale ba s co espond o 25 μm. (b) Flow cy ome y analysis o A549 cells exposed o ehicle (PBS) and he diffe en UiO-66-NH
2
:
X
−
@R6G o mula ions a 25 pM o NMOFs o o 2.5 μgZ mL
−1
. His og ams ep esen expe imen s done in iplica e. (c) Quan ifica ion o R6G fluo -
escence om flow cy ome y analysis in (b). Ba s ep esen means ± SD (n=3).
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This jou nal is © The Royal Socie y o Chemis y 2022 Nanoscale
Open Access A icle. Published on 25 Ap il 2022. Downloaded on 5/11/2022 11:25:20 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence.
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