Ci a ion: Blázquez-Ma ín, A.;
Ruiz-Ba dillo, A.; Ve de-Ses o, E.;
I u ospe, A.; A be, A.; Pomposo, J.A.
Towa d Long-Te m-Dispe sible,
Me al-F ee Single-Chain
Nanopa icles. Nanoma e ials 2023,13,
1394. h ps://doi.o g/10.3390/
nano13081394
Academic Edi o : An onios Kela akis
Recei ed: 24 Ma ch 2023
Re ised: 12 Ap il 2023
Accep ed: 14 Ap il 2023
Published: 18 Ap il 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
nanoma e ials
A icle
Towa d Long-Te m-Dispe sible, Me al-F ee Single-Chain
Nanopa icles
Agus ín Blázquez-Ma ín1, Aina a Ruiz-Ba dillo 1, Es e Ve de-Ses o 1,2,*, Amaia I u ospe 1, A an xa A be 1
and JoséA. Pomposo 1,2,3,*
1Cen o de Física de Ma e iales (CSIC, UPV/EHU)-Ma e ials Physics Cen e (MPC),
20018 Donos ia-San Sebas ián, Spain; [email p o ec ed] (A.A.)
2IKERBASQUE-Basque Founda ion o Science, 48009 Bilbao, Spain
3Depa men o Polyme s and Ad anced Ma e ials: Physics, Chemis y and Technology, Uni e si y o he
Basque Coun y UPV/EHU, 20018 Donos ia-San Sebas ián, Spain
*Co espondence: ma iaes e [email p o ec ed] (E.V.-S.); [email p o ec ed] (J.A.P.)
Abs ac :
We epo he ein on a new pla o m o syn hesizing s able, ine , and dispe sible me al- ee
single-chain nanopa icles (SCNPs) ia in amolecula me al- aceless azide–alkyne click chemis y.
I is well known ha SCNPs syn hesized ia Cu(I)-ca alyzed azide–alkyne cycloaddi ion (CuAAC)
o en expe ience me al-induced agg ega ion issues du ing s o age. Mo eo e , he p esence o me al
aces limi s i s use in a numbe o po en ial applica ions. To add ess hese p oblems, we selec ed a bi-
unc ional c oss-linke molecule, sym-dibenzo-1,5-cyclooc adiene-3,7-diyne (DIBOD). DIBOD has wo
highly s ained alkyne bonds ha allow o he syn hesis o me al- ee SCNPs. We demons a e he
u ili y o his new app oach by syn hesizing me al- ee polys y ene (PS)-SCNPs wi hou signi ican
agg ega ion issues du ing s o age, as demons a ed by small-angle X- ay sca e ing (SAXS) expe -
imen s. No ably, his me hod pa es he way o he syn hesis o long- e m-dispe sible, me al- ee
SCNPs om po en ially any polyme p ecu so deco a ed wi h azide unc ional g oups.
Keywo ds:
single-chain nanopa icles (SCNPs); me al- ee click chemis y; in amolecula c oss-
linking; sym-dibenzo-1,5-cyclooc adiene-3,7-diyne (DIBOD); s ain-p omo ed azide
−
alkyne cycload-
di ion (SPAAC)
1. In oduc ion
Single-chain nanopa icles (SCNPs), o med by he olding/collapse o disc e e indi-
idual polyme chains ia in a-chain in e ac ions, a e ul a-small so pa icles (3–30 nm)
ha ha e a a ie y o applica ions (ca alysis, sensing, d ug deli e y, nanocomposi es) [
1
–
9
].
Conce ning he na u e o he in a-chain in e ac ions, pe manen SCNPs esul when co-
alen bonds a e in ol ed in he in amolecula collapse o he p ecu so chain, whe eas
e e sible SCNPs a e ob ained by in a-chain olding h ough non-co alen in e ac ions o
dynamic co alen bonds [
10
–
15
]. Al hough e e sible SCNPs espond o ex e nal s imuli
(e.g., empe a u e, sol en s, pH) and ind applica ions in enzyme mimic y and d ug deli -
e y [
16
–
20
], pe manen SCNPs a e mo e app op ia e o applica ions in which long- e m
s abili y is a p e equisi e such as ca alysis o all-polyme nanocomposi es (APNCs) [
21
–
25
].
Among he di e en echniques de eloped o syn hesize pe manen SCNPs endowed
wi h obus in a-chain co alen bonds, hose based on “click” chemis y a e ega ded as
a ac i e [
26
]. Since i s disco e y by Sha pless [
27
] and Meldal [
28
] in 2002, Cu(I)-ca alyzed
azide–alkyne coupling (CuAAC)— he a che ypical “click” eac ion [
29
]—has gained eno -
mous in e es in a b oad numbe o ields, including pha maceu ical science and ma e ials
science. One o he au ho s o his pape pionee ed he use o CuAAC o SCNP con-
s uc ion a ew yea s a e i s disco e y [
30
–
32
]. CuAAC is a apid, high-yielding eac ion
ha can be conduc ed a oom empe a u e ( . .) using dilu ed concen a ions o eac an s,
making i compa ible wi h many o he unc ional g oups. Howe e , he esul ing iazole
Nanoma e ials 2023,13, 1394. h ps://doi.o g/10.3390/nano13081394 h ps://www.mdpi.com/jou nal/nanoma e ials
Nanoma e ials 2023,13, 1394 2 o 13
g oup o med on CuAAC is p one o complex Cu ions om he ca alys . In his sense, he
emo al o me al aces om polyme s and SCNPs is signi ican ly mo e complica ed han
in he case o wa e -soluble low-molecula -weigh subs ances and, consequen ly, se e ely
limi s p ac ical applica ions due o po en ial oxici y, colo a ion, and s abili y issues in he
esul ing ma e ials [
33
,
34
]. On he one hand, he in e e ence o Cu ions wi h many biolog-
ical p ocesses ( o ins ance, me al ions can s ongly bind o guanosine bases, dis up ing
he double-helical s uc u e o DNA) [
35
] and a ious elec ical/op oelec onic phenom-
ena [
36
] is well-known. On he o he hand, me al ions in some mac omolecule–me al
complexes end o agg ega e, o ming nanoscale ionic domains due o hei elec os a ic o
dipole–dipole in e ac ions [
37
]. In he case o SCNPs, hese e ec s lead o he i e e sible
agg ega ion o SCNPs in o mul i-chain nanopa icles du ing s o age in he solid s a e [
38
].
Consequen ly, when SCNPs con aining limi ed amoun s o Cu ions a e dissol ed a e
s o age, agg ega es wi h signi ican ly la ge sizes han indi idual SCNPs a e obse ed.
A p omising app oach o a oid any esidual me al ions while e aining he ad-
an ages o click chemis y is o use me al- ee e sions, such as he s ain-p omo ed
azide
−
alkyne cycloaddi ion (SPAAC) de eloped by Be ozzi [
39
] o co alen modi ica-
ion o biomolecules in li ing sys ems. SPAAC u ilizes s ained cyclooc ynes ha ha e
a dec eased ac i a ion ene gy o eac wi h azides in a click-like ashion, elimina ing he
need o a Cu(I) ca alys . This ype o me al- ee click chemis y has been applied in se e al
ields, om dynamic
in i o
imaging [
40
] o cellulose modi ica ion [
41
] and he unc ion-
aliza ion o su aces [
42
], among o he s. In his wo k, in o de o syn hesize me al- ee
SCNPs ha a e ee om agg ega ion issues du ing s o age in he solid s a e, we u n ou
a en ion o a bi unc ional c oss-linke molecule, sym-dibenzo-1,5-cyclooc adiene-3,7-diyne
(DIBOD), wi h wo highly s ained alkyne bonds(Figu e 1). DIBOD was i s syn hesized
by Sondheime e al. in 1974 [
43
]. The use o DIBOD o luo escence labeling o azido-
glycoconjuga es on cell su aces was pionee ed by Hosoya e al. in 2010 [
44
]. Mo e ecen ly,
his bi unc ional c oss-linke molecule has been employed o he e icien cons uc ion o
a ious ings [
45
] and cage-shaped polyme s [
46
]. Howe e , o he bes o ou knowledge,
DIBOD has no ye been in es iga ed as an in a-chain c oss-linke o me al- ee SCNP
cons uc ion.
Nanoma e ials 2023, 13, x FOR PEER REVIEW 2 o 13
science and ma e ials science. One o he au ho s o his pape pionee ed he use o Cu-
AAC o SCNP cons uc ion a ew yea s a e i s disco e y [30–32]. CuAAC is a apid,
high-yielding eac ion ha can be conduc ed a oom empe a u e ( . .) using dilu ed con-
cen a ions o eac an s, making i compa ible wi h many o he unc ional g oups. How-
e e , he esul ing iazole g oup o med on CuAAC is p one o complex Cu ions om
he ca alys . In his sense, he emo al o me al aces om polyme s and SCNPs is signi -
ican ly mo e complica ed han in he case o wa e -soluble low-molecula -weigh sub-
s ances and, consequen ly, se e ely limi s p ac ical applica ions due o po en ial oxici y,
colo a ion, and s abili y issues in he esul ing ma e ials [33,34]. On he one hand, he in-
e e ence o Cu ions wi h many biological p ocesses ( o ins ance, me al ions can s ongly
bind o guanosine bases, dis up ing he double-helical s uc u e o DNA) [35] and a ious
elec ical/op oelec onic phenomena [36] is well-known. On he o he hand, me al ions in
some mac omolecule–me al complexes end o agg ega e, o ming nanoscale ionic do-
mains due o hei elec os a ic o dipole–dipole in e ac ions [37]. In he case o SCNPs,
hese effec s lead o he i e e sible agg ega ion o SCNPs in o mul i-chain nanopa icles
du ing s o age in he solid s a e [38]. Consequen ly, when SCNPs con aining limi ed
amoun s o Cu ions a e dissol ed a e s o age, agg ega es wi h signi ican ly la ge sizes
han indi idual SCNPs a e obse ed.
A p omising app oach o a oid any esidual me al ions while e aining he ad-
an ages o click chemis y is o use me al- ee e sions, such as he s ain-p omo ed az-
ide−alkyne cycloaddi ion (SPAAC) de eloped by Be ozzi [39] o co alen modi ica ion
o biomolecules in li ing sys ems. SPAAC u ilizes s ained cyclooc ynes ha ha e a de-
c eased ac i a ion ene gy o eac wi h azides in a click-like ashion, elimina ing he need
o a Cu(I) ca alys . This ype o me al- ee click chemis y has been applied in se e al
ields, om dynamic in i o imaging [40] o cellulose modi ica ion [41] and he unc ion-
aliza ion o su aces [42], among o he s. In his wo k, in o de o syn hesize me al- ee
SCNPs ha a e ee om agg ega ion issues du ing s o age in he solid s a e, we u n ou
a en ion o a bi unc ional c oss-linke molecule, sym-dibenzo-1,5-cyclooc adiene-3,7-
diyne (DIBOD), wi h wo highly s ained alkyne bonds(Figu e 1). DIBOD was i s syn-
hesized by Sondheime e al. in 1974 [43]. The use o DIBOD o luo escence labeling o
azido-glycoconjuga es on cell su aces was pionee ed by Hosoya e al. in 2010 [44]. Mo e
ecen ly, his bi unc ional c oss-linke molecule has been employed o he efficien con-
s uc ion o a ious ings [45] and cage-shaped polyme s [46]. Howe e , o he bes o ou
knowledge, DIBOD has no ye been in es iga ed as an in a-chain c oss-linke o me al-
ee SCNP cons uc ion.
Figu e 1. S uc u e o sym-dibenzo-1,5-cyclooc adiene-3,7-diyne (DIBOD).
To illus a e he u ili y o DIBOD as an ex e nal bi unc ional c oss-linke molecule o
he syn hesis o me al- ee SCNPs ia SPAAC, we i s syn he ized a polys y ene (PS)-
based p ecu so deco a ed wi h azide unc ional g oups. Nex , we in es iga ed he eac-
ion condi ions o p oduce PS-SCNPs using DIBOD molecules as in a-chain c oss-linke s.
Th ough small-angle X- ay sca e ing (SAXS) expe imen s, we ound ha his new ap-
p oach leads o me al- ee PS-SCNPs wi hou signi ican agg ega ion issues du ing s o -
age. Rema kably, he new syn he ic me hod epo ed in his wo k is use ul o he p epa-
a ion o long- e m-dispe sible, me al- ee SCNPs om po en ially any polyme p ecu so
deco a ed wi h azide unc ional g oups.
Figu e 1. S uc u e o sym-dibenzo-1,5-cyclooc adiene-3,7-diyne (DIBOD).
To illus a e he u ili y o DIBOD as an ex e nal bi unc ional c oss-linke molecule
o he syn hesis o me al- ee SCNPs ia SPAAC, we i s syn he ized a polys y ene
(PS)-based p ecu so deco a ed wi h azide unc ional g oups. Nex , we in es iga ed he
eac ion condi ions o p oduce PS-SCNPs using DIBOD molecules as in a-chain c oss-
linke s. Th ough small-angle X- ay sca e ing (SAXS) expe imen s, we ound ha his
new app oach leads o me al- ee PS-SCNPs wi hou signi ican agg ega ion issues du ing
s o age. Rema kably, he new syn he ic me hod epo ed in his wo k is use ul o he
p epa a ion o long- e m-dispe sible, me al- ee SCNPs om po en ially any polyme
p ecu so deco a ed wi h azide unc ional g oups.
2. Resul s and Discussion
Scheme 1illus a es he p ocedu e ollowed in his wo k o he syn hesis o me al- ee
PS-SCNPs ia SPAAC using DIBOD molecules as in a-chain c oss-linke s.
Nanoma e ials 2023,13, 1394 3 o 13
Nanoma e ials 2023, 13, x FOR PEER REVIEW 3 o 13
2. Resul s and Discussion
Scheme 1 illus a es he p ocedu e ollowed in his wo k o he syn hesis o me al-
ee PS-SCNPs ia SPAAC using DIBOD molecules as in a-chain c oss-linke s.
Scheme 1. Illus a ion o he syn hesis o me al- ee PS-SCNPs, 3, a . . om a PS-based p ecu so
deco a ed wi h azide unc ional g oups (2).
2.1. Syn hesis and Cha ac e iza ion o he Polys y ene-Based P ecu so Deco a ed wi h Azide
Func ional G oups (2)
In he i s s ep, we syn he ized a andom copolyme o s y ene (S) and chlo ome hyl
s y ene (CMS) ia e e sible addi ion- agmen a ion chain- ans e (RAFT) polyme iza-
ion (see Scheme 1) [47]. The esul ing poly(S-co-CMS) copolyme , 1, con aining 27 mol%
o CMS uni s—as es ima ed by p o on nuclea magne ic esonance (1H NMR)—showed a
weigh -a e age molecula weigh (Mw) o 100.5 kDa and a na ow dispe si y (Ɖ) o 1.15,
as de e mined by size exclusion ch oma og aphy (SEC) equipped wi h iple de ec ion
(RI, MALS, and VIS, see Sec ion 3.2.1). The SEC/RI ace o 1 is illus a ed in Figu e 2. The
hyd odynamic adius (Rh) o 1 in THF was Rh (VIS) = 8.2 nm.
Figu e 2. SEC aces (RI de ec o ) o poly(S-co-CMS), 1, and poly(S-co-AMS), 2.
Subsequen ly, 1 was subjec ed o an azida ion p ocedu e [47] (NaN3, DFMF, 24 h) a
80 °C o gi e he polys y ene (PS)-based p ecu so deco a ed wi h –N3 (ins ead –Cl) unc-
ional g oups: poly(S-co-AMS), 2 (see Scheme 1). The SEC/RI ace o 2 is shown in Figu e
2, wi h alues o Mw (MALS) = 129.1 kDa, Ð = 1.18, and Rh (VIS) = 9.2 nm. We a ibu ed
0
0.2
0.4
0.6
0.8
1
6.577.588.599.5
No malized RI signal (a.u.)
SEC e en ion ime (min.)
21
Scheme 1.
Illus a ion o he syn hesis o me al- ee PS-SCNPs,
3
, a . . om a PS-based p ecu so
deco a ed wi h azide unc ional g oups (2).
2.1. Syn hesis and Cha ac e iza ion o he Polys y ene-Based P ecu so Deco a ed wi h Azide
Func ional G oups (2)
In he i s s ep, we syn he ized a andom copolyme o s y ene (S) and chlo ome hyl
s y ene (CMS) ia e e sible addi ion- agmen a ion chain- ans e (RAFT) polyme iza ion
(see Scheme 1) [
47
]. The esul ing poly(S-co-CMS) copolyme ,
1
, con aining 27 mol% o
CMS uni s—as es ima ed by p o on nuclea magne ic esonance (
1
H NMR)—showed a
weigh -a e age molecula weigh (M
w
) o 100.5 kDa and a na ow dispe si y (Ð) o 1.15, as
de e mined by size exclusion ch oma og aphy (SEC) equipped wi h iple de ec ion (RI,
MALS, and VIS, see Sec ion 3.2.1). The SEC/RI ace o
1
is illus a ed in Figu e 2. The
hyd odynamic adius (Rh) o 1in THF was Rh(VIS) = 8.2 nm.
Nanoma e ials 2023, 13, x FOR PEER REVIEW 3 o 13
2. Resul s and Discussion
Scheme 1 illus a es he p ocedu e ollowed in his wo k o he syn hesis o me al-
ee PS-SCNPs ia SPAAC using DIBOD molecules as in a-chain c oss-linke s.
Scheme 1. Illus a ion o he syn hesis o me al- ee PS-SCNPs, 3, a . . om a PS-based p ecu so
deco a ed wi h azide unc ional g oups (2).
2.1. Syn hesis and Cha ac e iza ion o he Polys y ene-Based P ecu so Deco a ed wi h Azide
Func ional G oups (2)
In he i s s ep, we syn he ized a andom copolyme o s y ene (S) and chlo ome hyl
s y ene (CMS) ia e e sible addi ion- agmen a ion chain- ans e (RAFT) polyme iza-
ion (see Scheme 1) [47]. The esul ing poly(S-co-CMS) copolyme , 1, con aining 27 mol%
o CMS uni s—as es ima ed by p o on nuclea magne ic esonance (1H NMR)—showed a
weigh -a e age molecula weigh (Mw) o 100.5 kDa and a na ow dispe si y (Ɖ) o 1.15,
as de e mined by size exclusion ch oma og aphy (SEC) equipped wi h iple de ec ion
(RI, MALS, and VIS, see Sec ion 3.2.1). The SEC/RI ace o 1 is illus a ed in Figu e 2. The
hyd odynamic adius (Rh) o 1 in THF was Rh (VIS) = 8.2 nm.
Figu e 2. SEC aces (RI de ec o ) o poly(S-co-CMS), 1, and poly(S-co-AMS), 2.
Subsequen ly, 1 was subjec ed o an azida ion p ocedu e [47] (NaN3, DFMF, 24 h) a
80 °C o gi e he polys y ene (PS)-based p ecu so deco a ed wi h –N3 (ins ead –Cl) unc-
ional g oups: poly(S-co-AMS), 2 (see Scheme 1). The SEC/RI ace o 2 is shown in Figu e
2, wi h alues o Mw (MALS) = 129.1 kDa, Ð = 1.18, and Rh (VIS) = 9.2 nm. We a ibu ed
0
0.2
0.4
0.6
0.8
1
6.577.588.599.5
No malized RI signal (a.u.)
SEC e en ion ime (min.)
21
Figu e 2. SEC aces (RI de ec o ) o poly(S-co-CMS), 1, and poly(S-co-AMS), 2.
Subsequen ly,
1
was subjec ed o an azida ion p ocedu e [
47
] (NaN
3
, DFMF, 24 h)
a 80
◦
C o gi e he polys y ene (PS)-based p ecu so deco a ed wi h –N
3
(ins ead –Cl)
unc ional g oups: poly(S-co-AMS),
2
(see Scheme 1). The SEC/RI ace o
2
is shown in
Figu e 2, wi h alues o M
w
(MALS) = 129.1 kDa, Ð= 1.18, and R
h
(VIS) = 9.2 nm. We
a ibu ed he sligh inc ease in R
h
o s e ic e ec s along he copolyme chain induced by
he eplacemen o he –Cl g oups wi h –N
3
moie ies. The o al eplacemen o –Cl moie ies
wi h –N
3
unc ional g oups was con i med by bo h
1
H and
13
C NMR spec oscopy, as
shown in Figu es 3and 4.
Nanoma e ials 2023,13, 1394 4 o 13
Nanoma e ials 2023, 13, x FOR PEER REVIEW 4 o 13
he sligh inc ease in Rh o s e ic effec s along he copolyme chain induced by he eplace-
men o he –Cl g oups wi h –N3 moie ies. The o al eplacemen o –Cl moie ies wi h –N3
unc ional g oups was con i med by bo h 1H and 13C NMR spec oscopy, as shown in Fig-
u es 3 and 4.
Figu e 3. (a) 1H NMR spec a o poly(S-co-CMS), 1, and poly(S-co-AMS), 2. (b) Enla ged egion be-
ween 3.8 and 4.8 ppm showing he shi o he peak co esponding o me hylene p o ons nex o –
Cl moie ies in 1 du ing ans o ma ion o me hylene p o ons nex o –N3 moie ies in 2. The comple e
shi obse ed con i ms he success ul o al eplacemen o –Cl moie ies in 1 wi h –N3 moie ies in 2.
Figu e 4. (a) 13C NMR spec um o poly(S-co-CMS), 1. (b) 13C NMR spec um o poly(S-co-AMS), 2.
No e he clea shi in he posi ion o he peak co esponding o he me hylene ca bon (deno ed as
a) du ing he eplacemen o all –Cl g oups wi h –N3 moie ies.
2.2. Syn hesis and Cha ac e iza ion o Me al-F ee Single-Chain Nanopa icles (3) om he
Polys y ene-Based P ecu so Deco a ed wi h Azide Func ional G oups (2)
Me al- ee PS-SCNPs we e syn hesized a . . ia SPAAC using DIBOD molecules as
in a-chain c oss-linke s, as depic ed in Scheme 1. We used a slow addi ion echnique, i s
in oduced by Hawke e al. [48], in which a solu ion o he p ecu so (i.e., 2) was added
a a low addi ion a e o a solu ion o he c oss-linke (i.e., DIBOD). A e he comple e
Figu e 3.
(
a
)
1
H NMR spec a o poly(S-co-CMS),
1
, and poly(S-co-AMS),
2
. (
b
) Enla ged egion
be ween 3.8 and 4.8 ppm showing he shi o he peak co esponding o me hylene p o ons nex o
–Cl moie ies in
1
du ing ans o ma ion o me hylene p o ons nex o –N
3
moie ies in
2
. The comple e
shi obse ed con i ms he success ul o al eplacemen o –Cl moie ies in 1wi h –N3moie ies in 2.
Nanoma e ials 2023, 13, x FOR PEER REVIEW 4 o 13
he sligh inc ease in Rh o s e ic effec s along he copolyme chain induced by he eplace-
men o he –Cl g oups wi h –N3 moie ies. The o al eplacemen o –Cl moie ies wi h –N3
unc ional g oups was con i med by bo h 1H and 13C NMR spec oscopy, as shown in Fig-
u es 3 and 4.
Figu e 3. (a) 1H NMR spec a o poly(S-co-CMS), 1, and poly(S-co-AMS), 2. (b) Enla ged egion be-
ween 3.8 and 4.8 ppm showing he shi o he peak co esponding o me hylene p o ons nex o –
Cl moie ies in 1 du ing ans o ma ion o me hylene p o ons nex o –N3 moie ies in 2. The comple e
shi obse ed con i ms he success ul o al eplacemen o –Cl moie ies in 1 wi h –N3 moie ies in 2.
Figu e 4. (a) 13C NMR spec um o poly(S-co-CMS), 1. (b) 13C NMR spec um o poly(S-co-AMS), 2.
No e he clea shi in he posi ion o he peak co esponding o he me hylene ca bon (deno ed as
a) du ing he eplacemen o all –Cl g oups wi h –N3 moie ies.
2.2. Syn hesis and Cha ac e iza ion o Me al-F ee Single-Chain Nanopa icles (3) om he
Polys y ene-Based P ecu so Deco a ed wi h Azide Func ional G oups (2)
Me al- ee PS-SCNPs we e syn hesized a . . ia SPAAC using DIBOD molecules as
in a-chain c oss-linke s, as depic ed in Scheme 1. We used a slow addi ion echnique, i s
in oduced by Hawke e al. [48], in which a solu ion o he p ecu so (i.e., 2) was added
a a low addi ion a e o a solu ion o he c oss-linke (i.e., DIBOD). A e he comple e
Figu e 4.
(
a
)
13
C NMR spec um o poly(S-co-CMS),
1
. (
b
)
13
C NMR spec um o poly(S-co-AMS),
2
.
No e he clea shi in he posi ion o he peak co esponding o he me hylene ca bon (deno ed as
a
)
du ing he eplacemen o all –Cl g oups wi h –N3moie ies.
2.2. Syn hesis and Cha ac e iza ion o Me al-F ee Single-Chain Nanopa icles (3) om he
Polys y ene-Based P ecu so Deco a ed wi h Azide Func ional G oups (2)
Me al- ee PS-SCNPs we e syn hesized a . . ia SPAAC using DIBOD molecules as
in a-chain c oss-linke s, as depic ed in Scheme 1. We used a slow addi ion echnique, i s
in oduced by Hawke e al. [
48
], in which a solu ion o he p ecu so (i.e.,
2
) was added
a a low addi ion a e o a solu ion o he c oss-linke (i.e., DIBOD). A e he comple e
addi ion o
2
in ca. 12 h, he mix u e emained (addi ionally) o 12 h unde s i ing o
allow he SPAAC o go o comple ion. Then, an app op ia e amoun o BzA was added o
end-cap any po en ial un eac ed alkyne g oup, and he mix u e was le unde s i ing o
an addi ional 24 h. A e concen a ing he eac ion mix u e,
3
was ob ained by p ecipi a ion
in cold E OH. Figu e 5shows he SEC/MALS aces o
2
,
3
be o e p ecipi a ion a e 48 h
o eac ion, and
3
a e p ecipi a ion, d ying, and e-dissolu ion. No change in he SEC
ace o he PS-SCNPs,
3
, was obse ed om he c ude o he d ied (and e-dissol ed)
Nanoma e ials 2023,13, 1394 5 o 13
samples. The PS-SCNPs,
3,
exhibi ed alues o M
w
(MALS) = 195.8 kDa, Ð= 1.21, and
R
h
(VIS) = 6.9 nm. The inc ease in M
w
obse ed was due o he inco po a ion o he
in a-chain c oss-linke DIBOD. The SEC esul s con i med he success ul o ma ion o
SCNPs wi h a hyd odynamic size (R
h
(VIS) = 6.9 nm) smalle han ha o he p ecu so
copolyme (R
h
(VIS) = 9.2 nm). We can exclude he p esence o mul i-chain agg ega es
since he SEC/MALS echnique is highly sensi i e o hei p esence, and hey should be
obse ed a a low SEC e en ion ime (<7 min.). Addi ionally, dynamic ligh sca e ing
(DLS) measu emen s also showed a signi ican educ ion in size (R
h
(DLS) = 7.5 nm) upon
he o ma ion o me al- ee PS-SCNPs ia SPAAC using DIBOD molecules as in a-chain
c oss-linke s, as shown in Figu e 6.
Nanoma e ials 2023, 13, x FOR PEER REVIEW 5 o 13
addi ion o 2 in ca. 12 h, he mix u e emained (addi ionally) o 12 h unde s i ing o
allow he SPAAC o go o comple ion. Then, an app op ia e amoun o BzA was added o
end-cap any po en ial un eac ed alkyne g oup, and he mix u e was le unde s i ing o
an addi ional 24 h. A e concen a ing he eac ion mix u e, 3 was ob ained by p ecipi a-
ion in cold E OH. Figu e 5 shows he SEC/MALS aces o 2, 3 be o e p ecipi a ion a e
48 h o eac ion, and 3 a e p ecipi a ion, d ying, and e-dissolu ion. No change in he
SEC ace o he PS-SCNPs, 3, was obse ed om he c ude o he d ied (and e-dissol ed)
samples. The PS-SCNPs, 3, exhibi ed alues o Mw (MALS) = 195.8 kDa, Ð = 1.21, and Rh
(VIS) = 6.9 nm. The inc ease in Mw obse ed was due o he inco po a ion o he in a-
chain c oss-linke DIBOD. The SEC esul s con i med he success ul o ma ion o SCNPs
wi h a hyd odynamic size (Rh (VIS) = 6.9 nm) smalle han ha o he p ecu so copolyme
(Rh (VIS) = 9.2 nm). We can exclude he p esence o mul i-chain agg ega es since he
SEC/MALS echnique is highly sensi i e o hei p esence, and hey should be obse ed
a a low SEC e en ion ime (<7 min.). Addi ionally, dynamic ligh sca e ing (DLS) meas-
u emen s also showed a signi ican educ ion in size (Rh (DLS) = 7.5 nm) upon he o -
ma ion o me al- ee PS-SCNPs ia SPAAC using DIBOD molecules as in a-chain c oss-
linke s, as shown in Figu e 6.
Figu e 5. SEC aces (MALS de ec o ) o poly(S-co-AMS), 2; me al- ee PS-SCNPs as syn he ized
wi hou isola ion, 3 C ude; and me al- ee PS-SCNPs a e isola ion, 3 D ied.
Figu e 6. Hyd odynamic size dis ibu ion o poly(S-co-AMS), 2, and me al- ee PS-SCNPs, 3, as de-
e mined by dynamic ligh sca e ing (DLS) measu emen s. P ecu so 2: Rh (DLS) = 10.1 nm. PS-
SCNPs, 3: Rh (DLS) = 7.5 nm.
0
0.2
0.4
0.6
0.8
1
6 6.5 7 7.5 8 8.5 9 9.5
No malized MALS signal (a.u.)
SEC e en ion ime (min.)
23 C ude
3 D ied
0
0.2
0.4
0.6
0.8
1
10 100
I
no m
DLS hyd odynamic size (nm)
32
Figu e 5.
SEC aces (MALS de ec o ) o poly(S-co-AMS),
2
; me al- ee PS-SCNPs as syn he ized
wi hou isola ion, 3 C ude; and me al- ee PS-SCNPs a e isola ion, 3 D ied.
Nanoma e ials 2023, 13, x FOR PEER REVIEW 5 o 13
addi ion o 2 in ca. 12 h, he mix u e emained (addi ionally) o 12 h unde s i ing o
allow he SPAAC o go o comple ion. Then, an app op ia e amoun o BzA was added o
end-cap any po en ial un eac ed alkyne g oup, and he mix u e was le unde s i ing o
an addi ional 24 h. A e concen a ing he eac ion mix u e, 3 was ob ained by p ecipi a-
ion in cold E OH. Figu e 5 shows he SEC/MALS aces o 2, 3 be o e p ecipi a ion a e
48 h o eac ion, and 3 a e p ecipi a ion, d ying, and e-dissolu ion. No change in he
SEC ace o he PS-SCNPs, 3, was obse ed om he c ude o he d ied (and e-dissol ed)
samples. The PS-SCNPs, 3, exhibi ed alues o Mw (MALS) = 195.8 kDa, Ð = 1.21, and Rh
(VIS) = 6.9 nm. The inc ease in Mw obse ed was due o he inco po a ion o he in a-
chain c oss-linke DIBOD. The SEC esul s con i med he success ul o ma ion o SCNPs
wi h a hyd odynamic size (Rh (VIS) = 6.9 nm) smalle han ha o he p ecu so copolyme
(Rh (VIS) = 9.2 nm). We can exclude he p esence o mul i-chain agg ega es since he
SEC/MALS echnique is highly sensi i e o hei p esence, and hey should be obse ed
a a low SEC e en ion ime (<7 min.). Addi ionally, dynamic ligh sca e ing (DLS) meas-
u emen s also showed a signi ican educ ion in size (Rh (DLS) = 7.5 nm) upon he o -
ma ion o me al- ee PS-SCNPs ia SPAAC using DIBOD molecules as in a-chain c oss-
linke s, as shown in Figu e 6.
Figu e 5. SEC aces (MALS de ec o ) o poly(S-co-AMS), 2; me al- ee PS-SCNPs as syn he ized
wi hou isola ion, 3 C ude; and me al- ee PS-SCNPs a e isola ion, 3 D ied.
Figu e 6. Hyd odynamic size dis ibu ion o poly(S-co-AMS), 2, and me al- ee PS-SCNPs, 3, as de-
e mined by dynamic ligh sca e ing (DLS) measu emen s. P ecu so 2: Rh (DLS) = 10.1 nm. PS-
SCNPs, 3: Rh (DLS) = 7.5 nm.
0
0.2
0.4
0.6
0.8
1
6 6.5 7 7.5 8 8.5 9 9.5
No malized MALS signal (a.u.)
SEC e en ion ime (min.)
23 C ude
3 D ied
0
0.2
0.4
0.6
0.8
1
10 100
I
no m
DLS hyd odynamic size (nm)
32
Figu e 6.
Hyd odynamic size dis ibu ion o poly(S-co-AMS),
2
, and me al- ee PS-SCNPs,
3
, as
de e mined by dynamic ligh sca e ing (DLS) measu emen s. P ecu so
2
:R
h
(DLS) = 10.1 nm.
PS-SCNPs, 3:Rh(DLS) = 7.5 nm.
The inco po a ion o DIBOD molecules as in a-chain c oss-linke s was con i med
by
1
H NMR and
13
C NMR spec oscopy, as illus a ed in Figu es 7and 8, espec i ely.
Addi ionally, he disappea ance o he in a- ed (IR) ib a ion band co esponding o he
azide unc ional g oup loca ed nea 2100 cm
−1
was clea ly isible in he FTIR spec um o
he me al- ee PS-SCNPs, 3, as illus a ed in Figu e 9.
Nanoma e ials 2023,13, 1394 6 o 13
Nanoma e ials 2023, 13, x FOR PEER REVIEW 6 o 13
The inco po a ion o DIBOD molecules as in a-chain c oss-linke s was con i med by
1
H NMR and
13
C NMR spec oscopy, as illus a ed in Figu e 7 and Figu e 8, espec i ely.
Addi ionally, he disappea ance o he in a- ed (IR) ib a ion band co esponding o he
azide unc ional g oup loca ed nea 2100 cm
−1
was clea ly isible in he FTIR spec um o
he me al- ee PS-SCNPs, 3, as illus a ed in Figu e 9.
Figu e 7.
1
H NMR spec a o me al- ee PS-SCNPs, 3. Peaks deno ed as g and h co espond o a o-
ma ic p o ons om he DIBOD in a-chain c oss-linke .
Figu e 8.
13
C NMR spec a o me al- ee PS-SCNPs, 3, showing he posi ions o he diffe en a oma ic
ca bons, including hose om he DIBOD in a-chain c oss-linke .
Figu e 7. 1
H NMR spec a o me al- ee PS-SCNPs,
3
. Peaks deno ed as
g
and
h
co espond o
a oma ic p o ons om he DIBOD in a-chain c oss-linke .
Nanoma e ials 2023, 13, x FOR PEER REVIEW 6 o 13
The inco po a ion o DIBOD molecules as in a-chain c oss-linke s was con i med by
1
H NMR and
13
C NMR spec oscopy, as illus a ed in Figu e 7 and Figu e 8, espec i ely.
Addi ionally, he disappea ance o he in a- ed (IR) ib a ion band co esponding o he
azide unc ional g oup loca ed nea 2100 cm
−1
was clea ly isible in he FTIR spec um o
he me al- ee PS-SCNPs, 3, as illus a ed in Figu e 9.
Figu e 7.
1
H NMR spec a o me al- ee PS-SCNPs, 3. Peaks deno ed as g and h co espond o a o-
ma ic p o ons om he DIBOD in a-chain c oss-linke .
Figu e 8.
13
C NMR spec a o me al- ee PS-SCNPs, 3, showing he posi ions o he diffe en a oma ic
ca bons, including hose om he DIBOD in a-chain c oss-linke .
Figu e 8. 13
C NMR spec a o me al- ee PS-SCNPs,
3
, showing he posi ions o he di e en a oma ic
ca bons, including hose om he DIBOD in a-chain c oss-linke .
Nanoma e ials 2023, 13, x FOR PEER REVIEW 7 o 13
Figu e 9. Disappea ance o he azide IR ib a ion band o p ecu so 2 upon eac ion wi h DIBOD
and he o ma ion o me al- ee SCNPs, 3.
Figu e 10a illus a es he he mal s abili y o p ecu so 2 and he PS-SCNPs, 3, as
de e mined by TGA measu emen s. The ini ial decomposi ion empe a u e o 2 was 220
°C, co esponding o he ex usion o dini ogen molecules om he azide g oups. The
ini ial decomposi ion empe a u e o 3 was ound o be 153 °C. We a ibu ed he lowe
he mal s abili y o 3 o he s ained na u e o he 1,10-dihyd odibenzo [3,4:7,8]cyclooc a
[1,2-d:5,6-d]bis([1,2,3] iazole) c oss-linking uni s (see Figu e 10b), which p omo ed hei
he mal decomposi ion when hea ing abo e 150 °C. Below his limi ing empe a u e, he
PS-SCNPs showed no weigh loss so one can conside 3 o be a s able ma e ial up o 150
°C.
Figu e 10. (a) Compa ison o he he mal s abili y o p ecu so 2 and he esul ing me al- ee SCNPs,
3. (b) The lowe he mal s abili y o 3 can be a ibu ed o he s ained na u e o he 1,10-dihyd odi-
benzo [3,4:7,8]cyclooc a [1,2-d:5,6-d]bis([1,2,3] iazole) uni s o he PS-SCNPs, as e ealed by he
con o ma ion ound a e ene gy minimiza ion (MMFF94 o ce ield, Chem3D).
19002000210022002300
T ansmi ance (a.u.)
Wa enumbe (cm-1)
2
3
Figu e 9.
Disappea ance o he azide IR ib a ion band o p ecu so
2
upon eac ion wi h DIBOD
and he o ma ion o me al- ee SCNPs, 3.
Nanoma e ials 2023,13, 1394 7 o 13
Figu e 10a illus a es he he mal s abili y o p ecu so
2
and he PS-SCNPs,
3,
as
de e mined by TGA measu emen s. The ini ial decomposi ion empe a u e o
2
was 220
◦
C,
co esponding o he ex usion o dini ogen molecules om he azide g oups. The ini ial
decomposi ion empe a u e o
3
was ound o be 153
◦
C. We a ibu ed he lowe he mal
s abili y o
3
o he s ained na u e o he 1,10-dihyd odibenzo [3,4:7,8]cyclooc a [1,2-d:5,6-
d’]bis([1,2,3] iazole) c oss-linking uni s (see Figu e 10b), which p omo ed hei he mal
decomposi ion when hea ing abo e 150
◦
C. Below his limi ing empe a u e, he PS-SCNPs
showed no weigh loss so one can conside 3 o be a s able ma e ial up o 150 ◦C.
Nanoma e ials 2023, 13, x FOR PEER REVIEW 7 o 13
Figu e 9. Disappea ance o he azide IR ib a ion band o p ecu so 2 upon eac ion wi h DIBOD
and he o ma ion o me al- ee SCNPs, 3.
Figu e 10a illus a es he he mal s abili y o p ecu so 2 and he PS-SCNPs, 3, as
de e mined by TGA measu emen s. The ini ial decomposi ion empe a u e o 2 was 220
°C, co esponding o he ex usion o dini ogen molecules om he azide g oups. The
ini ial decomposi ion empe a u e o 3 was ound o be 153 °C. We a ibu ed he lowe
he mal s abili y o 3 o he s ained na u e o he 1,10-dihyd odibenzo [3,4:7,8]cyclooc a
[1,2-d:5,6-d]bis([1,2,3] iazole) c oss-linking uni s (see Figu e 10b), which p omo ed hei
he mal decomposi ion when hea ing abo e 150 °C. Below his limi ing empe a u e, he
PS-SCNPs showed no weigh loss so one can conside 3 o be a s able ma e ial up o 150
°C.
Figu e 10. (a) Compa ison o he he mal s abili y o p ecu so 2 and he esul ing me al- ee SCNPs,
3. (b) The lowe he mal s abili y o 3 can be a ibu ed o he s ained na u e o he 1,10-dihyd odi-
benzo [3,4:7,8]cyclooc a [1,2-d:5,6-d]bis([1,2,3] iazole) uni s o he PS-SCNPs, as e ealed by he
con o ma ion ound a e ene gy minimiza ion (MMFF94 o ce ield, Chem3D).
19002000210022002300
T ansmi ance (a.u.)
Wa enumbe (cm-1)
2
3
Figu e 10.
(
a
) Compa ison o he he mal s abili y o p ecu so
2
and he esul ing me al- ee
SCNPs,
3
. (
b
) The lowe he mal s abili y o
3
can be a ibu ed o he s ained na u e o he 1,10-
dihyd odibenzo [3,4:7,8]cyclooc a [1,2-d:5,6-d’]bis([1,2,3] iazole) uni s o he PS-SCNPs, as e ealed
by he con o ma ion ound a e ene gy minimiza ion (MMFF94 o ce ield, Chem3D).
2.3. Long-Te m S abili y agains Agg ega ion o Me al-F ee Polys y ene-Single-Chain
Nanopa icles (3)
The long- e m s abili y agains agg ega ion o he me al- ee PS-SCNPs was in es i-
ga ed using small angle X- ay sca e ing (SAXS) measu emen s in THF solu ion a high
dilu ion. Re e ence SAXS measu emen s we e aken o de e mine he o m ac o o p ecu -
so
2
and he me al- ee PS-SCNPs,
3,
a e syn hesis (see Figu e 11). Analysis o he da a
shown in Figu e 11 in e ms o he gene alized Gaussian coil unc ion [
49
] p o ided he
alues o he adius o gy a ion (R
g
) and scaling exponen (
ν
) o
2
and
3
in solu ion a high
dilu ion. P ecu so
2
showed a size o R
g
= 14.1 nm and
ν
= 0.59, a scaling exponen alue
ha was in ag eemen wi h he expec ed Flo y alue o a linea chain in good sol en
(
νF≈
3/5). On he o he hand, he eshly syn hesized me al- ee PS-SCNPs exhibi ed a
educ ion in bo h he size and scaling exponen : R
g
= 9.1 nm and
ν
= 0.46. These alues
we e in ag eemen wi h p e iously epo ed da a on he single-chain olding o syn he ic
polyme s o SCNPs [1,38].
Nanoma e ials 2023,13, 1394 8 o 13
Nanoma e ials 2023, 13, x FOR PEER REVIEW 8 o 13
2.3. Long-Te m S abili y agains Agg ega ion o Me al-F ee Polys y ene-Single-Chain
Nanopa icles (3)
The long- e m s abili y agains agg ega ion o he me al- ee PS-SCNPs was in es i-
ga ed using small angle X- ay sca e ing (SAXS) measu emen s in THF solu ion a high
dilu ion. Re e ence SAXS measu emen s we e aken o de e mine he o m ac o o p e-
cu so 2 and he me al- ee PS-SCNPs, 3, a e syn hesis (see Figu e 11). Analysis o he
da a shown in Figu e 11 in e ms o he gene alized Gaussian coil unc ion [49] p o ided
he alues o he adius o gy a ion (Rg) and scaling exponen (ν) o 2 and 3 in solu ion a
high dilu ion. P ecu so 2 showed a size o Rg = 14.1 nm and ν = 0.59, a scaling exponen
alue ha was in ag eemen wi h he expec ed Flo y alue o a linea chain in good sol-
en (νF ≈ 3/5). On he o he hand, he eshly syn hesized me al- ee PS-SCNPs exhibi ed
a educ ion in bo h he size and scaling exponen : Rg = 9.1 nm and ν = 0.46. These alues
we e in ag eemen wi h p e iously epo ed da a on he single-chain olding o syn he ic
polyme s o SCNPs [1,38].
Figu e 11. SAXS esul s e ealing: (a) he o m ac o o p ecu so 2, and (b) he o m ac o o he
me al- ee SCNPs, 3, as syn he ized. Values o Rg and ν we e ob ained h ough i s o he expe i-
men al P(Q) s. Q da a o a gene alized Gaussian coil unc ion. P ecu so 2: Rg = 14.1 nm, ν = 0.59.
PS-SCNPs, 3: Rg = 9.1 nm, ν = 0.46.
Figu e 12 shows he SAXS esul s ob ained o he SCNPs, 3, in THF dilu e solu ion
a e 2 mon hs o s o age. Figu e 12a co esponds o s o age in he same solu ion, whe eas
Figu e 12b co esponds o s o age in he solid s a e and he edispe sion o he SCNPs in
THF. Analysis o he da a in e ms o a gene alized Gaussian coil unc ion e ealed no
signi ican changes (wi hin he expe imen al unce ain ies) in Rg o ν wi h espec o he
alues obse ed o he PS-SCNPs, 3, as syn he ized (Figu e 11b). Only in he case o s o -
age in he solid s a e was a mino inc ease in Rg obse ed, al hough he scaling exponen
did no change signi ican ly. Hence, unlike he case o CuAAC in which la ge agg ega es
a e ypically ound a e p olonged s o age, he syn hesis o he me al- ee SCNPs ia
SPAAC using DIBOD molecules as in a-chain c oss-linke s can p e en i e e sible
mul i-chain agg ega ion issues.
Figu e 11.
SAXS esul s e ealing: (
a
) he o m ac o o p ecu so
2
, and (
b
) he o m ac o o he
me al- ee SCNPs,
3
, as syn he ized. Values o R
g
and
ν
we e ob ained h ough i s o he expe imen al
P(Q) s. Q da a o a gene alized Gaussian coil unc ion. P ecu so
2
:R
g
= 14.1 nm,
ν
= 0.59. PS-SCNPs,
3:Rg= 9.1 nm, ν= 0.46.
Figu e 12 shows he SAXS esul s ob ained o he SCNPs,
3,
in THF dilu e solu ion
a e 2 mon hs o s o age. Figu e 12a co esponds o s o age in he same solu ion, whe eas
Figu e 12b co esponds o s o age in he solid s a e and he edispe sion o he SCNPs in
THF. Analysis o he da a in e ms o a gene alized Gaussian coil unc ion e ealed no
signi ican changes (wi hin he expe imen al unce ain ies) in R
g
o
ν
wi h espec o he
alues obse ed o he PS-SCNPs,
3
, as syn he ized (Figu e 11b). Only in he case o s o age
in he solid s a e was a mino inc ease in R
g
obse ed, al hough he scaling exponen did
no change signi ican ly. Hence, unlike he case o CuAAC in which la ge agg ega es a e
ypically ound a e p olonged s o age, he syn hesis o he me al- ee SCNPs ia SPAAC
using DIBOD molecules as in a-chain c oss-linke s can p e en i e e sible mul i-chain
agg ega ion issues.
Nanoma e ials 2023, 13, x FOR PEER REVIEW 9 o 13
Figu e 12. SAXS o m ac o o : (a) 3 a e 2 mon hs o s o age in THF solu ion (Rg = 8.7 nm, ν = 0.46),
and (b) 3 a e 2 mon hs o s o age in he solid s a e (Rg = 12.0 nm, ν = 0.47).
We a e cu en ly in es iga ing o he azide-con aining p ecu so s besides PS o e al-
ua e he scope o he SPAAC me hod using DIBOD molecules as in a-chain c oss-linke s
o he syn hesis o long- e m-dispe sible, me al- ee SCNPs. The esul s will be epo ed
a ano he ime.
3. Ma e ials and Me hods
3.1. Ma e ials
S y ene (S, ≥99%), sodium azide (NaN3, ≥99%), N,N-dime hyl o mamide (DMF,
≥99.9%), and 1,1′-azobis(cyclohexaneca boni ile) (ACHN, ≥98%) we e pu chased om
Sigma-Ald ich (Mad id, Spain). Chlo ome hyl s y ene (CMS, ≥90%) and sym-dibenzo-1,5-
cyclooc adiene-3,7-diyne (DIBOD, > 98%) we e supplied by TCI. Te ahyd o u an (THF,
GPC g ade) and me hanol (MeOH, analy ical g ade) we e pu chased om Scha lau (Ba -
celona, Spain). Benzyl azide (BzA, 94%), S-cyanome hyl-S-dodecyl i hioca bona e
(CDTC, ≥97%), and e hanol (E OH, 96% / ) we e supplied by The mo Scien i ic (Eindho-
en, Ne he lands), S em (Bischheim, F ance), and PanReac AppliChem (Ba celona,
Spain), espec i ely. Unless o he wise speci ied, all eagen s we e used as ecei ed wi h-
ou u he pu i ica ion. To emo e he inhibi o , S and CMS we e pu i ied by being
passed h ough a column packed wi h ac i a ed basic alumina.
3.2. Techniques
3.2.1. Size-Exclusion Ch oma og aphy (SEC)
SEC measu emen s we e pe o med a 30 °C on an Agilen 1200 sys em equipped
wi h PLgel 5 µm Gua d and PLgel 5 µm MIXED-C columns, and iple de ec ion: a diffe -
en ial e ac i e index (RI) de ec o (Op ilab Rex, Wya , Toulouse, F ance), a mul i-angle
lase ligh sca e ing (MALS) de ec o (MiniDawn T eos, Wya , Toulouse, F ance), and a
iscosime ic (VIS) de ec o (ViscoS a -II, Wya , Toulouse, F ance). Da a analysis was pe -
o med using ASTRA So wa e ( e sion 6.1) om Wya . THF was used as an eluen a a
low a e o 1 mL/min. A alue o dn/dc = 0.186 was used o poly(S-co-CMS), 1, poly(S-
co-AMS), 2, and he me al- ee PS-SCNPs, 3.
Figu e 12.
SAXS o m ac o o : (
a
)
3
a e 2 mon hs o s o age in THF solu ion (R
g
= 8.7 nm,
ν
= 0.46),
and (b)3a e 2 mon hs o s o age in he solid s a e (Rg= 12.0 nm, ν= 0.47).
We a e cu en ly in es iga ing o he azide-con aining p ecu so s besides PS o e alua e
he scope o he SPAAC me hod using DIBOD molecules as in a-chain c oss-linke s o
he syn hesis o long- e m-dispe sible, me al- ee SCNPs. The esul s will be epo ed a
ano he ime.
Nanoma e ials 2023,13, 1394 9 o 13
3. Ma e ials and Me hods
3.1. Ma e ials
S y ene (S,
≥
99%), sodium azide (NaN
3
,
≥
99%), N,N-dime hyl o mamide (DMF,
≥
99.9%), and 1,1
0
-azobis(cyclohexaneca boni ile) (ACHN,
≥
98%) we e pu chased om
Sigma-Ald ich (Mad id, Spain). Chlo ome hyl s y ene (CMS,
≥
90%) and sym-dibenzo-
1,5-cyclooc adiene-3,7-diyne (DIBOD, > 98%) we e supplied by TCI. Te ahyd o u an
(THF, GPC g ade) and me hanol (MeOH, analy ical g ade) we e pu chased om Scha lau
(Ba celona, Spain). Benzyl azide (BzA, 94%), S-cyanome hyl-S-dodecyl i hioca bona e
(CDTC,
≥
97%), and e hanol (E OH, 96% / ) we e supplied by The mo Scien i ic (Eind-
ho en, Ne he lands), S em (Bischheim, F ance), and PanReac AppliChem (Ba celona,
Spain), espec i ely. Unless o he wise speci ied, all eagen s we e used as ecei ed wi hou
u he pu i ica ion. To emo e he inhibi o , S and CMS we e pu i ied by being passed
h ough a column packed wi h ac i a ed basic alumina.
3.2. Techniques
3.2.1. Size-Exclusion Ch oma og aphy (SEC)
SEC measu emen s we e pe o med a 30
◦
C on an Agilen 1200 sys em equipped wi h
PLgel 5
µ
m Gua d and PLgel 5
µ
m MIXED-C columns, and iple de ec ion: a di e en ial
e ac i e index (RI) de ec o (Op ilab Rex, Wya , Toulouse, F ance), a mul i-angle lase
ligh sca e ing (MALS) de ec o (MiniDawn T eos, Wya , Toulouse, F ance), and a iscosi-
me ic (VIS) de ec o (ViscoS a -II, Wya , Toulouse, F ance). Da a analysis was pe o med
using ASTRA So wa e ( e sion 6.1) om Wya . THF was used as an eluen a a low a e
o 1 mL/min. A alue o dn/dc = 0.186 was used o poly(S-co-CMS),
1
, poly(S-co-AMS),
2
,
and he me al- ee PS-SCNPs, 3.
3.2.2. Nuclea Magne ic Resonance (NMR)
1
H NMR and
13
C NMR spec a we e eco ded a oom empe a u e on B uke (Mad id,
Spain) spec ome e s ope a ing a 400 MHz, using CDCl3as sol en .
3.2.3. Dynamic Ligh Sca e ing (DLS)
DLS measu emen s (numbe dis ibu ion) we e aken a oom empe a u e on a
Mal e n Ze asize Nano ZS (Camb idge, Uni ed Kingdom) appa a us.
3.2.4. Fou ie T ans o m In a-Red (FTIR) Spec oscopy
FTIR spec a we e eco ded a oom empe a u e on a JASCO 3600 (Mad id, Spain)
FTIR spec ome e .
3.2.5. The mog a ime ic Analysis (TGA)
TGA measu emen s we e pe o med on a Q500-TA Ins umen s (Ce danyola del Valles,
Spain) appa a us a a hea ing a e o 10 ◦C/min unde a ni ogen a mosphe e.
3.2.6. Small-Angle X- ay Sca e ing (SAXS)
SAXS expe imen s we e conduc ed on he Rigaku (Ba celona, Spain) 3-pinhole PSAXS-
L equipmen o he Ma e ials Physics Cen e , ope a ing a 45 kV and 0.88 mA. The
Mic oMax-002+ X- ay Gene a o Sys em is composed o a mic o ocus sealed ube sou ce
module and an in eg a ed X- ay gene a o uni , which p oduces CuK
α
ansi ion pho ons
o wa eleng h
λ
= 1.54 Å. The ligh pa h and he sample chambe in his equipmen a e
unde acuum. The sca e ed X- ays a e de ec ed on a wo-dimensional mul iwi e X- ay
De ec o (Gab iel (Ba celona, Spain) design, 2D-200X) and con e ed o one-dimensional
sca e ing cu es by adial a e aging. This gas- illed p opo ional ype de ec o o e s a
200 mm diame e ac i e a ea wi h ca. 200-mic on esolu ion. A e adial in eg a ion, he
sca e ed in ensi ies we e ob ained as a unc ion o momen um ans e Q = 4
πλ−1
sin
θ
,
whe e
θ
is hal he sca e ing angle. Recip ocal space calib a ion was pe o med using
sil e behena e as s anda d. The sample- o-de ec o dis ance was 2 m, co e ing a Q- ange
