As ea u ed in:
Showcasing collabo a i e esea ch om he Ins i u e
o Ad anced Ma e ials (Cas ellón, Spain), he A agón
Nanoscience and Ma e ials Ins i u e (Za agoza, Spain) and
he Uni e si y o No ingham (U.K.).
Decoupling manu ac u ing om applica ion in addi i e
manu ac u ed an imic obial ma e ials
We epo a new concep in he addi i e manu ac u ing
o an imic obial de ices made using polyme izable ionic
liquid sca olds, whe eby hei p oduc ion can be e ec i ely
sepa a ed om hei unc ional an imic obial p ope ies.
A apid and con ollable pos -p in ing cu ing p ocess
ini ia es he in-si u o ma ion o sil e nanopa icles wi hin
he polyme ic ma ix, con e ing bac e icidal ac i i y o he
de ice.
Co e illus a ion by Isabel F anco Cas illo.
Regis e ed cha i y numbe : 207890
sc.li/bioma e ials-science
See Sco G. Mi chell, Vic o Sans
e al. , Bioma e . Sci. , 2021, 9 , 5467.
Bioma e ials
Science
sc.li/bioma e ials-science
Volume 9
Numbe 16
21 Augus 2021
Pages 5387-5704
ISSN 2047-4849
PAPER
Dong-Hyun Kim e al.
Magne o mi ochond ial dys unc ion media ed cance cell
dea h using in acellula magne ic nano- ansduce s
Bioma e ials
Science
COMMUNICATION
Ci e his: Bioma e . Sci., 2021, 9,
5467
Recei ed 19 h Ma ch 2021,
Accep ed 10 h May 2021
DOI: 10.1039/d1bm00430a
sc.li/bioma e ials-science
Decoupling manu ac u ing om applica ion in
addi i e manu ac u ed an imic obial ma e ials†
Dominic J. Wales, ‡
a
Sa a Mi alles-Comins,§
b
Isabel F anco-Cas illo,
c,d
Jamie M. Came on,
e
Qun Cao,
a
E no Ka jalainen,
a
Jesum Al es Fe nandes,
G aham N. New on,
e
Sco G. Mi chell *
c,d
and Vic o Sans *
b
3D p in able ma e ials based on polyme ic ionic liquids (PILs)
capable o con olling he syn hesis and s abilisa ion o sil e
nanopa icles (AgNPs) and hei syne gis ic an imic obial ac i i y
a e epo ed. The in e ac ion o he ionic liquid moie ies wi h he
sil e p ecu so enabled he con olled in si u o ma ion and s abil-
isa ion o AgNPs ia ex ended UV pho o educ ion a e he p in ing
p ocess, hus demons a ing an effec i e decoupling o he de ice
manu ac u ing om he on-demand gene a ion o nanoma e ials,
which a oids he po en ial aging o he nanoma e ials h ough oxi-
da ion. The p in ed de ices showed a mul i- unc ional and une-
able mic obicidal ac i i y agains G am posi i e (B. sub ilis) and
G am nega i e (E. coli) bac e ia and agains he mould Aspe gillus
nige . While he polyme ic ma e ial alone was ound o be bac e -
ios a ic, he AgNPs con e ed bac e icidal p ope ies o he
ma e ial. Combining PIL-based ma e ials wi h unc ionali ies, such
as in si u and pho oac i a ed on-demand ab ica ed an imic obial
AgNPs, p o ides a syne gis ic unc ionali y ha could be ha nessed
o a a ie y o applica ions, especially when coupled o he
eedom o design inhe en o addi i e manu ac u ing echniques.
In oduc ion
An imic obial esis ance is a signi ican global heal h chal-
lenge a ound he wo ld.
1
A po en ial solu ion ha has enjoyed
signi ican in e es a e ma e ials wi h inhe en an imic obial
p ope ies.
2
One example is sil e nanopa icles (AgNPs),
which exhibi b oad an imic obial po en ial,
3,4
and can be sup-
po ed in a wide ange o polyme s and hyd ogels.
5–8
Such
ma e ials can be ab ica ed in o my iad unc ional shapes and
de ices by u ilising he design eedoms affo ded by 3D p in -
ing,
9
and indeed he e a e many examples o AgNP suppo ing
ma e ials ha ha e been 3D p in ed.
10–17
Typically, he AgNPs
a e ei he dispe sed in he esin be o e 3D p in ing, o he
AgNPs a e o med du ing he 3D p in ing p ocess, and hen
he ma e ials can be used immedia ely. Howe e , he dis-
ad an age o his app oach is ha i does no allow o une-
able ab ica ion o he AgNPs so he efficacy o he de ice is ai-
lo ed o he pa icula applica ion a he poin o use.
The e o e, o main ain maximal an imic obial efficacy,
whils s ill allowing o a esponsi e bespoke pe sonalised
heal hca e app oach enabled by he inhe en eedoms
affo ded by 3D p in ing, decoupling o he manu ac u e o he
3D p in ed pa and he o ma ion o AgNPs is needed. The e
a e some examples o 3D p in ed ma e ials con aining AgNPs,
whe ein he ab ica ion is decoupled om he AgNP o -
ma ion, bu hey ypically equi e pos -p in ing ea men s,
like ene gy-in ensi e hea ing s eps,
18
o imme sion o he
p in ed pa in a educing solu ion.
19
A desi able al e na i e is a polyme ic ma ix ha can be 3D
p in ed, has he equi ed mechanical p ope ies and also
main ains he sil e p ecu so in he oxidised o m o enable a
con olled educ i e o ma ion o he AgNPs a he poin o
need. Fan ino e al. ha e demons a ed on-demand pho o-
†Elec onic supplemen a y in o ma ion (ESI) a ailable: UV-Vis spec um o a 3D
p in ed PIL1 pa be o e pos -p in ing UV pho o educ ion ea men ; XPS spec-
um, s ess–s ain cu es, empo al UV-Vis spec a acking pho o educ ion, and
abula ed mechanical da a o pho o educed 3D p in ed PIL1 pa s; UV-Vis spec-
um o pho o educed analogous homogeneous non-polyme isable liquid IL
sys em; UV-Vis spec a o pho o educed 3D p in ed PIL2 and PIL3 pa s; pho o-
g aphs o PIL2 and PIL3 3D p in ed pa s be o e and a e pho o educ ion; em-
po al UV-Vis spec a o pho o educed 3D p in ed PIL2 aged in ambien ligh . See
DOI: 10.1039/d1bm00430a
‡P esen Add ess: Hamlyn Cen e, Ins i u e o Global Heal h Inno a ion,
Impe ial College London, Sou h Kensing on Campus, London, SW7 2AZ, Uni ed
Kingdom.
§These au ho s ha e con ibu ed equally.
a
Facul y o Enginee ing, Uni e si y o No ingham, Uni e si y Pa k, No ingham,
NG7 2RD, UK
b
Ins i u e o Ad anced Ma e ials (INAM), Uni e si a Jaume I, 12071 Cas ellon,
Spain. E-mail: [email p o ec ed]
c
Ins i u o de Nanociencia y Ma e iales de A agón (INMA-CSIC), CSIC-Uni e sidad de
Za agoza, c/Ped o Ce buna 12, 50009 Za agoza, Spain
d
CIBER de Bioingenie ía, Bioma e iales y Nanomedicina, Ins i u o de Salud Ca los
III, 28029 Mad id, Spain. E-mail: [email p o ec ed]
e
GSK Ca bon Neu al Labo a o y, Uni e si y o No ingham, Jubilee Campus,
No ingham, NG8 2GA, UK
School o Chemis y, Uni e si y o No ingham, Uni e si y Pa k, No ingham,
NG7 2RD, UK
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educ i e o ma ion o AgNPs wi hin pho ocu ed polye hylene
glycol diac yla e (PEGDA), which was decoupled om he 3D
p in ing s ep. Howe e , hei sys em appea ed o offe limi ed
abili y o s abilise AgNPs, and nanopa icle agg ega ion and
mig a ion we e obse ed.
20
One sui able class o ma e ials o
achie e supe io s abilisa ion abili y a e poly(ionic liquids),
also known as polyme ic ionic liquids (PILs), which a e poly-
elec oly es,
21
o med om polyme isable ionic liquid mono-
me s. PILs possess analogous p ope ies o bulk ionic liquids
in solu ion,
22,23
and also enable exquisi e uning o he
polyme p ope ies h ough a nea -in ini e combina ion o
ca ions and anions, oge he wi h he b oad ange o a chi ec-
u e and mo phology a ia ions inhe en o polyme ic
ma e ials.
24
The e a e many examples o poly(ionic liquid)
unc ional ma e ials such as po ous elec o-op ic ilms,
25
ion
conduc i e polyme ilms,
26–29
dispe san s,
30
and
ac ua o s.
31,32
Fu he mo e, PILs can also be 3D p in ed using
a ange o echniques, such as inkje p in ing,
33
DLP,
34,35
and
mic os e eoli hog aphy.
36
In addi ion, he abili y o ionic
liquids and PILs o s abilise and suppo me al nanopa icle
p ecu so sal s and nanopa icles has been well demon-
s a ed.
37
Finally, ILs and PILs can be designed so as o
possess inhe en b oad an imic obial p ope ies, agains bac-
e ia and ungi.
38–41
The e o e, we p opose he use o 3D p in able poly(ionic
liquids) o ab ica e mul i unc ional ma e ials ha ea u e
h ee key p ope ies; (i) on-demand pho o educ i e o ma ion
o he AgNPs wi hin he ma e ial ha is decoupled om he
3D p in ing s ep; (ii) an an imic obial effec o he PIL back-
bone; combined wi h (iii) he b oad an imic obial ac ion
affo ded by AgNPs. Va pho opolyme isa ion is selec ed as he
3D p in ing echnique o p oo o concep , since i allows he
apid o mula ion o polyme isa ion o mula ions and he
gene a ion o p ecise, complex geome ies.
He ein we epo he de elopmen o no el 3D-p in able
ma e ials based on wo diffe en polyme ic ionic liquids (PILs)
unc ionalised wi h sil e nanopa icles (AgNPs), we compa e
and con as he o ma ion and s abilisa ion o he sil e nano-
pa icles in he wo o mula ions and in es iga e he an i-
mic obial p ope ies. The abili y o each PIL o s abilise ac i e
nanoma e ials ia pho o educ ion is demons a ed by effec i e
decoupling o he ab ica ion om he on-demand gene a ion
o low polydispe si y AgNPs by subsequen exposu e o UV
i adia ion. Fu he mo e, he uneable an imic obial ac i i y o
he AgNP@PIL p in ed de ices agains G am-posi i e Bacillus
sub ilis (B. sub ilis) and G am-nega i e Esche ichia coli (E. coli)
bac e ia, and Aspe gillus nige (A. nige ) and Cladospo ium cla-
dospo ioides (C. cladospo ioides) ungi is p esen ed.
Resul s and discussion
In his wo k, se e al o mula ions based on combina ions o
diffe en IL based monome s (mIL) and c oss-linke s we e
in es iga ed (Fig. 1A & B), wi h he aim o de eloping an op i-
mised ink composi ion o p in ing AgNP@PIL ma e ials.
Ini ial 3D p in ing expe imen s used he digi al ligh p oces-
sing (DLP) echnique, employing a Li le RP p in e wi h a DLP
p ojec o o gene a e he UV ligh equi ed o polyme isa ion.
La e 3D p in ing expe imen s we e pe o med wi h a newe
liquid c ys al display (LCD) based p in e (Elegoo Ma s). The
la e allowed o ma ginally be e esolu ion, and had a
smalle oo p in , bu no diffe ences o he p in ing p ocess
we e no iced. The i s unable 3D p in ing ink o mula ion
was based on he polyme isable monome ic IL (mIL) p ecu so
1-bu yl-3- inylimidazolium bis( i luo ome hane)sul onimide
([BVIM][NT
2
]) (mIL1, Fig. 1A), which was syn hesised om
1-bu yl-3- inylimidazolium b omide ([BVIM][B ]) ia anion
me a hesis as desc ibed in he ESI.†
34,35
mIL1 was chosen as
he polyme isable ionic liquid componen as i has been p e-
iously demons a ed as a sui able poly(ionic liquid) (PIL) o
s abilising and solubilising hyb id o ganic–ino ganic polyoxo-
me ala es, and o ms mechanically esis an polyme s when
3D p in ed.
34,35
Based on ou p e ious expe ience, he ini ial
ink o mula ion (PIL1, Fig. 1B) consis ed o mIL1 (80 mol%),
he c osslinke 1,4-bu anediol diac yla e (20 mol%) and he
o ganic pho oini ia o diphenyl(2,4,6- ime hylbenzoyl)phos-
phine oxide (TMDPO, 1 mol%) and was a clea , e y pale
yellow iscous liquid. This ink composi ion was chosen as i
was p e iously ound o be he mos effec i e o 3D-p in ing
o PILs wi h he DLP echnique. To his baseline o mula ion
sil e hexa luo oan imona e (AgSbF
6
,∼1 w %) sal was solubil-
ised and s abilised.
The pho opolyme isa ion o he PIL1 o mula ion esul ed
in well-de ined clea and e y pale yellow PIL pa s (Fig. 2A,
0 min). The pale-yellow colou o he 3D p in ed PIL1 pieces is
a ibu ed o he polyme , hus sugges ing ha he encapsu-
la ed sil e was s ill in he Ag
+
s a e, despi e exposu e o he
ligh du ing he 3D p in ing s ep. Fu he mo e, he UV-Visible
spec a o he as p in ed pieces con i med he absence o an
abso p ion peak in he egion λ≈375–475 nm, a ibu able o
he LSPR band o he AgNPs (Fig. S1†),
42
bu does show he
expec ed abso p ion peak ea u es o he pho oini ia o
TMPDO.
43
Then upon exposu e o UV ligh , he pho o-
educ ion o Ag
+
o Ag(0) was ini ia ed and he colou o he
p in ed pa s changed o e ime o inc easingly da ke shades
o ambe (Fig. 2A), and TEM analysis con i med he p esence
o AgNPs (Fig. 2A, inse ). The p esence o Ag(0) was u he
con i med by XPS cha ac e isa ion, which e ealed he
expec ed peaks in he spec um o Ag 3d
3/2
and Ag 3d
5/2
(Fig. S2A†).
44
Indeed, he sligh asymme y o he Ag 3d
5/2
peak owa d highe binding ene gies could be due o a con o-
lu ed peak ha co esponds o agg ega ed Ag nanopa icles,
which u he co obo a es he p esence o AgNPs.
45
Mechanical analysis o pa s exposed o UV pho o educ ion
o diffe en imes indica ed an inc ease in ensile s iffness
wi h inc easing UV exposu e ime (Fig. S2B†). Addi ionally, he
co esponding an ∂ s. empe a u e cu es e ealed he T
g
alues (de ined as he x-axis alues a each cu e maximum)
also inc eased wi h inc easing UV exposu e ime (Fig. 2B), he
seconda y peak a low empe a u es can be a ibu ed o local
chain mo ions o side g oup mo emen s in he polyme
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chains.
46
The mechanical p ope ies o he PIL1 pieces a e
summa ised in Fig. S2D.†FT-IR s udies du ing UV exposu e
o e ime e ealed only a small dec ease in he in ensi y o he
C C s e ch peak a ∼1660 cm
−1
a e 5 minu es o UV
exposu e, and no u he change upon u he UV exposu e
(Fig. 2C). The peak ea u e a ∼1660 cm
−1
can also be a ibu-
ed o he C C bond in he imidazole ing,
47–49
and hus he
in ensi y o his peak would no change upon u he UV
exposu e. The e o e, i is p oposed ha he inc ease in ensile
s iffness and T
g
upon inc eased UV exposu e ime is due o he
con inued o ma ion o AgNPs and/o agg ega ion o AgNPs,
bo h o which con ibu e o modi ying he mechanical p o-
pe ies o polyme ma ices.
50
UV-Vis ansmission spec-
oscopy expe imen s du ing he UV pos -p in ing ea men
u he con i med he o ma ion o AgNPs due o he p esence
o a shoulde peak cen ed a λ≈450 nm inc easing in abso -
bance, which is a ibu ed o he AgNP plasmon. Fo compa i-
son, an analogous liquid IL homogeneous sys em was p e-
pa ed, whe e Ag(SbF
6
) (1 w %) was added o non-polyme isa-
ble 1-bu yl-3-me hylimidazolium bis( i luo ome hane)sul oni-
mide and pho o- educed unde UV ligh (λ= 395 nm). In his
case, he plasmon a ising due o he p esence o AgNPs, was
also obse ed as a shoulde in he UV-Vis spec um (see
Fig. S3†). In e es ingly, changing he monome o 1-(3-hyd oxy-
Fig. 1 This figu e p o ides an o e all isual summa y o he wo k in his pape , and also shows he ollowing specific esul s: (A) The componen s
employed o he de elopmen o he 3D p in able PIL-based o mula ions; (B) able summa ising he o mula ions explo ed and a quali a i e assess-
men o he p ope ies o he ma e ials de eloped. Unless indica ed o he wise, 80 : 20 mol : mol, 1 mol% PI, 1 w % AgSbF
6
; (C) example CAD design
ha was employed as a benchma k o 3D-p in ing; (D) 3D-p in ed pa wi h PIL3 o mula ion wi hou Ag educ ion; (E) 3D p in ed pa wi h PIL3
o mula ion wi h AgNPs gene a ed by pos p in ing UV i adia ion. P in ed pa ; (F) he UV-Visible peak ea u e a ibu ed o he AgNP plasmon in
3D-p in ed and UV-exposed PIL3 cen ed a 422 nm; and (G) ansmission elec on mic og aph o a 3D p in ed and UV-exposed PIL3 pa showing
he p esence o AgNPs dispe sed wi hin he PIL ma ix.
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p opyl)-3- inylimidazolium bis( i luo ome hane)sul onimide
[POHVIm][NT
2
](mIL2, Fig. 1B) in he o mula ion, whils
keeping he es o he o mula ion cons an (PIL2), led o
be e esol ed plasmons a e pos -cu ing wi h a well-de ined
plasmon (dis inc abso p ion peak, a he han a shoulde
ea u e) wi h a maximum a λ= 413 nm (Fig. S4,†blue cu e).
Howe e , i was de e mined ha LCD-based p in ing o he
PIL2 o mula ion esul ed in excessi ely igid samples, which
de o med and c acked du ing he polyme isa ion p ocess. I
was hypo hesised ha he p esence o he hyd oxyl g oup o
he ca ion, combined wi h he use o 1,4-BDA as c oss-linking
agen , had an in luence on he mechanical p ope ies o he
esul ing ma e ial, which in u n affec ed he p in abili y.
Ins ead, use o poly(e hyleneglycol) diac yla e (PEGDA, M
n
=
575 g mol
−1
) esul ed in much mo e lexible ilms (he ein
PIL3, Fig. 1B), which showed sui able mechanical p ope ies
o he polyme isa ion and 3D p in ing o sui able geome ies
wi h accep able esolu ion (Fig. 1C–E and Fig. S5†). The co es-
ponding UV-Visible peak ea u e a ibu able o AgNPs was
well-de ined and cen ed a 422 nm (Fig. 1F and Fig. S4,† ed
cu e) and he AgNPs we e well dispe sed wi hin he PIL
ma ix (Fig. 1G). Now ha a sui able composi ion o PIL ink
had been de e mined, aging expe imen s we e pe o med o
de e mine he s abili y o he AgNP@PIL pa s o e ime.
To examine he pho os abili y o he AgNP@PIL ma e ials,
PIL2 3D-p in ed pa s we e s o ed ei he unde ambien ligh -
ing o in da k condi ions. I was obse ed ha AgNP@PIL2
ma e ials, which had been gene a ed by pos -cu ing pho o e-
duc ion exhibi ed pho o-aging when exposed o ambien ligh
in e ms o an inc ease in he magni ude o he abso bance
peak a ibu ed o AgNPs, sugges ing he con inued pho o e-
duc i e o ma ion o AgNPs (Fig. S6†). The s abili y o 3D-
p in ed AgSbF
6
-con aining PILs o pho o educ ion was hen
in es iga ed and i was de e mined ha s o age in da k con-
di ions was equi ed o p e en pho o-aging. When non-pho o-
educed PIL2 pieces we e exposed o h ee weeks o ambien
ligh a peak (cen ed a ∼450 nm) in he UV-Visible ans-
mission spec a de eloped, sugges ing he o ma ion o AgNPs
(Fig. 3A). In con as , non-pho o educed 3D p in ed PIL2
pieces s o ed in he da k p oduced no such obse able abso -
bance ea u e ha could be a ibu ed o AgNPs (Fig. 3B).
Thus, o use o hese ma e ials as po en ial an imic obial
ma e ials/de ices, s o age in da k condi ions demons a es he
hypo hesis ha i is possible o decouple he manu ac u ing o
de ices by 3D p in ing om he pho o educ ion o ac i e
AgNPs.
The p esence o hyd ophobic poly(ionic liquids) combined
wi h AgNPs make PILs in e es ing and highly applicable candi-
da es o an imic obial su ace coa ings o p e en bio ilm o -
ma ion. Ionic liquids a e gaining ele ance as hey p esen
some desi able cha ac e is ics highly ele an in he clinical
use, such as imp o ed bac e ial esis ance, an ibio ilm p o-
pe ies, and he possibili y o being 3D-p in ed as speci ic
den al objec s, among o he s.
41,51
The an imic obial ac i i y o
he PILs epo ed he ein we e e alua ed agains non-pa ho-
genic E. coli and B. sub ilis and wo moulds (A. nige and
Fig. 2 (A) The PIL1 3D p in ed pa s exhibi ed diffe en colou s depending on he exposu e ime o UV ligh (λ= 395 nm, 36 W, . .) wi h ( ed inse ) a
ansmission elec on mic og aph showing he p esence o nanopa icles; (B) The an ∂ s. empe a u e cu es, whe e he T
g
alues (defined as he
x-axis alues a each cu e maximum) inc ease wi h inc easing UV exposu e ime; and (C) FT-IR spec um demons a ing li le o no change in he
C C peak upon inc easing UV exposu e ime ( he cu e colou s a e he same as pe he legend inse in (B). This sugges s ha u he polyme isa ion
o unpolyme ised C C bonds in he ma e ials is no con ibu ing o he inc eases in ensile s iffness and T
g
upon UV exposu e.
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C. cladospo ioides) ia zone o inhibi ion (Ki by-Baue ) es s
and cell p oli e a ion assays, as well as bio ilm inhibi ion
s udies. In gene al, PIL and AgNP@PIL samples ac o inhibi
mic obial g ow h, bu AgNP-con aining PILs (AgNP@PIL) a e
mo e effec i e a educing bac e ial iabili y.
In he i s ins ance, a zone o inhibi ion es o PIL1 and
AgNPs@PIL1 e ealed ha only he AgNP@PIL1 displayed an
inhibi ion halo agains E. coli and B. sub ilis (Fig. 4A). This
inhibi ion o bac e ial g ow h a ound he sample is due o he
diffusion o he Ag
+
ions in o he aga , which is he mos
impo an ac o in he an imic obial ac i i y o he AgNPs
agains bac e ia.
51
No inhibi ion halo was ound wi h he PIL1
samples due o he lack o sil e . A delay halo—cha ac e ised
o he mycelium g ow h bu a lack o spo ula ion—was ound
a ound AgNP@PIL1 incuba ed in he pla e inocula ed wi h
A. nige . No inhibi ion no delay halo was ound wi h he PIL1
sample, and nei he PIL1 no AgNP@PIL1 esul ed in
mycelium o spo ula ion inhibi ion o C. cladospo ioides
(Fig. 4B).
In o de o de e mine he an ibac e ial ac i i y o he
samples in solu ion, he bac e ial g ow h in cell cul u e media
in he p esence o he samples was moni o ed by op ical
densi y. PIL1 ep essed he g ow h o E. coli and B. sub ilis in
liquid media o e a 24 hou pe iod bu , in e es ingly, he
co esponding educ ion in bac e ial cell iabili y was lowe
o he G am-posi i e B. sub ilis (Fig. 5A). Since hyd ophobic
an imic obial compounds o en ac by dis up ing, depola -
ising, and des abilising bac e ial cell memb anes,
52,53
ou
hypo hesis was ha he bac e ios a ic PIL samples and he
associa ed s ess o he cell memb ane p o okes inc eased p o-
li e a ion in his species. Fu he mo e, B. sub ilis can o m p o-
ec i e endospo es ha a e able o ole a e ha sh en i on-
men al condi ions, hus ano he hypo hesis is ha he p es-
ence o he PIL wi hou sil e can s imula e he p oduc ion o
endospo es by B. sub ilis, po en ially explaining he diffe ences
compa ed wi h he g ow h o E. coli. This was con i med by
En i onmen al Scanning Elec on Mic oscopy (ESEM), which
showed a high deg ee o spo ula ion in B. sub ilis (Fig. 5C). On
he o he hand, he AgNPs@PIL1 a ian was ound o educe
bac e ial cell g ow h o E. coli and B. sub ilis by 86% and 75%,
espec i ely, as expec ed, due o he an imic obial effec o
sil e . B o h and aga dilu ion me hods a e s anda d me hods
used o de e mine he an imic obial effec o he compounds,
bu hese assays a e s anda dised o soluble compounds.
Hence, pe o ming a su ace an imic obial assay is essen ial
when using compounds which a e mean o be applied as a
coa ing o , like in his case, hey a e c ea ed di ec ly as a 3D
objec . He e, a modi ied Japanese Indus ial S anda d Z
2801 me hod was used o de e mine he su ace an imic obial
ac i i y o PIL1 and AgNP@PIL1 (Table 1). PIL1 displayed a
bac e ial educ ion o 100% and 89% o E. coli and B. sub ilis,
espec i ely, while AgNP@PIL1 educed 100% o he bac e ial
cell coun . Subcul u es o he supe na an s con i med he bac-
e icidal effec o he AgNP@PIL1, while PIL1 only displayed
bac e icidal effec agains E. coli. These esul s a e commensu-
a e wi h he esul s in liquid medium and he ESEM images
(Fig. 5A), whe e he samples con aining Ag displayed a highe
an imic obial effec , wi h B. sub ilis being he mos esis an
s ain. The diffe ences in an imic obial ac i i y be ween he
solu ion and he su ace ac i i y es s show ha he an i-
mic obial p ope ies a e based p ima ily on su ace con ac -
Fig. 3 (A) AgNPs@PIL2 non- educed and hen aged unde ambien ligh highligh ing he de elopmen o a peak ea u e a ∼450 nm sugges ing he
o ma ion o AgNPs; and (B) AgNPs@PIL2 non- educed and hen aged unde da k condi ions, showing no AgNP plasmon. In all cases he samples
we e exposed o ambien a mosphe e.
Fig. 4 (A) Zone o Inhibi ion es o PIL1 and AgNPs@PIL1 agains wo
bac e ial s ains (E. coli and B. sub ilis) and (B) wo moulds (A. nige and
C. cladospo ioides).
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killing a he han he elease o an ac i e componen , such as
Ag
+
ions, in o he local en i onmen .
51
E alua ion o he an i-
bac e ial ac i i y o he PIL3 o mula ion yielded simila
esul s, howe e , AgNP@PIL3 was ound o possess he highes
deg ee o inhibi ion a e 24 hou s o incuba ion, esul ing in
a educ ion o a ound 95% o bo h bac e ial s ains (Fig. 5B).
Conclusions
He ein, he capabili y o no el 3D-p in ed ma e ials consis ing
o polyme ic ionic liquids (PILs) o he con olled on-demand
syn hesis, and subsequen s abilisa ion, o sil e nanopa icles
(AgNPs) has been demons a ed. I was de e mined ha he
na u e o he ionic liquid moie ies affec ed he in si u o -
ma ion o he AgNPs and enabled he con olled o ma ion
and s abilisa ion o AgNPs ia ex ended UV pho o educ ion
a e he 3D p in ing p ocess. This demons a ed an effec i e
decoupling o he 3D p in ing ab ica ion s ep om he on-
demand gene a ion o AgNPs wi h na ow size dispe si y by
subsequen exposu e o UV i adia ion. Upon u he in es i-
ga ion, i was de e mined ha he s uc u e o he ca ion o
he monome ionic liquid had a p o ound effec on he o -
ma ion o AgNPs, wi h a disc e e UV-Vis peak ea u e a ibu-
ed o he plasmon esonance o well- o med disc e e AgNPs
wi hin he PILs o med om mIL2 (AgNPs@PIL3), in con as
o a b oad shoulde ea u e measu ed in he UV-Vis spec um
o AgNPs@PIL1. Thus, h ough ca e ul choice o monome
ionic liquid, a sui able c osslinke o a ou able mechanical
p ope ies, and a o mula ion ha had p e iously been shown
o be effec i e o 3D p in ing, he ab ica ion o ma e ials/
de ices decoupled om on-demand pho o- educ i e o ma ion
o AgNPs o enhanced an imic obial ac i i y was ealised.
Indeed, he p in ed de ices showed a mul i- unc ional and
uneable mic obiocidal ac i i y agains G am posi i e (B. sub i-
lis) and G am nega i e (E. coli) bac e ia and agains he ungus
A. nige . PIL-based ma e ials we e ound o be bac e ios a ic,
whe eas AgNPs@PIL ma e ials possessed enhanced an i-
mic obial ac i i y and bac e icidal p ope ies agains he es ed
s ains.
This wo k ep esen s he i s example o he no el combi-
na ion o he decoupled in si u pho o educ i e ab ica ion o
sil e nanopa icles om he ab ica ion ia 3D p in ing o he
PIL-based ma e ials. This in si u gene a ion o nanos uc u ed
an imic obial ma e ials opens new ou es o he de elopmen
o 3D p in ed de ices esis an o bio ilm o ma ion, o appli-
ca ions in he biomedical, ood echnology and heal hca e
indus ies. Fu he mo e, he nea in ini e combina ions o
ca ions and anions possible in PIL-based ma e ials allows o
u he uning and enhancemen o he syne gis ic an i-
mic obial p ope ies, especially when combined wi h he
eedom o design inhe en o addi i e manu ac u ing
echniques.
Fig. 5 (A) Bac e ial cell iabili y o E. coli and B. sub ilis incuba ed wi h PIL1 and AgNP@PIL1 and (B) PIL3 and AgNP@PIL3. (C) ESEM images o PIL1
and AgNP@PIL1 inocula ed wi h E. coli and B. sub ilis. Bo h PIL1 and AgNP@PIL1 affec E. coli mo phology, dis up ing he cell memb ane. On he
o he hand, PIL1 s imula es B. sub ilis spo ula ion, while he AgNP@PIL1 p e en s colonisa ion o he polyme ic su ace due o a highe bac e icidal
effec .
Table 1 Bac e ial educ ion (%) a e 24 hou s incuba ion o e he
samples
Bac e ial educ ion
E. coli B. sub ilis
PIL1 100% 89%
AgNP@PIL1 100% 100%
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Expe imen al and me hods
Gene al expe imen al in o ma ion
Unless o he wise s a ed, all eagen s we e pu chased om
Sigma-Ald ich and used wi hou u he pu i ica ion. BDA
(90%) and 1- inylimidazole we e used a e pu i ica ion by
passing h ough a sho column o basic alumina (aluminium
oxide).
The 3D p in e s used in his s udy we e a Li leRP 3D
p in e and an Elegoo Ma s. The Li le RP 3D p in e was
equipped wi h an Ace P1500 p ojec o (3D p in e ligh
sou ce) ha was i ed wi h an OSRAM P-VIP high p essu e
(>200 ba ) Hg apou lamp (210 W). The Elegoo Ma s p in ed
was equipped wi h a 2560 × 1440 2 K HD masking LCD, illumi-
na ed wi h 40 W UV LED backligh (λ= 405 nm). The ligh
sou ces in bo h p in e s we e bo h ∼3000 lumens. UV– is
spec a we e collec ed using an Agilen Ca y 5000 UV– is–NIR
spec opho ome e . FT-IR spec a we e collec ed on a B uke
Alpha i ed wi h a Pla inum ATR module.
1
H NMR spec a
we e eco ded on a B uke AVX400 (400 MHz) spec ome e a
ambien empe a u e.
13
C NMR spec a we e eco ded on a
B uke AVX400 (101 MHz) spec ome e a ambien empe a-
u e. En i onmen al Scanning Elec on Mic oscopy (ESEM)
da a was collec ed on a Quan a FEG-250 (FEI Company) ield
emission SEM o high- esolu ion imaging wo king in ESEM
mode using a GSED de ec o unde high ela i e humidi y con-
di ions. T ansmission elec on mic oscopy (TEM) measu e-
men s we e pe o med using a JEOL 2000-FX ope a ed wi h an
accele a ing ol age o 200 kV. The samples we e p epa ed
using ul amic o omy p ocess, whe e sample slices o ew
nanome es hickness we e deposi ed on TEM g ids. The
mechanical analyses we e pe o med wi h a TA Q800 DMA
using a ension clamp. The moduli measu emen s as a unc-
ion o empe a u e we e measu ed using a s ain ampli ude o
0.1% and equency o 1 Hz. The samples we e cooled o
−50 °C and s abilised a −50 °C o 5 min. Finally, he sample
was hen hea ed o 200 °C a he a e o 5 °C min
−1
. The
moduli we e measu ed du ing he hea ing un. Fo he s ess–
s ain measu emen s, he samples we e i s equilib a ed a
25 °C o 5 min and hen he o ce was amped om 0 o 18 N
a he a e o 1 N min
−1
and he s ess–s ain cu es we e
eco ded.
Syn hesis o ionic liquids
Full syn hesis de ails and cha ac e isa ion da a a e gi en a he
end o he ESI.†
P ocess o 3D p in ing imidazolium PIL ne wo ks
The p ocess o 3D p in ing imidazolium PIL pieces ab ica ed
wi h 80 mol% [BVIM][NT
2
](mIL1) ionic liquid is gi en as an
example. The s e eoli hog aphy ab ica ion began by slicing a
3D compu e aided design model in o indi idual images o
p ojec ing on o he pho ocu able ink. Addi i e manu ac u ing
so wa e, C ea ion Wo kshop RC36, was used o c ea e slices o
a desi ed hickness (50 μm pe laye ), con ol he p ojec ion
ime o he images by he p ojec o , and he mo emen o he
build pla e o he Li leRP FlexVa . Fo he Elegoo Ma s 3D
p in e CHITUBOX so wa e was employed. Then he p in e
a was illed wi h he p emixed PIL1 ink, which con ained
80 mol% [BVIM][NT
2
], 20 mol% 1,4-bu anediol diac yla e
(BDA), 1 mol% diphenyl(2,4,6- ime hylbenzoyl)phosphine
oxide (pho oini ia o ) and 1 w % sil e hexa luo oan imona e
(AgSbF
6
). Then he build pla e was lowe ed in o he ink agains
he anspa en ilm o he bo om o he a . The so wa e
hen con olled he p ojec o o show a black backg ound wi h
whi e images o each o he laye s which we e o be pho o-
cu ed. The UV ligh passed h ough he anspa en bo om o
he a . Each laye (50 μm) was cu ed o 8 s. A e 8 s, he
p in e showed a black backg ound (negligible ligh was p o-
jec ed in o he pho ocu able ink) whils he build pla e was
au oma ically li ed by 50 μm. Then he so wa e loaded he
image o he nex laye and u ned “on” he UV ligh by
showing he nex whi e colou image. This p ocess was
epea ed un il he objec was comple ely ab ica ed. A e he
objec was ab ica ed, he objec was isola ed and copiously
washed wi h isop opyl alcohol o emo e any uncu ed ink.
UV–Vis abso bance measu emen s o 3D-p in ed pieces
Fo collec ion o he UV– is abso bance spec a o he 3D
p in ed specimen, be o e and a e i adia ion wi h UV ligh ,
he specimen was i s affixed o a me al holde exposed o UV
ligh o a de e mined ixed amoun o ime, hen a UV– is
spec um o his specimen was collec ed using he Ca y 5000
UV– is–NIR spec opho ome e . Once he UV-Vis spec um
was collec ed, he p ocedu e was epea ed un il a o al UV ligh
exposu e o he desi ed ime was eached.
Mic oo ganisms and g ow h condi ions
Two bac e ial s ains we e es ed in he an ibac e ial assays:
Esche ichia coli DH5αas a G am-nega i e model and Bacillus
sub ilis 1904-E as a G am-posi i e model. Two moulds om
he “Colección Española de Cul i os Tipo (CECT)”we e es ed
in he an i ungal assays: Aspe gillus nige CECT 2088 and
Cladospo ium cladospo ioides CECT 2111. Fungal spo e suspen-
sions we e s o ed in 0.1% Tween, 20% glyce ol a −80 °C p io
o use. The ollowing cul u e media was used: T yp one Soy
Aga (TSA) (The mo Scien i ic™), Sabou aud Dex ose Aga
supplemen ed wi h chlo amphenicol (SDA) (Scha lab, S. L.), as
solid media; Lu ia–Be ani (LB) and Nu ien B o h (NB)
(Scha lab, S. L.) as liquid media. The mic obial g ow h con-
di ions a e summa ised in he Table 2.
Aga diffusion es
A modi ied Ki by Baue disk diffusion echnique was used.
The 3D p in ed samples (3 mm-diame e ci cles o PIL and
AgNPs@PIL) we e i s s e ilised wi h e hanol 70% and hen
hey we e placed in solid media p e iously inocula ed wi h a
bac e ial lawn (10
7
CFU mL
−1
)o E. coli o B. sub ilis. A e
24 hou s o incuba ion a 37 °C, he inhibi ion halo a ound
he samples was obse ed.
Fo he an i ungal assay, he 3D p in ed samples (20 mm-
diame e ci cles o PIL and AgNPs@PIL) we e s e ilised wi h
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e hanol 70% and hen placed in solid media p e iously inocu-
la ed wi h a spo e solu ion (10
4
conidia mL
−1
)o A. nige o
C. cladospo ioides. A e 96 hou s o incuba ion a he co es-
ponding empe a u e (see Table 2), he inhibi ion halo a ound
he samples was obse ed.
Bac e ial cell p oli e a ion assay
Bac e ial g ow h was eco ded measu ing he op ical densi y
(OD) o he samples a 600 nm o e a 24 hou pe iod using a
mic opla e eade (The mo Scien i ic MULTISKAN GO). Resul s
we e compa ed wi h he OD a ia ion o a con ol cul u e con-
aining only E. coli o B. sub ilis. The da a o all con ol expe i-
men s and an ibac e ial assays a e based on a o al o six
epea s o e i y he ep oducibili y o he esul s and o calcu-
la e he mean alues and he s anda d de ia ion; each expe i-
men was epea ed a minimum o h ee imes o e i y he
esul s be ween diffe en inocula ions. The samples (PIL and
AgNPs@PIL), p e iously s e ilised wi h e hanol 70%, we e
placed in a 24-well pla e inocula ed wi h 1 mL o he inoculum
(1 × 10
7
CFU mL
−1
o E. coli o B. sub ilis), incuba ed a 37 °C,
and he an ip oli e a i e effec s induced by he samples we e
obse ed by plo ing op ical densi y (OD) s. ime. Bac e ial
g ow h was eco ded measu ing he op ical densi y (OD) o
100 µL collec ed om each well o he 24-well pla e, in a
96-well pla e. Measu emen s a 600 nm we e pe o med using
a mic opla e eade , each hou du ing he i s 8 hou s and a
24 hou s as a inal poin . To main ain he inal olume (1 mL)
du ing he expe imen , he 100 µL sub ac ed om he wells
om he 24-well pla e we e eplaced wi h 100 µL o medium.
Resul s we e compa ed wi h he OD a ia ion o a con ol
cul u e con aining only bac e ia (E. coli o B. sub ilis).
S a is ical analysis was pe o med using unpai ed - es . P
alues <0.05 we e conside ed signi ican . Single s a s deno e
0.01 < P< 0.05.
Su ace an imic obial ac i i y
The su ace an imic obial ac i i y was s udied using a modi-
ied JIS Z 2801 s anda d. The p o ocol las s h ee days. On he
i s day, a bac e ial suspension o 1 × 10
7
CFU mL
−1
was p e-
pa ed and hen 50 µL o his suspension was added o e he
su ace o he samples o in e es (2 × 2 cm) and o e a e e -
ence sample wi h no an imic obial ac i i y (2 × 2 cm) a e he
s e ilisa ion o hese samples wi h e hanol 70%. In o de o
e en he con ac su ace, a co e slip was pu abo e he 50 µL o
bac e ial suspension in all he samples. The samples we e
incuba ed in a humid chambe a 37 °C o 24 h. A e he 24 h
o incuba ion, he bac e ia o e he samples we e ex ac ed
in o liquid medium by pu ing he samples in o a 50 ml alcon
wi h 15 mL o cul u e media and shaking hem one minu e
wi h a o ex. The cul u e media wi h he ex ac ed bac e ia
was hen dilu ed and sown in aga pla es, which we e incu-
ba ed a 37 °C du ing 24 h. On he las day, he CFU o he cul-
i a ed aga pla es we e coun ed. The pe cen age o bac e ial
log educ ion was ob ained using he ollowing o mula:
Reduc ion ¼logðc u e e ence sampleÞ
logðc u an imic obial sampleÞ
En i onmen al scanning elec on mic oscopy (ESEM) o he
bac e ia incuba ed o e he samples
The effec o he samples (PIL and PIL@AgNPs) on he mo -
phology o he bac e ia was s udied by ESEM. The samples
we e s e ilised wi h e hanol 70% and inocula ed wi h 100 μL
o a 10
4
CFU mL
−1
bac e ial suspension. Then he inocula ed
samples we e incuba ed in a humid chambe a 37 °C o 24 h.
A e he incuba ion ime, he samples we e insed wi h dis-
illed s e ile wa e and isualised on a Quan a FEG-250 (FEI
Company) ield emission SEM o high- esolu ion imaging
wo king in ESEM mode using a GSED de ec o .
Conflic s o in e es
The e a e no con lic s o decla e.
Acknowledgemen s
Gene ali a Valenciana (CIDEGENT/2018/036), UJI (UJI-B2020-
44) and he Agencia Valenciana o Inno acion (INNCON/2020/
14) a e g a e ully acknowledged o unding ha pa ially
unded his wo k. SMC hanks he Gene ali a Valenciana o a
doc o al schola ship unding (ACIF/2020/338). Financial
suppo om Minis e io de Ciencia Inno ación y
Uni e sidades (Spain) h ough P oyec o I+D+i PID2019-
109333RB-I00. We hank he Beacons o Excellence:
P opulsion Fu u es and G een Chemicals o he Uni e si y o
No ingham o inancial suppo ; he Nanoscale and
Mic oscale Resea ch Cen e –UoN. J. M. C., V. S., and G. N. N.
hank he Le e hulme T us (RPG-2016-442). IFC hanks
Gobie no de A agón o a doc o al schola ship. Fu he inan-
cial suppo om Fondo Social Eu opeo-Gobie no de A agón
(E15_20R) and CIBER-BBN is g a e ully acknowledged. The
au ho s would like o acknowledge he Labo a o io de
Table 2 Mic oo ganisms and g ow h condi ions
Mic oo ganism Solid medium Liquid medium Incuba ion empe a u e (°C) P e-inoculum incuba ion
Bac e ia E. coli DH5αTSA LB 37 24 hou s
B. sub ilis TSA NB 37 24 hou s
Fungi A. nige SDA —35 4 days
C. cladospo ioides SDA —25 4 days
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