Academic Edi o s: Yen Wei and
Jiujiang Ji
Recei ed: 28 June 2025
Re ised: 1 Augus 2025
Accep ed: 5 Augus 2025
Published: 8 Augus 2025
Ci a ion: Slepiˇcka, P.; Labíko á, I.;
F ýdlo á, B.; Pagáˇco á, A.; Slepiˇcko á
Kasálko á, N.; Sajdl, P.; Š o ˇcík, V.
Coppe –PLLA-Based Biopolyme
W inkle S uc u es o Enhanced
An ibac e ial Ac i i y. Polyme s 2025,
17, 2173. h ps://doi.o g/10.3390/
polym17162173
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
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A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/
licenses/by/4.0/).
A icle
Coppe –PLLA-Based Biopolyme W inkle S uc u es o
Enhanced An ibac e ial Ac i i y
Pe Slepiˇcka 1,* , I a Labíko á 1, Bá a F ýdlo á 1, Ane a Pagáˇco á 2, Nikola Slepiˇcko á Kasálko á 1,
Pe Sajdl 3and Václa Š o ˇcík 1
1Depa men o Solid S a e Enginee ing, Uni e si y o Chemis y and Technology P ague,
166 28 P ague, Czech Republic; [email p o ec ed] (I.L.); [email p o ec ed] (B.F.);
[email p o ec ed] (N.S.K.); acla [email p o ec ed] (V.Š.)
2Depa men o Biochemis y and Mic obiology, Uni e si y o Chemis y and Technology P ague,
166 28 P ague, Czech Republic; [email p o ec ed]
3Depa men o Powe Enginee ing, Uni e si y o Chemis y and Technology P ague,
166 28 P ague, Czech Republic; pe [email p o ec ed]
*Co espondence: pe [email p o ec ed]
Abs ac
The inc easing p e alence o an ibio ic- esis an bac e ia has in ensi ied he need o inno a-
i e an ibac e ial su aces, pa icula ly in biomedical applica ions. T adi ional app oaches
o en ely on chemical agen s alone, which may lead o diminishing e icacy o e ime. To
add ess his, we in es iga ed he de elopmen o a no el an ibac e ial su ace by combining
he inhe en an imic obial p ope ies o coppe wi h an enginee ed su ace opog aphy
on a biopolyme ma ix. A coppe –poly-L-lac ic acid (Cu-PLLA) composi e sys em was
ab ica ed using spu e ing deposi ion ollowed by con olled he mal ea men o in-
duce w inkle-like mic o- and nanos uc u es on he su ace. The su ace mo phology was
cha ac e ized using scanning elec on mic oscopy (SEM) and a omic o ce mic oscopy
(AFM), con i ming he o ma ion o hie a chical w inkle pa e ns. The chemical composi-
ion and dis ibu ion o coppe we e analyzed ia ene gy-dispe si e X- ay spec oscopy
(EDS). An ibac e ial pe o mance was assessed agains bo h G am-nega i e Esche ichia coli
and G am-posi i e S aphylococcus au eus using s anda d colony coun educ ion assays.
The Cu-PLLA w inkled su aces demons a ed signi ican ly enhanced bac e icidal ac i i y
compa ed wi h la PLLA and coppe - ee con ols, a inding a ibu ed o a syne gis ic
e ec o mechanical memb ane dis up ion and coppe -media ed chemical oxici y. These
indings sugges ha biopolyme –me al hyb id su aces wi h enginee ed opog aphy o e
a p omising s a egy o de eloping nex -gene a ion an ibac e ial ma e ials sui able o
biomedical and clinical use.
Keywo ds: biopolyme ; poly-L-lac ic acid (PLLA); w inkle s uc u e; coppe composi es;
su ace mo phology; an ibac e ial p ope ies
1. In oduc ion
The de elopmen o ad anced bioma e ials wi h inhe en an ibac e ial unc ional-
i y has become a necessi y in mode n medicine, pa icula ly due o he ala ming ise
in an ibio ic- esis an bac e ial s ains. Implan able medical de ices, wound d essings,
and issue-enginee ing sca olds a e o en subjec o bac e ial con amina ion, leading o
in ec ion, delayed healing, o implan ailu e. Consequen ly, mul i unc ional su aces ha
p e en bac e ial coloniza ion while suppo ing mammalian cell compa ibili y a e highly
Polyme s 2025,17, 2173 h ps://doi.o g/10.3390/polym17162173
Polyme s 2025,17, 2173 2 o 19
desi able [
1
,
2
]. Among he s a egies de eloped o achie e an ibac e ial ac i i y, su ace
opog aphy enginee ing and me al inco po a ion ha e eme ged as wo o he mos e ec i e
and e sa ile app oaches. Recen s udies ha e shown ha speci ic mic o- and nanoscale
su ace pa e ns, pa icula ly hose mimicking na u al an imic obial su aces (such as in-
sec wings o sha k skin), can signi ican ly inhibi bac e ial adhesion and g ow h h ough
mechanical dis up ion o bac e ial memb anes [
3
,
4
]. Among biodeg adable polyme s, poly-
L-lac ic acid (PLLA) has been widely s udied due o i s biocompa ibili y, biodeg adabili y,
and FDA app o al o a ious biomedical uses [
5
,
6
]. Howe e , i s inhe en ly smoo h and
chemically ine su ace o en lacks he p ope ies necessa y o de e bac e ial coloniza-
ion; ecen ad ancemen s ha e sough o enhance PLLA’s unc ionali y by in oducing
su ace modi ica ions such as plasma ea men s, and speci ic echniques such as imp o ed
phase sepa a ion c ea e pa e ns [
7
–
10
] ha can modula e bo h bac e ial and euka yo ic
cell esponses.
W inkling ins abili y is a bo om-up, sel -o ganizing app oach in which une en expan-
sion o su aces wi h di e en mechanical p ope ies leads o w inkle o ma ion [
11
]. This
phenomenon is obse ed when he esidual s ess exceeds a c i ical alue, igge ed by
a ious ac o s such as he mal e ec s [
12
,
13
], sol en swelling [
14
], mechanical s e ching,
o con ac ion [
15
,
16
]. Fo ins ance, hea ing a hin polyme –me al bilaye ilm induces
w inkling due o misma ched he mal expansion coe icien s be ween he laye s [
13
,
17
].
S udies ha e demons a ed ha hea ing below he glass ansi ion empe a u e (Tg) o
polyme s does no al e he su ace mo phology, while empe a u es abo e Tg esul in he
desi ed w inkle o ma ion [
18
]. Simila s udies ha e in es iga ed sys ems wi h a ious
combina ions o polyme s and me als, including gold [19] and pla inum [20].
The an imic obial p ope ies o coppe we e i s documen ed in Egyp ian medical
ex s om 2200 o 2600 BC, whe e i was used o wa e s e iliza ion and wound ea -
men [
21
]. Today, coppe nanopa icles a e ecognized o hei po en an imic obial
p ope ies, inding signi ican applica ions in an ibac e ial su aces and medicine. These
nanopa icles exhibi s ong ac i i y agains bac e ia, i uses, and ungi, making hem
e ec i e agains pa hogens, including d ug- esis an mic oo ganisms such as me hicillin-
esis an S aphylococcus au eus (MRSA) [
22
–
25
]. In addi ion, ecen s udies show ha coppe
e ec i ely inac i a es he COVID-19 i us [
26
]. In he ield o elec onics, coppe is mo e
cos e ec i e han p ecious me als and o e s excellen elec ical conduc i i y, making i an
ideal ma e ial o a ious applica ions [
27
,
28
]. This dual applicabili y unde sco es he e -
sa ili y in bo h medical and elec onic ields. The biopolyme w inkles can be inely uned
in wa eleng h and ampli ude [
29
] o physically in e e e wi h bac e ial cell memb anes
while main aining a conduci e en i onmen o mammalian cell adhesion and p oli e -
a ion. No ably, w inkled su aces ha e demons a ed educed su ace a ea a ailable o
bac e ial adhesion and inc eased local cu a u e, which enhances an ibac e ial e ec s ia
mechanical s ess on bac e ial memb anes [
30
]. Coppe -based nanocomposi es ha e shown
e icacy agains bo h G am-posi i e and G am-nega i e bac e ia, and hei in eg a ion in o
polyme ic bioma e ials is a p omising s a egy o in ec ion- esis an su aces [
31
]. Recen
esea ch sugges s ha such hyb id ma e ials can ou pe o m hei indi idual componen s,
o e ing enhanced an imic obial esis ance, mechanical s abili y, and long- e m bio unc ion-
ali y [
32
]. Mo eo e , s udies ha e con i med ha con olled coppe elease om polyme
ma ices can be ailo ed o maximize bac e icidal ac ion while minimizing cy o oxici y o
hos issues [33,34].
Se e al s udies aimed a modi ying/enhancing he an ibac e ial p ope ies o PLA-
based composi es ha e been published. Elec ospun PLA/ZnO composi e ilms ha e shown
enhanced an ibac e ial p ope ies and been applied in he ood indus y [
35
]. The an ibac-
e ial, c ys alliza ion, and mechanical p ope ies o PLA we e modi ied wi h he addi ion
Polyme s 2025,17, 2173 3 o 19
o chi osan-based ma e ials [
36
]. A combina ion o alkaline ea men and PLA/ -CNT
composi e coa ing in luenced he co osion, biocompa ibili y, and an ibac e ial ac i i y o
Mg alloy [
37
]. The biomass ca bon do s and he composi e nano ibe memb anes o biomass
ca bon do s and PLA, along wi h hei pho oca aly ic deg ada ion and bio-an imic obial
p ope ies, we e s udied in [
38
]. Lee a al. p epa ed an an ibac e ial PLA/Mg compos-
i e wi h enhanced mechanical and biological pe o mance o biodeg adable o hopedic
implan s [
39
]. Sil e nanopa icles we e used in ae ogels o imp o e hei an ibac e ial
p ope ies [
40
]. Rome o e al. p epa ed an ibac e ial and biocompa ible polyme ic compos-
i es wi h coppe zeoli e ille and coppe oxide nanopa icles [
41
]. Gene al s a egies o he
p epa a ion o polylac ic acid composi es wi h UV esis ance and an ibac e ial p ope ies
we e in oduced in [42].
The Cu-PLLA w inkle su aces de eloped in his s udy ha e s ong po en ial o
applica ions in a a ie y o ields in which bac e ial con amina ion poses a c i ical isk.
No ably, hese su aces could be implemen ed in biomedical de ices such as ca he e s,
su gical ools, wound d essings, and implan able componen s [
43
] in which in ec ion
p e en ion is pa amoun and in which sil e is also es ed [
44
]. Beyond he medical sec o ,
applica ions in ood packaging, wa e il a ion memb anes, and high-con ac su aces
in public o indus ial se ings (e.g., doo handles, ailings, o ouchsc eens) a e also
p omising. The in eg a ion o coppe as an an imic obial agen , combined wi h he physical
bac e icidal e ec s o nanoscale opog aphies, o e s a dual-ac ion mechanism ha may
educe eliance on an ibio ics and chemical disin ec an s. F om a cos pe spec i e, he
ab ica ion me hod employed— he mal w inkling combined wi h spu e ing—is ela i ely
s aigh o wa d and compa ible wi h exis ing indus ial p ocesses [
45
]. Compa ed wi h
adi ional an ibac e ial coa ings, such as hose based solely on sil e nanopa icles o
complex polyme –an ibio ic conjuga es, he Cu-PLLA app oach is mo e cos e ec i e due
o he lowe ma e ial cos s o coppe and he po en ial o scalable pa e ning wi hou he
need o li hog aphy o clean oom condi ions. Addi ionally, he use o biodeg adable PLLA
as he base polyme aligns wi h sus ainable ma e ial s a egies and may lowe end-o -li e
disposal cos s. O e all, his sys em demons a es a a o able balance be ween pe o mance,
ma e ial cos , and manu ac u abili y, suppo ing i s easibili y o eal-wo ld adop ion
ac oss bo h high- ech and commodi y p oduc sec o s.
E en de ails o he an ibac e ial p ope ies o coppe in di e en ma e ials, such as
ac yloni ile s y ene ac yla e [
31
], and o he polyme ic nanocomposi es, he combina ion o
coppe and poly-L-lac ic acid enhanced wi h su ace w inkling, ha e no been published o
da e, o he bes o ou knowledge. Despi e g owing in e es in his a ea, ew s udies ha e
sys ema ically explo ed coppe -in eg a ed PLLA su aces wi h w inkled mic oa chi ec u e
o an ibac e ial applica ions. Unde s anding he in e play be ween su ace mo phology,
chemical composi ion, and biological esponse emains c ucial. The p esen wo k add esses
his gap by de eloping and cha ac e izing w inkled Cu–PLLA su aces and e alua ing hei
an ibac e ial e icacy agains bo h E. coli and S. au eus, which se e as ep esen a i e models
o G am-nega i e and G am-posi i e bac e ial s ains, espec i ely. The use o w inkle
enginee ing combined wi h bime allic o monome allic coppe deposi ion and sol en -
based shaping echniques o e s a no el pla o m o he nex gene a ion o in ec ion-
esis an bioma e ials.
2. Ma e ials and Me hods
2.1. Ma e ials
Biopolyme poly-L-lac ic acid (PLLA) (densi y 1.25 g
·
cm
−3
, glass ansi ion empe -
a u e T
g
= 60
◦
C, c ys allini y 60–70%, hickness 50
µ
m; Good ellow L d., Hun ingdon,
UK, elonga ion a b eak 6%, ensile modulus 3.5 GPa) was used in he expe imen s. The
Polyme s 2025,17, 2173 4 o 19
polyme oil was modi ied ia coppe spu e ing using Quo um 300T ES sys em (Quo um
Technologies, Laugh on, UK). The ypical spu e ing condi ions we e oom empe a u e,
spu e ime 50–400 s, a gon gas wi h 99.997% pu i y a a p essu e o 1 Pa, and spu e ing
cu en o 20–60 mA.
2.2. Analy ical Me hods
2.2.1. A omic Fo ce Mic oscopy
The su ace mo phology and oughness o he p is ine and ea ed ilms we e ex-
amined wi h a omic o ce mic oscopy (AFM) using a Dimension ICON (B uke Co p.,
Bille ica, MA, USA). The samples we e analyzed in Scan-Assys
®
mode using a ni ide
le e SCANASYST-AIR (B uke Co p., Bille ica, MA, USA) wi h a Si ip (sp ing cons an
o 0.4 N
·
m
−1
). NanoScope Analysis 1.80 so wa e was applied o da a p ocessing. The
su ace oughness (R
a
) ep esen s he a i hme ic mean o he absolu e alues o he heigh
de ia ions measu ed om he cen al plane; RMS ep esen s he oo mean squa e a ia ion.
2.2.2. Scanning Elec on Mic oscopy
The mo phology o he sample su aces was also cha ac e ized wi h a scanning elec on
mic oscope FIB-SEM LYRA3 GMU (Tescan, B no, Czech Republic). The accele a ion ol age
was se o 10 kV. The elemen al composi ion was measu ed using ene gy-dispe si e X- ay
spec oscopy (EDS, analyze X-ManN, 20 mm
2
SDD de ec o , Ox o d Ins umen s, Ox o d,
UK), while he accele a ing ol age o SEM-EDS analysis was se o 10 kV.
2.2.3. X-Ray Pho oelec on Spec ome y
The elemen al composi ion on he ma e ial su ace was analyzed wi h X- ay pho o-
elec on spec oscopy (XPS) using a spec ome e ESCAP obeP (Omic on Nano echnology
L d., Taunuss ein, Ge many). As a sou ce, a monoch oma ic X- ay a an ene gy o 1486.7 eV
was used. The a omic concen a ions o he elemen s we e de e mined om he indi idual
peak a eas using CasaXPS so wa e 2.3.17PR 1.1.
2.2.4. We abili y
The we abili y o he s udied samples was de e mined by measu ing he con ac
angles (CA,
θ
) using a goniome e (Ad ex Ins umen s, B no, Czech Republic) connec ed
o he SEE Sys em 7.6 p og am. Analysis o CA was pe o med a oom empe a u e wi h
8
µ
L d ops o dis illed wa e using a T ans e pe e
®
au oma ic pipe e (B and, We heim,
Ge many) a 6 di e en posi ions o 3 samples in pa allel and pe pendicula di ec ions.
Subsequen ly, he d ops we e pho og aphed and e alua ed a 3 ma ked poin s. Es ima ion
o su ace ene gy was also based on he measu emen wi h he SEE Sys em. Two liquids
(wa e and glyce ol) we e used o he measu emen s. On he base o he Owens–Wend
me hod, he alues o con ac angles we e e alua ed. The measu emen was ca ied ou a
oom empe a u e. Values o su ace ene gy we e consequen ly e alua ed wi h he O igin
8.0 so wa e.
2.3. An ibac e ial Tes ing
The an ibac e ial ac i i y o he samples was de e mined using he so-called d ip es
pe o med wi h Esche ichia coli ep esen ing G am-nega i e bac e ia and S aphylococcus
au eus ep esen ing G am-posi i e bac e ia. F om aga pla es o E. coli and S. au eus
s ains, one colony each was ans e ed in o 20 and 5 mL o liquid Lu ia–Be ani (LB)
medium, espec i ely. The inocula we e hen incuba ed o e nigh in an o bi al shake a
37
◦
C. The ollowing day, he bac e ial suspensions we e dilu ed wi h phospha e-bu e ed
saline (PBS) o a concen a ion o app oxima ely 1.5
×
10
4
bac e ia/mL o E. coli and
1.3 ×104bac e ia/mL
o S. au eus. The es samples we e dipped in iplica es in o 2 mL o
Polyme s 2025,17, 2173 5 o 19
dilu ed bac e ial suspension and s a ically incuba ed a labo a o y empe a u e. A bac e ial
suspension wi hou added sample was p epa ed and incuba ed in he same manne o
con ol bac e ial g ow h, and a PBS solu ion alone was used o con ol con amina ion. A e
2 and 24 h, he samples we e mixed, and 5 d ops o 25
µ
L each we e d opped on o PCA
pla es (pla e coun aga ). These pla es we e hen cul u ed a 28
◦
C un il colonies we e
isible and sui able o coun ing in he samples o con ol bac e ial g ow h. The numbe o
colony- o ming uni s (CFUs) was hen de e mined and compa ed wi h he CFU coun in
he bac e ial g ow h con ol. The expe imen was conduc ed unde s e ile condi ions.
3. Resul s
3.1. A omic Fo ce Mic oscopy
The su ace mo phology o spu e ed poly-L-lac ic acid oil is shown in Figu e 1. Two
di e en Cu hicknesses on PLLA oil a e p esen ed; o he sake o cla i y, he de ailed
mo phology is o he 3
×
3
µ
m
2
hickness only. As is e iden om his igu e, no signi ican
change in su ace mo phology is associa ed wi h he inc easing hickness (spu e ing ime
o Cu). The inse o he igu e ep esen s he squa e o 300
×
300 nm
2
, whe e he globula
pa e n o med on he su ace is e iden .
Figu e 1. Su ace mo phology o p is ine PLLA, spu e ed PLLA wi h Cu: le , spu e ing cu en
20 mA and spu e ing ime 400 s; igh , spu e ing cu en 40 mA and spu e ing ime 200 s.
I is e iden om he alues o su ace a e age oughness and e ec i e su ace a ea
ha he coppe spu e ing does no change he su ace mo phology signi ican ly. I is also
impo an o say ha we did no in oduce he su ace mo phology o p is ine PLLA, since
no signi ican changes compa ed wi h spu e ed su aces we e obse ed, excep ha he
de ailed su ace mo phology o p is ine PLLA lacks a globula pa e n.
Al hough he spu e ed globula s uc u e is p esen , a much “smoo he ” su ace and
smalle globules compa ed wi h hose obse ed, e.g., o gold [
46
] we e de ec ed. The Cu-
Polyme s 2025,17, 2173 6 o 19
spu e ed PLLA oils wi h di e en hicknesses o Cu we e subsequen ly hea - ea ed a he
PLLA glass ansi ion empe a u e o one hou . Ou p e iously acqui ed esul s con i med
ha , wi h inc easing hickness o noble me al, a w inkle-like pa e n is induced on he
su ace o he noble me al–PLLA sys em. This phenomenon was o he i s ime con i med
o Cu on PLLA. Wha is connec ed wi h his phenomenon? Du ing he hea ea men ,
when i is close o he glass ansi ion empe a u e o PLLA, a bilaye o coppe -enhanced
PLLA is o med on he su ace o he bulk PLLA oil. This bilaye has some speci ic
p ope ies, such as inc eased he mal conduc i i y. The e o e, du ing his p ocedu e, and
a e he emo al o he Cu-enhanced sample om he o en du ing he cooling p ocedu e
(a a he apid phenomenon conside ing he sys em and he ma e ial—wi hin ens o ms),
w inkling ins abili y occu s. Compa ed wi h he bulk oil, he enhanced he mal ea men
induces he w inkle pa e n, which is di ec ly connec ed o he amoun o coppe and,
he e o e, he hickness o he coppe laye on he PLLA oil.
W inkling occu s in a ilm suppo ed by a so subs a e due o he de o ma ion mis-
ma ch once he comp essi e s ess exceeds a h eshold alue. Va ious me hods associa ed
wi h he mal deposi ion, plasma, ul a iole /ozone ea men , mechanical s e ching, and
sol en swelling [
18
,
19
,
47
,
48
] ha e been de eloped o induce o de ed w inkles; in ou
case, he mal ea men was applied. On he basis o he p inciple o minimizing he
o al po en ial ene gy, when he cooling p ocedu e is applied o he su ace [
47
,
48
], he
polyme /me al bilaye di e s om he emaining bulk due o be e hea conduc i i y, and
a w inkle pa e n is o med. The w inkle wa eleng h depends on ilm hickness and he
elas ic moduli o ilms and subs a es, while he w inkle ampli ude is ela ed o no only
ilm hickness and he elas ic moduli o ilms/subs a es bu also comp essi e s ain, and
he biaxial o uniaxial ilm o ien a ion may also play an impo an ole [19,20].
As shown in Figu e 2, he w inkling appea s when he he mal ea men is applied
o he PLLA, in acco dance wi h he e ec desc ibed in he p e ious pa ag aph. The
ampli ude o he w inkle pa e n and he densi y o he w inkles a e s ongly dependen on
he hickness o he me al laye (and, he e o e, he spu e ing cu en and ime). Wi h he
inc easing hickness o he Cu laye , he w inkle pa e n is mo e p onounced a e he hea
ea men p ocedu e. Wi h an inc easing amoun o me al p esen on he PLLA, he su ace
oughness and he su ace e ec i e a ea also inc ease, bo h o he inc easing spu e ing
ime and he spu e ing cu en , as is e iden om Figu e 2. The w inkling ins abili y
leading o he inducemen o he su ace w inkle pa e n may also signi ican ly a ec he
an ibac e ial p ope ies o p epa ed su aces, as desc ibed below. Addi ional AFM scans
o a 40 mA cu en and di e en spu e ing imes a e in oduced in Figu e S1.
3.2. Scanning Elec on Mic oscopy and Ene gy Dispe si e Analysis
The signi ican change in su ace mo phology induced by he combina ion o Cu
spu e ing on he PLLA su ace and subsequen hea ea men a a empe a u e close
o he glass ansi ion empe a u e o PLLA biopolyme was also con i med by scanning
elec on mic oscopy analysis. Fo compa ison, we i s analyzed he samples wi h di e en
hicknesses o Cu nanolaye s on he PLLA su ace (20 mA and 40 mA, 200 s), as shown
in Figu e 3. As expec ed, he spu e ing o he hin me al nanolaye s did no induce any
signi ican changes on he su ace o he PLLA biopolyme . The globula pa e n s uc u e
isible in he AFM analysis esul s is no clea ly isible in he SEM images.
Polyme s 2025,17, 2173 7 o 19
Figu e 2. Su ace mo phology o spu e ed PLLA wi h Cu and subsequen ly hea - ea ed (1 h, 60
◦
C):
spu e ing cu en s o 20 mA and 40 mA (100 s and 200 s). The e ec i e su ace a ea (S), a e age
su ace oughness (Ra), and oo mean squa e oughness (RMS) a e shown.
The su ace mo phology o he hea - ea ed PLLA wi h deposi ed coppe is shown in
Figu e 4. We selec ed o he compa ison he same spu e ing imes and di e en spu e ing
cu en s o 20 mA and 40 mA, he “40 mA” sample ha ing app oxima ely doubled he Cu
laye on i s su ace.
Figu e 4clea ly indica es ha , wi h an inc easing amoun o Cu on he su ace, he
pe iod o he ipple pa e n inc eases a e hea ea men . As shown in he igu e, he
homogenei y o he su ace ipple also inc eased, and in co ela ion wi h he AFM images,
he pe iod o he pa e n inc eased app oxima ely in he same a io as he deposi ed Cu
hickness (double he hickness, double he pa e n pe iod).
Polyme s 2025,17, 2173 8 o 19
Figu e 3. Su ace mo phology (SEM image y) o spu e ed PLLA wi h Cu: spu e ing cu en 20 mA
and 200 s (A); spu e ing cu en 40 mA and 200 s (B).
Figu e 4. Su ace mo phology (SEM image) o spu e ed PLLA wi h Cu: le , spu e ing cu en
20 mA and 200 s; igh , 40 mA and 200 s, subsequen ly hea - ea ed a 60
◦
C o 1 h. Inse image
ep esen s always he 3 mic on scan o pa icula sample.
We also de e mined he su ace chemis y o bo h he Cu-deposi ed and subsequen ly
hea - ea ed PLLA su aces. As Figu e 5clea ly shows, e en o a e y hin Cu laye , we
de ec ed 5.2 w . % o Cu on he spu e ed su ace. Wi h an inc easing spu e ing cu en , he
concen a ion o Cu inc eased, in good co ela ion wi h he inc easing spu e ing cu en .
The su ace deposi ed wi h 40 mA exhibi ed a Cu concen a ion o 10.9%, while o 60 mA,
we de ec ed a Cu concen a ion o 19.1%. As is ob ious om hese da a, he oxygen
concen a ion also dec eases, which indica es ha a non-oxidized laye is o med on he
PLLA su ace. Howe e , he oxida ion o Cu is discussed below in he sec ion de o ed o
he XPS analysis.
Polyme s 2025,17, 2173 9 o 19
Figu e 5. Su ace chemis y (EDS spec a) o spu e ed PLLA wi h Cu: spu e ing cu en 20 mA,
40 mA, and 60 mA wi h spu e ing ime 200 s.
The majo phenomenon ha occu ed du ing he hea ea men o he Cu-spu e ed
PLLA was he w inkle pa e n o ma ion. This was connec ed wi h he o ma ion o a
Cu-PLLA composi e bilaye , as discussed abo e. The e o e, we also aimed o s udy he
su ace chemis y changes when he ipple o ma ion is de ec ed. The EDS spec a o he
same se o samples desc ibed in he p e ious pa ag aph a e p esen ed in Figu e 6 o he
hea - ea ed specimens. Al hough he w inkle pa e n is induced, he amoun o Cu in he
w inkled su ace laye is no signi ican ly al e ed; we obse ed a maximum dec ease om
19.1 o 16.7 w . % o he sample deposi ed wi h he highes cu en 60 mA. We ha e o
no e ha he abili y o EDS analysis o acqui e su ace elemen al concen a ion is up o
se e al hund eds o nm; he e o e, al hough he e was spa ial edis ibu ion o Cu om he
su ace in o he ipple composi e, no signi ican di e ences we e expec ed. As s a ed abo e,
he only excep ion was he sample wi h he la ges amoun o Cu on he su ace (60 mA),
whe e he la e al pe iod o he w inkles was he highes ; in his case, some o he coppe
was di used a om he su ace, and a lowe Cu concen a ion was obse ed. A a he
di e en si ua ion can be expec ed as we analyze he su ace laye and su ace elemen al
concen a ion, which will be desc ibed in he nex sec ion de o ed o he XPS analysis o
bo h deposi ed and w inkled su ace pa e ns.
Polyme s 2025,17, 2173 16 o 19
bac e ial cell: he cell wall, cy oplasmic memb ane, nucleic acids, and in acellula p o eins.
Cu ions can des abilize he pep idoglycan laye (especially in G am-posi i e bac e ia),
dis up memb ane po en ial, and induce oxida i e s ess h ough he gene a ion o eac i e
oxygen species (ROS). These e ec s collec i ely lead o memb ane up u e and cell lysis [
57
].
This mul i- a ge , mul i-phase mechanism o coppe ’s an ibac e ial ac ion con ibu es o
i s ela i ely low suscep ibili y o he de elopmen o bac e ial esis ance [
58
], making i a
p omising ma e ial o an imic obial su ace design and biomedical applica ions.
4. Conclusions
This s udy demons a es he success ul de elopmen o a no el an ibac e ial su ace
based on w inkled coppe –poly-L-lac ic acid (Cu-PLLA) composi es. The su ace modi ica-
ion, achie ed h ough coppe spu e ing ollowed by con olled he mal ea men a he
glass ansi ion empe a u e o PLLA, esul ed in he o ma ion o hie a chical w inkle-like
s uc u es ha enhance an ibac e ial e icacy. Comp ehensi e su ace cha ac e iza ion using
SEM, AFM, EDS, and XPS e ealed a clea co ela ion be ween coppe laye hickness, w in-
kle mo phology, and su ace chemis y. No ably, he w inkle o ma ion was con i med o
be a esul o bilaye ins abili y, ini ia ed by he he mal esponse o he Cu-PLLA in e ace
and u he go e ned by he hickness o he coppe laye .
Despi e he lack o signi ican su ace oughness a ia ion be o e hea ea men , he
in oduc ion o he mal w inkling signi ican ly inc eased he e ec i e su ace a ea and
mo phological he e ogenei y. XPS analysis con i med ha , while some coppe di uses
deepe in o he polyme ma ix du ing w inkling, a subs an ial concen a ion emains
on he su ace, main aining bac e icidal ac i i y. Impo an ly, he an ibac e ial assays
conduc ed agains bo h Esche ichia coli and S aphylococcus au eus demons a ed ha he
syne gy be ween coppe ’s inhe en an imic obial p ope ies and he opog aphical cues
p o ided by he w inkle pa e ns leads o supe io bac e icidal pe o mance.
Among he es ed condi ions, he su aces wi h hicke coppe laye s and hose sub-
jec ed o he mal ea men showed he mos signi ican educ ion in bac e ial iabili y.
In pa icula , he comple e supp ession o bac e ial colonies on samples wi h highe Cu
con en (e.g., 60 mA, 200 s) and hea - ea ed w inkled su aces unde sco es he c i ical ole
o enginee ed su ace a chi ec u e in enhancing an imic obial e iciency. Mo eo e , he
obse a ion ha e en hinne Cu-coa ed su aces p o ided long-las ing an ibac e ial e ec s
a e 24 h highligh s he s abili y and p ac ical po en ial o hese hyb id su aces.
In conclusion, he o ma ion o w inkle pa e ns on Cu-PLLA su aces no only p o-
ides a mechanical means o dis up bac e ial memb anes bu also ensu es sus ained
exposu e o coppe ions, c ea ing a dual-mode an ibac e ial mechanism. These indings
posi ion Cu-PLLA w inkled composi es as p omising candida es o use in biomedical
applica ions, including as implan coa ings, wound d essings, and an ibac e ial packaging
ma e ials. Fu u e wo k may ocus on long- e m s abili y and biocompa ibili y s udies and
on ex ending his app oach o o he biopolyme /me al sys ems o b oade applicabili y.
Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a : h ps:
//www.mdpi.com/a icle/10.3390/polym17162173/s1, Figu e S1. Su ace mo phology o spu e ed
PLLA wi h Cu and subsequen ly hea ea ed (1 h, 60
◦
C): spu e ing cu en 40 mA (50 s and 300 s).
The e ec i e su ace a ea (S), a e age su ace oughness (Ra), and oo mean squa e oughness
(RMS) a e shown; Figu e S2. The su ace elemen al chemis y (XPS spec a) o spu e ed p is ine
PLLA, PLLA spu e ed wi h Cu, he spu e ing cu en 40 mA wi h spu e ing ime 50 s and 300 s,
and he sample spu e ed wi h 40 mA o 50 s and subsequen ly hea ea ed a 60
◦
C o 1 h (T);
Figu e S3. We abili y (con ac angle) o spu e ed PLLA wi h Cu and hea ea ed o 1 h and 60
◦
C:
spu e ing cu en s 20–60 mA wi h spu e ing imes om 50 s o 400 s; he samples we e measu ed
a e deposi ion (day 1) and aged o 6 days.
Polyme s 2025,17, 2173 17 o 19
Au ho Con ibu ions: Me hodology, I.L., A.P. and P.S. (Pe Sajdl); alida ion, P.S. (Pe Slepiˇcka)
and B.F.; o mal analysis, V.Š. and N.S.K.; in es iga ion, B.F., A.P. and P.S. (Pe Slepiˇcka); da a
cu a ion, V.Š. and P.S. (Pe Sajdl); w i ing—o iginal d a p epa a ion, P.S. (Pe Slepiˇcka), I.L. and
B.F.; w i ing— e iew and edi ing, N.S.K. All au ho s ha e ead and ag eed o he published e sion
o he manusc ip .
Funding: This wo k was suppo ed by he P ojec OP JAK Mebiosys, No. CZ.02.01.01/00/22_008/
0004634, o he Minis y o Educa ion, You h and Spo s, which is co- unded by he Eu opean Union.
This wo k was suppo ed by he g an GACR unde p ojec 22-04006S.
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : The da a a e a ailable a h ps://doi.o g/10.5281/zenodo.15872583.
Con lic s o In e es : The au ho s decla e no con lic s o in e es .
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