Oc obe 20 - 22, 2021, B no, Czech Republic, EU
ANTIMICROBIAL PROPERTIES OF POLYMERIC NANOFIBROUS MEMBRANES
CONTAINING FERROUS SULPHATE
Zuzana KRCHNACKOVA, Dusan KIMMER, Lenka LOVECKA, Mi osla a KOVAROVA,
Hana PISTEKOVA, Daniela VESELA, I o VINCENT, Muhammad YASIR,
Vladimi SEDLARIK
Cen e o Polyme Sys ems, Tomas Ba a Uni e si y in Zlin, Czech Republic, EU, [email p o ec ed]
h ps://doi.o g/10.37904/nanocon.2021.4347
Abs ac
This pape epos s on nano ibe s o polyme s doped wi h e ous sulpha e (FeSO4) possibly in
combina ion wi h qua e na y ammonium sal (QAS) p epa ed by elec ospinning. Th ee ypes o
polyme s (poly inylidene luo ide/PVDF, polylac ic acid/PLA and polyu e hane/PU) wi h good
elec ospinning p ocessabili y and good mechanical p ope ies o nano ibe s we e chosen. The
p epa ed nano ib ous memb anes we e cha ac e ised in e ms o mo phology o he ib es assessed by
SEM, and he po e sizes we e de e mined by po ome y. The leaching es showed a i m ancho ing o
he addi i e in he nano ibe s uc u e. An imic obial ac i i y was moni o ed a e 0 h, 4 h and 24 h using
S aphylococcus au eus (CCM 4516) and Klebsiella pneumoniae (CCM 4415) s ains. Fu he mo e,
ae osol il a ion e icycy was de mined and also quali y ac o o il a ion.
The memb ane p epa ed om PU doped wi h FeSO4 showed he bes an ibac e ial e iciency. The
po osi y and mo phology o he nano ib ous memb ane e ec i ely con ibu ed o he apping o
mic oo ganisms. This sys em was also e alua ed as he mos sui able o he elec ospinning p ocess
om he e ec i i y poin o iew. Spinning ang dopping p ocess o p epa a ion is easily applica bole o
he he indus ial condi ions o p oduc ion which is e y impo a n aspec o his submi ed scien i ique
wo k.
Keywo ds: An imic obial p ope ies, doped nano ibe s, FeSO4, il a ion
1. INTRODUCTION
In ecen yea s, polyme ic nano ibe s ha e been demons a ed as e ec i e ool o ae osol il a ion
applica ions. They ha e a g ea abili y o ae osol il a ion and apping mic oo ganisms ha ha e
became ex emely ele an due o he COVID-19 occu ence [1]. The size o SARS-COV-2 i us is 60–
140 nm wi h nanospikes coa ed on i s sphe ical i al capsid/en elop wi h heigh s o 9–12 nm [2]. The
mos bene icial p ope ies in his ield o s udy a e: high su ace a ea o weigh a io, low densi y, high
po e olume and besides o he also small po e size [3]. As inc ease equi emen o an imic obial e ec
ac oss nano ibe applica ions i needs o be men ion also ano he aluable ad an age; possibili y o hei
ela i ely easy su ace coa ing o doping wi h a ious species, which can p o ide nano ibe s wi h speci ic
p ope ies, including an imic obial e ec s [4].
As demands o unc ionali y o nano ibe s memb ane a e mo e and mo e complex, many esea ch
g oups ha e p epa ed al eady wide ange o su ace modi ied nano ibe s, including an imic obial [5].
Howe e , i needs o be said ha some o me hods a e e y complica ed, expensi e o no s able du ing
usage ime o memb ane. The e o e, in ou s udy we de eloped easy p ocedu e o an imic obial
nano ibe s doping wi h FeSO4 possibly in combina ion wi h QAS as an imic obial agen s. Ou me hod
o spinning and doping o nano ibe s is easily applicable in condi ons o indus ial p oduc ion.
Oc obe 20 - 22, 2021, B no, Czech Republic, EU
In ou wo k, a e de ailed cha ac e iza ion o p epa ed ma ials he an imic obial e ec was e alua ed on
wo species o bac e ia, S aphylococcus au eus and Klebsiella pneumoniae. Apa om he pa hogenic
e ec s o hese wo bac e ial s ains, ano he eason o es ing hem is ha hey a e undamen ally
di e en in he a angemen o ou e memb anes. So ob ained esul s p o ide gene al in o ma ion on
he in e ac ion be ween a biocide compound and o he G am-posi i e and G am-nega i e bac e ia [6].
Fu he mo e, ae osol il a ion e icacy a di e en pa icle sizes was p o ed ia au oma ed il e es e
Model 3160. An ideal ai il e should ha e high pa icle- emo al e iciency, low-p essu e d op, and a
long li e ime as well as high quali y ac o numbe . [7]
Nano ib ous memb anes doped wi h an ac i e an ibac e ial componen di ec ly in he mass o nano ibe s
a e he nex s ep owa ds p oduc s ha ac i ely elimina e mic oo ganisms and hus p o ec human
heal h.This wo k ocused on de eloping nano ib ous memb ane doped wi h an imic obial compounds
o ae osol il a ion, which is he nex s ep owa ds p oduc s ha ac i ely elimina e mic oo ganisms and
hus p o ec human heal h.
2. MATERIALS AND METHODS
We p epa ed h ee polyme ic nano ib ous memb anes based on PVDF, PU, and PLA deposi ed on
polyp opylene (PP) non-wo en as collec ing suppo . All ypes o success ully elec ospun nano ibe s
we e doped ia 1 % o FeSO4. Then PU nano ibe s we e doped wi h bo h 1 % o FeSO4 and also 7.5
% o QAS inco po a ed in o PU chains. Basic weigh s o all p epa ed nano ibe s we e a a maximum o
1 g/cm2, as shown in Table 1.
Table 1 Basic weigh s o all p epa ed nano ib ous ma e ials
Sample
Basic weigh (g/cm2)
PVDF/FeSO4
0.77
PLA/FeSO4
0.56
PU/FeSO4
0.45
PU/FeSO4/QAS
1.00
Polyme ic nano ibe s we e p epa ed by elec ospinning de ice SpinLine 40 (SPUR, Czech Republic)
equipped wi h wo se s o mo ing nano ibe s o ming je s. The applied ol age du ing he spinning
p ocess was 75 kV, and he dis ance be ween elec odes was 19 cm o all samples. O he pa ame e s
o polyme ic solu ion we e di e en o each sample. Summa isa ion o hese pa ame e s is comple ed
in Table 2.
Table 2 Summa iza ion o pa ame e s o polyme ic solu ions.
Sample
Sol en
Basic weigh (g/cm2)
Viscosi y (Pa.s)
Conduc i i y (µS/cm)
PVDF/FeSO4
DMF
0.77
1.60
25.10
PLA/FeSO4
DMF/ace on
0.56
0.75
110.3
PU/FeSO4
DMF/H2O
0.45
3.25
120.20
PU/FeSO4/QAS
DMF/H2O
1.00
0.80
310.00
The mo phology o he elec ospun nano ibe s was obse ed using scanning elec on mic oscopy
(SEM; Vega 3, Tescan, Czech Republic). The a e age diame e o he elec ospun nano ibe s was
de e mined by analysing he SEM images using a cus om code image analysis p og am. As well as he
po e size o nano ib ous ma e ials was pe o med by po ome y de ice (SPUR, Czech Republic)
acco ding o ASTM F316-03.The an imic obial ac i i y o he p epa ed nano ibe ’s ma s was es ed
Oc obe 20 - 22, 2021, B no, Czech Republic, EU
acco ding o he es ing me hod o he an ibac e ial ac i i y o ex iles using S aphylococcus au eus
(CCM 4516) and Klebsiella pneumoniae (CCM 4415) s ains. Then an imic obial ac i i y was calcula ed
ia ISO 22196:2011.
An imi obial ac i i y (A) calcula ion:
A = log N0 - log Nx
Whe e:
x - incuba ion ime wi h bac e ial suspension (0, ½, 4 a 24h)
A - an ibac e ial ac i i y o sample
N - numbe o iable bac e ia (CFU/cm2)
The samples we e cu in o 20 x 20 mm squa es and he polyp opylene co e oil was also cu in o
20 x 20 mm. P io o es ing, he samples we e disin ec ed wi h UV adia ion o 30 minu es on bo h
sides (s e iliza ion in an au ocla e o disin ec ion wi h e hanol wasn’ possible due o po en ial damage
o sample). The co e oils we e disin ec ed wi h 70 % e hanol. The s e iled samples we e p epa ed in
iplica ed and pu in o he s e ile glass pe i dishes and co e ed by 0.1 ml o bac e ial suspension, and
his was co e ed wi h PP oil. P epa ed samples we e incuba ed o 4h and 24h a 35 °C wi h a ela i e
humidi y o 95 %. Samples in ime 0h was immedia ely p ocessed. T iplica es o one sample including
oil we e ans e ed in o he s e ile PP con aine . Neu aliza ion medium (SCDLP) was added in a o al
olume 15 ml (5 ml pe sample). Incuba ion ime was 30 s. Then all saples we e decimally dilu ed nad
mixed wi h aga media (PCA) and incuba ed o 24h a 35 °C.
3. RESULTS AND DISCUSSION
The samples we e es ed a e op imising he spinning p ocess o all he polyme solu ions leading o
epe able s uc u es. The SEM images in Figu e 1 show he po ous s uc u e o he p epa ed
nano ib ous ma e ials wi h andomly o ien ed nano ibe s ha ing a smoo h su ace wi hou beads-like o
any o he mo phological de ec s. Al hough he elec ospinning p ocess was adequa ely op imised, in he
case o sample PU/FeSO4 he p oduced nano ibe s we e no s aigh and we e angled. The ib es also
had a la ge diame e compa ed o he o he ma e ials. These de iciencies we e co ec ed by adding 7.5
% QAS o he polyme ic solu ion con aining 1 % FeSO4. On he con a y, PVDF and PLA polyme ic
nano ibe s we e s aigh e en wi hou adding QAS in o he solu ion.
Figu e 1 SEM images o ou di e en ypes o elec ospun nano ibe s: PVDF doped wi h 1 % o
FeSO4, PLA doped wi h 1 % o FeSO4, PU doped wi h 1 % o FeSO4 and PU doped wi h 1 % FeSO4 in
combina ion wi h 7.5 % o QAS
Oc obe 20 - 22, 2021, B no, Czech Republic, EU
Among o he cha ac e isa ion me hods, he measu emen o ib e diame e is c ucial o p edic ing he
p ope ies o nano ibe s uc u es. The diame e o ib es depends on he su ace ension, low a e,
iscosi y and elec ic conduc i i y o polyme solu ion [8]. Figu e 2 ep esen s he ib e diame e
dis ibu ion o he elec ospun ib es on o he non-wo en PP subs a e, indica ing a ange o diame e
be ween 0.16 µm and 0.33 µm. The PVDF/FeSO4 sample shows he la ges a e age nano ibe
hickness, which is due o he low conduc i i y o he polyme solu ion. Howe e , his sample also
con ains a signi ican p opo ion o ib es wi h a diame e o a ound 0.1 µm, which could imp o e he
il a ion p ope ies o he ma e ial. Fo polyu e hane samples, he addi ion o QAS shi s he dis ibu ion
o ib e diame e s owa ds lowe alues and he a e age ib e diame e alue d ops om
0.33 o 0.16 µm. Usage o nanome ic ib es has p o en ad an ageous in ai il a ion since hei small
diame e , and high su ace o olume a ios can enhance he cap u e o pa icles h ough in e cep ion.
Figu e 2 Nano ibe s diame e dis ibu ions o PVDF/ FeSO4 (a e age disme e 0.33 µm), PLA/ FeSO4
(0.16 µm), PU/ FeSO4 (0.33 µm) and PU/ FeSO4/QAS (0.18 µm)
Ano he impo an measu emen o e ealing he p ope ies o p epa ed samples was po ome y. The
low po ome y echnique is a use ul ool o es ing nano ibe s uc u es. Figu e 3 p esen s he po e
size dis ibu ions o all es ed samples. While he dis ibu ions o PVDF and PLA ha e a wid h o 0.75 µm
and 0.85 µm, espec i ely, and a mean po e size o 0.92 µm and 0.84 µm, he wid h o he dis ibu ion
cu e o PU/FeSO4 is up o 1.6 µm and he mean po e size eaches 2 µm. Howe e , using a
combina ion o FeSO4 and QAS esul s in a signi ican na owing o he dis ibu ion (wid h o 0.64 µm)
and a dec ease in he mean po e size o 0.91 µm. The na owe po e size dis ibu ion sugges s ha he
ma e ial will be mo e homogeneous. The mo e homogenous nano ibe laye means lowe p essu e d op,
which is a cha ac e is ic playing a signi ican ole in he applica ion o elec ospun nano ibe s in ai
il a ion [9].
Oc obe 20 - 22, 2021, B no, Czech Republic, EU
Figu e 3 Flow po ome y o spun nano ibe s doped wi h FeSO4
A e de ailed cha ac e isa ion o p epa ed nano ib ous ma e ials, we p oceeded o an imic obial ac i i y
es s acco ding o -SO 22196:2011. An imic obial p ope ies we e in es iga ed using S. au eus and
K. pneumoniae as model mic oo ganisms. A e ea men ime wi h bac e ial solu ion (4h, 24h) inal
CFU o bac e ia was coun ed and con e ed o an imic obial ac i i y (A) desc ibed abo e. Then i was
possible o e alua e p ecisely he e icacy o an imic obial ac i i y o he es ed ma e ials. Table 3
desc ibes he exac a e o e ec i i y o an imic obial ac i i y acco ding o he A alues gi en in Figu e 4.
I seems ha hyd ophilici y/hyd ophobici y has a signi ican in luence on he an imic obial ac i i y o he
polyme because elec ospun nano ibe s om PVDF and PLA polyme s ha e low an imic obial ac i i y
compa ed wi h mo e hyd ophilic PU nano ibe s. Fu he mo e, adding QAS o polyme solu ion shows
an essen ial e ec on bac e ia elimina ion. The an imic obial e ec is s onges in he case o bo h
bac e ial s ains (A ≥ 3).
Figu e 4 An imic obial ac i i y o p epa ed nano ib ous ma e ials using S. au eus and K. pneumoniae
as model mic oo ganisms, and e alua ion o an imic obial ac i i y (A) a e 4h and 24h

Oc obe 20 - 22, 2021, B no, Czech Republic, EU
Table 3 Explana ion o e ec i i y o an imic obial ac i i y (A) alues
E icacy o an ibac e ial p ope ies
Value o an imic obial ac i i y A
Weak
1 < A < 2
Signi ican
2 ≤ A< 3
S ong
A ≥ 3
4. CONCLUSION
Elec ospun nano ibe s o e a numbe o ad an ages when used in ai il e s. The e a e ex ensi e
s udies ha demons a e imp o emen in il e a ion e iciency wi hou inc easing p essu e d op. In his
esea ch, we success ully doped a ious nano ib ous ma s (PVDF, PLA, PU) designed o ai il a ion
pu poses wi h po en ial an imic obial species FeSO4 and QAS.
Cha ac e isa ion analyses such as SEM, po ome y o measu emen o he diame e o nano ibe s we e
pe o med. These cha ac e is ics may help o elucida e co ela ions be ween an imic obial ac i i y and
he na u e and s uc u e o he nano ib ous ma e ial. Changes in conduc i i y o elec ospinning polyme
solu ions signi ican ly impac he diame e and he dis ibu ion o nano ibe ’s diame e s as well as
po osi y o nano ib ous ma e ial and i s dis ibu ion. Fu he mo e, we p o ed ha adding QAS o
PU/FeSO4 polyme solu ion inc eased he an imic obial e ec , which was obse ed o a o al pe iod o
24h. P epa ed nano ib ous ma e ials wi h he bes e ec agains bac e ia, i.e. PU/FeSO4 and
PU/FeSO4/QAS, was selec ed o a u he s udy dealing wi h op imising o nanos uc u e p ope ies o
ai il a ion.
ACKNOWLEDGEMENTS
The au ho s g a e ully acknowledge inancial suppo om he con ac esea ch Plas 2020 -
VaV by companies Plas ikářský klas s. z. and SPUR a.s. and by he Minis y o Educa ion,
You h, and Spo s o he Czech Republic (g an no. RP/CPS/2020/002).
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