Electrospun Polylactic Acid-Based Fibers Loaded with
Multifunctional Antibacterial Biobased Polymers
A. Chiloeches, R. Cuervo-Rodríguez, Y. Gil-Romero, M. Fernández-García, C. Echeverría,*
and A. Muñoz-Bonilla*

Cite This: ACS Appl. Polym. Mater. 2022, 4, 6543−6552 Read Online

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ABSTRACT: Here, we report the development of antibacterial and compostable electrospun polylactic acid (PLA) fibers by
incorporation of a multifunctional biobased polymer in the process. The multifunctional polymer was synthesized from the bio-
sourced itaconic acid building block by radical polymerization followed by click chemistry reaction with hydantoin groups. The
resulting polymer possesses triazole and hydantoin groups available for further N-alkylation and chlorination reaction, which provide
antibacterial activity. This polymer was added to the electrospinning PLA solution at 10 wt %, and fiber mats were successfully
prepared. The obtained fibers were surface-modified through the accessible functional groups, leading to the corresponding cationic
triazolium and N-halamine groups. The fibers with both antibacterial functionalities demonstrated high antibacterial activity against
Gram-positive and Gram-negative bacteria. While the fibers with cationic surface groups are only effective against Gram-positive
bacteria (Staphylococcus epidermidis and Staphylococcus aureus), upon chlorination, the activity against Gram-negative Escherichia coli
and Pseudomonas aeruginosa is significantly improved. In addition, the compostability of the electrospun fibers was tested under
industrial composting conditions, showing that the incorporation of the antibacterial polymer does not impede the disintegrability of
the material. Overall, this study demonstrates the feasibility of this biobased multifunctional polymer as an antibacterial agent for
biodegradable polymeric materials with potential application in medical uses.
KEYWORDS: poly(lactic acid), polyitaconates, biobased polymers, antimicrobial fibers, triazolium, N-halamine, compostability

1. INTRODUCTION
Microbial contamination on surface contacts of solid materials
constitutes an important source of infections not only in
biomedical devices, implants, and prostheses but also in other
fields such as the food industry.1,2 Microbial adhesion onto
such surfaces and subsequent biofilm formation, together with
the emergence of antibiotic resistance, has become one of the
more critical public health concerns.3−5 Then, surface
disinfection and sterilization processes (physical and chemical)
in hospitals, food processing facilities, or even in the domestic
environment are crucial to reduce and control microbial
infections. Typically, both physical and chemical methods are
used. Application of chemical disinfectants, such as alcohols,
chlorines, or quaternary amines, are effective treatments that,
however, have several disadvantages, such as the need for high

concentration to achieve sterility standards and the potential
hazards to humans and the environment. For instance,
common quaternary ammonium compounds such as the
disinfectant benzalkonium chloride which generates bacterial
resistance,6 have been found in sediments and soils.7 Similarly,
the ethylene oxide chemical sterilization method presents
potential hazards, as ethylene oxide is toxic, carcinogenic, and
allergenic and can be present after the process.8 On the other

Received: June 2, 2022
Accepted: August 18, 2022
Published: August 26, 2022

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hand, physical methods such as gamma irradiation, UV-
irradiation, and steam sterilization can alter the properties of
the materials, in particular polymer materials commonly used
for biomedical or packaging applications.9−11 This is even
more critical in biodegradable biopolymers such as polylactic
acid (PLA), which typically have hydrolytic and thermal
sensitivity, and sterilization can alter the properties of the
materials.12,13 Nowadays, PLA is widely used as a sustainable
alternative to conventional plastics14,15 in many applications,
including the manufacture of biomedical devices and food
packing. Indeed, a large part of plastic waste comes from these
sectors, and therefore, the use of biodegradable and renewable
polymers in such applications represents a great alternative to
protect the environment.16,17

New perspectives to prevent or reduce surface contami-
nation and bacterial growth on these bioplastic materials,
maintaining safety requirements and long-term activity, are still
challenged. Incorporation of antimicrobial biodegradable
polymers into bioplastics is considered a valuable alternative,
as these materials retain high long-term antimicrobial activity,
do not leakage easily, have low toxicity and low susceptibility
for developing resistance, and are able to degrade after their
useful lifetime.18−22 Recently, our group has developed
antibacterial biobased polymers derived from itaconic acid
bearing cationic azolium groups derived from vitamin thiamine
(B1). These polymers demonstrate excellent antibacterial
activity against Gram-positive bacteria, negligible toxicity to
human cells, and compostability.23,24 In addition, these
antibacterial cationic copolymers have been studied as active
additives in biodegradable films based on poly(butylene
adipate-co-terephthalate) for packaging applications.24 How-
ever, the activity of these systems against Gram-negative
bacteria is limited. Herein and based on our previous itaconate
derivatives, we synthesize a biodegradable antibacterial
polymer with multifunction, including contact-killing and
release-killing mechanisms, with the purpose of improving
the antibacterial activity and being more effective against
Gram-negative bacteria. The multifunctional itaconate polymer
was designed to combine in its structure two functional groups,
cationic azolium and N-halamine groups. While the cationic
groups in the polymer kill bacteria by physically damaging the
cell membrane, the activity of N-halamine groups is due to the
release of oxidative halogen, which can react with the
appropriate biological receptors, affecting cell metabolism.25

This antibacterial biobased polymer was subsequently
employed to impart antibacterial character to PLA fiber mats
obtained by electrospinning in order to achieve “fully
biodegradable” material. Electrospun fibers, widely used for
food packages and biomedical materials such as face masks or
wound dressing,26−28 have a large surface-to-volume ratio so,
in principle, low quantities of antimicrobial polymer would be
needed to achieve satisfactory results.29

2. EXPERIMENTAL PART
2.1. Materials. Itaconic acid (IA, ≥ 99%), propargyl alcohol

(≥99%), 4-(dimethylamino) pyridine (DMAP, ≥ 99%), N,N′-
dicyclohexylcarbodiimide (DCC, 99%), hydroquinone (99%), 5,5-
dimethylhydantoin (97%), 1-bromo-2-chloroethane (98%), potassium
hydroxide (KOH, 90%), sodium azide (NaN3, ≥99%), sodium iodide
(NaI, 99.5%) di-tert-butyl dicarbonate (Boc2O, 99%), copper(I)
chloride (CuCl, ≥99.995%), N,N,N′,N″,N″-pentamethyldiethylenetri-
amine (PMDTA, 99%), iodomethane (MeI, 99.5%), trifluoroacetic
acid (TFA, 99%), tert-butanol (≥99.5%), acetic acid (≥99.7%),
neutral aluminum oxide, sodium bicarbonate (NaHCO3, ≥99.7%),

magnesium sulfate anhydrous (MgSO4, ≥99.5%), fluorescein sodium
salt, sodium thiosulfate (Na2S2O3, 99%), cetyltrimethylammonium
chloride (CTAC, ≥98%), iodine standard solution, anhydrous
tetrahydrofuran (THF, 99.9%), and anhydrous N,N-dimethylforma-
mide (DMF, 99.8%) were purchased from Merck and used as
received. The radical initiator 2,2′-azobisisobutyronitrile (AIBN,
98%) was purchased from Acros and was recrystallized twice from
methanol. PLA (6202D) was provided by NatureWorks. All the
organic solvents were of AR grade; dichloromethane (DCM), THF,
DMF, ethanol (EtOH), isopropyl alcohol (iPrOH), hexane, diethyl
ether, and chloroform (CHCl3) were purchased from Scharlau; ethyl
acetate (EtOAc) was purchased from Cor Quiḿica S.L.; toluene was
purchased from Merck; sulfuric acid (H2SO4) was purchased from
Panreac. Deuterated chloroform (CDCl3), water (D2O), and dimethyl
sulfoxide (DMSO-d6) were acquired from Sigma-Aldrich. Cellulose
dialysis membranes (CelluSep T1) were purchased from Membrane
Filtration Products, Inc.
For the antibacterial assay: phosphate buffered saline powder (pH

7.4) and sodium chloride solution (NaCl suitable for cell culture,
BioXtra) were purchased from Sigma-Aldrich. BBL Mueller−Hinton
broth used as a microbial growth medium in the determination of the
minimum inhibitory concentration (MIC) was acquired from Becton,
Dickinson and Company, and the 96 well microplates were purchased
from BD Biosciences. Columbia agar (5% sheep blood) plates were
obtained from Fisher Scientific. American Type Culture Collection
(ATCC): Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853),
Escherichia coli (E. coli, ATCC 25922), Staphylococcus epidermidis (S.
epidermidis, ATCC 12228), and Staphylococcus aureus (S. aureus,
ATCC 29213) were used as bacterial strains and were purchased from
Oxoid.
2.2. Synthesis of poly(di(prop-2-yn-1-yl)) Itaconate, P(PrI).

The clickable polymer P(PrI) was synthesized as previously
described.23 First, the monomer di(prop-2-yn-1-yl) itaconate (PrI)
bearing clickable alkyne groups was synthesized via condensation
reaction of itaconic acid with propargyl alcohol. Subsequently, the
monomer PrI was polymerized by conventional radical polymerization
with 5 mol % of the AIBN initiator, at a total concentration of 2 M in
anhydrous DMF, at 70 °C under a nitrogen atmosphere for 24 h. The
polymer P(PrI) was isolated by precipitation in isopropanol and dried
overnight under vacuum at room temperature to afford a white solid
(71% yield, Mn = 6700 g/mol, Mw/Mn = 1.58). 1H NMR (400 MHz,
CDCl3, δ, ppm): 4.67 (4H, −CH2C�CH), 2.49 (2H, −CH2C�
CH), 1.99−1.00 (8H, CH2−CO and −CH2-chain).
2.3. Synthesis of tert-Butyl 3-(2-azidoethyl)-5,5-dimethyl-

hydantoin-1-carboxylate, Boc-DMH-N3. First, 3-(2-azidoethyl)-
5,5-dimethylhydantoin was synthesized through nucleophilic sub-
stitution reaction between 5,5-dimethylhydantoin and 1-bromo-2-
chloroethane,30 followed by transformation of chlorine groups to
azide groups via a substitution reaction. Briefly, 5,5-dimethylhydan-
toin (20.0 g, 156 mmol) and potassium hydroxide (8.7 g, 156 mmol)
were dissolved in 100 mL of ethanol in a round-bottomed flask. Then,
1-bromo-2-chloroethane (26 mL, 312 mmol) was added, and the
mixture was heated under reflux for 6 h. The reaction was cooled to
room temperature, and the solvent was removed by rotary
evaporation. The resulting white solid was dissolved in EtOAc and
washed with water and NaHCO3 solution (10%) by several extraction
steps. The organic fractions were dried over anhydrous magnesium
sulfate, filtered, and evaporated under reduced pressure. The white
solid was further purified by recrystallization in isopropanol/toluene
(9:1) to afford DMH-Cl (yield 48%). 1H NMR (400 MHz, CDCl3, δ,
ppm): 6.31 (s, 1H, −NH−), 3.85 (t, 2H, −CH2−Cl), 3.74 (t, 2H,
−CH2−CH2−Cl), 1.46 (s, 6H, −CH3).
Later, chlorine-substituted hydantoin (DMH-Cl) (5.0 g, 26 mmol)

was dissolved in DMF and deoxygenated by purging with argon.
Sodium iodide (3.9 g, 26 mmol) and an excess of sodium azide (2.4 g,
37 mmol) were dissolved in 6 mL of water. Then, both solutions were
mixed and heated at 80 °C under an argon atmosphere for 3 h. The
resultant mixture was concentrated under reduced pressure and, then,
was extracted with EtOAc and washed repeatedly with NaCl aqueous
solution. The organic extract was separated, dried over MgSO4, and

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filtered, and the solvent was removed under reduced pressure to
afford the product DMH-N3 as a colorless oil (yield: 88%). 1H NMR
(400 MHz, CDCl3, δ, ppm): 6.57 (s, 1H, −NH−), 3.70 (t, 2H,
−CH2−N3), 3.52 (t, 2H, CH2−CH2−N3), 1.44 (s, 6H, −CH3).
Subsequently, the amine group of the DMH-N3 product was

protected by di-tert-butyl dicarbonate. DMH-N3 (0.9 g, 4.6 mmol),
Boc2O (1.3 g 6 mmol), DMAP (56 mg, 0.46 mmol), and 25 mL of
DCM were added in a round bottom flask. Then, the solution was
stirred at room temperature for 4 h. The reaction mixture was washed
with water several times. The organic layer was dried over Mg2SO4
and filtered, and the solvent was evaporated by rotary evaporation.
The residue was purified by silica gel column chromatography using
diethyl ether as eluent to give the protected product Boc-DMH-N3
(yield: 95%). 1H NMR (400 MHz, CDCl3, δ, ppm): 3.75 (t, 2H,
−CH2−N3), 3.56 (t, 2H, CH2−CH2−N3), 1.64 (s, 6H, −CH3), 1.57
(s, 9H, −CH3 Boc). 13C NMR (100 MHz, CDCl3, δ, ppm): 174.9
(N−C�O), 151.8 (N−(C�O)−N), 148.4 (N−(C�O)−O), 84.4
(Cquat, Boc), 60.3 (Cquat, hydantoin), 48.3 (−CH2−N3), 38.2 (CH2−
CH2−N3), 28.2 (−CH3, Boc), 23.2 (−CH3, hydantoin). FTIR: 2097
cm−1 associated with the azide group.
2.4. Synthesis of Poly(bis((1-(2-(3-(tert-butoxycarbonyl)-4,4-

dimethyl-2,5-dioxoimidazolidin-1-yl)ethyl)-1H-1,2,3-triazol-4-
yl)methyl)itaconate), P(Boc-DMHI). The incorporation of the
hydantoin Boc-DMH-N3 into the clickable itaconate polymer P(PrI)
was conducted by click chemistry Cu(I)-catalyzed azide−alkyne
cycloaddition (CuAAC). In a typical procedure, a mixture of polymer
P(PrI) (1.50 g, 7.3 mequiv of alkyne groups), Boc-DMH-N3 (5.40 g,
18.2 mmol), PMDTA (300 μL, 1.45 mmol), and CuCl (0.072 g, 0.72
mmol) were dissolved in 40 mL of CHCl3. The mixture was stirred at
40 °C for 24 h and then passed through a neutral alumina column.
The resulting polymer P(Boc-DMHI) was isolated by precipitation in
ethanol, and the degree of modification was almost quantitative
(yield: 98%). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 8.08 (2H, H-
triazole), 5.04 (4H, O−CH2-triazole), 4.61 (4H, CH2−N triazole),
3.85 (4H, CH2−N hydantoin), 1.49 (s, 18H, −CH3 Boc), 1.45 (s,
12H, −CH3), 2.66−1.00 (8H, CH2−CO and −CH2-chain).
2.5. N-Alkylation of P(Boc-DMHI) and Deprotection Re-

action. Synthesis of Cationic Polymer P(DMHI-Q). The polymer
P(Boc-DMHI) was modified by N-alkylation reaction with iodo-
methane (MeI) leading to the corresponding cationic itaconate
polymer. The polymer (1.50 g, 3.75 mequiv of triazole groups) was
dissolved in 25 mL of anhydrous DMF, and then, a large excess of
MeI was added (1.2 mL, 18.75 mmol; ratio triazole groups/alkyl
iodide ≈ 1:5). The mixture was deoxygenated with argon for 15 min,
sealed, and then stirred at 70 °C for one week to achieve a high
degree of modification. The resulting cationic polymer was purified by
precipitation into n-hexane followed by dialysis against distilled water
and finally was isolated by freeze-drying. The degree of quaternization
was almost quantitative. Subsequently, the deprotection reaction was
performed. For this purpose, the polymer was dissolved in
trifluoroacetic acid (5 mL) and stirred at 50 °C for 2 h. After cooling
down to room temperature, the cationic polymer P(DMHI-Q) was
purified by dialysis against distilled water and then lyophilized to yield
a white solid (96%). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 9.10
(2H, H-triazole), 5.44 (4H, O−CH2-triazole), 4.78 (4H, CH2−N
triazole), 4.37 (6H, N+CH3 triazole), 3.85 (4H, CH2−N hydantoin),
1.29 (s, 12H, −CH3), 2.66−1.00 (8H, CH2−CO and −CH2-chain).
2.6. Chlorination of P(DMHI-Q). Synthesis of Polymer

P(DMHICI-Q). Initially, tert-butyl hypochlorite was synthesized.30

Briefly, sodium hypochlorite solution (250 mL, 10 wt %) was placed
in a round-bottomed flask and cooled in an ice bath. A solution of
tert-butyl alcohol (10 mL) and glacial acetic acid (15.5 mL) was
added under stirring. After 10 min, the organic layer of the mixture
was extracted with a separatory funnel, and the aqueous layer was
discarded. The organic layer was washed successively with an aqueous
solution of NaHCO3 10% and water, dried over CaCl2, and filtered
(yield: 73%).
The chlorination of the P(DMHI-Q) polymer to afford the

P(DMHICl-Q) multifunctional polymer was carried out using tert-
butyl hypochlorite as follows: P(DMHI-Q) (1.50 g, 4.85 mequiv of

hydantoin groups) and tert-butyl hypochlorite (2.25 mL, 19.4 mmol)
were dissolved in 20 mL of H2O/t-butanol (1/4 v/v). Then, the
mixture was stirred in dark for 24 h at room temperature and dried
under a vacuum with a rotary evaporator to obtain the final N-
halamine polymer P(DMHICl-Q) in a quantitative amount. The
polymer was characterized by Fourier transform infrared (FTIR), and
the oxidative chlorine (Cl+) content of the polymer was quantified
through the iodometric/thiosulfate titration method.31 Briefly, 1.8 mg
of P(DMHICl-Q) was dissolved in 1 mL of 1% aqueous potassium
iodide solution for 10 min to form I2 (the color of the solution
changed to yellow). Then, 100 μL of 1% starch solution was added to
the polymer solution that changed to blue. This solution was titrated
with 0.5 mN sodium thiosulfate solution (the color of the solution
changed from blue to colorless). The Cl+ content of the polymer was
calculated as follows

35.45 N V
2 m

10Cl (ppm) 6= × ×
×

×+

where Cl+ (ppm) is the weight percentage of the oxidative chlorine in
the polymer, N (equiv/L) is the normality, V (L) is the volume of the
titrant sodium thiosulfate, and m (g) is the weight of the polymer.
2.7. Electrospinning Process. Electrospinning solutions were

prepared by dissolving the polymeric blends [PLA/P(DMHI) or
P(DMHICl-Q)] in a 90/10 v/v mixture of CHCl3/DMF at a
concentration of 18% w/v. Electrospun polymeric fibers were
prepared from these solutions using a homemade electrospinner in
a horizontal configuration equipped with a syringe needle connected
to a high voltage power. The polymer solutions were fed at 1 mL h−1.
The electrospun fiber mats were collected in a grounded aluminum
foil collector located perpendicular at 12 cm from the needle tip by
applying a voltage of 16 kV. Those conditions were selected from the
results obtained in our previous works.32,33 The obtained electrospun
samples, PLA/P(DMHI) and PLA/(DMHICl-Q), were dried for 48 h
under a vacuum to remove any potential residual solvent before their
use.
2.8. N-Alkylation and Chlorination of the Fibers. Prepara-

tion of Antimicrobial Fiber Mats. PLA/P(DMHI) fiber mats were
subjected to N-alkylation and chlorination to obtain antimicrobial
functional fibers containing triazolium and halamine groups at the
surface. First, the PLA/P(DMHI) electrospun mat was cut into
several pieces (1 × 1 cm2), and each of them was incubated in 1 mL
of methanol. Then, a large excess of methyl iodide (200 μL) was
added. The N-alkylation reaction was left for 10 days with constant
shaking at 37 °C to ensure a complete reaction. After that period, the
mats were washed with methanol several times to remove any residual
reagent to afford PLA/P(DMHI-Q) fibers. Surface charge determi-
nation was performed following a method previously described in the
literature.34,35 A Mat sample of 1 × 1 cm2 (2 cm2 surface area) was
placed in 10 mL of 1 wt % aqueous sodium fluorescein solution for 10
min. After that, the sample was rinsed extensively with distilled water
and sonicated to remove residual fluorescein. Then, the fluorescein
was desorbed from the surface of the sample by treating the mat with
3 mL of 0.1 wt % CTAC solution for 20 min with shaking at 300 rpm.
Subsequently, the amount of fluorescein obtained in the supernatant
was determined by UV−vis spectroscopy (Lamda 35, PerkinElmer) in
a solution prepared by adding 10% v/v of 100 mM phosphate buffer
(pH 8.0). The absorbance of the resulting solution was measured at
501 nm, and the concentration of fluorescein was calculated with an
extinction coefficient36 of 77 M−1 cm−1 assuming a relatioship of 1:1
for fluorescein to each accessible cationic triazolium group.
Next, the chlorination reaction of PLA/P(DMHI-Q) fibers was

carried out to form N-halamine functional groups at the surface of the
cationic fibers. For this reaction, the pieces of fibers obtained after
alkylation were immersed in a diluted bleach solution (10% v/v) at
room temperature for 2 h. Then, the samples were rinsed with a
copious amount of water to reach pH 7 and dried to afford PLA/
P(DMHICl-Q) fibers. The chlorine amount in the functionalized
fibers was determined by the iodometric/thiosulfate titration method
as described above31 using mat pieces of 1 × 1 cm2 (∼2.7 mg).

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2.9. Characterization. NMR spectra were recorded on a Bruker
Avance III HD-400AVIII spectrometer at room temperature using
solvents CDCl3 and DMSO-d6. FTIR spectra were obtained with a
PerkinElmer Spectrum Two instrument equipped with an attenuated
total reflection module. Size exclusion chromatography measurements
were performed on a Waters Division Millipore system equipped with
a Waters 2414 refractive index detector. DMF stabilized with 0.1 M
LiBr (Sigma-Aldrich, >99.9%) was used as eluent at a flow rate of 1
mL min−1 at 50 °C. The calibration was made with poly(methyl
methacrylate) standards (Polymer Laboratories LTD). The morphol-
ogy of electrospun fibers of PLA and PLA/itaconate polymers was
studied using a scanning electron microscope (SEM) Philips XL30
with an acceleration voltage of 25−10 kV. The samples were coated
with gold prior to scanning. SEM images were analyzed using NIH
ImageJ software and measuring at least 100 fibers of each sample from
different SEM images. Microhardness (MH) of the electrospun fibers
was measured with a Vickers indentor attached to a Leitz
microhardness tester. A contact load of 0.96 N and a time of 25 s
were employed.
2.10. Antibacterial Assays. The antibacterial activity of the

PLA/P(DMHICl-Q) fibers was measured following the E2149-13a
standard method from the American Society for Testing and Materials
(ASTM)37 against P. aeruginosa, E. coli, S. epidermidis, and S. aureus.
First, bacterial cells were cultured on 5% sheep blood Columbia agar
plates for 24 h at 37 °C. Then, the bacterial suspensions were
prepared in saline using the McFarland turbidity scale and further
diluted to 106 colony-forming units (cfu) mL−1 with PBS. Next, PLA/
P(DMHICl-Q) fiber mats (1 × 1 cm2) were placed in sterile falcon
tubes containing 1 mL of the tested inoculum and 9 mL of PBS to
reach a working solution of ∼105 cfu mL−1. Control experiments were
also performed in the presence of PLA fiber, and also in the absence
of mats. The suspensions were shaken at 120 rpm for 30 min or 24 h.
After this period, the colonies were counted by the plate counting

method, and the reduction percentage was calculated in comparison
to the control. The measurements were made at least in triplicate.
2.11. Disintegrability under Composting Conditions Test.

The disintegrability test under simulated composting conditions of
the prepared PLA and PLA/P(DMHICl-Q) fiber mats was performed
at the laboratory scale level following the ISO 20200 standard.38 The
synthetic wet compost was prepared by mixing 45 wt % of solid
synthetic wet waste [10% of compost (Compo, Spain), 30% rabbit
food, 10% starch, 5% sugar, 4% corn oil, 1% urea, and 40% sawdust]
and 55 wt % of water contained in a perforated plastic box as
composter reactors. Then, several pieces of mats from each sample
(previously weighted) were placed in textile meshes buried in the
compost and subjected to an aerobic disintegration process at 58 °C.
Samples were withdrawn periodically, cleaned with distilled water,
dried in an oven at 37 °C under a vacuum for 24 h, and reweighed.
The disintegration degree was calculated by normalizing the sample
weight, on different days of composting, to the initial weight. The
samples were characterized by SEM and visual inspection.

3. RESULTS AND DISCUSSION
3.1. Synthesis and Characterization of the P(DMHICl-

Q) Antibacterial Polymer. In previous studies, biobased and
biodegradable polymers derived from itaconic acid containing
azolium antibacterial groups were prepared from a clickable
polyitaconate developed by our group.23 These biopolymers
bearing cationic thiazolium and triazolium groups demon-
strated excellent antibacterial activity against Gram-positive
bacteria and negligible toxicity to human cells but were poorly
active against Gram-negative bacteria. Based on our previous
finding, herein, we design and synthesize a multifunctional
antibacterial polyitaconate bearing azolium and N-halamine

Figure 1. Synthesis route of the antibacterial biobased polymer P(DMHICl-Q).

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groups to achieve systems with a broad antimicrobial spectrum.
Figure 1 displays the synthetic route followed for the
preparation of the antibacterial polymer P(DMHICl-Q).
Clickable polymer derived from itaconic acid, P(PrI), was
employed as a platform to prepare multifunctional polymers by
azide−alkyne coupling reactions (CuAAC). For this purpose,
Boc-protected 5,5-dimethylhydantoin functionalized with azide
group (Boc-DMH-N3) was incorporated onto the polymer via
CuAAC click chemistry, leading to triazole linkages. Sub-
sequently, N-alkylating reaction of the triazole groups with
methyl iodide provides the corresponding cationic itaconate
derivatives. Each of these steps consisted of very efficient
reactions and proceeded with high yields and a high degree of
conversion of functional groups, as confirmed by FTIR and 1H
NMR (see Supporting Information). After the N-Boc
deprotection step, the chlorination reaction changes the
amide group of hydantoin into N-halamines.
Figure 2 shows the FTIR spectra of the P(DMHICl-Q) and

their precursors. In the spectrum of P(PrI), the bands at 3283

and 2128 cm−1 assigned to the alkyne C−H and C�C
stretching vibrations, respectively, are clearly observed. After
the click reaction of P(PrI) with the protected 3-(2-
azidoethyl)-5,5-dimethylhydantoin (Boc-DMH-N3), new
bands appear in the spectrum. It can be seen the band at
1810 cm−1 is associated with the ν (C�O) of the Boc group
and the band at 1734 cm−1 due to the imide and ester C�O
stretching vibrations. Subsequently, N-alkylation and depro-
tection reactions provide the cationic polymer P(DMHI-Q), in
whose spectrum the bands assigned to the Boc group disappear
and the vibrational band of the imide carbonyl bond is shifted
to 1709 cm−1. Also, a new band at 1581 cm−1 corresponding to
the ν(C�N+) clearly emerges, due to the formation of
triazolium groups. The last chlorination step provides a shift of
the imide carbonyl group to 1718 cm−1 as a result of the
electron-withdrawing effect of the oxidative chlorine.39

It should be mentioned that synthetic t-butyl hypochlorite
was used for chlorination instead of commercial bleach
because, in this way, the polymer can be easily purified, as
the excess of t-butyl hypochlorite is removed under vacuum.40

The content of oxidative chlorine in the polymer was
iodometrically titrated and resulted in 7134 ppm for the

tested polymer solution. This value corresponds to a degree of
chlorination of 9%.
3.2. Preparation of Antimicrobial Electrospun Fibers

based on PLA/P(DMHICl-Q). Next, the multifunctional
biobased polymer P(DMHICl-Q) was employed to impart
antibacterial activity to PLA electrospun fibers. First, the
preparation of antimicrobial fibers by electrospinning techni-
que was attempted from CHCl3/DMF solutions of PLA/
P(DMHICl-Q) polymeric blends at a 90/10 ratio. However,
the antibacterial polymer P(DMHICl-Q) containing cationic
and halamine groups was not soluble in the solvent mixture,
and its incorporation as a component of the fibers by the
electrospinning process would conduct to maldistribution,
aggregation, or leakage problems. It is well known that
electrospinning of multiple components using one nozzle is
critical and requires careful optimization of the systems.
Indeed, this cationic polymer with halamine groups was not
soluble in any solvent compatible with PLA or with the
electrospinning process and therefore can hardly be electro-
spun in a direct way. For this reason, we follow an alternative
approach consisting of the use of neutrally charged polymer
resulting from the deprotection of P(Boc-DMHI). The
P(DMHI) polymer was incorporated into the CHCl3/DMF
solvent mixture at 10 wt % together with PLA 90 wt %, at a
total polymer concentration of 18 wt %. Then, PLA-based
fibers loaded with the polymer P(DMHI), bearing triazole and
hydantoin groups, were prepared by electrospinning leading to
antimicrobial precursor fibers. SEM images, shown in Figure 3,
illustrate the morphology of the electrospun fiber mats
compared to PLA fibers obtained under similar conditions. It
is clearly observed that the incorporation of the P(DMHI)
polymer affects the morphology of the fibers as it could modify
the viscosity and conductivity of the solution, resulting in
electrospun fibers with nonuniform size and shape. While the
morphology of the electrospun PLA fibers was smooth,
uniform, and bead-free, with a diameter of 4.2 ± 0.9 μm, the
fiber mats loaded with P(DMHI) are nonuniform in size, with
a lower average diameter of 2.3 ± 1.5 μm. Nevertheless,
reasonable fiber mats can be obtained by this approach.
The fibers loaded with P(DMHI) polymer precursor were

subsequently surface functionalized to provide antimicrobial
activity. For this purpose, the N-alkylation reaction of the
triazole groups was carried out with iodomethane to afford the
corresponding cationic triazolium groups [P(DMHI-Q)],
followed by chlorination of the hydantoin groups with bleach
to provide N-halamine functionalities [P(DMHICl-Q)]. In this
case, bleach was employed for the chlorination reaction
because the washing steps of the fibers can be easily performed
with water. The successful quaternization reaction on the
surface was confirmed by measuring the accessible cationic
units able to bind fluorescein. In this experiment, the
fluorescein adsorbed on the surface, which corresponds to
accessible positive surface charges, was desorbed with CTAC
and quantified by UV−vis spectroscopy. The estimated
cationic groups were found to be 1.4 × 1015 N+/cm2, a value
near that previously determined for other contact-active
surfaces.34,41 Likewise, the active chlorine content of the
fiber mat (1 × 1 cm2, 2.7 mg), determined via the iodometric
titration method, was calculated to be 1654 ppm, an amount
enough, in principle, to impart activity to the surface, as has
been demonstrated in other N-halamine-functionalized surfa-
ces.31 These experiments demonstrated the successful

Figure 2. FTIR spectra of the antibacterial biobased polymer
P(DMHICl-Q) and its precursors P(PrI), P(Boc-DMHI), and
P(DMHI-Q).

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functionalization of the fibers with both antimicrobial active
groups.
3.3. Mechanical Properties. Microhardness measure-

ments were carried out on the obtained PLA and PLA/
P(DMHICl-Q) fiber mats to evaluate how the incorporation of
the polyitaconate derivative and the posterior modification
affects the mechanical behavior of PLA material. PLA fiber
mats exhibit an MH value of 154 ± 5 MPa, near other values
found in the literature for PLA materials;42−44 however, in
PLA/P(DMHICl-Q) fibers, the MH increases to 171 ± 6
MPa. As expected, the addition of a glassy polymer such as this
polyitaconate derivative results in a slight increase in the
hardness of the materials, with values similar to PLA-based
composites materials obtained by the incorporation of
reinforcing fillers.43,44 It is also worth noting that the fiber
diameter decreases with the incorporation of P(DMHICl-Q),
which could also contribute to the differences in the
mechanical properties.
3.4. Antibacterial Efficacy. Next, the antibacterial activity

of the chlorinated and quaternized fiber mats, PLA/
P(DMHICl-Q), was evaluated against Gram-positive, S.
epidermidis and S. aureus, and Gram-negative bacteria, P.

aeruginosa and E. coli, and compared with the quaternized
fibers before chlorination, PLA/P(DMHI-Q), to analyze the
combined action of both functionalities. In the antibacterial
test, fiber mats of 1 × 1 cm2 were inoculated in 10 mL of
bacterial suspension (106 cfus). Controls of PLA fibers alone
and inoculum without fibers were also tested. The control
samples did not provide any noticeable reduction of bacteria
after 24 h, whereas the fibers containing the antibacterial
polyitaconate provoke great reduction (see Figure 2 of
Supporting Information). Table 1 summarizes the percentage
reduction of bacterial viable counts related to controls upon
contact with antibacterial fibers after 30 min or 24 h. The
results indicate that the fibers functionalized with cationic
triazolium groups after 30 min of contact time are only
effective against Gram-positive bacteria, causing 0.59 and 0.75-
Log reductions of S. epidermidis and S. aureus, respectively.
These films need a large contact time to achieve high bacterial
reduction, 5-Log reductions, as described for other anti-
bacterial systems based on cationic polymers acting through a
contact kill mechanism, in which prolonged contacts are
necessary to affect bacteria that are not in contact with the
surface.45 This antibacterial efficacy is considerably reduced

Figure 3. SEM micrographs and their corresponding histograms of fiber diameters of (a) PLA and (b) PLA/P(DMHI) fiber mats.

Table 1. Antibacterial Activity Expressed as the Percentage of Bacterial Reduction (%) and Log Reduction (Log) of PLA/
P(DMHI-Q) and PLA/P(DMHICl-Q) Against, S. epidermidis, S. aureus, P. aeruginosa, and E. coli After 30 min or 24 h of
Contact Time

S. epidermidis S. aureus P. aeruginosa E. coli

% Log % Log % Log % Log

PLA/P(DMHI-Q) (30 min) 75 ± 4 0.6 ± 0.1 82 ± 0.6 0.8 ± 0.1
PLA/P(DMHI-Q) (24 h min) 99.999 ± 0.001 5.0 ± 0.3 99.999 ± 0.001 5.0 ± 0.1 88 ± 3 0.9 ± 0.1
PLA/P(DMHICl-Q) (30 min) 99.999 ± 0.001 5.0 ± 0.2 99.999 ± 0.001 5.0 ± 0.1 90 ± 1 1.0 ± 0.2 99.0 ± 0.5 2.0 ± 0.2
PLA/P(DMHICl-Q) (24 h) 99.999 ± 0.001 5.0 ± 0.3 99.999 ± 0.001 5.0 ± 0.2 93.2 ± 0.6 1.2 ± 0.2 98.9 ± 0.6 2.0 ± 0.1

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against P. aeruginosa, a Gram-negative bacteria, by 0.90-Log,
whereas no effect on E. coli was observed after 24 h of contact
time. Remarkably, the chlorination of the fibers considerably
improves their activity for both Gram-positive and Gram-
negative bacteria. Against Gram-positive bacteria, the chlori-
nated fibers yield 5-Log reduction after only 30 min of contact,
whereas they cause 1.01-Log reduction of P. aeruginosa and 2-
Log reduction of E. coli. The effectivity of these chlorinated
PLA/P(DMHICl-Q) fibers practically reaches the highest
results after the shortest time tested, 30 min.
Thus, the incorporation of N-halamine with cationic

triazolium groups demonstrates to be a good approach to
achieving a broadened antibacterial spectrum of the fibers,
improving the activity against both Gram-positive and Gram-
negative bacteria.
3.5. Disintegrability. To study the compostability of the

antimicrobial polymeric fibers, a disintegration test was

conducted under simulated industrial composting conditions
at 58 °C according to the ISO 20200 standard.38 A qualitative
evaluation of the physical disintegration of the fiber mats as a
function of composting time was performed by taking
photographs and by SEM measurements (Figure 4),
confirming the biodisintegrable character of PLA and PLA-
loaded mats in less than 90 days.
From the visual appearance of the samples, it is noticeable

that the PLA/P(DMHICl-Q) sample turns yellowish on day 7,
whereas the coloration becomes evident in PLA on day 11.
This change in color may be due to hydrolytic degradation,46,47

in addition to the presence of sawdust. After 11 days under
composting conditions, both samples exhibited considerable
surface deformation and fractures, due to physical and/or
chemical degradation of the polymers that cause a loss of
flexibility. The PLA/P(DMHICl-Q) sample starts to turn dark
brown, suggesting that the disintegration of this sample is

Figure 4. Visual appearance and SEM images of the tested PLA and PLA/P(DMHICl-Q) fiber mats over time under composting conditions.

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faster than the degradation of the PLA mat. From day 25, only
small pieces of mats were recovered, mostly smaller than 2 mm
in size. At this point it is difficult to separate the polymeric
sample from compost particles and, according to the ISO
20200 standard, pieces smaller than 2 mm should be discarded.
Finally, after 42 days, samples reached a level of disintegration
where no visible polymeric fragments could be easily
recovered. Nevertheless, SEM images of the compost particles
on day 42 reveal the presence, adhered on the surface, of small
pieces of polymeric fibers in the range of micrometric scale (in
diameter and length), demonstrating the existence of micro-
plastic particles when apparently the fiber mats are completely
disintegrated. Indeed, from SEM micrographs of the mats
obtained at different composting times, a considerable fracture
of fibers leading to a reduction of the length scale to micron
can be appreciated. Figure 5a showed magnified SEM images
of the PLA and PLA/P(DMHICl-Q) fibers taken at large
incubation composting period, 42 days, in which is appreciated
fiber with lengths smaller than 10 μm. Also, during the
degradation process, a reduction in the fiber diameter is
observed. Figure 5b displays the average fiber diameter as a
function of composting time. In both samples, PLA and PLA/
P(DMHICl-Q), the fiber diameter diminishes with the
incubation periods, as previously observed in PLA fiber mats
biodegraded under simulated physiological conditions.48 From
SEM images of the samples obtained at different incubation
times, as seen from visual observation, it seems that the
disintegration process is a bit faster in PLA fibers loaded with
the antimicrobial polymer P(DMHICl-Q), probably due to the
smaller fiber diameter and, therefore, its larger surface area.
The disintegration degree was also analyzed in terms of mass

loss as a function of disintegration time (Figure 6). The
process seems to be slightly accelerated in the PLA/

P(DMHICl-Q) samples, as previously observed in photo-
graphs and SEM micrographs. However, statistical analysis
using one-way ANOVA followed by Tukey’s test (p < 0.05)
indicates the degradation profile of PLA and PLA/P(DMHICl-
Q) fibers are not significantly different.

4. CONCLUSIONS
In this study, antibacterial and compostable PLA-based fibers
were successfully fabricated by incorporating a multifunctional
biobased polymer derived from itaconic acid bearing N-
halamine and triazolium antibacterial groups, P(DMHICl-Q).
In the first attempt, the antibacterial polymer was synthesized
by the attachment of both functionalities onto a polyitaconate
derivative following efficient and straightforward approaches.

Figure 5. (a) SEM image of PLA and PLA/P(DMHICl-Q) fibers taken after 42 days of composting incubation. (b) Average fiber diameter of PLA
and PLA/P(DMHICl-Q) fiber mats over time under composting conditions.

Figure 6. Disintegration degree of PLA and PLA/P(DMHICl-Q)
fiber mats over time under composting conditions.

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However, the polymer exhibits poor solubility in common
solvents used in the electrospinning process of PLA, which
makes the preparation of homogeneous fibers very difficult. As
an alternative, a precursor of such a polymer obtained before
N-alkylation and chlorination, P(DMHI), was employed and
added directly to the electrospinning PLA solution. Sub-
sequent surface functionalization of the accessible triazole and
hydantoin groups provides PLA-based fibers with the ability to
efficiently inactivate Gram-positive and Gram-negative bac-
teria. It was demonstrated that the presence of cationic
triazolium groups at the surface only provides high efficacy
against Gram-positive bacteria, while their combination with
N-halamine groups extends the effectiveness also to Gram-
negative bacteria. Remarkably, due to the small diameter, and
therefore high surface area, a minor amount of fibers loaded
with antimicrobial is needed for pathogen inactivation. The
compostability of the antibacterial electrospun fibers was also
tested under simulated industrial composting conditions. It
was observed that the incorporation of the antimicrobial
biobased polymer apparently did not compromise PLA
disintegration under aerobic composting conditions. However,
a deep analysis of the compost samples proves the presence of
microplastics in both samples (PLA and antimicrobial loaded
PLA) after the biodisintegration test following the ISO 20200
standard protocol. Then, more studies are needed in this
regard. In addition, these multifunctional biobased polymeric
derivatives can be employed as an additive or component not
only in electrospun fibers but could also be used in the
preparation of films or other devices by different processing
methods such as melt extrusion and additive manufacturing in
which the solubility problem is avoided.

■ ASSOCIATED CONTENT
*sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsapm.2c00928.

1H NMR spectra of the P(Boc-DMHI) and P(DMHI-
Q) polyitaconate derivatives and photographs of the
agar plates of E. coli (Gram-negative bacteria) and S.
epidermidis (Gram-positive bacteria) bacterial colonies
after spreading inoculum (at different dilutions) in the
previous contact with fibers (PDF)

■ AUTHOR INFORMATION
Corresponding Authors

C. Echeverría − Instituto de Ciencia y Tecnología de Polímeros
(ICTP-CSIC), 28006 Madrid, Spain; Interdisciplinary
Platform for Sustainable Plastics Towards a Circular
Economy-Spanish National Research Council (SusPlast-
CSIC), 28006 Madrid, Spain; orcid.org/0000-0002-
0734-766X; Email: cecheverria@ictp.csic.es

A. Muñoz-Bonilla − Instituto de Ciencia y Tecnología de
Polímeros (ICTP-CSIC), 28006 Madrid, Spain;
Interdisciplinary Platform for Sustainable Plastics Towards a
Circular Economy-Spanish National Research Council
(SusPlast-CSIC), 28006 Madrid, Spain; orcid.org/0000-
0003-0210-1246; Email: sbonilla@ictp.csic.es

Authors
A. Chiloeches − Instituto de Ciencia y Tecnología de
Polímeros (ICTP-CSIC), 28006 Madrid, Spain; Escuela

Internacional de Doctorado de la Universidad Nacional de
Educación a Distancia (UNED), 28015 Madrid, Spain

R. Cuervo-Rodríguez − Facultad de Ciencias Químicas,
Universidad Complutense de Madrid, 28040 Madrid, Spain

Y. Gil-Romero − Hospital Universitario de Móstoles, 28935
Madrid, Spain

M. Fernández-García − Instituto de Ciencia y Tecnología de
Polímeros (ICTP-CSIC), 28006 Madrid, Spain;
Interdisciplinary Platform for Sustainable Plastics Towards a
Circular Economy-Spanish National Research Council
(SusPlast-CSIC), 28006 Madrid, Spain; orcid.org/0000-
0003-2061-0351

Complete contact information is available at:
https://pubs.acs.org/10.1021/acsapm.2c00928

Notes
The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS
This work was funded by the MICINN (PID2019-104600RB-
I00), Agencia Estatal de Investigación (AEI, Spain), Fondo
Europeo de Desarrollo Regional (FEDER, EU), and CSIC
(LINKA20364). A.C. acknowledges MICIU for his FPU
fellowship FPU18/01776.

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