molecules

Article
Bud-Poplar-Extract-Embedded Chitosan Films as
Multifunctional Wound Healing Dressing
Carla Russo 1,† , Miranda Piccioni 2,† , Maria Laura Lorenzini 3 , Chiara Catalano 2 , Valeria Ambrogi 3 ,
Rita Pagiotti 2 and Donatella Pietrella 1, *

1 Medical Microbiology Unit, Department of Medicine and Surgery, University of Perugia, Piazzale Sereni,
Building D, 4th Floor, 06129 Perugia, Italy
2 Biochemical Sciences and Health Unit, Department of Pharmaceutical Sciences, University of Perugia,
Via del Giochetto, 06122 Perugia, Italy
3 Pharmaceutical Technology Unit, Department of Pharmaceutical Sciences, University of Perugia,
Via del Liceo 1, 06123 Perugia, Italy
* Correspondence: [email protected]
† These authors equally contributed to this work.

Abstract: Wounds represent a major global health challenge. Acute and chronic wounds are sensitive
to bacterial infection. The wound environment facilitates the development of microbial biofilms,
delays healing, and promotes chronic inflammation processes. The aim of the present work is the
development of chitosan films embedded with bud poplar extract (BPE) to be used as wound dressing
for avoiding biofilm formation and healing delay. Chitosan is a polymer with antimicrobial and
hydrating properties used in wound dressing, while BPE has antibacterial, antioxidative, and anti-
inflammatory properties. Chitosan-BPE films showed good antimicrobial and antibiofilm properties
against Gram-positive bacteria and the yeast Candida albicans. BPE extract induced an immunomod-
ulatory effect on human macrophages, increasing CD36 expression and TGFβ production during
Citation: Russo, C.; Piccioni, M.;
Lorenzini, M.L.; Catalano, C.; M1/M2 polarization, as observed by means of cytofluorimetric analysis and ELISA assay. Significant
Ambrogi, V.; Pagiotti, R.; Pietrella, D. antioxidant activity was revealed in a cell-free test and in a human neutrophil assay. Moreover,
Bud-Poplar-Extract-Embedded the chitosan-BPE films induced a good regenerative effect in human fibroblasts by in vitro cell mi-
Chitosan Films as Multifunctional gration assay. Our results suggest that chitosan-BPE films could be considered a valid plant-based
Wound Healing Dressing. Molecules antimicrobial material for advanced dressings focused on the acceleration of wound repair.
2022, 27, 7757. https://doi.org/
10.3390/molecules27227757 Keywords: bud poplar resin extract; chitosan; wound healing; antibiofilm activity; antioxidant
Academic Editors: Silvia Fialová and activity; anti-inflammatory activity
Pavel Mučaji

Received: 2 September 2022

Accepted: 7 November 2022
1. Introduction
Published: 10 November 2022
Skin is the largest organ of the body. It protects from external insults such as ultraviolet
Publisher’s Note: MDPI stays neutral radiation, toxic chemical compounds, and microorganisms. A wound is defined as the
with regard to jurisdictional claims in interruption of the anatomic skin tissue; it may be classified as acute or chronic depending
published maps and institutional affil-
on the healing time. While an acute wound may heal completely within 8–12 weeks,
iations.
chronic wounds usually take longer periods (more than 12 weeks) with a high possibility
of recurrence. Chronic wounds represent a major global health challenge. Diabetic foot
ulcers are among the most common complications of diabetes mellitus, with a lifetime
Copyright: © 2022 by the authors.
incidence of up to 15% among the diabetic population [1]. In 2017, 425 million people
Licensee MDPI, Basel, Switzerland.
worldwide, or 8.8% of adults aged 20–79 years, were estimated to have diabetes. If the
This article is an open access article
current trend continues, by 2045, 629 million people aged 20–79 years will have diabetes.
distributed under the terms and Venous insufficiency and peripheral neuropathy lead to loss of sensitivity in the feet, which
conditions of the Creative Commons degenerates into chronically infected lesions known as diabetic foot ulcers. Moreover,
Attribution (CC BY) license (https:// pressure ulcers are a serious problem for all bed-bound and chair-bound patients. Wound
creativecommons.org/licenses/by/ infections may also occur in burn victims, patients with traumatic wounds, and patients
4.0/). with surgical wounds. Both acute and chronic wounds are sensitive to bacterial infection,

Molecules 2022, 27, 7757. https://doi.org/10.3390/molecules27227757 https://www.mdpi.com/journal/molecules

Molecules 2022, 27, 7757 2 of 21

which can also elicit a systemic response. This leads to the retardation or inhibition of
the healing process. The wound environment facilitates the development of microbial
biofilms. Biofilms are found on the surface of the skin, and there is much evidence to
suggest their involvement in the delay of wound healing and the chronic inflammation
process [2]. The prevention of biofilm formation is the goal of wound treatment because
the standard protocols based on topical and systemic administration are often unable to
remove biofilms [3].
Wound dressings are designed to protect the wound from the external environment
and to absorb wound exudate during the healing process. Many types of materials have
been utilized to develop wound dressings and been commercialized in the market. Bio-
derived polymers such as polysaccharides have been tested in wound dressing develop-
ment; they are biocompatible, nonimmunogenic, and have anti-microbial properties [4].
Chitosan is a biocompatible and biodegradable polymer with antimicrobial and hy-
drating properties [5], obtained from the partial deacetylation of the natural polymer chitin.
Chitosan has been approved for wound dressing applications. Chitosan nanoparticles
show a positive surface charge and mucoadhesive properties that allow their adherence to
mucus membranes and the release of the drug payload in a sustained release manner [6].
Chitosan-based dressings offer several advantages over traditional materials employed in
the wound healing process [7]. Chitosan-based dressing materials are considered suitable
for clinically problematic wounds, as they facilitate hemostasis, enhance wound healing,
and have antimicrobial properties. Moreover, chitosan showed low toxicity and immune-
stimulatory activity [8]. Chitosan displays excellent film-forming capability and finds many
applications in oral mucosal, buccal, transdermal, sublingual, and periodontal delivery
systems as a carrier for drugs and bioactive agents ranging from small molecule-like antibi-
otics, nucleic acid, and proteins [9]. The incorporation of bioactive components in wound
dressings could improve wound dressing performance [10]. Propolis is one of the biocidal
molecules used with chitosan. Moreover, chitosan is similar to glycosaminoglycans with
similar morphological and mechanical properties to collagen; these features make it an
excellent alternative for tissue engineering applications.
Bees use propolis to keep a low concentration of bacteria and fungi in the hive. Thus,
the action against microorganisms is an essential characteristic of propolis exploited by
man since ancient times. It is still one of the remedies in the Balkan states for the treatment
of wounds and burns [11]. Propolis is widely used in traditional medicine for its antiseptic,
antibacterial, antioxidative, anti-inflammatory, and antitumor properties [12–15].
A twenty percent extract of Malaysian propolis in chitosan nanoparticles showed
an antibiofilm effect against Enterococcus faecalis. The chitosan–propolis nanoformulation
downregulated the expression of cytolysin and biofilm-forming genes, rendering the bacte-
ria susceptible to treatment, suggesting that the nano formulation is an ideal antibiofilm
agent [16]. Moreover, propolis promotes skin wound healing by stimulating epithelial
re-generation, modulating extracellular matrix deposition, and facilitating the formation of
granulation tissue [17].
The antimicrobial components of propolis are different in distinct geographic regions.
For example, in European samples, flavonoids and cinnamic acid derivatives have been
observed, and in Brazilian propolis, diterpenic acids and prenylated coumaric acids have
been identified [11]. The composition of propolis is chemically related to that of the bud
exudates from which it derives. In temperate climates, bees are thought to collect propolis
mainly from Populus but also from Betula (birch), Salix (willow), Alnus (alder), and Aesculus
(horse chestnut) [18]. Bud exudates of Populus species (cottonwood, poplar, aspen) are
the main propolis plant sources. Poplar buds are coated with a viscous substance, an
exudate, which has been reported to contain phenolic compounds (terpenoids, flavonoid
aglycons and their chalcones) and phenolic acids and their esters [19]. Poplar buds have
been used as a remedy in traditional medicine. Leaf buds were used as an expectorant and
in the treatment of dermatitis, while leaves and bark have demonstrated antirheumatic
properties. The leaf buds of Populus nigra, P. canadensis, and P. balsamifera have shown
 (terpenoids, flavonoid aglycons and their chalcones) and phenolic acids and their esters
[19]. Poplar buds have been used as a remedy in traditional medicine. Leaf buds were
Molecules 2022, 27, 7757 used as an expectorant and in the treatment of dermatitis, while leaves and bark have 3 of 21
demonstrated antirheumatic properties. The leaf buds of Populus nigra, P. canadensis, and
P. balsamifera have shown antioxidant properties [20]. Moreover, Wang et al. showed that
P. canadensis bud
antioxidant extracts
properties have
[20]. significant
Moreover, anti-inflammatory
Wang effects
et al. showed that by inhibiting
P. canadensis budIL-6, IL-
extracts
10, MCP-1, and TNF-α secretion and blocking NF-κB activation [21].
have significant anti-inflammatory effects by inhibiting IL-6, IL-10, MCP-1, and TNF-α
This and
secretion workblocking
aims toNF-κB
develop eco-friendly
activation [21]. films based on chitosan-containing bud
poplar resin
This extracts
work aims to
to be used as
develop a wound dressing.
eco-friendly films based on chitosan-containing bud poplar
resin extracts to be used as a wound dressing.
2. Results
2. Results
In our recently published work, we have characterized and analyzed the extract of
poplar Inbuds
our recently published
that showed work, anti-inflammatory,
antioxidant, we have characterized and and analyzed
antibiofilm the extract
activity of
against
poplar
the buds that showed
Gram-negative antioxidant,
bacterium anti-inflammatory,
Pseudomonas and antibiofilm
aeruginosa [13]. activity
In the present against
work, the
the Gram-negative
same bacterium Pseudomonas
extract was incorporated into chitosan aeruginosa [13]. were
films. The films In thefirst
present work, the same
characterized, and
extract was incorporated into chitosan films. The films were first characterized,
then their antimicrobial, antibiofilm, antioxidant, anti-inflammatory, and regenerative and then
their antimicrobial, antibiofilm,
properties were determined. antioxidant, anti-inflammatory, and regenerative properties
were determined.
2.1. Characterization of Chitosan–Bud Poplar Resin (CBPR) Films
2.1. Characterization of Chitosan–Bud Poplar Resin (CBPR) Films
The photographic images of the prepared films are reported in Figure 1. As can be
The photographic
observed, chitosan film images of the prepared
was transparent films are
and clearer thanreported
the otherin Figure 1. As can
films, which werebe
observed, chitosan film was transparent and clearer than the other films, which were
gradually darker at the higher end of the bud poplar extract concentration. A good
gradually darker at the higher end of the bud poplar extract concentration. A good
dispersion of the extract was obtained up to 1% concentration, and at 2%, aggregates of
dispersion of the extract was obtained up to 1% concentration, and at 2%, aggregates of the
the extract were observed. Chitosan film thickness was 60 ± 10 μm, and it was not affected
extract were observed. Chitosan film thickness was 60 ± 10 µm, and it was not affected by
by the addition of the bud poplar extract.
the addition of the bud poplar extract.

Figure 1. Photographs of the prepared films.

Figure 1. Photographs of the prepared films.
The ATR FT-IR spectra of the CBPR 2%, chosen as a model film, the plain chitosan
film,The andATRthe FT-IR spectraextract
bud poplar of the are
CBPR 2%, chosen
reported as a model
in Figure 2. Chitosanfilm, thefilmplain chitosan
showed the
film, and the bud poplar extract are reported in Figure 2.
typical polymer absorptions bands. The large band at 3270 cm is associated withChitosan − 1film showed the typical
-OH
polymer
and NHabsorptions bands. The large band at 3270 cm−1 is associated −1 with -OH and
cm−NH 1 are2
2 stretching vibrations, the absorption peaks at 1634 cm and 1554
stretching vibrations, the absorption peaks at 1634 cm −1 and 1554 cm−1 are assigned to the
assigned to the NHCO (amide I) and -NH bending (amide II), respectively, and finally
NHCO (amide I)
the absorption and at
bands -NH2922bending
cm−1 and (amide
2869II),
cmrespectively,
−1 are relative andto CHfinally the absorption
2 bending [22]. The
bands at 2922 cm −1 and 2869 cm−1 are relative to CH2 bending [22]. The bud poplar extract
bud poplar extract spectrum shows the typical hydrogen-bonded OH stretch of phenolic
spectrum
compounds shows the typical
at 3336 hydrogen-bonded
cm−1 . Other bands can beOH stretchatof2917
observed phenolic
cm−1compounds
and 2849 cmat−13336 due
cm −1
to the. Other bands can
asymmetric andbe observedstretching
symmetric at 2917 cmof CH
−1 and2, 2849 cmcmdue, to
at 1157 −1 − 1 the asymmetric
attributable to C-Oandand
symmetric
C-OH vibration,stretching
at 1448 cm−
of CH atdue
2, 1 1157tocm
C-H vibration, 1512 cm−and
−1, attributable to C-O C-OH
1 , 1604 cm−vibration,
1 , and 1630 atcm
1448−1 ,
cm −1 due to to
attributed C-H vibration,
aromatic 1512
ring cm , 1604 cmand
−1
deformations, −1 , and 1630 cm
at 1681 cm−,1attributed
−1
due to C=O to aromatic
stretching ringof
deformations, and at 1681
flavonoids and lipids [23,24]. cm −1 due to C=O stretching of flavonoids and lipids [23,24].

The
Thechitosan
chitosan andand CBPR 2% films’ films' spectra
spectra had
hadalmost
almostthe thesame
samepatterns,
patterns,and andnonoshifts
shiftsof
of chitosan-typical
chitosan-typical absorption
absorption bandsbandscould could be observed,
be observed, indicating indicating
the absence theof absence
interactionsof
interactions
between propolis between andpropolis
chitosan.and chitosan.
In CBPR 2%, In CBPR 2%,
spectrum spectrum
absorption absorption
bands of propolisbands of
could
propolis could not
not be observed, be observed,
possibly due to the possibly due to the low
low concentration concentration
of bud poplar extract of in
budthepoplar
film.
extract Inin the film.
Figure 3, the surface appearance of neat chitosan film and CBPR 2% film are reported.
As shown, the surface of the chitosan film was quite homogeneous, and after incorporation
of bud poplar extract, no change in microstructure could be observed.
Molecules 2022, 27, 7757 4 of 21

Molecules2022,
Molecules 27,7757
2022,27, 7757 4 of2121
4 of

Figure 2. ATR‒FT IR spectra of chitosan film, CBPR 2%, and bud poplar extract.

In Figure 3, the surface appearance of neat chitosan film and CBPR 2% film are
reported. As shown, the surface of the chitosan film was quite homogeneous, and after
incorporation of IR
Figure 2. ATR-FT bud poplar
spectra extract, no
of chitosan change
film, in microstructure
CBPR 2%, could be observed.
and bud poplar extract.
Figure 2. ATR‒FT IR spectra of chitosan film, CBPR 2%, and bud poplar extract.

In Figure 3, the surface appearance of neat chitosan film and CBPR 2% film are
reported. As shown, the surface of the chitosan film was quite homogeneous, and after
incorporation of bud poplar extract, no change in microstructure could be observed.

Figure
Figure3.3.SEM
SEMmicrographs
micrographsof
ofChitosan
Chitosan(A)
(A)and
andCBPR
CBPR 2%
2% (B).
(B).

CBPR
CBPRfilmsfilmswerewere characterized
characterizedfor forwater
waterabsorption
absorptioncapability
capabilityand
andbudbudpoplar
poplarresin
resin
in vitro release. Water absorption capacity is an important property for a
in vitro release. Water absorption capacity is an important property for a wound dressing wound dressing
material,
material, as ititabsorbs
as absorbs wound
wound exudates,
exudates, avoiding maceration and enhancing
wound wound
Figure 3. SEM micrographs of Chitosan (A) avoiding
and CBPRmaceration
2% (B). and enhancing healing.
healing.
All films showed a good and fast hydration capacity (Figure 4). After 30 min, hydration
All
wasCBPR
in thefilms
range
films showed a goodThe
of 120–140%.
were characterized and fast
presence
for hydration
water capacity
ofabsorption
bud poplar resin(Figure
not 4).
did and
capability After
decrease
bud 30 resin
the
poplar min,
water
hydration
sorption was in
capability; the range
rather, of
the120–140%.
best The
hydration presence of
percentage bud poplar
values wereresin did
obtained
in vitro release. Water absorption capacity is an important property for a wound dressing not decrease
with CBPR
the
1% water
and CBPR
material, sorption
as it 2% capability;
films.
absorbs woundrather, the best
exudates, hydration
avoiding percentage
maceration andvalues were obtained
enhancing wound
with CBPR
healing.Bud 1%
poplar and CBPR
resin 2%
was films.
released very slowly, and after 24 h, the percentage of the
release was 13%, 17%, and 37% for CBPR 2%, CBPR 1%, and CBPR
All films showed a good and fast hydration capacity (Figure 4). After 30 min, 0.5% films, respectively
(Figure 5A),
hydration wascorresponding
in the range ofto 8 µg/mL,The
120–140%. 5.5presence
µg/mL, and 5 µg/mL
of bud (Figure
poplar resin did5B).
notAfter 24 h,
decrease
the release showed only a very small increase, though no plateau was
the water sorption capability; rather, the best hydration percentage values were obtainedreached. The initial
faster release of bud poplar resin can be explained by the release of the BPE extract fraction
with CBPR 1% and CBPR 2% films.
present on the surface of the films, which was released faster than that present deeper in
the films.
Molecules 2022, 27, 7757 5 5ofof 21
21

Figure 4. Percentage of hydration of the CBPR films as a function of time. Results are reported as an
average ± SD.

Bud poplar resin was released very slowly, and after 24 h, the percentage of the
release was 13%, 17%, and 37% for CBPR 2%, CBPR 1%, and CBPR 0.5% films, respectively
(Figure 5A), corresponding to 8 µg/mL, 5.5 µg/mL, and 5 µg/mL (Figure 5B). After 24 h,
the release showed only a very small increase, though no plateau was reached. The initial
faster release of bud poplar resin can be explained by the release of the BPE extract fraction
present
Figure
Figure 4. on
4. the surface
Percentage
Percentage of of the films,
of hydration
hydration thewhich
of the was as
CBPR films
CBPR released faster
a function thanResults
of time. that present
Results deeper
are reported
are reported in
as an
the films.
average ±±SD.
SD.

Bud poplar resin was released very slowly, and after 24 h, the percentage of the
release was 13%, 17%, and 37% for CBPR 2%, CBPR 1%, and CBPR 0.5% films, respectively
(Figure 5A), corresponding to 8 µg/mL, 5.5 µg/mL, and 5 µg/mL (Figure 5B). After 24 h,
the release showed only a very small increase, though no plateau was reached. The initial
faster release of bud poplar resin can be explained by the release of the BPE extract fraction
present on the surface of the films, which was released faster than that present deeper in
the films.

Figure
Figure 5. In vitro
5. In vitro bud
bud poplar
poplar resin
resin release
release from
from CBPR
CBPR films.
films. Data
Data are
are expressed
expressed as
as percentage
percentage of
of
release
release (A)
(A) and
and as
as relative
relative mg/mL
mg/mL of of bud
bud poplar
poplar resin
resin extract
extract in
in the
the release
release medium
medium (B)
(B) with
with inset
inset
first 4 h of test (C).

2.2. Antimicrobial Activity of CBPR Films

2.2. Antimicrobial Activity of CBPR Films
After CBPR films were characterized, their antimicrobial activity was determined
After CBPR films were characterized, their antimicrobial activity was determined
with a Kirby–Bauer radial diffusion assay (Table 1). Chitosan films induce a contact
with a Kirby–Bauer radial diffusion assay (Table 1). Chitosan films induce a contact
inhibition halo for all microorganisms, while an evident inhibition zone is recorded in
inhibition halo for all microorganisms, while an evident inhibition zone is recorded in all
all microorganisms treated with the CBPR 2% films. The inhibition zone increase was
microorganisms
Figure treated
5. In vitro bud with
poplar therelease
resin CBPRfrom
2% films.
CBPR The
films.inhibition zone increase
Data are expressed was dose-
as percentage of
dose-dependent with respect to BPE in the film. These data are very interesting because
release (A) and as relative mg/mL of bud poplar resin extract in the release medium (B) with inset
they suggested that in the areas in direct contact with the film, there was no microbial
first 4 h of test (C).
proliferation, and in the presence of the poplar bud extract the antimicrobial activity could
reach
2.2. deeper layers.
Antimicrobial Activity of CBPR Films
After CBPR films were characterized, their antimicrobial activity was determined
with a Kirby–Bauer radial diffusion assay (Table 1). Chitosan films induce a contact
inhibition halo for all microorganisms, while an evident inhibition zone is recorded in all
microorganisms treated with the CBPR 2% films. The inhibition zone increase was dose-
 dependent with respect to BPE in the film. These data are very interesting because they
suggested that in the areas in direct contact with the film, there was no microbial
proliferation, and in the presence of the poplar bud extract the antimicrobial activity could
Molecules 2022, 27, 7757 reach deeper layers. 6 of 21

Table 1. Antimicrobial activity evaluated by Kirby–Bauer assay. The diameter of the inhibition zone
reported
Table is the mean ofactivity
1. Antimicrobial three measures
evaluatedofby
three independent
Kirby–Bauer experiments.
assay. The diameter of the inhibition zone
reported is the mean of three measures ofHalo
Inhibition threeDiameter
independent
(mm)experiments.
Treatments
Staphylococcus aureus Staphylococcus epidermidis Pseudomonas aeruginosa Candida albicans
Inhibition Halo Diameter (mm)
Chitosan
Treatments 7.5 7.5 6 7.5
Staphylococcus aureus Staphylococcus epidermidis Pseudomonas aeruginosa Candida albicans
CBPR 0.5% 7 7.5 7 7.5
Chitosan 7.5 7.5 6 7.5
CBPR 1% 8 10 7.5 7.5
CBPR 0.5% 7 7.5 7 7.5
CBPR 2%
CBPR 1% 88.5 10 11.5 7.5 8 7.5 8
Gentamicin/Fluconazole
CBPR 2% 8.522 11.5 28 8 24 8 8
Gentamicin/Fluconazole 22 28 24 8

The impact of biofilms on chronic wound infection and their involvement in late
Theisimpact
healing of biofilms[25].
well established on chronic
Startingwound
from thisinfection and their
assumption, the involvement
ability of the in late
CBPR
healing is well established [25]. Starting from this assumption, the ability
films to slow down the formation of the biofilm and their ability to disperse already- of the CBPR
films to microbial
formed slow down the formation
biofilms of the biofilm
were evaluated. All filmsand their
were ability
active to disperse
during already-
the formation of
formed microbial biofilms were evaluated. All films were active during the formation
S. aureus, S. epidermidis, and C. albicans biofilms (Figure 6); the antibiofilm activity was of
S. aureus, S. epidermidis, and C. albicans biofilms (Figure 6); the antibiofilm
always significant with respect to the untreated sample. Chitosan films were able to activity was
always significant
inhibit the biofilmwith respect of
formation to the untreated sample.
staphylococci, Chitosan
while the filmsofwere
addition budable to inhibit
poplar resin
the biofilm formation of staphylococci, while the addition of bud poplar resin extracts at
extracts at 0.5%, 1%, and 2% did not affect this activity, although a tendency to reduce the
0.5%, 1%, and 2% did not affect this activity, although a tendency to reduce the biofilm
biofilm of S. aureus was evident. No effect was observed on biofilm formation or
of S. aureus was evident. No effect was observed on biofilm formation or dispersion of
dispersion of Gram-negative bacterium Pseudomonas aeruginosa. Interestingly, chitosan
Gram-negative bacterium Pseudomonas aeruginosa. Interestingly, chitosan films were able to
films were able to disperse preformed S. epidermidis and C. albicans biofilms, while films
disperse preformed S. epidermidis and C. albicans biofilms, while films containing chitosan
containing chitosan and BPE were able to increase the dispersion of all preformed biofilms
and BPE were able to increase the dispersion of all preformed biofilms of S. aureus and
of S. aureus and Candida yeasts. The CBPR 1% and 2% dispersed the preformed biofilm of
Candida yeasts. The CBPR 1% and 2% dispersed the preformed biofilm of S. aureus better
S. aureus better than chitosan film. The CBPR 2% film was able to significantly increase
than chitosan film. The CBPR 2% film was able to significantly increase the dispersion of
the dispersion of the Candida biofilm compared to the biofilm treated with chitosan film.
the Candida biofilm compared to the biofilm treated with chitosan film.

Effectofofchitosan
Figure 6. Effect chitosanand andCBPRCBPRfilms
filmsononthe
theformation
formation andand dispersion
dispersion of of S. aureus
S. aureus (A),(A),
S.
epidermidis
S. (B),
epidermidis P. P.
(B), aeruginosa
aeruginosa(C),(C),and
andC.C.albicans
albicansbiofilm
biofilm(D).
(D).The
The data
data are
are expressed asas aa mean
mean± ± SD
of
of the
the percentage
percentage of of the
the biofilm
biofilm massmass (n
(n =
= 12).
12). The
The statistical
statistical analysis
analysis was
was performed
performed with
with two-tailed
two-tailed
Student’s
Student’s t-test.
t-test. **pp≤≤0.05,
0.05,****p p≤ ≤
0.01 (chitosan-
0.01 (chitosan-and CBPR-film-treated
and CBPR-film-treatedcells vs. vs.
cells untreated
untreatedcells), #p
cells),
≤# p0.05 (CBPR-film-treated cells vs. chitosan-treated cells).
≤ 0.05 (CBPR-film-treated cells vs. chitosan-treated cells).

2.3. Regenerative Activity of CPBR Films

Wound healing is a complex and dynamic process. Ulcers are caused by a prolonged
inflammatory process, persistent infections produced by microbial biofilms, and failure
of the epidermal and/or dermal cells to respond to the reparatory stimuli. Chitosan
is approved as safe by the European Medicines Agency and the U.S. Food and Drug
Molecules 2022, 27, 7757 7 of 21

Administrations for wound dressing applications [6]. Cytotoxicity of CBPR films was
tested on human dermal fibroblast (HuDe) and human keratinocyte (NCTC2544) cell lines.
The results reported in Table 2 show that all tested CBPR films were not cytotoxic after
four hours of contact with fibroblasts, while after 24 h, the 1% and 2% BPE decreased
the viability of cells by 27% and 55%, respectively. Keratinocytes were more sensitive to
chitosan films than were the fibroblasts; after 4 h of contact, CPBR 1% and 2% reduced
the viability of cells by 45% and 64%, respectively, and after 24 h a reduction of 70% was
observed. The CPBR 0.5% film was not cytotoxic for either of the two cell lines after 4 h
and 24 h.

Table 2. Cytotoxicity of CBPR films on human fibroblasts and keratinocytes. Results are expressed as
percentage of live cells with respect to untreated cells, assumed to be 100. The results are expressed
as mean ± SD of four different measures.

Hude NCTC2544
4h 24 h 4h 24 h
Untreated 100 ± 12 100 ± 9 100 ± 25 100 ± 39
Chitosan 85 ± 30 85 ± 4 68 ± 30 65 ± 17
CBPR 0.5% 73 ± 23 67 ± 26 125 ± 33 57 ± 14
CBPR 1% 84 ± 25 63 ± 4 ** 53 ± 28 * 27 ± 19 *
CBPR 2% 67 ± 40 45 ± 26 * 36 ± 8 ** 29 ± 22 *
The statistical analysis was performed with a two-tailed Student’s t-test. * p ≤ 0.05, ** p ≤ 0.01 (film treated cells
vs. untreated cells).

The regenerative capability of bud poplar extract (BPE) on human dermal fibroblasts
(HuDe) was assessed by a mobility assay that used a silicone apparatus (Ibidi Micro Insert
4-well dishes) to grow the cells of the dermis at a predefined distance. The removal of
the silicone support allowed quantifying the closure of spaces (Figure 7A) in the presence
or absence of CBPR 0.5%. Cell distances were monitored by microscopy and measured
for 7 days (Figure 7B,C). The results showed that BPE at low concentrations induces a
regenerative activity by chitosan films on fibroblasts of the human dermis. The effect ©s
already evident after 24 h, and it is maintained until the closure of the septum at 7 days.

2.4. Anti-Inflammatory Activity of BPE

Given the known immunomodulatory properties of propolis, we analyzed how the
BPE that can be released by CBPR films can influence the polarization of macrophages
towards an anti-inflammatory M2 phenotype. To this end, we tested a model of human
macrophage cell line THP-1 stimulated with phorbol-12-myristate-13-acetate (PMA) [26].
PMA treatment, which activates protein kinase C (PKC), induces a greater degree of
differentiation in THP-1 cells as reflected by increased adherence and expression of surface
markers associated with macrophage differentiation. During this process, the cultured
cells without PMA (PMAr) increased their cytoplasmic-to-nuclear ratio, mitochondrial and
lysosomal numbers, and altered differentiation-dependent cell surface markers in a pattern
similar to MDM (monocyte-derived macrophages). Moreover, PMAr cells showed relative
resistance to apoptotic stimuli and maintained levels of the differentiation-dependent
antiapoptotic protein Mcl-1, similar to MDM [26]. At the end of the treatment, we obtained
an M0 phenotype. To evaluate the direct immunomodulatory effect of BPE, M0 cells
were treated with BPE extract (5, 25, and 50 µg/mL). No cytotoxicity of BPE extract was
observed on M0 and M1 cells (Figure S1, Supplementary Material). The polarization was
evaluated by the cytofluorimetric determination of the membrane marker receptors of the
two populations: B7 molecules (CD80 and CD86) for the inflammatory phenotype M1
and CD36 for the M2 anti-inflammatory phenotype. The results in Figure 8 show that the
extract was not able to induce polarization of M0; even BPE at a concentration of 50 µg/mL
was able to slightly reduce the expression of CD36. However, B7 molecule expression is
not affected by the extract.
 (HuDe) was assessed by a mobility assay that used a silicone apparatus (Ibidi Micro Insert
Molecules 2022, 27, 7757 8 of 21
4-well dishes) to grow the cells of the dermis at a predefined distance. The removal of the
silicone support allowed quantifying the closure of spaces (Figure 7A) in the presence or
absence of CBPR 0.5%. Cell distances were monitored by microscopy and measured for 7
was
days monitored
(Figure under theThe
7B,C). microscope
results(20× magnification)
showed that BPE for 7atdays
lowand photos were taken
concentrations (B). Thea
induces
Molecules 2022, 27, 7757 distances between the frames were recorded and the results are expressed as mean ± SD of8 of the21
regenerative activity by chitosan films on fibroblasts of the human dermis. The effect ©s
measurements by microscope (n = 6) carried out in two individual experiments (C). the statistical
already evident after 24 h, and it is maintained until the closure of the septum at 7 days.
analysis was performed with a two-tailed Student’s t-test. * p ≤ 0.05 (chitosan- and CBPRfilm-treated
cells vs. untreated cells).

2.4. Anti-Inflammatory Activity of BPE

Given the known immunomodulatory properties of propolis, we analyzed how the
BPE that can be released by CBPR films can influence the polarization of macrophages
towards an anti-inflammatory M2 phenotype. To this end, we tested a model of human
macrophage cell line THP-1 stimulated with phorbol-12-myristate-13-acetate (PMA) [26].
PMA treatment, which activates protein kinase C (PKC), induces a greater degree of
differentiation in THP-1 cells as reflected by increased adherence and expression of
surface markers associated with macrophage differentiation. During this process, the
cultured cells without PMA (PMAr) increased their cytoplasmic-to-nuclear ratio,
mitochondrial and lysosomal numbers, and altered differentiation-dependent cell surface
markers in a pattern similar to MDM (monocyte-derived macrophages). Moreover, PMAr
cells showed relative resistance to apoptotic stimuli and maintained levels of the
differentiation-dependent antiapoptotic protein Mcl-1, similar to MDM [26]. At the end of
the treatment, we obtained an M0 phenotype. To evaluate the direct immunomodulatory
effect of BPE, M0 cells were treated with BPE extract (5, 25, and 50 μg/mL). No cytotoxicity
Figure
Figure
of 7.
7. Regenerative
BPE extractRegenerative
was observedeffect of
of chitosan
effect on chitosan
M0 and M1 films
films on (Figure
on
cells human dermal
human dermal fibroblasts. The
fibroblasts.
S1, Supplementary The regenerative
regenerative
Material). The
activity
activity assay
assay was
was performed
performed in
in Micro-Insert
Micro-Insert 4-well
4-well dishes
dishes (A),
(A), the
the distances
distances between
between the
the frames
frames
polarization was evaluated by the cytofluorimetric determination of the membrane
was monitored
marker under
receptors of the
themicroscope (20× magnification)
two populations: B7 moleculesfor 7 days
(CD80and and
photos were taken
CD86) (B).
for the
The distances between
inflammatory phenotypethe frames
M1 and wereCD36
recorded
for and
the the
M2results mean ± SD of
are expressed asphenotype.
anti-inflammatory Thethe
measurements
results by 8microscope
in Figure show that(nthe = 6)extract
carriedwas
out not
in two
ableindividual
to induce experiments (C).of
polarization theM0;
statistical
even
analysis
BPE at awas performed with
concentration a two-tailed
of 50 µg/mL was Student’s t-test.
able to * p ≤ 0.05
slightly (chitosan-
reduce and CBPRfilm-treated
the expression of CD36.
cells vs. untreated cells).
However, B7 molecule expression is not affected by the extract.

Figure 8. Effect of BPE on macrophages polarization. THP-1-derived macrophages were treated

Figure 8. Effect of BPE on macrophages polarization. THP-1-derived macrophages were treated for
for 24 h with the indicated concentration of BPE. After incubation, cells were labelled with FITC
24 h with the indicated concentration of BPE. After incubation, cells were labelled with FITC mouse
mouse antihuman
antihuman CD36 (A)CD36 and(A)PE
andmouse
PE mouse antihuman
antihuman B7 (B).
B7 (B). Labelled
Labelled cells
cells wereanalyzed
were analyzedby by
cytofluorimeter. Results are expressed as Mean Intensity Fluorescence (MIF). The statistical
cytofluorimeter. Results are expressed as Mean Intensity Fluorescence (MIF). The statistical analysisanalysis
wasperformed
was performedwith
withaatwo-tailed
two-tailedStudent’s t-test.**pp<<0.05
Student’st-test. 0.05(BPE-treated
(BPE-treatedcells
cellsvs.
vs.untreated
untreatedcells).
cells).

To evaluate the anti-inflammatory activity, M0 were treated for 24 h with BPE extract
before or after LPS/IFNγ stimulation to induce the M1 phenotype. Pretreatment of M0 with
BPE at 5–25–50 µg/mL before stimulation with LPS/IFNγ did not regulate the expression
of the proinflammatory B7 molecules (Figure 9A), but instead significantly increased the
Molecules 2022, 27, 7757 9 of 21

To evaluate the anti-inflammatory activity, M0 were treated for 24 h with BPE extract
Molecules 2022, 27, 7757 before or after LPS/IFNγ stimulation to induce the M1 phenotype. Pretreatment of9 M0 of 21
with BPE at 5–25–50 μg/mL before stimulation with LPS/IFNγ did not regulate the
expression of the proinflammatory B7 molecules (Figure 9A), but instead significantly
increased
expression the
ofexpression of the anti-inflammatory
the anti-inflammatory M2 receptor
M2 receptor CD36 (FigureCD36 (Figure
9C). When 9C). When
macrophages
macrophages were first polarized towards M1 phenotype, BPE treatment did not
were first polarized towards M1 phenotype, BPE treatment did not significantly affect the
significantly affect the expression of B7 or CD36
expression of B7 or CD36 molecules (Figure 9B,D). molecules (Figure 9B,D).

Figure 9.
Figure Effectof
9. Effect of BPE
BPE pretreatment
pretreatment (A,C)
(A,C) and
and posttreatment
posttreatment (B,D)
(B,D) on
on macrophages’
macrophages’ polarization
polarization
induced by LPS/IFNγ.
induced LPS/IFNγ. THP-1-derived macrophages
macrophageswerewerepretreated
pretreatedforfor
2424
h with the the
h with indicated con-
indicated
concentrations
centrations of BPEof BPE before
before LPS/IFNγ
LPS/IFNγ stimulation
stimulation (A,C)(A,C) or posttreated
or posttreated withafter
with BPE BPELPS/IFNγ
after LPS/IFNγ
(B,D).
(B andstimulation,
After D). After stimulation, cells were
cells were labelled labelled
with FITC with
mouse FITC mouse antihuman
antihuman B7 and PE B7 and antihuman
mouse PE mouse
antihuman
CD36. Labelled cells were analyzed by cytofluorimeter. Results are expressed as Mean as
CD36. Labelled cells were analyzed by cytofluorimeter. Results are expressed Mean
Intensity
Intensity Fluorescence (MIF). The statistical analysis was performed with a two-tailed Student’s t-
Fluorescence (MIF). The statistical analysis was performed with a two-tailed Student’s t-test. * p < 0.05
test. * p < 0.05 (BPE+LPS/IFNγ-treated cells vs. LPS/IFNγ-treated cells).
(BPE+LPS/IFNγ-treated cells vs. LPS/IFNγ-treated cells).

To
To further
further verify
verify thethe immunomodulatory
immunomodulatory activity activity ofof the
the extract,
extract, we
we evaluated
evaluated the the
secretion of proinflammatory and anti-inflammatory
secretion of proinflammatory and anti-inflammatory cytokines by macrophages cytokines by macrophages
stimulated
stimulated with BPE
with BPE before beforepolarization.
M1–M2 M1–M2 polarization.
BPE at BPE5, 25,at 5,
and25,50
and 50 µg/mL
µg/mL were
were notnot able
able to
to induce the proinflammatory cytokine TNFα (Figure 10A). Regarding
induce the proinflammatory cytokine TNFα (Figure 10A). Regarding the production of the the production of
the anti-inflammatory
anti-inflammatory IL-10,
IL-10, we we verified
verified thatthat poplar
poplar budbud extract
extract is not
is not ableable to stimulate
to stimulate the
the production
production of cytokine
of the the cytokine (Figure
(Figure 10B), 10B), although
although an increase
an increase is observed
is observed with the with the
highest
highest
dose useddose(50 used (50 µg/mL).
µg/mL). Secondly,
Secondly, we analyzed
we analyzed the production
the production of TGFβ1;
of TGFβ1; the extract
the extract at a
at a concentration of 50 μg/mL was able to stimulate the production of
concentration of 50 µg/mL was able to stimulate the production of the cytokine by the M0 the cytokine by the
M0
cellscells (Figure
(Figure 10C),10C), suggesting
suggesting an M2 anpolarization.
M2 polarization.Ansorge Ansorge et al.described
et al. have have described
a similara
similar
patternpattern
analyzinganalyzing the of
the effect effect of propolis
propolis and itsand its constituents
constituents on human
on human T regulatory
T regulatory cells
cells
and and observed
observed that that
TGFβ1 TGFβ1 production
production was was increased
increased [27]. [27].

2.5. Antioxidant Activity of BPE and CBPR Films

Bud poplar extract showed strong antioxidant activity due to its abundant active poly-
phenols [15,18]. Moreover, it is known that the antioxidant activity of propolis contributes
to its protective effects on skin diseases; for example, it was reported that propolis could
alleviate cell damage in fibroblasts by suppressing intracellular ROS production. Moreover,
a lower concentration of free radicals has been detected in burn wounds treated with
propolis [15]. In the first series of experiments, we analyzed the antioxidant ability of our
BPE by means of neutralizing the free DPPH radical. BPE at a concentration of 500 µg/mL
showed a slight scavenging ability versus the positive control ascorbic acid (Figure 11).
 Molecules 2022, 27, 7757 10 of 21
Molecules 2022, 27, 7757 10 of 21

Figure
Figure 10.10. Effect
Effect of of
BPEBPE
onon TNF-α
TNF-α (A),
(A), IL-10
IL-10 (B),
(B), TGFβ1
TGFβ1 (C)(C) production
production byby macrophages.
macrophages. THP-1-
THP-1-
derived macrophages were stimulated with BPE. Data represent the mean ± standard
derived macrophages were stimulated with BPE. Data represent the mean ± standard deviation (SD) deviation (SD)
of ofthree
threeindependent
independent experiments.
experiments.The Thestatistical analysis
statistical was was
analysis performed with awith
performed two-tailed Student’s
a two-tailed
Student’s
t-test. * pt-test. * p(BPE-treated
< 0.05, < 0.05, (BPE-treated
cells vs.cells vs. untreated
untreated cells). cells).

In a second
2.5. Antioxidant seriesofofBPE
Activity experiments,
and CBPRwe analyzed the antioxidant ability of chitosan film
Films
by means of neutralizing the free DPPH radical. Chitosan film itself showed antioxidant
Bud poplar extract showed strong antioxidant activity due to its abundant active
activity, while only CBPR 2% significantly increased the scavenging activity of chitosan to
poly-phenols [15,18]. Moreover, it is known that the antioxidant activity of propolis
70% (Figure 12).
contributes to its protective effects on skin diseases; for example, it was reported that
propolis could alleviate cell damage in fibroblasts by suppressing intracellular ROS
production. Moreover, a lower concentration of free radicals has been detected in burn
wounds treated with propolis [15]. In the first series of experiments, we analyzed the
antioxidant ability of our BPE by means of neutralizing the free DPPH radical. BPE at a
concentration of 500 μg/mL showed a slight scavenging ability versus the positive control
ascorbic acid (Figure 11).
Molecules
Molecules2022,
2022,27,
27,7757
7757 11 11
of of
2121

Figure 11. BPE radical scavenging capacity by DPPH method. Results are expressed as percentage
of DPPH scavenging activity. Data represent the mean ± standard deviation (SD) of two
independent experiments performed in triplicate. The statistical analysis was performed with a two-
tailed Student’s t-test. * p < 0.05, ** p < 0.01 (BPE-, ascorbic-acid- treated cells vs. ethanol-treated
cells).

Figure
Figure a11.
In11. BPEradical
BPE
second radical scavenging
seriesscavenging capacity
capacity
of experiments, webyby DPPHmethod.
DPPH
analyzed method. Resultsare
Results
the antioxidant are expressed
expressed
ability asaspercentage
percentage
of chitosan film
ofofDPPH
DPPH scavenging activity.
activity. Data
Data represent
represent the the
meanmean
± ± standard
standard deviation
deviation
by means of neutralizing the free DPPH radical. Chitosan film itself showed antioxidant (SD) of(SD)
two of two
indepen-
independent experiments
dent experiments
activity, while only performed
performed
CBPR 2% in triplicate.
in significantly The
triplicate. The increasedstatistical analysis
statistical analysis was performed
was performed
the scavenging with
activity with a two-
ofachitosan
two-tailed
tailed
to 70%Student’s
Student’s t-test.
t-test.12).
(Figure * p <**0.05,
* p < 0.05, ** p (BPE-,
p < 0.01 < 0.01 ascorbic-acid-
(BPE-, ascorbic-acid-
treated treated
cells vs. cells vs. ethanol-treated
ethanol-treated cells).
cells).

In a second series of experiments, we analyzed the antioxidant ability of chitosan film

by means of neutralizing the free DPPH radical. Chitosan film itself showed antioxidant
activity, while only CBPR 2% significantly increased the scavenging activity of chitosan
to 70% (Figure 12).

Figure
Figure12.12.
Scavenging
Scavengingactivity of chitosan
activity andand
of chitosan CBPR.
CBPR.TheThe
datadata
are expressed as a as
are expressed mean ± SD±
a mean ofSD
theof
percentage of theof
the percentage scavenging activity
the scavenging (n = 6).(nThe
activity = 6).statistical analysisanalysis
The statistical was performed with a with
was performed two- a
tailed Student’s
two-tailed t-test. t-test.
Student’s * p ≤ * 0.05, ** p **≤ p0.01
p ≤ 0.05, (film-treated
≤ 0.01 sample
(film-treated vs.vs.
sample control), # p# p≤ ≤0.05
control), 0.05
(chitosan/CBPR-film treated samples vs. chitosan-treated samples).
(chitosan/CBPR-film treated samples vs. chitosan-treated samples).

Total
TotalROS
ROSproduction
productionbybyhuman
humanneutrophils
neutrophils stimulated
stimulated with
with phorbol-12-myristate-
phorbol-12-myristate-
Figure 12. Scavenging
13-acetate (PMA) was tested by chemiluminescence assay. Neutrophils aactivated
13-acetate (PMA) wasactivity
tested of
bychitosan and CBPR. Theassay.
chemiluminescence data are expressed asactivated
Neutrophils mean ± by
SDPMA
byofPMA
the
percentage
produce of the scavenging
luminol-dependent activity (n = 6).
chemiluminescence The statistical
profilesanalysis
followingwas performed
ROSROS with
production a two-
afteraf-
produce luminol-dependent chemiluminescence profiles following production
tailed Student’s t-test. * p ≤ 0.05, ** p ≤ 0.01 (film-treated sample vs. control), # p ≤ 0.05
the addition of the stimulus. The results showed that bud poplar resin
ter the addition of the stimulus. The results showed that bud poplar resin extract was extract was able to
(chitosan/CBPR-film treated samples vs. chitosan-treated samples).
reduce ROS production by neutrophils activated with PMA in a dose-dependent
able to reduce ROS production by neutrophils activated with PMA in a dose-dependent manner
(Figure
manner 13). This result is very encouraging, as it demonstrates that the activity of DPPH
Total(Figure 13). This
ROS production result
by humanis very encouraging,
neutrophils as it demonstrates
stimulated that the activity
with phorbol-12-myristate-
scavenging
of DPPH of the extract
scavenging of is
theenhanced
extract byenhanced
is its abilitybytoitslower ROS
ability to concentration
lower ROS in the
concentra-
13-acetate (PMA) was tested by chemiluminescence assay. Neutrophils activated by PMA
supernatants
tion in the of human PMN,
supernatants cells recruited
of chemiluminescence
human in therecruited
PMN, cells infectioninsitetheduring the inflammation
infection site during the
produce luminol-dependent profiles following ROS production after
process.
inflammation process.
the addition of the stimulus. The results showed that bud poplar resin extract was able to
As observed with the DPPH test, chitosan film is able to reduce the production of
reduce ROS production by neutrophils activated with PMA in a dose-dependent manner
ROS. However, CBPR films decreased the ROS concentration in BPE in a dose-dependent
(Figure 13). This result is very encouraging, as it demonstrates that the activity of DPPH
manner (Figure 14).
scavenging of the extract is enhanced by its ability to lower ROS concentration in the
supernatants of human PMN, cells recruited in the infection site during the inflammation
process.
 Molecules 2022, 27, 7757 12 of 21

Molecules
Molecules2022, 27,27,
2022, 7757
7757 12 of 21 21
12 of

Figure 13. In vitro neutralization of ROS produced by human PMA-stimulated PMN. Results are
expressed in relative luminescence units (RLU). Data represent the mean of three independent
experiment performed in triplicate for BPE at two different concentrations. The statistical analysis
was performed with a two-tailed Student’s t-test. * p < 0.05 (BPE-treated cells vs. untreated cells).

Figure
FigureAs In In
13.13. vitro
vitro
observed
neutralization
neutralization
with the DPPH ofof ROS
ROS produced
produced
test, chitosan byby human
human
film
PMA-stimulated
PMA-stimulated
is able to reduce the
PMN.
PMN. Results
Results
production
are
areof
expressed
expressed in in relative
relative luminescence
luminescence units
units (RLU).
(RLU). Data
Data represent
represent thethe mean
mean of ofthree
threeindependent
independent
ROS. However, CBPR films decreased the ROS concentration in BPE in a dose-dependent
experiment
experiment performed
performed in in
triplicate forfor
triplicate BPE
BPE at at
two
two different
differentconcentrations.
concentrations. The
The statistical analysis
statistical analysis
manner (Figure 14).
was performed
was performed with
witha two-tailed
a two-tailed Student’s
Student’s t-test. * p* <p 0.05
t-test. < 0.05(BPE-treated
(BPE-treatedcells vs.vs.
cells untreated
untreated cells).
cells).

As observed with the DPPH test, chitosan film is able to reduce the production of
ROS. However, CBPR films decreased the ROS concentration in BPE in a dose-dependent
manner (Figure 14).

Figure 14.
Figure Antioxidantactivity
14. Antioxidant activityofofdifferent
differentchitosan/BPE
chitosan/BPEfilms
filmsononROS
ROSproduction
productionininhuman
human
neutrophils.Results
neutrophils. Results
areare expressed
expressed as RLU
as RLU (relative
(relative luminescence
luminescence units).units). The are
The results results are ex-
expressed
as mean ±as
pressed of the±measurements
SDmean made under the
SD of the measurements mademicroscope (nmicroscope
under the = 6) carried out
(n =in6)two individual
carried out in
experiments.
two individual experiments.

FigureThen,
14. Antioxidant
Then, activityactivity
the antioxidant
antioxidant of different
activityofof
BPEchitosan/BPE
BPEon human
on humanfilms on ROS
fibroblasts and
fibroblastsproduction in human
keratinocytes stimu-
and keratinocytes
neutrophils. Results
lated with with are
IFNγ IFNγ expressed
and histamine as RLU (relative
was analyzed. luminescence
BPE, by units).
themselves, The results
did notdidare expressed
induce the ROS
stimulated and histamine was analyzed. BPE, by themselves, not induce
as mean ± SD of the measurements made under the microscope (n = 6) carried out in two individual
production
the in humanindermal
ROS production human fibroblasts (Figure 15A).
dermal fibroblasts However,
(Figure 15A).BPE at the concentration
However, BPE at the
experiments.
of 5 µg/mL, but
concentration of 5not at 50 µg/mL,
µg/mL, but not reduced the ROS
at 50 µg/mL, formation
reduced induced
the ROS by IFNγ/histamine
formation induced by
treatment. These
IFNγ/histamine results
treatment.suggest
These a dual
results activity
suggest of
a the
dualextract at
activity different
of the concentrations.
extract at differentIt
Then, the antioxidant activity of BPE on human fibroblasts and keratinocytes
seems, in fact, that
concentrations. at lower
It seems, in doses, the anti-inflammatory
fact, that activity predominates, activity
while at
stimulated with IFNγ and histamine wasatanalyzed.
lower doses,
BPE, bythe anti-inflammatory
themselves, did not induce
higher doses the
predominates, proapoptotic
while at higher and antiproliferative
doses the activity
proapoptotic anddescribed in the literature
antiproliferative is
activity
the ROS production in human dermal fibroblasts (Figure 15A). However, BPE at the
evident [28].
described in the
concentration of 5literature
µg/mL, butis evident
not at [28].
50 µg/mL, reduced the ROS formation induced by
IFNγ/histamine treatment. These results suggest a dual activity of the extract at different
concentrations. It seems, in fact, that at lower doses, the anti-inflammatory activity
predominates, while at higher doses the proapoptotic and antiproliferative activity
described in the literature is evident [28].
 Human keratinocytes (NCTC 2544) produce a high basal level of ROS; stimulation
with BPE reduced in a dose-dependent manner the production of radicals (Figure 15B).
Similar results have been reported by Kim et al. that showed the reduced generation of
Molecules 2022, 27, 7757 13 of 21
ROS in human keratinocytes pretreated with propolis [29]. The effect could be ascribed to
chrysin as suggested by Wu et al., although it was not detected in our extract [30].

Figure
Figure15.
15.Effect of of
Effect BPE on on
BPE intracellular ROSROS
intracellular production
productionof HuDe (A) and
of HuDe (A) NCTC
and NCTC 2544 (B)
2544cells.
(B) The
cells.
experiment was performed
The experiment was performed in quadruplicate
in quadruplicate for for
each substance.
each substance.TheThestatistical
statisticalanalysis
analysiswas was
performed
performedwith
withaa two-tailed Student’st-test.
two-tailed Student’s t-test.**pp<<0.05
0.05(BPE-treated
(BPE-treated cells
cells vs.vs. untreated
untreated cells);
cells); $ p $< p0.05
<
0.05 (BPE-treated cells vs. IFNγ+-HIST-treated
(BPE-treated cells vs. IFNγ+-HIST-treated cells). cells).

3. Discussion
Human keratinocytes (NCTC 2544) produce a high basal level of ROS; stimulation
withKeeping
BPE reduced in a dose-dependent
the wound manner
environment as sterile asthe production
possible helps toof prevent
radicalsthe
(Figure 15B).
entrance
ofSimilar results haveinto
microorganisms beenthe
reported
woundbyand Kimthe
et al.seeding
that showed the reduced
of biofilm generation
infections. Woundof
ROS in human
disinfection doeskeratinocytes
not eliminatepretreated with propolis
microorganisms. Wound [29]. The effect
dressing is ancould be ascribed
important tool forto
chrysin as suggested by Wu et al., although it was not detected in our extract
accelerating healing and avoiding microbial colonization [31]. Many plant compounds [30].
with antioxidant, anti-inflammatory, and antimicrobial properties could be of great
3. Discussion
benefit for wound healing using bioactive wound dressings [32].
Keeping
Our the wound
data about environmentactivity
the antimicrobial as sterileof as
BPEpossible
extracthelps
agreetowith
prevent
thosethe entrance
obtained byof
microorganisms into the wound and the seeding of biofilm infections.
Popova et al., who observed better activity of propolis towards Gram-positive bacteria Wound disinfection
does not eliminate
compared microorganisms.
to Gram-negative bacteriaWound dressing isPopova
[11]. Moreover, an important
et al. tool
[11]for accelerating
analyzed the
healing and avoiding microbial colonization [31]. Many plant compounds
antibacterial activity of propolis from different regions of Turkey, confirming with antioxidant,
the
anti-inflammatory,
importance and antimicrobial
of the amount properties
of phenolic compounds,could be of great benefit
flavones, for woundfor
and flavanones healing
the
using bioactive wound dressings [32].
antibacterial activity of poplar propolis. The extract used in the film contains about 25%
Our data about the antimicrobial activity of BPE extract agree with those obtained
of the total flavonoid [13]. These results are in line with those observed by Torlak et al.
by Popova et al., who observed better activity of propolis towards Gram-positive bacteria
[33] and Eskandarinia et al. [23]. The antibacterial efficacy of chitosan–propolis-coated
compared to Gram-negative bacteria [11]. Moreover, Popova et al. [11] analyzed the
polypropylene films has been studied against foodborne pathogens Bacillus cereus,
antibacterial activity of propolis from different regions of Turkey, confirming the importance
Cronobacter sakazakii, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella
of the amount of phenolic compounds, flavones, and flavanones for the antibacterial activity
typhimurium, and Staphylococcus aureus [17,34–36]. Chitosan-coated film exhibited a broad
of poplar propolis. The extract used in the film contains about 25% of the total flavonoid [13].
spectrum of antibacterial activity; the incorporation of ethanol propolis extract enhanced
These results are in line with those observed by Torlak et al. [33] and Eskandarinia et al. [23].
antibacterial activity against all pathogens tested [33]. The recent work of Eskandarinia et
The antibacterial efficacy of chitosan–propolis-coated polypropylene films has been studied
al. described the antimicrobial activity of cornstarch, hyaluronic acid, and propolis wound
against foodborne pathogens Bacillus cereus, Cronobacter sakazakii, Escherichia coli O157:H7,
dressing [23]. Film dressing containing propolis exhibited higher antibacterial activity
Listeria monocytogenes, Salmonella typhimurium, and Staphylococcus aureus [17,34–36]. Chitosan-
against S. aureus,
coated film E. acoli,
exhibited broadand S. epidermidis
spectrum in comparison
of antibacterial with
activity; the the cornstarch
incorporation and
of ethanol
cornstarch/hyaluronic acid dressings. Moreover, our data align with the results
propolis extract enhanced antibacterial activity against all pathogens tested [33]. The recent described
inwork
a recent study that demonstrated
of Eskandarinia et al. describedthat chitosan–propolis
the antimicrobial nanoparticles
activity of cornstarch, were able to
hyaluronic
reduce the biofilm formation of Staphylococcus epidermidis [16].
acid, and propolis wound dressing [23]. Film dressing containing propolis exhibited higher
Recently, activity
antibacterial propolisagainst
has been S. used as E.
aureus, ancoli,
active
andcomponent in wound
S. epidermidis biomaterials
in comparison withforthe
its antimicrobial
cornstarch effects, but to the best
and cornstarch/hyaluronic of our knowledge,
acid dressings. Moreover, the antibiofilm,
our data align withanti-
the
inflammatory, and cell proliferative activities of chitosan–BPE films
results described in a recent study that demonstrated that chitosan–propolis nanoparticles have not been
evaluated
were able [23,24,37–42]. When formulated
to reduce the biofilm formation ofin corn-starch-based
Staphylococcus wound
epidermidis [16].dressing with
hyaluronic acid, the proliferation of mouse fibroblasts was observed,
Recently, propolis has been used as an active component in wound biomaterials but this was due forto
its
antimicrobial effects, but to the best of our knowledge, the antibiofilm, anti-inflammatory,
and cell proliferative activities of chitosan–BPE films have not been evaluated [23,24,37–42].
When formulated in corn-starch-based wound dressing with hyaluronic acid, the prolifera-
tion of mouse fibroblasts was observed, but this was due to the presence of hyaluronic acid
and actually decreased in films containing propolis [30]. This latter result is in contrast with
the regenerative activity of our BPE in chitosan film. However, we used human dermal
Molecules 2022, 27, 7757 14 of 21

fibroblasts, while in the previous study, murine fibroblasts from subcutaneous adipose
tissue were used. Moreover, contrary to Paramasivan et al., who have described an antipro-
liferative activity of chitosan–dextran gel on human fibroblasts [43], the chitosan included
in our film showed no activity on the proliferation of human fibroblasts, confirming that
the active CBPR–chitosan film could be an excellent dressing for wounds.
Regarding the anti-inflammatory activity of BPE, Bueno-Silva et al. reported the effect
of propolis on the expression of B7 molecules; they stimulated murine macrophages with
Brazilian red propolis extract and demonstrated an up-regulation of the costimulatory
molecules [14]. The apparent discrepancy with our data is due to the different composition
of the propolis compared to bud poplar extract and the cells used; indeed, the authors have
tested the propolis extracts on murine macrophages, while in our study, the effect of the
extract on human macrophages was analyzed. To date, as far as we know, this is the first
work studying the activity of poplar bud extract on the polarization of human M1/M2
macrophages, confirmed by the production of the anti-inflammatory cytokine TGFβ1, the
slight increase of IL-10, and decrease of the proinflammatory cytokine TNFα.
In a recent work on propolis originating in different countries, Conti et al. [44] demon-
strated that Brazilian and Cuban propolis increase TNFα production by human monocytes,
while Mexican propolis decreases the basal production of the cytokine. The authors at-
tributed this difference to the different composition of the propolis; in particular, the major
compounds found in Brazilian, Cuban, and Mexican propolis samples were artepillin C,
isoflavonoids, and pinocembrin, respectively.
Macrophages exposed to LPS are characterized by an IL-10-high and IL-12-low pheno-
type and promote type II responses; they have been called M2b [45]. In presence of IL-10,
macrophages polarize towards the M2c phenotype that is involved in immunoregulation,
matrix deposition, and tissue remodeling.
The antioxidant activity of propolis has been widely studied; propolis varieties of
different geographic locations have shown an antioxidant activity between 20% and 67%
at the concentration of 20 µg/mL, showing that they are more active than our extract [46].
Kurek-Górecka et al. [47] reported antioxidant activity of propolis at concentrations much
higher than that used in our experiments. Finally, Bonamigo et al. [48] reported antioxidant
activity of propolis extracts with IC50 values between 50 and 180 µg/mL.
The addition of BPE to chitosan films improved the antioxidant activity in a dose-
dependent way. The flavonoid content (pinocembrin and pinostrobin) of poplar bud
extracts has been shown to be responsible for its antioxidant effect [19,20]. Pinocembrin is
present in our extract to a greater extent than in propolis [13], thus confirming its excellent
antioxidant activity.

4. Materials and Methods

4.1. Preparation of Chitosan–Bud Poplar Resin (CBPR) Films
Chitosan film was prepared by dispersing 1.5% (w/w) chitosan in a solution of 0.5%
(v/v) acetic acid, 0.1% (v/v) glycerin, and 1% (w/w) ethanol 96◦ . Bud poplar resin ex-
tracts (BPE) in 85% ethanol were graciously provided by ABOCA S.p.A. (Arezzo, Italy).
Poplar buds were grounded by an electronic blender before extraction. Samples were then
centrifuged; supernatants were recovered, concentrated, and lyophilized. Samples were
maintained at 4 ◦ C. A stock solution of each bud poplar extract was prepared in ethanol at
a concentration of 100 mg/mL and maintained at 4 ◦ C. The extract was characterized as
previously described [13].
Films containing bud poplar resin were prepared by mixing 1.5% chitosan with an
aqueous solution of 0.5% (v/v) acetic acid, 0.1% (v/v) glycerin, and 1% (w/w) ethanol 96◦ ,
in which an appropriate amount of bud poplar resin extract had been previously dissolved.
Films containing 0.5%, 1%, and 2% w/w BPE were prepared. These are henceforth referred
to CBPR 0.5%, CBPR 1%, CBPR 2%.
The mixture was stirred for 24 h until a homogeneous dispersion was obtained. Subse-
quently, the air in the dispersion was removed by using a vacuum pump, and the mixture
Molecules 2022, 27, 7757 15 of 21

(20 g) was seeded in Petri dishes (diameter of 10 cm) and left to rest for 24 h. Films were
then dried at 50 ◦ C for 6 h.

4.2. ATR-FT-IR
ATR-FT-IR spectra were recorded in transmittance using a Spectrophotometer Shimadz
QATR-S IR Spirit A552. The wavenumber range was from 4.000 to 400 cm–1 with a
resolution of 4 cm−1 . All spectra were rationed against the spectra of an empty cell.

4.3. Field Emission Scanning Electron Microscopy (FE-SEM)

FE-SEM examined the morphology of the films through a LEO 1525 ZEISS instrument
(Oberkochen, Germany). The acceleration potential voltage was maintained at 1 keV.
Samples were placed onto carbon-tape-coated aluminum stubs. The stubs were sputter-
coated with chromium before imaging by a high-resolution sputter (Quorum Technologies,
East Essex, UK). The coating was performed at 20 mA for 20 s.

4.4. Water Absorption of Films

Film squares of 2 × 2 cm were stabilized in a dryer in the presence of a saturated
solution of magnesium nitrate (RH 53%). Before the assay, the films were sterilized and
weighed (W1), then films were immersed in Petri dishes containing a simulated wound
fluid (SWF 0.02 M calcium chloride, 0.4 M sodium chloride, 0.08 M tris hydroxymethyl,
aminomethane in deionized water, pH 7.5) [49,50]. The capsules were then incubated
at 37 ◦ C (±0.1). At fixed time points, the films were recovered from the fluid, and after
absorption of excess liquid, they were weighed again (W2). Hydration was determined by
calculating the change in weight using the following formula:

% Hydration. = [(W2t − W1)/W1] × 100

W1 = weight of the initial dried film

W2t = weight of the wet film at different time points (t)
Tests were made in triplicate, and results are reported as an average ± SD.

4.5. Release Test

Bud poplar resin release studies were performed by immersing circular films of
CBPR 2% (2 cm diameter) in 10 mL of a mixture composed of SWF/ethanol 80/20 v/v.
The presence of ethanol guaranteed sink conditions. The test was performed at 37 ◦ C
under mild stirring (80 rpm). At fixed time intervals, aliquots (1 mL) of supernatant were
removed and replaced by a fresh dissolution medium. Bud poplar resin concentration was
determined by UV spectrophotometry using an Agilent 8453 UV–Vis spectrophotometer at
a maximum absorbance of λmax = 290 nm [37]. The experiment was done in triplicate, and
results are reported as an average.

4.6. Microorganisms
The microbial strains used in this study were the two Gram-positive bacteria
Staphylococcus aureus (ATCC 29213) and Staphylococcus epidermidis (ATCC 35984), the Gram-
negative Pseudomonas aeruginosa (ATCC 15692), and the yeast Candida albicans (SC5314).
The bacterial cultures were maintained in Muller–Hinton agar (MHA). The day before the
test, one colony was inoculated in Mueller–Hinton broth (MHB) and incubated for 24 h at
37 ◦ C. Candida cells from stock cultures in Sabouraud dextrose agar (SDA) with 50 µg/mL
chloramphenicol were grown in Sabouraud broth at 37◦ C for 24 h. Microbial cells were
harvested by centrifugation, washed, counted by spectrophotometric analysis (600 nm),
and brought to the desired concentration in the appropriate culture medium.
Molecules 2022, 27, 7757 16 of 21

4.7. Kirby–Bauer Assay

The antimicrobial activity of CBPR films was measured by Kirby–Bauer assay in MHA
(Gram-positive S. aureus and S. epidermidis, Gram-negative P. aeruginosa) or in SDA Agar
(Candida) Petri dishes.
Microbial suspensions were prepared from an 18 h culture in a liquid medium. The
microorganisms were recovered in sterile phosphate buffer (PBS) and brought to a concen-
tration of 108 CFU/mL.
Dishes were seeded with the different microbial strains by sliding (rotating the plate
◦
60 three times). The 6 mm diameter UV-sterilized film disks were hydrated with 20 µL of
sterile water to facilitate both the placement on the surface of Petri dishes seeded with the
microorganisms and to promote the release of the active ingredients from the film. A film
of only chitosan was used as a negative control. Filter paper disks containing gentamicin
(30 µg, Oxoid) for bacterial strains and fluconazole (25 µg, Oxoid) for C. albicans were used
as positive controls. After 24 h of incubation at 37 ◦ C, the microbial growth inhibition halos
were measured. The extent of inhibition (80% growth reduction) was expressed as the
diameter of the halo zone in mm.

4.8. Biofilm Determination

The ability of CBPR films to inhibit the growth of bacterial biofilms of S. aureus,
S. epidermidis, P. aeruginosa, and C. albicans was evaluated as previously described [51] with
some modifications. Bacterial cells were grown on MHB broth medium for bacteria or
SAB broth for yeast overnight at 37 ◦ C. The following day, microorganisms were diluted
in 2% sucrose MHB or 2% sucrose SAB at the concentration of 106 –107 cells/mL and
inoculated (100 µL) in a 96-well polystyrene flat-bottom plate. Chitosan-based films 6 mm
in diameter, previously sterilized with UV rays, were layered on microbial suspensions,
and finally, 100 µL of medium were added to each well. Gentamicin (20 µg/mL) and
fluconazole (20 µg/mL) were added as a positive control for bacterial strains and C. albicans,
respectively. Plates were incubated for 24 h at 37◦ C. For dispersion study, biofilms were
grown for 24 h in a 96-well plate, then CBPR films were added and incubated for a further
24 h at 37 ◦ C. After removal of the biofilm, it was quantified by crystal violet staining [51].
The absorbance was measured at 570 nm. All samples were tested in quadruplicate, and
each experiment was repeated at least twice. The data were expressed as a mean ± SD of
the percentage of the biofilm mass calculated with respect to untreated biofilm.

4.9. Cytotoxicity Assay

After treatment with CBPR films, the viability of HuDe (human dermis fibroblast cell
line, Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, BS PRC
41) and NCTC 2544 (human skin keratinocytes, Istituto Nazionale per la Ricerca sul Cancro
HL97002), maintained in RPMI 1640 medium plus 10% FCS and penicillin 100 IU/mL and
streptomycin 100 µg/mL (cRPMI), was measured using the ViaLight Plus Kit (Lonza, ME,
USA). Cytotoxicity effect of BPE (5, 25, and 50 µg/mL) was determined on THP-1 cells
(human monocyte-macrophages) in cRPMI, and the percentage of live cells was determined
by ViaLight Plus Kit.

4.10. Human Macrophages Polarization M1/M2

The THP-1 cell line was maintained in cRPMI in a humidified incubator containing
5% CO2 at 37 ◦ C. THP-1 cells were seeded at the concentration of 2 × 105 /tube in 1 mL of
cRPMI and were differentiated into macrophages (M0) using 50 ng/mL (80 nM) of PMA
for 48 h. The PMA-containing media was aspirated gently from the activated M0, and cells
were incubated in fresh cRPMI for a further 5 days. To study the immune-modulating
effect of poplar bud extracts, M0 were treated with BPE (5, 25, 50 µg/mL) for 24 h. After
incubation, cell suspensions were centrifuged, supernatants were recovered and stored at
−20 ◦ C for cytokine determination, and pellets were suspended in PBS and transferred to a
96-well plate where cells were fixed with paraformaldehyde (PFA) 2% (100 µL for each well)
Molecules 2022, 27, 7757 17 of 21

for 15 min at room temperature. After fixation, the plate was centrifuged, and cells were
labelled at 4 ◦ C for 30 min with specific antibody mouse antihuman CD36 for M1 phenotype
and mouse antihuman CD80 and CD86 for M2 phenotype in 100 µL of fluorescent buffer
(PBS + 2% FBS + 0.1% sodium azide). After labelling, cells were re-suspended in 100 µL
of focusing fluid and transferred in tubes containing other 900 µL of focusing fluid. All
samples were read at the flow cytometer (Attune NxT, Life Technology, Thermo Fisher
Scientific, Milan, Italy).

4.11. Cytokine Determination

Culture supernatants were recovered and stored at −20 ◦ C until cytokine determina-
tion. TNFα, TGFβ1, and IL-10 were determined using an immune assay Enzyme-Linked
ImmunoSorbent Assay (ELISA) (U-Cytech Biosciences, Utrecht, The Netherlands). The
ELISA test was performed in three days. On the first day, a 96-well ELISA plate was
prepared by adding a specific coating antibody and then was incubated overnight at 4 ◦ C.
The day after, the coating antibody was removed, and the plate was washed 4 times with
wash buffer (0.05% Tween-20 in PBS). Blocking buffer (10% bovine serum albumin in PBS)
was added to the plate, then the plate was incubated for 1 h at 37 ◦ C. The blocking buffer
was removed, and specific standards and samples were added to the wells. The plate was
incubated at 4 ◦ C overnight. On the last day, the standards and samples were removed,
and the plate was washed 4 times with wash buffer. Specific detection antibody was added
to the wells, and the plate was incubated for 1 h at 37 ◦ C. The detection antibody was
removed, the plate was washed 4 times, and the SSP conjugate antibody was added to
the wells. After 1 h of incubation at 37 ◦ C, the plate was again washed 4 times, and 3, 30 ,
5, 50 -tetramethylbenzidine solution was added to the wells. After 20 min in darkness at
room temperature, the solution turned blue; the stop solution was added, and the color
became yellow. The plate was then read after 30 min at 450 nm (Infinite M200pro, Tecan,
Milan, Italy).

4.12. Human Polymorphonuclear (PMN) Cells Isolation

Heparinized venous blood was obtained from a buffy coat gently provided by the
Blood Bank of the Ospedale della Misericordia of Perugia. All donors have been informed
and they signed the consensus form (MO-SIT_06) approved by Ethics Committee CEAS
(Comitato Etico Aziende Sanitarie) (Rev. 3 Ottobre 2014) in which they authorize the use
of their sample for research studies. Heparinized venous blood was diluted with RPMI
1640 (Gibco-BRL). Human PMN were recovered by density gradient centrifugation over
Ficoll-Hypaque Plus (Amersham Pharmacia Biotech, Milan, Italy), counted, and adjusted
to the desired concentration.

4.13. Evaluation of ROS Production by Chemiluminescence Assay

Antioxidant activity was evaluated by chemiluminescence assay according to the
previous report [13] with small modifications. PMN were then stimulated with 2 × 10−8 M
PMA in the absence or presence of CBPR films. The chemiluminescence produced by the
cells was monitored for 20 min in a luminometer (Infinite M100 Tecan). The light output
was recorded as RLU (relative photons light units). Each film was tested in triplicate in two
separate experiments.

4.14. DCF Method for Detection of Intracellular ROS

The normal human keratinocyte cell line NCTC 2544 and the human dermis fibroblast
cell line HuDe were routinely maintained in cRPMI and incubated at 37 ◦ C in a humidified
incubator containing 5% CO2 . The medium was changed every 2–3 days. For experiments,
the cells were trypsinized and plated either in 96-well plates (2 × 105 cells/mL) or in a final
volume of 100 µL for 24 h.
Measurements of intracellular ROS levels in NCTC2544 and HuDe cells were made
using 20 , 70 -dichlorodihydrofluorescein diacetates (DCFH2-DA). DCFH2-DA and its aceto-
Molecules 2022, 27, 7757 18 of 21

methyl ester H2DCFDA-AM can diffuse through the cell membrane and become enzy-
matically hydrolyzed by intracellular esterase to produce nonfluorescent DCFH2 (20 , 70 -
dichlorodihydrofluorescein), which is ionic in nature and therefore trapped inside the
cell. The oxidation of DCFH2 by intracellular ROS, mainly H2 O2 , HO•, ROO•, NO•, and
ONOO− results in fluorescent 20 , 70 -dichlorofluorescein (DCF), which stains cells [52].
Measuring the fluorescence emitted from the DCF at 535 nm, after being excited at 485 nm,
allowed determination of the amount of ROS formed after the oxidation process.
Cell samples were incubated in the presence of 10 µM DCFH2-DA at 37 ◦ C in the dark
for 30 min. After this time, they were centrifuged at 1200 rpm to remove the extracellular
DCFH2-DA and then suspended in a new medium. The cells were exposed to 200 U/mL
of IFNγ and 10− 4 M of histamine in the presence or absence of bud poplar extract (5 and
50 µg/mL) for 24 h and lysed in 1 N NaOH on the following day.

4.15. DPPH Assay

The antioxidant activity of chitosan or CBPR films was evaluated by using the
2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay as previously de-
scribed [13]. When the DPPH reacts with an antioxidant compound, which can donate
hydrogen, it is reduced. The color changes from intense violet to light yellow. The ab-
sorbance was read at 517 nm using a UV-VIS spectrophotometer (Infinite M100 Tecan).
Films, hydrated with 100 µL of water, were incubated with 100 µL of the DPPH radical
(25 µg/mL) in ethanolic solution. Absorbance was measured after 30 min. The negative
control was prepared with water and DPPH in the absence of the disk. The following
formula was used to determine the antioxidant activity:

A% = 100 − [(Abs sample-Abs blank) × 100]/Abs control

4.16. In Vitro Fibroblast Migration Assay

Micro-Insert 4-well dishes (Ibidi) were used for the regenerative activity assay. The
HuDe cells were counted and seeded at a concentration of 5 × 105 /mL (15 µL in each well).
The supports with the cells were incubated at 37 ◦ C for 2 h to allow the cells to adhere to
the plate. Then, 2.5 mL of cRPMI were added, and cells were incubated for 48 h to reach
confluence. After the formation of the monolayer, the silicone insert was removed with
sterile tweezers. In the following days, CBPR films were added for 24 h at 37 ◦ C. Films were
removed, and the cell growth was monitored under the microscope (20× magnification) for
7 days until the space between the frames was closed. The distances between the frames
were recorded, and photos were taken every day.

4.17. Statistical Analysis

Differences between CBPR-treated biofilm and untreated biofilm were compared using
the Student’s t-test (two-tailed). * p-values of <0.05 were considered significant.

5. Conclusions
The antioxidant and antimicrobial effects of chitosan-based films embedded with bud
poplar resins were investigated. With the increase in the prevalence of wounds in almost
every country, there is a growing interest in the search for natural healing agents with low
cost and toxicity for the treatment of wounds and ulcers. There is indeed a great demand
for wound dressing. Considering the overall results, skin application of chitosan films
loaded with BPE seems to be a promising vegan approach to prevent and control wounds
and acute and chronic infections.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules27227757/s1, Figure S1: Citotoxicity of BPE on human
monocyte-macrophages. Results are expressed as percentage of live cells with respect to untreated
cells, considered 100. The results are expressed as mean ± SD of three different measures.
Molecules 2022, 27, 7757 19 of 21

Author Contributions: Conceptualization, D.P., V.A. and R.P.; investigation, D.P., C.R., C.C. and
M.L.L.; data curation, D.P., C.R. and M.P.; writing—original draft preparation, review, and editing,
D.P. and V.A.; supervision, C.R., M.P. and V.A.; All authors have read and agreed to the published
version of the manuscript.
Funding: This work was supported by a grant from the University of Perugia (Ricerca di base 2019).
Institutional Review Board Statement: All blood donors have been informed and they signed
the consensus form (MO-SIT_06) approved by Ethics Committee CEAS (Comitato Etico Aziende
Sanitarie) (Rev. 3 Ottobre 2014).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: All data used to support the findings of this study are available from
the corresponding author upon request.
Acknowledgments: The authors thank ABOCA SPA for providing the bud poplar extracts and
Alessandro Di Michele of the Department of Physics and Geology of the University of Perugia (Italy)
for SEM micrographs.
Conflicts of Interest: The authors declare that there are no conflicts of interest regarding the publica-
tion of this paper.

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