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Iemma and O. Vittorio, J. Mater. Chem. B, 2019, DOI: 10.1039/C9TB00871C.

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DOI: 10.1039/C9TB00871C

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Journal of Materials Chemistry B Accepted Manuscript

Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Combining antioxidant hydrogels with self-assembled
microparticles for multifunctional wound dressings †
Mariagrazia di Luca,‡,a,b,c Manuela Curcio,‡,d Emanuele Valli,‡,e,f Giuseppe Cirillo,*,d Florida Voli,e
Maria Eugenia Butini,b,c Annafranca Farfalla,d Elvira Pantuso,d Antonella Leggio,d Fiore Pasquale
Nicoletta,d Arianna Tavanti,a Francesca Iemmad and Orazio Vittorioe,f,g

A multi-functional composite to be employed as dressing material was prepared by combining hydrogel and microparticle
systems. For the synthesis of hydrogel counterpart, a free radical polymerization was carried out using a Gelatin-Curcumin
conjugate, previously obtained through immobilized laccase catalysis, and polyethylene glycol dimethacrylate as functional
element, plasticizer, and crosslinker. Hydrogel was found to possess high water affinity, biocompatibility, and ability to
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reduce the H2O2 induced oxidative stress on MRC-5 cells by 30%. The spherical microparticle system (mean diameter of 1.75
µm) was prepared by self-assembly of a Keratin-methacrylated polyethylene glycol-40 stearate derivative synthesized by a
free radical reaction. The final composite, prepared by absorption of microparticles on hydrogel system, was found to be
effective as support for enhanced cell growth (3.5 times). Furthermore, a reduction of methicillin-resistant Staphylococcus
aureus proliferation by 1 log10 CFU was reached taking advantage of the sustained release of the antimicrobial Quercetin.

injuries involves a wound cleaning process followed by

1. Introduction antibiotic prophylaxis,20, 21 since open wounds have a potential
for serious bacterial infections, which, in turn, may lead not only
Wound healing is a natural and dynamic process, involving
to inappropriate healing, associated with clinical signs such as
multiple integrated events at both cellular and molecular level,
pain, erythema, edema, and exudate development, but also to
aimed at restoring the structural and functional features of a
long term disabilities.22-24 Furthermore, an over-generation of
damaged tissue (e.g. skin).1-3 In order to maintain favorable
free radical species at the healing site can delay the healing
conditions at the wound site and facilitate tissue regeneration,
process due to serious damages of surrounding tissues, thus the
each step of the healing process, including inflammation,
administration of antioxidants has been proven to have
proliferation, and remodeling,4, 5 can be targeted by the use of
beneficial effects.25, 26 Finally, the use of functional components
active dressing materials.6-8 An ideal active dressing should
promoting cell growth has gained particular interest in
promote a rapid and painless healing by coupling the basic
designing active dressing materials.27
characteristics of conventional dressing systems (e.g. the
Here we propose a hydrogel/microparticle composite as
absence of cytotoxicity and the maintenance of a moist
dressing materials with tailored activities, with the ultimate aim
environment with physiologic pH and temperature values)9-11
to combine the properties of the single components in a multi-
with the functional activities of smart materials such as the
functional device. Hydrogels have been proven to be valuable
prevention of microbial infections,12-14 protection from toxic
materials for wound dressing due to their high biocompatibility
effects of Reactive Oxygen Species (ROS),15-17 and amelioration
and non-antigenicity, flexibility, and capability to maintain a
of tissue regeneration.18, 19 An appropriate management of
moist environment at the wound site promoting the autolytic
debridement of necrotic tissues and the healing process.28, 29
a. Department of Biology, University of Pisa. Microparticles offered solutions to the burst release of payloads
b. Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität
Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Center for
from hydrogel-based drug delivery systems, ensuring a more
Musculoskeletal Surgery, Charitéplatz 1, 10117 Berlin, Germany. sustained release over time.30-32 For the synthesis of either
c. Berlin-Brandenburg Center for Regenerative Therapies, Charité –
hydrogel or microparticle counterpart, we choose fibrous
Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany.
d. Department of Pharmacy, Health and Nutritional Sciences, University of Calabria protein materials, namely Gelatin (Gel) and Keratin (Ker), owing
– 87036 – Rende (CS) – Italy. E-mail: giuseppe.cirillo@unical.it to their high chemical versatility and similarity to natural
e. Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney, NSW,

Australia 2031.
f. 6School of Women’s and Children’s Health, Faculty of Medicine, UNSW Sydney,
NSW, Australia 2052.
g. ARC Centre of Excellence for Convergent BioNano Science and Technology,
extracellular matrix.33-36 Hydrogel support (HGC) was
synthesized by covalent incorporation of a Gelatin-Curcumin
bioconjugate (Gel-CUR) into an acrylic network to take

Australian Centre for NanoMedicine, UNSW Sydney, NSW, Australia 2052. advantage from the peculiar features of Gel and Curcumin
‡ Equal contribution. (CUR) in wound healing: gelatin-based hydrogels possess,
†Electronic Supplementary Information (ESI) available. See DOI:
10.1039/x0xx00000x.
indeed, biocompatibility, non-immunogenicity, and capability

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to promote homeostasis and preserve epidermal growth factor
from proteolysis,37-40 while CUR has been proven to facilitate
wound healing by quenching free radical species.41, 42
Moreover, the microparticle counterpart consisted in self
assembling lipidized Keratin (l-Ker) microparticles loaded with
Quercetin (Q), designed to combine the favorable properties of
Ker for regenerative medicine applications (e.g. improved cell
attachment and proliferation),43-45 with the significant anti-
bacterial activities of Q against the microbial species involved in
most of the wound infections.46, 47
2. Experimental section
2.1. Synthesis of Gel-Cur conjugate
The synthesis of Gel-Cur conjugate was as follows: Gel (300 mg,
Ph Eur, Bloom 160) was dissolved in a mixture (20 mL) of
DMSO/phosphate buffer solution (10-3 mol L-1, pH 6.8) 25/75
vol/vol, then CUR (36 mg) and immobilized Laccase (0.12 U),
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prepared as previously reported,48 were added and allowed to
react at 37 °C under 70 rpm. After the reaction time (12 h), the
Gel-Cur conjugate was subjected to dialysis (dialysis tubes of 6–
27/32″ Medicell International LTD, MWCO: 12,000-14,000 Da)
against i) reaction medium and ii) distilled water to remove the
unreacted CUR and the residual DMSO, respectively, and then
freeze-dried (Micro Modulyo, Edwards Lifesciences, USA) to
obtain vaporous solids. The complete removal of unreacted
CUR was checked by High-Pressure Liquid Chromatography
(HPLC) analysis of the dialysis medium on a Jasco PU-2089 Plus
liquid chromatography equipped with a Rheodyne 7725i
injector (fitted with a 20 μL loop), a Jasco UV-2075 HPLC
detector operating at 420 nm, Jasco-Borwin integrator (Jasco
Europe s.r.l., Milan, Italy). The stationary phase consisted in a
Tracer Excel 120 ODS-A column particle size 5 μm, 15 x 0.4 cm
(Barcelona, Spain); while methanol at a flow rate of 1.0 mL min−1
was used as the mobile phase.49
1H-NMR spectra (300 MHz, DMSO-d ) were recorded on a
6
Bruker Avance 300 (Bruker Italy, Milan, Italy).
2.2. Characterization of Gel-Cur conjugate
2.2.1. Determination of free amino groups. The determination of
free amino groups in Gel-CUR vs Gel was performed as
follows:50 2,4,6-trinitrobenzenesulfonic acid solution (0.5 mL,
0.01 % w/v) was added to NaHCO3 (1.0 mL, 0.1 mol L-1, pH 8.5)
containing Gel-CUR or Gel (2.0 mg mL-1), mixed in the dark and
allowed to stand at 37 °C for 4 h in a water bath under shaking.
Finally, reaction was terminated by adding HCl (25 μL, 1 mol L-1)
and SDS (50 μL, 0.35 mol L-1) and the absorbance measured at
335 nm on a V-530 Jasco UV-Vis spectrophotometer operating
with 1.0 cm quartz cells (Jasco Europe, Milan, Italy).
The amount (mg) of CUR in Gel-Cur was calculated according to
the following Equation (1):
𝐴0 ― 𝐴1
𝑚𝑔𝐶𝑈𝑅 = × 𝑛0𝑁𝐻2 × 𝑀𝑊𝐶𝑈𝑅 (1)
𝐴0
2 | J. Mater. Chem. B, 2019, 00, 1-3
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where A0 and A1were the absorbance of Gel and Gel-Cur; 𝒏𝟎𝑵𝑯𝟐
View Article Online
and 𝑴𝑾𝑪𝑼𝑹 the amount of NH2 groups in Gel, and the
DOI: 10.1039/C9TB00871C
molecular weight of CUR, respectively.
Journal of Materials Chemistry B Accepted Manuscript
2.2.2. Trolox equivalent antioxidant capacity. The
determination of trolox equivalent antioxidant capacity (TEAC)
was performed according to the literature with slight
modifications.51 Gel-Cur conjugate (7.0 10-4 mg mL-1) was added
to 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
(ABTS) in a 0 - 1.23 10-4 mol L-1 concentration range and
incubated at 37°C for 6 min in the dark. Then, the absorbance
determined at 734 nm and TEAC calculated according to the
following Equation (2):
𝐶
𝑇𝐸𝐴𝐶 = 1.9 ∙ [𝐶𝑈𝑅] (2)
Here, [CUR] is the CUR equivalent concentration (mol L-1) in the
sample, 1.9 is the number of molecules that can be scavenged
per trolox and C, the maximal amount of ABTS scavenged by the
tested concentration of the antioxidant, was calculated by
plotting the reduction of ABTS concentration against its initial
concentration according to the following Equation (3):
𝑦 = 𝐶(1 ― 𝑒 ―𝑏𝑥) (3)
Where y is the reduction in ABTS concentration, and x is the
initial ABTS concentration.
All chemicals were from Merck/Sigma Aldrich, Germany.
2.3. Synthesis of functional hydrogels
Hydrogel system (HGC) was synthesized according to the
following procedure: Gel-Cur (300 mg) was dissolved in distilled
water (4.0 mL) in the presence of Acrylamide (AAm, 356 mg),
and polyethylene glycol dimethacrylate 750 (PEGDMA, 310 mg).
After purging with N2, ammonium persulfate (50 mg) was
added, the solution poured in a reaction cell consisting of two
10 × 10 cm2 glass plates separated with Teflon spacers
(thickness 0.8 mm) and brought together by binder clips and
then thermo-polymerized at 40°C in an oven for 24 h. Blank
hydrogel (HG) was prepared in the same conditions when native
Gel was used instead of Gel-Cur. The obtained hydrogels were
extensively washed with distilled water to remove unreacted
species and then dried under vacuum.
All chemicals were from Merck/Sigma Aldrich, Germany.
2.4. Characterization of functional hydrogels.
2.4.1. Determination of swelling profiles and antioxidant
properties. Specimens (1 cm2) were cut from each sample,
placed in a sintered glass filter (porosity G3), weighted, and
immersed into the swelling medium (PBS 10-3 M, pH 7.4) at
37°C. At suitable time intervals, excess water was removed,
samples blotted with a tissue to remove surface moisture, and
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weighed. The water content percentage (WR) was expressed
by the following Equation (4):
𝑊𝑠 ― 𝑊𝑑
𝑊𝑅 = 𝑊𝑑 × 100 (4)
Where Wd and Ws are the weight of dried and swollen sample,
respectively.
The determination of TAEC value was performed as reported
above by incubating the ABTS solutions with suitable amount of
HGC and HG samples (3.0 mg).
All chemicals were from Merck/Sigma Aldrich, Germany.
2.4.2. Cytotoxicity studies. The cytotoxic effects of HGC and HG
and the ability to protect from H2O2 induced toxicity were
assessed on human fibroblast lung cells MRC-5 (ATCC, In Vitro
Technologies Pty. Ltd., Australia).
Cells were cultured in Dulbecco's Modified Eagle's Medium
(DMEM) supplemented with 10% FBS, and 1% L-glutamate and
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grown as a monolayer at 37 °C in 5% CO2, and then seeded into
96-well plates (100 µL per well) at a predetermined density
(~20,000 cells) to achieve 90% confluency by the endpoint of
the assay.
For the cytotoxicity determinations, HGC and HG were
pulverized, suspended in DMEM + FCS 10% in a concentration
range 0 – 5.0 mg mL-1 and incubated with MRC-5 cells.
For the antioxidant assay, pulverized HGC and HG (20 mg mL-1)
were incubated with H2O2 in PBS (2.0 mL, 20 mmol L-1) for 1.5 h
in the dark at room temperature. Then, tubes were centrifuged
at 1500 rpm for 5 minutes and the supernatant used for cell
treatments.
Cell viability was determined after incubation at 37 °C, 5% CO2
for 72 (cytotoxicity) or 4 (antioxidant assay) hours by measuring
resazurin reduction (excitation 530 nm, emission 590 nm) on a
Versamax microplate reader (Molecular Devices, Sunnyvale, CA,
USA).52
All chemicals were from Merck/Sigma Aldrich, Germany.
2.5. Preparation of microparticle and composite system
Microparticle system (PK) were prepared by the method
previously described for the preparation of β-casein micelles
with slight modifications.53 Briefly, lipidized Keratin derivative
(5.0 mg), synthesized according to a procedure published
elsewhere,54 were added to 2.45 mL PBS solution (10-3 M, pH
7.4), and then 50 µL DMSO were added with a final stirring at
room temperature for 3 h. Microparticles were employed
without further purification because the amount of DMSO (2.0
% vol/vol) is not harmful to cells.55
The composite systems PKHGC and PKHG were prepared by
allowing HGC and HG (5.0 mg) to swell in 0.25 mL of PK prepared
as above reported.
SEM images were recorded on NOVA NanoSEM 200
(ThermoFisher Scientific, Hillsboro, OR, USA) with an
acceleration voltage of 10 kV after depositing samples onto self-
adhesive conducting carbon tape (Plano GmbH, Wetzlar,
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ARTICLE
Germany) and coating with gold to a thickness ofView
about
Article300 Å
Online
using a sputter coater. DOI: 10.1039/C9TB00871C
2.6. Evaluation of MRC-5 proliferation on composite materials
Journal of Materials Chemistry B Accepted Manuscript
To evaluate the MRC-5 proliferation on composite materials,
cells were plated in clear transparent 96-well plates at
optimized cell densities of 104 cells per well. To ensure cell
attachment, cells were seeded 24 h before being treated with
PKHGC or PKHG (5.0 mg) for 72h. The effect of the treatments on
cell viability was assessed by counting the viable cell via the
Trypan Blue exclusion method as previously described.56 Cell
viability assays were performed in the presence of 10% FBS.
Similarly, Neuroblastoma KELLY cells genetically engineered to
stably express GFP (KELLY GFP positive) were treated with the
same amount of PKHGC or PKHG and then imaged by microscopy.
2.7. Quercetin loading and in vitro release experiments
Q (Merck/Sigma Aldrich, Germany) was loaded into
microparticle (PK) and hydrogel formulations (HGC and HG) at a
20% (w/w) ratio, by involving Q solution in DMSO in either PK
preparation or hydrogel swelling. The final DMSO concentration
was 10% vol/vol in all cases. Furthermore, when HGC and HG
were used, samples were finally dried to constant weight under
vacuum. Loaded samples were coded Q@PK, Q@HGC, Q@HG,
Q@PKHGC, and Q@PKHG.
The release experiments were performed in phosphate
buffered saline (10-3 mol L-1) at pH 7.4 by inserting loaded carrier
in PBS (1.5 mL) into a dialysis bag (MWCO: 12,000-14,000 Da),
and dialyzed against fresh DMSO solution in PBS (13.5 mL, 10%
vol/vol). At suitable time intervals, the amount of Q in the
release media was determined by UV-Vis at 373 nm.57
2.8. Determination of antimicrobial activity
2.8.1. Storage and culture of bacterial strain and antimicrobial
agent. The bacterial stock of methicillin-resistant
Staphylococcus aureus (MRSA) ATCC 43300 was maintained in
cryovial bead preservation systems (Microbank; PRO-LAB
DIAGNOSTICS, Richmond Hill, ON, Canada) at -80 °C. Bacterial
strain was cultivated on Trypticase Soy Agar (TSA) (VWR
Chemicals, Leuven, Belgium) for 18 h at 37 °C. Inoculum size was
prepared according to a McFarland standard turbidity of 0.5 (λ
= 565 ± 15 nm) and determined by Colony Forming Unit (CFU)
counting. A stock solution of 1.5 mg mL-1 Q (Merck/Sigma
Aldrich, Germany) in 50% DMSO was freshly prepared before
each experiment.
2.8.2. Antibacterial activity of free quercetin. The minimal
inhibiting concentration (MIC) of Q against planktonic MRSA
was evaluated using broth microdilution following the CLSI
guidelines.58 Briefly, a standard inoculum of 1-5 × 105 CFUs mL-
1 was prepared in cation adjusted Müller Hinton Broth (CAMHB)
(Becton, Dickinson and Company, Le Pont de Claix, France) and
treated with two-fold serial dilutions of Q. Next, microdilution
tubes were incubated at 37 °C for 18 h. The MIC was defined as
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ARTICLE Journal of Materials Chemistry B

the lowest concentration of antimicrobial that completely
inhibited the growth of the MRSA strain, as detected by unaided
eye. To evaluate the bactericidal activity of Q, samples showing
no turbidity from the previous experiment were plated for
colony counting after, 18 h-incubation at 37°C (plating
PKHG, Q@PKHGC, and Q@PKHG) were added toVieweach tube
Article Online
containing bacteria and then incubatedDOI:at 37 °C for 24 h. A
10.1039/C9TB00871C
positive control consisting of untreated bacteria (no hydrogel)
was included as growth control, as well as gels incubated in
sterile CAMHB and sterile CAMHB alone as negative controls.

Journal of Materials Chemistry B Accepted Manuscript

detection limit = 50 CFUs mL-1). The minimum bactericidal
concentration (MBC) was defined as the lowest antimicrobial
concentration that determined a reduction ≥ 3log10 in CFUsmL-
1, as compared to the initial inoculum (T ), according to CLSI
0
guideline for antimicrobial testing.58
2.8.3. Antibacterial activity of loaded quercetin. The
antibacterial activity of Q@PKHGC and Q@PKHG was assessed by
macrobroth dilution in order to evaluate the number of viable
bacteria remaining after quercetin/curcumin exposure, as
previously reported.59 Briefly, a standard inoculum of 1-5 × 105
CFUs mL-1 was prepared in CAMHB/DMSO (10% vol/vol) in test
tubes (final volume 1mL). Next, 5.0 mg of each sample (PKHGC,
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After incubation, aliquots of each sample were serially diluted
and plated for colony counting, after 24 h-incubation at 37 °C
(plating detection limit = 50 CFUs mL-1).
2.9. Statistical analysis
Experiments were performed in triplicate and results were in
agreement within the relative standard deviation. One-way
analysis of variance was performed to assess the significance of
the differences among data, and Tukey–Kramer post-test was
used to compare the data from different treatments (P < 0.05
was considered statistically significant).

Fig. 1. Schematic representation of the synthesis of multi-functional hydrogel/microparticle composite.

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3. Results and Discussion View Article Online

DOI: 10.1039/C9TB00871C
The fabrication of multi-functional hydrogel/microparticle
composite is sketched in Fig. 1 and involved: i) the synthesis of the
protein conjugates, Gel-CUR (Fig. 1A) and l-Ker (Fig. 1B), ii) the

Journal of Materials Chemistry B Accepted Manuscript

preparation of hydrogel (HGC, Fig. 1C) and Quercetin-loaded
microparticles (Q@PK, Fig. 1D), and iii) the combination of HGC and PK
in the final device (Fig. 1E).

3.1. Synthesis and characterization of protein conjugates

Gel and Ker were modified in order to insert specific features:
antioxidant properties were conferred to Gel by conjugation with
CUR, while the conjugation of methacrylated polyethyleneglycol-40
stearate (PEG40STMA) to Ker allowed obtaining amphiphilic
derivative (l-Ker).
Gel-CUR was synthesized via a heterogeneous enzyme catalysis
where Cur, oxidized by the laccase residues immobilized into acrylate
polymer network (HL), reacted with the nucleophilic groups (mainly
Fig. 2. 1H-NMR spectra of Gel, Cur and Gel-Cur conjugate.
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amino groups) in the side chain of the Gel (Fig. 1A).60-62 The use of
immobilized laccase was previously successfully exploited for the
derivatization of polysaccharide and protein materials with
polyphenols species,63-66 with the advantages to address the
requirements of a green environment (e.g. absence of any trace of
toxicity) and obtain high functionalization degree and purity of the
final bioconjugates.67-69 The conjugation between Cur and Gel was
proved by the presence of the characteristic CUR signals in the
aromatic resonance area of the Gel-Cur 1H-NMR spectra (Fig. 2).
Furthermore, the Uv-Vis spectra showed a shift of the Cur absorption
peak from 466 to 404 nm upon conjugation (Fig. S1).
Since the conjugation reaction mainly involved the NH2 groups of Gel,
their determination in Gel vs Gel-Cur by TNBS assay was used to
calculate the amount of linked Cur, obtaining a value of 79 mg per g
of conjugate. Finally, the determination of the trolox equivalent
antioxidant capacity (TEAC) by testing the ABTS quenching activity, 51
clearly proved that the conjugation procedure did not affect the
antioxidant power, with TEAC values being 2.43 ± 0.49 and 3.30 ±
0.48 for free and conjugated Cur, respectively (p>0.05).
l-Ker was prepared by conjugation of hydrolyzed Ker with
PEG40STMA via a free radical reaction, as previously reported for the
preparation of self-assembling delivery vehicles for targeted release
of lipophilic anticancer drugs.54
In detail, PEG40STMA was prepared via trans-esterification of PEG40ST
with methacrylic anhydride (MA) using pyridine as a catalyst, and
then reacted with Ker thiol groups preliminarily activated by S2O8−
radicals (Fig. 1B). This approach allowed the preparation of l-Ker with
a substitution degree of 27% and a critical aggregation concentration
(CAC) of 10 µg mL-1, as per Ellman’s test and Pyrene fluorescence
assay, respectively.54
3.2. Synthesis and characterization of hydrogel systems
As reported in literature, gelatin gels possess low stability, and thus
different strategies have been developed to improve the thermal and
mechanical properties of gelatin-based polymer networks.70-72 We
previously reported on the possibility to covalently incorporate
gelatin into acrylate hydrogels via free radical polymerization, using
AAm as plasticizing co-monomer and PEGDMA as crosslinking agent,
obtaining materials with high water affinity (WR of around 250 %)
proposed for healing applications.59, 73 Here, following the same
approach, Gel-Cur was successfully employed for the preparation of
HGC (0.8 mm thickness) (Fig. 1C), optimizing the reagent ratio in order
to maximize the water affinity while avoiding hydrogel breakage
upon drying.

Fig. 3. SEM images of HGC (A); PK (B) and PKHGC (C). Calibration bars denote 20 µm.

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Fig. 4. MRC-5 viability after 4 h incubation with H2O2 pre-mixed with HG (■) or
HGC (■).
As a control sample, HG was synthesized by replacing Gel-Cur with
native Gel. HGC was characterized by a rough surface, as per SEM
investigation (Fig. 3A), and increased swelling profile over time, with
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a maximum WR of 990% after 24 h (Fig. S2). The higher water affinity
recorded for HGC compared to HG matrix (maximum WR of 710%) can
be attributed to the interference of Cur into the polymerization
mechanism: Cur moieties, indeed, acted as quenching agent for the
growing polymer chains, resulting in a reduced crosslinking degree.74
This finding was further supported by the determination of ultimate
tensile strength, which was reduced by 25% compared to the sample
prepared in the absence of Cur (22.5 and 30.0 MPa for HGC and HG,
respectively). The key requirement of absence of cytotoxicity was
assessed by checking the viability of Human foetal lung fibroblasts
MRC-5, a widely recognized human skin equivalent for in vitro assays,
well matching with the EU regulations encouraging replacement of
animal models (EU Directive 2010/63/EU).75, 76 Such cells, by virtue
of their specific metabolic features and high sensibility to almost any
types of chemical species, were found to be useful for the
classification and risk assessment studies of chemicals and
biomedical devices.77-80 After incubation of HGC and HG in
concentrations ranged from 1.25 to 5.0 mg mL-1 (corresponding to
the highest amount of samples to be suspended in cell culture media)
for 72 h, viability values higher than 97 % were recorded in all
conditions (Fig. S3), proving the high biocompatibility of the
proposed hydrogel systems.
In consideration of the recorded antioxidant performance of Gel-Cur
conjugate, we tested the ability of HGC formulations to protect
wounds from oxidative stress produced by H2O2, which is well known
to cause high mortality of MRC-5 cells.81.
At first, the maintenance of Gel-Cur antioxidant properties was
confirmed by determining the HGC TEAC value, which was found to
be 1.48 ± 0.48 with HG showing no ability to quench ABTS radical; and
then the MRC-5 viability upon exposure to H2O2 was assessed.
For the latter determination, MRC-5 viability was assessed after
treatment with H2O2 previously incubated with either HGC or HG,
proving that HGC was able to reduce the oxidative stress by 30% (H2O2
equivalent IC50 value increased from 186 to 246 µmol L-1). As
expected, no interference was detected when HG was used (Fig. 4).
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View Article Online
DOI: 10.1039/C9TB00871C
Journal of Materials Chemistry B Accepted Manuscript
Fig. 5. Fluorescence microscopy image of GFP positive KELLY cells cultured
on non-fluorescent PKHGC.
3.3. Synthesis and characterization of Keratin microparticles
We explored the suitability of l-Ker for the preparation of the
microparticle system (PK- Fig. 1D) able to release the antimicrobial
agent Q, by taking advantages from the ability to both encapsulate
lipophilic agents and to promote cell proliferation.54 Due to its
amphiphilic behavior, l-Ker self-assemblies in water media with the
hydrophobic blocks (mainly stearyl residues) oriented toward the
inner core and the hydrophilic portions of Ker and PEG towards the
external environment. Morphological analysis and measurement of
particle size distribution, performed by SEM analysis (Fig. 3B),
showed spherical microparticles with a smoot and uniform surface
(see inset of Fig. 3B) and a mean diameter of approximately 1.75 µm.
Q@PK was obtained by performing the self-assembly process in the
presence of a Q solution in DMSO (saturated solution, 30 mg mL-1), a
methodology widely reported in the literature for the encapsulation
of different lipophilic drugs in protein self-assembling systems.82, 83
3.4. Synthesis and characterization of assembled devices
HGC and PK were assembled to form the final PKHGC composite (Fig.
1E), with SEM analysis showing PK embedded into hydrogel matrix as
a confirmation of the effective absorption onto HGC without
modification of either shape or size patterns (Fig. 3C). The ability to
induce cell proliferation plays a key role in assessing the performance
of a functional dressing device, thus facilitating the healing
process.84, 85 In our experiments, PKHGC was found to promote cell
viability, with the number of MRC-5 cells increasing by 3.5 times
compared to the control after 72 h incubation. PK acted as the
functional element for cell proliferation, since un-assembled HGC did
not showed any effect on cell viability (Fig. S3).
Furthermore, the incubation of PKHGC with cells transfected with a
Green Fluorescent Protein (GFP) has been used as noninvasive
approach for assessing the cells activation in a healing process.86, 87
In our case, Neuroblastoma KELLY-GFP cells were used because they
are easy to transfect88 and possessed proved ability to fast migrate
in a wound-healing assay.89 Inverted fluorescence microscope
analyses revealed the presence of fluorescent cells on the non-
fluorescent hydrogel support, confirming the ability of growing GFP
cells to colonize the device (Fig. 5).
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Journal of Materials Chemistry B ARTICLE

Here, kR the release rate constant. View Article Online

To underline the effect of each component (hydrogel, l-Ker, Cur) on
DOI: 10.1039/C9TB00871C
the release kinetics, Q was also loaded on PKHG, PK, HGC and HG.
Release data are shown in Fig. 6 and Table 1 together with t1/2 values,
representing the time required for reaching 50% of Fmax and being

Journal of Materials Chemistry B Accepted Manuscript

calculated by the following Equation 8, 9 for the reversible first- or
second-order kinetics, respectively:

𝑭𝒎𝒂𝒙
𝒕𝟏𝟏/𝟐 = 𝒌𝑹 𝒍𝒏𝟐 (8)

𝛼
𝑡21/2 = 2𝑘𝑅ln (3 ― 2𝐹𝑚𝑎𝑥) (9)

The proposed model is suitable for describing Q release from all

Fig. 6. Q release profile from Q@PK (●); Q@PKHGC (♦); Q@PKHG (◊); Q@HGC
(■); Q@HG (□), showing the ability of microparticle system to extend the
release over time compared to un-assembled hydrogels.
Published on 10 June 2019. Downloaded on 6/11/2019 3:41:00 AM.
carriers, with microparticles significantly modifying the release
kinetics form a reversible first-order, recorded for Q@HG and Q@HGC
samples, to a reversible second-order in the Q@PKHGC,Q@PKHG, and
Q@PK cases (see R2 values in Table 1). The comparison between
Q@HG and Q@HGC behavior proved that the higher water affinity of

HGC resulted in a fast Q release (t1/2 of 36 vs 27 min).

Furthermore, microparticles were found to affect the Q release by
To investigate the suitability of the PKHGC composite as delivery
reducing the Fmax values recorded in both Q@HG and Q@HGC cases (~
vehicle for the antimicrobial agent Q, the release profile was
95% in 300 min), to around 80% in 3500 min. More interestingly, the
analyzed in terms of physico chemical affinity towards the carrier vs
Q release was significantly extended over time, with t1/2 moved to
the surrounding media. According to a mathematical model available
265 min for Q@PK. A further reduction of the releasing rate was
in the literature,90 this phenomenon can be described by the α
recorded when Q@PK were absorbed onto the hydrogels, with a
parameter (Equation 5):
lower swelling degree (HG) resulting in a more evident effect (t1/2 of
𝐹𝑚𝑎𝑥 500 and 347 min for Q@PKHG and Q@PKHGC, respectively. Overall,
𝛼 = 1 ― 𝐹𝑚𝑎𝑥 (5) the obtained results proved the suitability of the proposed
hydrogel/microparticle composite as Q delivery device, and allowed
where Fmax is the maximum value of relative release (Mt/M0).By this us to hypothesize its potential applicability in wound healing in view
approach, reversible first- or second-order kinetics equations of the possibility to have a sustained Q release over time.
(Equation 6, 7) can be applied: An appropriate dressing material should be able to minimize the risk
of infection, by protecting wounds from the proliferation of
( )𝑡)
𝑘𝑅
𝑀𝑡 ― 𝐹𝑚𝑎𝑥 pathogenic bacteria.91, 92 Here, we evaluated the ability of Q loaded
𝑀0 = 𝐹𝑚𝑎𝑥(1 ― 𝑒 (6) PKHGC to inhibit/reduce the growth of MRSA, since it is widely
reported that this flavonoid possesses antimicrobial properties. In
𝑘𝑅

𝑀𝑡 𝐹𝑚𝑎𝑥(𝑒
( ) ― 1)
2
𝛼
𝑡 addition, Q is contextually less toxic and provides fewer side effects
= (7) compared to that of conventional antimicrobial agents.46
𝑀0 𝑘𝑅

1 ― 2𝐹𝑚𝑎𝑥 +𝑒
( ) 2
𝛼
𝑡

Table 1. Kinetic parameters as per applied mathematical modelling.

Parameter Q@HG Q@HGC Q@Pk Q@PkHG Q@PkHGC

R2 0.9680 0.9989 0.8709 0.9774 0.8890

Fmax 0.94 0.98 0.79 0.75 0.76
𝑴𝒕
= 𝑭𝒎𝒂𝒙(𝟏 ― 𝒆 ― (𝒌𝑹/𝑭𝒎𝒂𝒙)𝒕
) α 15.7 49.0 4.00 3.00 3.17
𝑴𝟎
kR (10 )
-2 1.80 2.50 0.23 0.12 0.19
𝒕𝟏𝟏/𝟐(min) 36 27 241 433 277
R2 0.9114 0.9574 0.9087 0.9898 0.9245
𝒌𝑹

𝑴𝒕 𝑭𝒎𝒂𝒙(𝒆
𝟐 ( ) ― 𝟏)
𝜶
𝒕 Fmax 0.98 0.99 0.85 0.82 0.84
= Α 49.0 99.0 5.67 4.56 5.25
𝑴𝟎 𝒌𝑹

𝟏 ― 𝟐𝑭𝒎𝒂𝒙 + 𝒆
( ) 𝟐
𝜶
𝒕 kR (10-2) 3.0 4.5 0.28 0.14 0.21
𝒕𝟐𝟏/𝟐 (min) 32 22 265 500 347

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ARTICLE Journal of Materials Chemistry B

View Article Online

DOI: 10.1039/C9TB00871C
by proving that each of the above mentioned requirements were met
by either hydrogel or microparticle counterparts in suitable in vitro
experiments. Overall results can be considered a valid starting point
for addressing the key needs of a functional healing device.

Journal of Materials Chemistry B Accepted Manuscript

Conflicts of interest
There are no conflicts to declare.

Acknowledgements
This work was supported by University of Calabria (Italy) funds
(ex 60%).
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Fig. 7. Antibacterial activity of different formulations releasing quercetin
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