Bacteria Live Dead Assay
We performed an additional test after antibacterial assay with gelatin
hydrogels to evaluate the amount of bacteria alive or dead on the
surface of samples. We used the BacLight Redox Sensor CTC Vitality Kit
(Molecular Probes). SYTO 24 green-fluorescent nucleic acid staining is
used at 0.001 mM to counting all bacteria and CTC red staining is used
at 50 mM to detect bacteria alive.
3. Results
3.1 Synthesis and characterization of PDA-PAR30 NPs
In this study, polydopamine nanoparticles (PDA NPs) were synthesized
using a protocol developed by Chassepot et al.(Chassepot & Ball, 2014)
In this work, the ability of proteins (either negatively or positively
charged) to stabilize and control the size of dopamine aggregates formed
in dopamine solutions upon oxidation in order to get functional
nanoparticles was demonstrated. In the current work, we have used a
chain-length controlled polycation, polyarginine with 30 residues
(PAR30), known to have a strong antimicrobial
activity.(Mutschler et al., 2016) Then we have synthesized
PDA-PAR30 nanoparticles by adding a
PAR30 solution (1 mg.mL-1) in contact
with different concentrations of dopamine hydrochloride solution ranging
from 0.2mg.mL-1 to 0.5 mg.mL-1 in
alkaline Tris Buffer (pH=8.5) for 24 hours under stirring (Scheme 1). We
first studied the influence of the dopamine concentration on the
nanoparticle stability. After 2 months in a closed vial, only the
formulation synthesized with 0.2mg/mL of dopamine hydrochloride
(PDA-PAR30 0.2) was still stable without apparent
sedimentation (Figure 1A). For the other formulations containing 0.5 and
0.3 mg.mL-1 of dopamine hydrochloride (noted as
PDA-PAR30 0.5 and PDA-PAR30 0.3) a total
sedimentation was observed after few days. The size of the aggregates
was analysed using dynamic light scattering (Figure S1). Aggregates at
the microscale were found for both formulations. The size of these
aggregates was estimated at about 2 µm and 4 µm for the
PDA-PAR30 0.5 and PDA-PAR30 0.3
formulations respectively. For the stable formulation,
PDA-PAR30 0.2, Transmission Electron Microscope (TEM)
analysis was performed to determine the size of the resulting
nanoparticles (Figure 1B). Nanoparticles with a narrow size distribution
of 3.9 ± 1.7 nm (mean diameter based on the measurement of 300
nanoparticles) were obtained. As a control, we also synthesized PDA NPs
using the same protocol but without any decoration but these particles
were not stable and formed aggregates between 1-10µm (Figure S1). The
only way to obtain stable PDA based NPs was to use polypeptides or
proteins to stabilize them. Thus, for the rest of the experimental work,
we used the stable nanoparticles obtained with a solution of 0.2 mg.
mL-1 of dopamine hydrochloride
(PDA-PAR30 0.2 condition). To further probe and
demonstrate the decoration of polydopamine NPs with chain-length
controlled polyarginine, zeta potential measurements were performed. It
is known that polydopamine is negatively charged above pH 5. Indeed, the
measurement on PDA NPs gave a zeta potential value of -22.7mV (Figure
1C). By adding PAR30 to the PDA particles, we obtained
zeta potential value of +30.3 mV. The surface charge of the NPs has
switched from negative to positive values as PAR30 is a
polycation. This demonstrates that the polycation was successfully
deposited on the surface of PDA NPs.
In the next step, we have investigated the antimicrobial properties of
PDA-PAR30 0.2 NPs to check if the PAR chains maintained
their biological activity once immobilized at the surface of PDA NPs.
Indeed, our previous studies demonstrated the need for the mobility of
PAR molecules in the coatings to exert their antimicrobial activity and
this mobility in the particles is not guaranteed.(Mutschler et al.,
2016) We have tested different NPs dilutions ranging from 1:10 to 1:100
from the initial particle concentration and incubated them with S.
aureus ( 8.105 CFU.mL-1) for 24
hours. As a control, we also synthesized unmodified PDA NPs and used
them in the same range of dilution against S. aureus . The results
are presented in Figure 2A. PDA-PAR30 0.2 NPs ranging
from 1:10 to 1:50 in dilution completely inhibit the bacterial growth
(100% growth inhibition) and then from the dilution 1 :100, we can
again observe a significant bacterial growth (40% of growth inhibition
for the dilution 1:100). Based on these results, we can deduce that the
MIC (Minimum inhibitory concentration) of these
PDA-PAR30 0.2 NPs is between 1:50 and 1:100. As it was
difficult to estimate the true concentration of NPs, based on the
absence of a precise knowledge about the dopamine
oxidation-oligomerization reaction yield, we have performed this
experiment on different batches of NPs coming from different synthesis
done in identical conditions. The MIC was always estimated between 1:50
and 1:100 (Figure S2) which means that the synthesis is reproducible,
and we can assume that we always obtain comparable amount of NPs of
similar size in each synthesis. As a control, experiments performed on
PDA NPs without the decoration of PAR30 show absence of
antimicrobial activity whatever the dilution used. This means that the
antimicrobial activity of the NPs can be attributed to the presence of
PAR30 and that PAR30 chains maintain
their biological activity after immobilization on PDA NPs.
After the evidence of the antimicrobial properties of the NPs, it was
necessary to check their cytotoxicity. For this purpose, we used Balb
3T3 cells as a well-established model (mouse fibroblasts).
PDA-PAR30 0.2 NPs at different dilutions in cell culture
medium were put in contact with an almost confluent layer of 3T3 cells
for 24 hours and the viability of these cells was then estimated with a
MTT assay and compared to a control (normal medium without NPs). A
material is defined as cytotoxic if a decrease of at least 30% in
viability compared to the control is observed according to ISO/EN
109935. The obtained results are presented in Figure 2B and images of
the cell morphology are given in Figure S3. As a control, we also
investigated the cytotoxicity of Tris Buffer at a dilution 1 :10 which
represents the highest concentration of Tris used in the experiments to
dilute NPs in the cell culture medium. At this concentration of Tris
Buffer, it was shown that the buffer was not cytotoxic. This indicates
that for the rest of the experiment, if a cytotoxic effect is monitored,
it can only be attributed to the presence of PDA-PAR nanoparticles.
Finally, it was demonstrated that for PDA-PAR30 0.2 NPs,
only the 1:10 dilution exhibits a cytotoxic behaviour with a decrease in
viability of about 90%. Hence, we can conclude that
PDA-PAR30 0.2 NPs particles are non-cytotoxic at
dilutions higher than 1:10.
3.2 Design and characterization of antimicrobial Gelatin hydrogels
loaded with PDA-PAR30 NPs
(Gel-PDA-PAR30)
We have previously demonstrated that decoration of PDA NPs with
PAR30 allows to confer antimicrobial properties. In the
second part of the study, we have incorporated these PDA decorated NPs
into gelatin hydrogels to develop an antimicrobial hydrogel that can be
further used as an implant surface coating to fight infection after
implantation while providing a degradable environment for remodelling.
For this purpose, PDA-PAR30 0.2 NPs were incorporated
into gelatin type A hydrogel without any dilution
(Gel-PDA-PAR30 and the hydrogels were then crosslinked
with microbial-Transglutaminase in order to reinforce their mechanical
properties and stability. This hydrogel Gel-PDA-PAR301:1 is depicted in Figure 3A and compared to gelatin hydrogel without
NPs (Gel). We observed a brown color for the hydrogel loaded with NPs
which is the characteristic colour of PDA. The presence of NPs at the
surface of the gelatin hydrogel was checked using SEM and it was found
that the particles were homogeneously distributed. However, we can
notice that once incorporated in the hydrogel, there is an increase of
particle size from few nanometers (as found for solution dispersed NPs,
Figure 1B) to almost 100 nm. This can be explained by the aggregation of
the NPs during the hydrogel formation (Figure 3B). We then investigated
the swelling properties of the composite hydrogels and compared them to
pure gelatin hydrogels (Figure 3C). It has been found that the addition
of NPs in the gelatin network had an effect on the swelling behaviour: a
significant increase with a value of 500% was monitored, compared to
pure gelatin hydrogel where a value of only about 300% was measured.
This could be explained by Donnan effect due to the high positive charge
density of the PAR covered NPs inducing a strong incorporation of
counteranions and hence of hydration water.(Rathna, Mohan Rao, &
Chatterji, 1996) Then, the release of PDA-PAR30 0.2 NPs
from the gelatin hydrogel was studied using fluorescently labelled NPs
with PAR30-FITC (Figure 3D). We can observe a burst
release of the NPs during the first hours with most of the NPs released
during the first 3 days. However, the hydrogel was still brown after 3
days of release (data not shown), which means that some of the NPs were
still present in the gelatin network even if no further release was
recorded.
Next, we investigated the rheological properties of the
gelatin-PDA-PAR30 hydrogel composite to determine the
influence of the addition of PDA-PAR30 NPs on the
mechanical properties. We first analysed the shear viscosity in solution
of the two formulations (Gel and Gel-PDA-PAR30) without
the crosslinking agent, microbial Transglutaminase (Figure 4A). The
shear viscosity of both formulations remained constant over a large
range of shear rates (from 0.1 to 1000 s-1). Moreover,
the addition of nanoparticles in the formulation did not affect the
shear viscosity since the values of both Gel and
Gel-PDA-PAR30 formulations were similar with values
ranging from 0.1 to 0.06 Pa.s depending on the shear rate applied. After
demonstrating that the addition of NPs in the formulation did not affect
their viscosity, we crosslinked both formulations with
microbial-transglutaminase and studied the viscoelastic properties of
the resulting hydrogels. The two hydrogels were crosslinked for at least
16 hours in situ in the rheometer to ensure complete crosslinking
of the network. Then we recorded the storage modulus of the two
hydrogels as a function of frequency (Figure 4B). Over the frequency
range tested (0.1 - 50 Hz), it was found that the storage modulus (G’)
of the two formulations increased slightly as a function of frequency.
In addition, we observed a higher storage modulus in the case of
Gel-PDA-PAR30 hydrogel composite with values between 8
to almost 11 kPa, whereas for the gelatin hydrogel the values were
between 5 and 7 kPa. Thus, the addition of NPs within PDA-PARhydrogel enhanced its viscoelastic properties. We can assume that the
NPs act as a filler in the structure of the hydrogel.
After characterization of the mechanical properties of the hydrogels, we
performed a stability study in different conditions (Figure 4C, D, E):
enzymatic stability in the presence of collagenase, stability in acidic
conditions (pH=5) as a model of infection and stability in physiological
pH at 7.4 in PBS. All these experiments were conducted at 37°C and the
mass of hydrogels were recorded as a function of time. For enzymatic
stability, hydrogels were incubated in 1 mL of collagenase solution (1
mg.mL-1) at 37°C. In Figure 4C, it is shown that the
two formulations, Gel or Gel-PDA-PAR30, were completely degraded after 2
hours with the same kinetics. For the stability at pH=5, hydrogels were
incubated in 1mL of citric buffer and the experiment was performed for 7
days with an estimation of mass loss at days 1, 4 and 7 (Figure 4D).
After the first day, the hydrogel containing PDA-PAR30NPs showed better stability (86% of remaining hydrogel versus 69 % for
the pure gelatin hydrogel). But during the next days, the degradation
was indistinguishable for both formulations. After 7 days, the
difference between the two hydrogels was not significant (64%
degradation for Gel-PDA-PAR30 vs 59% for Gel). The last
stability experiment was also conducted for 7 days in PBS buffer at
pH=7.4 (Figure 4E) and the results showed the same degradation profile
between the two formulations with values of remaining hydrogel after 7
days of about 55% (56% for Gel-PDA-PAR30 vs 53% for
Gel). With this set of experiments, we can conclude that the addition of
PDA-PAR30 NPs in the gelatin structure only increases
the viscoelastic properties of the hydrogel (increase of G’, Figure 4B)
but did not change the stability of the resulting hydrogels compared to
pure gelatin hydrogels.
Once the hydrogel composites were characterized, their antimicrobial
activity starting from a dilution 1:1 (no dilution) and until 1 :50 was
tested against S. aureus (Figure 5A). Hydrogels were prepared in
PBS but as the NPs were prepared in Tris Buffer, the final hydrogel
composite was prepared in a PBS/Tris mix. It was found that
Gel-PDA-PAR30 hydrogels exhibit complete antimicrobial
activity with a 100% growth inhibition after 24 hours in contact withS. aureus until a dilution of 1:4. For higher dilutions (1:10 and
1:50), the antimicrobial activity was lost because the NPs were probably
too diluted in the hydrogel to maintain a biological activity. SEM
analyses were also performed to compare the behaviour of
Gel-PDA-PAR30 (1:4) vs Gel (Figure 5B). After 24 hours
of contact with S. aureus , the bacteria fully colonized gelatin
hydrogel and started to form a biofilm whereas with the NPs loaded
hydrogel almost no bacteria were found. Zooming on the
Gel-PDA-PAR30 surface allows to show the presence of
particles at the surface of the hydrogel which confer the antimicrobial
activity.
These results are also in agreement with the metabolic activity of
bacteria, using CTC/Syto 24 staining where no viable bacteria were found
on the hydrogel loaded with NPs (Figure S4). For final applications, the
gel formulation with NPs diluted in a ration 1:4 seems to be optimal.
Then, the cytotoxicity of the formulation Gel-PDA-PAR30.
has been tested with Balb 3T3 cells using extraction method following
ISO Standard 10993-5 recommendations. As the hydrogel composite was
prepared in a PBS/Tris mix, we have first tested the cytotoxicity of
pure gelatin hydrogels prepared in either Tris or PBS and then the
cytotoxicity of hydrogel prepared in different ratios of PBS/Tris
(Figure S5). It was found that none of the configurations tested
exhibited cytotoxicity with viability up to 70% maintained in all
cases. Then, we prepared Gel-PDA-PAR30 hydrogel
composites starting from a dilution 1 :4 and until 1:50 (NPs were also
diluted from 1:4 to 1:50 in the hydrogel) and studied their
cytotoxicity. None of the formulations tested were cytotoxic and a
viability up to 85% was maintained (Figure 6). The resulting cell
morphology is given in Figure S6 and there was no evidence of anormal
cell shape. The formulation 1:4 was tested towards Balb 3T3 cells and
its viability was estimated to be around 90%. Hence we can conclude
that this formulation was not cytotoxic. Thus, we have designed a
powerful antimicrobial hydrogel ony based on natural and bioderived
materials, i.e gelatin, polydopamine and polyarginine. An
adequate formulation has been obtained both in term of antimicrobial
activity with 100% bacteria growth inhibition and in term of
biocompatibility.
4. Discussion
4.1 Polyarginine decorated PDA NPs are stable and antimicrobial
Polydopamine NPs-based chemistry is versatile and should open the road
to develop bioactive biomaterials with the possibility to decorate
nanoparticles with multiple bioactive agents enabling us the design of
hydrogel composites with bioactive properties. As these particles have a
wide range of inherent properties as a potential substrate for
photothermal effect or as quenchers and catalysts, they can be used in
many biomedical applications including cancer therapy and stimuli
responsive drug delivery. Adding a layer of bioactive agents on this
structure creates a tool that is highly modular and can have
applications in incorporating theranostic delivery and biosensing for
tissue engineering and regenerative medicine applications. In this
study, our main goal was to create stable polydopamine based
nanoparticles with antimicrobial properties and to do that we have used
a known polypeptide antibiotics (polyarginine).
In our study, we have first demonstrated that the addition of a
sufficient amount of PAR30 to a sufficiently diluted
solution of dopamine, 0.2 mg.mL-1, in Tris buffer
allows to produce small PDA-PAR and stable nanoparticles having an
average diameter of 3.9 ± 1.7 nm and displaying a positive zeta
potential of about +30.3 mV compared to pristine PDA nanoparticles
having a zeta potential close to -23 mV in the same conditions (Figure
1D).
Polyarginine, a positively charged polymer, has been originally used as
a highly efficient cell penetrating peptide for gene delivery
applications. Beyond this cell penetrating properties, due to its
polycationic nature, polyarginine is a widely used component to build
polyelectrolyte multilayers. Recently, it has also been shown that
PAR18 and also its D-enantiomer has neuroprotective
properties.(Meloni et al., 2019) As mentioned before, polyarginine also
has antimicrobial activity whose efficacy is related to the mobility of
PAR chains within polyelectrolyte structures.(Dong et al., 2016) In
solution, a wide range of polyarginine chain lengths have shown
antimicrobial activity; however in film configurations where
polyarginine was in interaction with polyanions (heparin, chondroitin
sulfate, alginate, hyaluronic acid) only a narrow range of chain length
and only films with HA were active. Thus, decoration of polyarginine on
PDA particles might have hindered the antimicrobial activity of
polyarginine due to the lack of mobility.
However, our experiments demonstrated that polyarginine chains on PDA
surfaces sustain their antimicrobial capacity. Indeed, the
PDA-PAR30 0.2 NPs display an antimicrobial activity
against S. aureus up to a dilution of about 1:50 to 1:100 (Figure
2A) whereas the PDA NPs are not antimicrobial whatever their dilution;
this demonstrates that the antimicrobial activity arises from the
presence of PAR on its surface and also that the particle formation does
not hinder the antimicrobial activity of PAR, excepting the cases of
thin layer-by-layer coatings where the antimicrobial properties depend
on the polyanion. This is due to the fact that, even though immobilised,
PAR chains are at the surface of the particles thus they are free to
electrostatically interact with bacterial membranes even though the
interaction is partially shared with the interaction with the PDA
surface. Our experimental results clearly demonstrated that such
interactions cannot overshadow the antimicrobial properties of
polyarginine.
4.2 PDA-PAR NPs incorporated hydrogels have improved viscoelastic
properties and antimicrobial capacity
The need for antimicrobial coatings for non-degradable implants has been
long recognized such with systems such as TYRXTMantimicrobial, degradable polymer pouch used for pacemakers in clinical
applications. The surface of non-degradable implants, can create an
immune-privileged zone that can facilitate the bacterial attachment and
the biofilm formation. For example, the rate of infections with fixation
devices were estimated between 2-5% The replacement of passive implants
with engineered artificial tissues can in one way alleviate this problem
as the porous nature of such scaffolds will enable the angiogenesis.
Consequently, the implanted structure will integrate with the host
immune system rather than being an isolated surface. But, on the other
hand, materials such as natural polymers (as for instance collagen,
gelatin, hyaluronic acid etc.,with certain exceptions like chitosan)
used for scaffolds generation can also boost the attachment and
proliferation of bacteria just as well that of the host tissue.
Moreover, potential use of engineered tissues in non-sterile wounds
would also necessitate an antimicrobial component. Previously, such
precautions has been tried to be put in place by either doping
hydroxyapatite with silver or copper to confer antimicrobial activity to
it(Sahithi et al., 2010; Stanić et al., 2011) or incorporation of silver
based nanoparticles in the scaffold formulations. (Saravanan et al.,
2011) Specifically, with gelatin foams crosslinked with genipin,
Yazdimamaghani et al. demonstrated antimicrobial activity againstE. coli and S. aureus by in situ formed silver
nanoparticles.(Yazdimamaghani et al., 2014) However, it has been shown
that silver nanoparticles are less cytotoxic compared to silver
sulfadazine which at high concentrations can demonstrate hepatotoxicity
in clinical settings.(Trop et al., 2006) Moreover, at the cellular
level, it has been shown that silver exhibits mild cytotoxicity against
mesenchymal stem cells, which are one of the major cell sources in
tissue engineering.(Samberg, Loboa, Oldenburg, & Monteiro-Riviere,
2012) Thus, alternative antimicrobial solution would be beneficial,
particularly in formats that would also contribute to the mechanical
properties of the overall scaffold in tissue engineering. The
nanoparticles developed in this study could potentially achieve these
two effects concomitantly in the context of hydrogels.
In this study, to demonstrate such utility of PDA-PAR NPs, we have used
gelatin hydrogels, which creates a highly amenable surface for bacterial
attachment as demonstrated by our results. The incorporation of the
particles in the gel structure resulted in an increase in G’ values
which indicates a slightly stiffer hydrogel due to the nanoparticle
based reinforcement. The incorporation of the particles in the gel
structures could decrease their mobility and as a result their
antimicrobial capacity; however, experiments showed that it was possible
to maintain antimicrobial activity of the NPs at dilutions below the
cytotoxic limits, the reason for this observation being the ability of
the hydrogels to release the NPs as demonstrated by the release
experiments. The relatively controlled manner of release of the NPs also
points out using the hydrogels as a potential delivery system for such
NPs.