1. Introduction
Tissue engineering and regenerative medicine use biomaterials, cells and
bioactive agents to engineer implantable structures to replace damaged
tissues and organs. In many cases, the size of the tissue to be replaced
is considerable and once in the body, the engineered tissue might have
immune privileged zones separated from the host circulation, which can
create local havens for bacterial attachment and biofilm formation. This
constitutes a similar situation to that of peri-implantitis (infections
around dental implants) which can affect 5 to more than 10% of dental
implants.(Koldsland, Scheie, & Aass, 2010) As tissue engineering
solutions become more and more common, there is a requirement for
inclusion of antimicrobial agents as a safety precaution. However, such
antimicrobial agents should not trigger the development of antimicrobial
resistance or any undesired side effects and preferably they should have
additional properties that can support the main function of the
scaffold. One of the potential ways of achieving such an effect is the
inclusion of nanoparticles functionalised with antimicrobial agents in
scaffolds.
Polydopamine (PDA) films are versatile coatings obtained through the
oxidation of dopamine(H. Lee, Dellatore, Miller, & Messersmith, 2007)
or other catecholamines(Kang, Rho, Choi, Messersmith, & Lee, 2009)
using either dissolved oxygen in basic solutions or other strong
oxidants.(Ponzio et al., 2016; Wei, Zhang, Li, Li, & Zhao, 2010)
Knowing that PDA displays many structural similarities with
eumelanin,(Meredith & Sarna, 2006) the black–brown pigment of the
skin, and that eumelanin grains display a structural hierarchy,(Clancy
& Simon, 2001) motivating us to explore specific controlled ways to
synthesize PDA to obtain well defined and stable nanoparticles. Since
eumelanin grains of the skin are always surrounded by proteins,(Mani,
Sharma, Tamboli, & Raman, 2001) the natural way to control the size of
PDA particles is to add proteins(Chassepot & Ball, 2014) or other
polymers(Arzillo et al., 2012; Mateescu, Metz-Boutigue, Bertani, &
Ball, 2016) to the dopamine solution during its oxidation. The
properties of transferrin capped PDA nanoparticles(Hauser et al., 2018)
and the photothermal properties of such nanoparticles(Han et al., 2016)
have already been explored. These inherent properties in term of
photothermal sensitivity and versatile surface chemistry have shown
great potential for biomedical applications. For example, PDA NPs
photothermal properties have been used recently to develop drug release
carriers for cancer therapy.(Poinard et al., 2018; Wang et al., 2019)
Moreover, the versatile surface chemistry of PDA NPs have allowed the
conjugation of various bioactive agents (proteins, polyelectrolytes)
though different mechanisms such as π-π stacking, electrostatic
interactions and nucleophile addition of amine to quinone (Michael
addition).(Ball, 2018) The possible functionalization of the PDA
nanomaterials combined with their low toxicity made them good candidates
to develop bioactive carriers with properties relevant for biomedical
purposes such as anti-cancer, pro-angiogenic, anti-inflammatory or
antimicrobial activities.(Chen et al., 2015; Chia-Che Ho, 2013; S. Li et
al., 2019)
We have recently demonstrated that chain-length controlled polyarginine
(PAR) can be used as an antimicrobial agent inside thin films without
having any adverse effect on mammalian cell behaviour at concentrations
several times higher than its minimal inhibitory
concentration.(Knopf-Marques et al., 2019; Mutschler et al., 2016) The
polycationic nature of polyarginine is the underlying reason for such
antimicrobial activity, as evidenced by the high frequency of arginine
residues in naturally occurring antimicrobial peptides. Moreover,
polyarginine has been shown to have cell penetration properties and has
been utilised for DNA and RNA delivery.(Hu, Lou, & Wu, 2019; Kim,
Davaa, Myung, & Park, 2010) Thus, incorporation of polyarginine in
tissue engineering scaffolds such as hydrogels would provide not only
the required antimicrobial properties, but it can also be used for gene
delivery within the scaffold to enhance their bioactive properties.
Hence PAR can be potentially used for decoration of PDA nanoparticles
while rendering them antimicrobial and by guaranteeing that the
decoration does not hinder its antimicrobial activity.
Hydrogels due to their intrinsic properties in term of high-water
absorption, viscoelastic properties and biocompatibility can closely
simulate properties of living tissues. In particular hydrogels made of
natural polymer-based components such as collagen, gelatin, chitosan or
alginate are of particular interest since they can mimic the natural
microenvironment of an extracellular matrix.(Hoffman, 2012; K. Y. Lee &
Mooney, 2001) For this reason, these materials represent good candidates
for biomedical applications such as drug delivery, wound dressing or
tissue engineering.(Hamedi, Moradi, Hudson, & Tonelli, 2018; Hunt,
Chen, van Veen, & Bryan, 2014; J. Li & Mooney, 2016; Mohamad, Loh,
Fauzi, Ng, & Mohd Amin, 2019; Zilberman, 2015) Nevertheless, their use
is still limited by their poor mechanical properties and their lack of
stability in physiological condition. To overcome these limitations,
multiple strategies have been proposed to reinforce hydrogels. These
strategies include i) the crosslinking of the hydrogel either chemically
(creation of covalent bonds between macromolecular chains), physically
(entanglement, ionic bonds, H-bonds) or biologically (nucleotide
pairing, self-assembly, enzymatic crosslinking)(Place, George, Williams,
& Stevens, 2009) or ii) the use of filler agents such as nanoparticles
or nanofibers.(Sahraro, Barikani, & Daemi, 2018; Sheffield, Meyers,
Johnson, & Rajachar, 2018; Zhou & Wu, 2011) With both strategies the
mechanical behaviour of the hydrogel could be improved but with the use
of fillers, the hydrogel’s bio-functionality can also be tuned by the
intrinsic properties of nanoparticles (such as silver NP which are
antimicrobial)(Ribeiro et al., 2017) or by functionalizing these
nanoparticles with bioactive molecules. Thus, in this context, PAR
decorated PDA particles can be used to render hydrogels antimicrobial
and change their mechanical properties simultaneously.
The risk of contamination is a serious problem in implantable devices as
infection can even lead to implant failure and in this aspect, most of
the hydrogels used in the biomedical field are also concerned by this
issue.(Salomé Veiga & Schneider, 2013) As a consequence, antimicrobial
strategies must be envisioned in all biomedical products development.(Ng
et al., 2014; Ribeiro et al., 2017; Yu et al., 2007) PDA based
nanoparticles have been previously utilized to elaborate,
biocatalysts,(El Yakhlifi, Ihiawakrim, Ersen, & Ball, 2018)
cell-targeting agents(W.-Q. Li et al., 2017) or theranostic agent(Dong
et al., 2016) but to our knowledge, protein capped PDA nanoparticles
have never been used for antimicrobial applications particularly in
conjugation with hydrogels. Hence, we aim to develop a gelatin based
hydrogel with antimicrobial properties and enhanced mechanical
properties through the loading of polydopamine nanoparticles decorated
with an antimicrobial agent, polyarginine. This gelatin based hydrogel
should provide the optimal microenvironment for cell encapsulation while
PAR decorated PDA particles will prevent bacterial contamination after
implantation. To achieve this, the following hypotheses must be
validated:
i) PDA NPs should demonstrate the feasibility of being decorated with
PAR,
ii) the immobilization of PAR at the surface of PDA NPs should not
interfere with antimicrobial properties of PAR,
iii) PAR decorated PDA particles should retain their antimicrobial
properties in gelatin hydrogels without affecting the biocompatibility
of the hydrogels.
2. Materials and Methods
2.1 Materials
Dopamine hydrochloride (Mw = 189,64 Da, H8502, CAS: 62-31-7),
Tris(hydroxymethyl) aminomethane (Mw = 121.1 Da, T-1503, CAS: 77-86-1)
and Gelatin Type A from porcine skin (Mw = 5-10 x 104Da) were purchased from Sigma-Aldrich (St Quentin Fallavier, France).
Poly(L-arginine hydrochloride) with 30 arginine residues
(PAR30, Mw = 5.8 kDa, CAS: 26982-20-7) was purchased
from Alamanda Polymers (US). Microbial-Transglutaminase (Activa, 86-135
units/g) was kindly provided by Ajinomoto (Japan).
2.2 Methods