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