**1. Introduction**

The use of growth factors in tissue engineering has been widely studied due to their ability to encourage healing and tissue growth [1]. A key example of an application where growth factors could provide significant benefit is for enhancing healing of chronic wounds. Chronic wounds are a significant issue in healthcare, severely affecting quality of life of affected people and contributing 2% to the total health care expenditure in countries such as Australia, the U.K. and the U.S.A. [2,3]. Applying growth factors to a wound site could promote healing by mimicking a healthy body's natural response to injury, i.e., by delivering the growth factors to a wound bed, the migration and proliferation of cells will be promoted. Of particular interest is fibroblast growth factor-2 (FGF-2) which is one of several biomolecules that are responsible for signalling cell migration and proliferation in the body [4]. FGF-2 is part of the 22-member FGF-family and has been shown to promote angiogenesis, cell proliferation, migration, and differentiation [5]. FGF-2 is commonly studied as a model growth factor in materials science studies for wound healing applications [6–10] and has been shown to reduce healing time [11,12]. However, there are limitations in regards to the delivery of growth factors, i.e., the growth factor must be protected from degradation and delivered at the optimal stage of healing at the right dose and for the correct duration [13].

Polyelectrolyte multilayers (PEMs) are surface coatings that offer potential advantages for delivering growth factors in many biomedical applications, such as vascular repair [14] and also in wound healing. When deployed for wound treatment, the architecture of the PEM film can be designed to give a burst release [15–17] or slow diffusion of the peptides from the film [18]. Delivery of target molecules can be partially controlled through changing the conditions of multilayer assembly including ionic strength [19], pH [16,20,21], temperature [22] and polyelectrolyte species [8]. In addition, PEMs can be applied to many surfaces including flat planes [23], nanoparticles [24] and nanocapsules [25,26], and three-dimensional (3D) porous sca ffolds [27]. Furthermore, growth factors often show no conformational change or reorganisation upon binding to polyelectrolytes in PEMs when absorbed from solution at room temperature [5,28,29].

The goal of our study into growth factor-loaded PEM films was two-fold. First, to create a multilayer that will act as a natural matrix for the growth factor. Second, to create a multilayer host film that could have potential for synergistic therapeutic e ffect. Both of these goals were addressed through the choice of the polymers used to create the multilayers. Our work has made use of two naturally occurring biopolymers as the two major multilayer components; fucoidan and chitosan. The latter of these two polymers finds widespread application and study in the area of would healing, due to its properties: non-toxic, biocompatible, biodegradable, and anti-fungal. Past studies have also shown that chitosan promotes fibroblast proliferation [30,31]

Fibroblast growth factor-2 (FGF-2) needs to be embedded within the PEM to ensure it is protected from degradation but can remain biologically active [32]. The heparin binding site of FGF-2 is a highly positive environment due to the basic amino acid groups present [28] and can interact with negatively charged sulfate groups in heparin and heparin-mimics, including fucoidan [33–38] to create a ternary complex with the growth factor cell receptors. This complex is required for FGF-2 to be bioactive [33] and has been shown to improve skin healing [39]. Combining fucoidan and growth factors has been attempted by other groups, and has been shown to improve angiogenesis [40,41] and cell proliferation [42]. A single previous example exists of fucoidan combined with a growth factor (vascular endothelial growth factor) in a multilayer film [43]. This study found improved anti-thrombic properties and re-endothelialisation of a decellurised heart valve in response to interaction with this multilayer system.

Furthermore, fucoidan can be pro-angiogenic [38], anti-inflammatory, anti-viral, and promotes anti-bacterial activity of other molecules [44,45]. In addition, it can be pro- or anti-coagulant depending on molecular weight [46], promotes cell proliferation and migration [35,39,47] and can be immuno-modulating depending on the molecular weight and structure [48–50]. Fucoidan also inhibits MMP-2 (matrix metalloproteinase-2), an enzyme that degrades type IV collagen, a major component of basement membranes upon which the epithelium is constructed [51]. Meanwhile, chitosan has been shown to o ffer protection of FGF-2 against denaturation from heat, proteolysis and acid [52]. Chitosan has been shown to accelerate healing in diabetic mice (chronic wound models), acting as a delivery method for FGF-2 to further promote healing [52,53].

We have used attenuated total internal reflectance Fourier transform infrared spectroscopy and quartz crystal microbalance with dissipation monitoring to investigate the fucoidan/chitosan polyelectrolyte multilayers as a potential reservoir for FGF-2. Additionally, the permeation of FGF-2 into the multilayer was compared to the permeation of lysozyme. Lysozyme is a small protein of similar size and charge to FGF-2 and has been shown to permeate into these multilayers in our previous work [54]. Our overall aim has been to determine how FGF-2 can be incorporated into multilayer films, either pre-prepared for deployment in biomaterials applications, or as a sink within a biofluid environment to harvest and protect released FGF-2, to allow it to survive for longer in the environment in which it needs to act.
