Free standing membranes made of biocompatible polyelectrolytes using the layer by layer method

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Abstract

The build up of biocompatible and biodegradable free standing polyelectrolyte multilayer membranes consisting in poly(l-lysine) and hyaluronic acid is presented. Different methods were applied for the film detachment from the underlying substrate leading to membranes with specific porosity and roughness as observed with confocal microscopy and atomic force microscopy. The possibility to functionalize these membranes is demonstrated by the incorporation of an enzyme, alkaline phosphatase, which displays constant biological activity during at least 1 week. These new free standing natural membranes could be an original way to build up bioactive patches carrying specific drugs. This open the route for the build up of “biopatches” able to possess specific biological properties and useful for future biomedical applications.

Introduction

The functionalization of surfaces with molecules, conferring specific properties, is one of the major concerns in surface science with possible applications in the field of microelectronics or for the development of biosensors. Methods like transfer from Langmuir monolayers [1], build up of self-assembled monolayers on noble metal surfaces [2] are very efficient to reach these goals. However, these methods are cumbersome, need special equipments and extreme care in the surface cleaning preceding surface functionalization. The deposition of polyelectrolyte multilayers [3], [4] through the layer by layer (LBL) method has stimulated a tremendous amount of studies aimed both to the understanding of the physicochemical mechanisms allowing this kind of self assemblies and the development of numerous applications. The build up of polyelectrolyte multilayers occurs through the alternate dipping of the substrate in polyanion and polycation solutions and is a consequence of a charge overcompensation of the substrate during the adsorption of the previous polyelectrolyte layer [5], [6]. This kind of surface self-assembly offers new opportunities in the modification of surfaces, since it constitutes a versatile technique, which can be applied to every kind of substrates that carry a surface charge at the physico-chemical conditions in which the deposition is performed. Moreover, it is possible to control deposited film thickness and roughness by changing either the number of bilayers deposited onto the substrate or the pH and ionic strength of the solutions from which polyelectrolytes are adsorbed [6], [7], [8], [9], [10], [11]. Two kinds of growing mechanisms have been found for these polyelectrolyte multilayers: (i) a linear growth in which the thickness and the amount of polyelectrolytes deposited per bilayer increases linearly with the number of deposited bilayers n [6], [12] and (ii) an exponential growth in which the thickness and the amount adsorbed increases exponentially with n [13], [14], [15], [16], [17]. This second kind of growing regime occurring with specific polycation/polyanion pairs is very attractive since it allows to reach thickness in the micrometer range after the deposition of only a limited number of bilayers [14] and these multilayers can act as a reservoir for the diffusion of free molecules [18]. This is obviously not the case with polyelectrolyte multilayers growing linearly: typically, the thickness reached after deposition of 10 bilayers is in the order of 100 nm. Also for such films, the thickness increment per bilayer increases with the ionic strength of the polyelectrolyte solution used [6], [19] up to a certain point after which the multilayer decomposes [20], [21].

Up to now, these polyelectrolyte multilayers were mostly studied to modify the surface properties of a substrate, but it appeared that the concept can also be used as a way to elaborate entirely new materials after removing them from the solid substrate onto which they were deposited. The first application of this free standing multilayers was hollow capsules [22] employed as microreactors [23], [24], [25], for immunoassay [26] or as a biocompatible drug carrier [27], [28], [29]. More recently, free standing polyelectrolyte multilayered membranes were produced using the LBL deposition of poly(diallyldimethylammonium bromide) and magnetite nanoparticles on a cellulose acetate substrate [30]. This flat polymeric substrate was subsequently dissolved in acetone yielding to the multilayers membranes. The mechanical strength of the membranes can be reinforced by the incorporation of montmorillonite clay platelets [31] or gold nanoparticles [32]. Kotov and co-workers also managed [33] to produce hybrid membranes made from poly(ethylene imine)/poly(acrylic acid) multilayers including single wall nanotubes. They described membranes with Young modulus reaching 220 MPa. Using a different approach [34], free standing membranes were obtained by the construction of an hybrid multilayer film on a substrate using four different synthetic polyelectrolytes A, B, C and D, and consisting in (A/B)m  (C/D)n multilayer film (m and n corresponding to the number of layer pairs). By changing the pH and the ionic strength, the (A/B)m assemblies are decomposed, whereas the (C/D)n assemblies remained stable and can be removed from the substrate as free standing membranes. All these membranes were composed of polyelectrolyte multilayers growing linearly.

Polyelectrolyte multilayers deposited on the surface of mesoporous membranes were shown to be able to allow for the separation of alcohol/water mixtures as well as the separation of a mixture of mono and divalent ions in aqueous solution [35], [36], [37], [38], [39]. Moreover, it has been shown recently that a membrane consisting in a polypeptide multilayer film with chiral centers (for example, poly(l-glutamic acid)/poly(l-lysine), (PGA/PLL)) was able to partition a mixture of l- and d-ascorbic acid [40].

All these observations clearly show that free standing membranes made from polyelectrolyte multilayers are promising new materials since it is possible to engineer their mechanical as well as their chemical properties. The aim of this article is to present the development of free standing polyelectrolyte multilayered membranes made of biocompatible and biodegradable polyelectrolytes. We use poly(l-lysine) a biodegradable polypeptide and hyaluronic acid (HA), which constitutes an important component of the extracellular matrix [41], [42] and which is known to play a major role in tissue hydratation, cell adhesion and also in lubricating functions. Hence, the multilayers membranes produced and characterized in the present study possess potential applications in wound healing, in arterial repairs and as dermal substitute. We will present various methods for the build up of these membranes, which lead to different thickness, porosity and stability. Moreover, we will functionalize the multilayers membranes to confer to them an enzymatic activity.

Section snippets

Preparation of polyelectrolyte solutions

Poly(l-lysine) (MW = 57,000 g mol−1) and fluorescein isothiocyanate labeled poly(l-lysine) (PLL-FITC, MW = 50,200 g mol−1) were purchased from Sigma (St. Quentin Fallavier, France) and hyaluronic acid (MW = 400,000 g mol−1) from BioIberica (Barcelona, Spain). Polyelectrolyte solutions were prepared by dissolution of the adequate amounts of PLL or HA in filtered 0.15 M NaCl solution (pH 6.2). The final polyelectrolyte concentrations were of 1 mg mL−1. All solutions were prepared using ultrapure water (Milli

Results

In order to obtain free standing polyelectrolyte multilayered membranes, we first built a poly(l-lysine)/hyaluronic acid multilayer on a polystyrene substrate by dipping successively this substrate in PLL and HA solutions. Between each step, a rinsing was performed by dipping the substrates in 0.15 M NaCl solution. The build up of such multilayer films has been extensively studied [14], [15]. It is of exponential nature so that films of several micrometers in thickness can easily be obtained.

Conclusions

Finally, we proposed here a new method to build biocompatible and biodegradable free standing polyelectrolyte multilayered membranes. The multilayer films consisting in poly(l-lysine) and hyaluronic acid have been deposited on the glass or polystyrene substrate. Two different methods are applied for detaching the film from the underlying substrate. For films built on polystyrene substrate, THF is used to dissolve the polystyrene substrate and leads to a membrane possessing micrometric holes.

Acknowledgement

The CLSM platform used in this study was co-financed by the Region Alsace, the CNRS, the Universite Louis Pasteur and the Association pour la Recherche sur le Cancer.

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