pH dependent growth of poly(L-lysine)/poly(L-glutamic) acid multilayer films and their cell adhesion properties
Introduction
There is an increasing interest in developing new coatings to improve biocompatibility and to prepare biomaterial surfaces that can either resist or enhance cellular adhesion by mimicking extracellular matrix components [1], [2] or even locally deliver drugs. The most important step toward this end concerns the improvement at the nanometer to micrometer scales of material surface properties [3] in order to control cellular responses such as adhesion, motility, spreading, growth and differentiation. This has been the focus of many studies since about 40 years. It has been shown that different parameters such as hydrophobicity and hydrophilicity [4], surface charge [5], [6], [7], roughness [8], surface free energy [9], [10] and topography [11] affect cellular adhesion. The definition of general rules is not straightforward and the observed cellular behaviors probably do not depend on a single parameter but on a complex combination of several factors.
In addition to being able to control the physico-chemical surface properties, it is of interest to establish easy handling methodologies available for various types of surfaces and for creating controlled topographical features. Typically, appropriate surface chemistries such as ethylene glycol grafting [12] or monolayer self-assembly [13] can be used in conjunction with patterning techniques to create both cell-resistant and cell-adherent surface domains. Alternatively, adhesive ligands such as RGD can be grafted to the deposited polymers [14].
Polyelectrolyte multilayers (PEM) constitute a new attractive way for creating biofunctionalized surface coatings. The layer-by-layer (LbL) technique consists in the alternate deposition of polyanions and polycations from aqueous solutions to build the multilayered films [15], [16]. These films have tunable properties in the thickness range from nanometer to micrometer [17], [18]. It has been shown that the film morphology, internal molecular structure and thickness depend largely on the processing conditions such as ionic strength [19], and pH of the polyelectrolyte or rinsing solution [20]. The films are not only stabilized by electrostatics interactions but also via hydrogen bonds [21], [22].
Of special importance for biomedical applications is the control of the chemical composition of the surface that can affect biological activity. Films made of polyamino-acids, i.e. poly(L-lysine) (PLL), poly(L-glutamic acid) (PGA), natural polyelectrolytes (e.g. hyaluronan (HA), alginate, chitosan, collagen) allow, for example, to create biomimetic architectures [18], [23], [24], [25]. Various compounds such as DNA, RNA, drugs, inorganic particles can be embedded in such films or deposited on the top of them.
A great advantage offered by PEM films is their ability to coat any type of material with any shape. Thus, PEM films have recently been deposited onto stainless steel [26], polydimethysiloxane (PDMS) [27], vascular stents made of NiTi [28], and onto biodegradable poly(L-lactic) acid matrices [29]. The geometry was not necessarily planar but also curved or spherical, as for titanium beads [30], or polystyrene and glass microspheres. In the case of films built on particles, the particle core can also be subsequently removed to form hollow capsules [25] or the films can be detached from the surface to give self-supported membranes [31].
Within the past few years, there has been an increasing interest for biomedical applications of PEM films. Fundamental and applied studies of PEM in terms of biological properties with respect to biological fluids or cells have been published (see paragraph 2). This includes, for example, the fabrication of non-adhesive barriers for vascular grafts [23], the fabrication of films with pro or anti-coagulant properties [24], the control of the cell adhesive properties of a film by varying the deposition pH [32], [33], the preparation of hollow capsules for drug release [25], the preparation of bacterial resistant film [34], [35]. Moreover and very interestingly, a further functionalization of the films can be achieved by inserting peptides covalently bonded to polyelectrolytes [36] or through protein embedding [37].
For the biomaterial field, biocompatibility is a major requirement: the material must be non-toxic to any living cell. Another requirement is that the material possesses chemical and physical properties that promote or avoid specific cell substrate interactions, i.e. either cell adhesion or non-adhesion depending on the final application.
Rubner and coworkers [32], [33] have recently demonstrated that PEM films based on synthetic polyelectrolytes (polyacrylic acid, polyacrylamide, and poly(allylamine) hydrochloride) can either be cell resistant (cytophobic) or cell adhesive (cytophilic) depending on the swelling capacities of the films, which are modulated by changing the deposition pH of the films. In fact, changing the deposition pH is known to considerably change the internal properties and thicknesses of the films [21], [38]. We have already investigated cell interaction with biodegradable (PGA/PLL) multilayers built at physiological pH and containing an increasing number of layer pairs. We found that the initial adhesion was always very low for PGA-ending films whereas it was very high and decreased with the number of deposited layers for PLL-ending films. But at the working pH (7.4), cells were always strongly adhering to PLL-ending films and poorly adhering to PGA-ending films. At the cellular scale, it would also be of interest to test whether the findings presented by the Rubner and coworkers [32], [33] remain valid for polyamino-acid based films. In other words, can a given polyelectrolyte system such a PGA/PLL, which is capable of forming ordered structures (α-helix and β-sheets) [39], be adhesive and anti-adhesive for cells depending on the deposition pH of the films?
The present study is first aimed at investigating the possibilities of building films of synthetic polypeptides, such as (PGA/PLL) films, over a large range of pHs in order to change their internal properties. Second, we will investigate whether these films can be used for cell culture experiments, i.e. whether they remain stable in a physiological medium. The third aim is to quantify by micromanipulation the short-term interaction of chondrosarcoma cells with the (PGA/PLL) films built at different pH.
Section snippets
Survey of the literature regarding cell adhesion studies on polyelectrolyte multilayer films
Studies dealing with the adhesion properties of cells on various PEM films are presented in Table 1. Cell adhesion and proliferation assays were initially performed on cell lineages [33], [36], [40], [41]. Thin films were found to be cell adhesive with some differences depending on the outermost layer of the film [40]. Recently, Rubner and coworkers [32], [33] showed that poly(acrylic acid)/poly(allylamine hydrocholoride) (PAH) multilayers can either be non-adhesive or adhesive for NR6WT
Polyelectrolyte solutions and coupling agent
Anionic PGA (MW = 54 kDa), cationic PLL (MW = 30.3 kDa), PEI (MW = 750 kDa), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfo-succinimide (sulfo-NHS) were purchased from Sigma-Aldrich. Sodium chloride (purity = 99.5%) was purchased from Fluka, and Sodium dodecyl sulfate (SDS) was purchased from Sigma. All solutions were prepared using ultrapure water (Milli-Q-plus system, Millipore) with a resistivity of 18.2 MΩ cm. Polyelectrolyte solutions were always freshly prepared by direct
Physico-chemical characteristics of the PEI–(PGA/PLL)i films built at different pHs
The buildup of the PEI–(PGA/PLL)10 multilayer films at different pH values in water was followed in situ step-by-step by OWLS (Fig. 1A). In a previous work [47], the PEI–(PGA/PLL)i multilayers built in a buffer containing 0.15 M NaCl at pH 7.4 have been characterized by in situ atomic force microscopy and by OWLS. The evolution of the thickness and of the adsorbed mass with the number of deposited layers was exponential. These layers formed extended structures that appear with a vermiculate
Conclusions
The short-term interaction of chondrosarcoma cells with (PGA/PLL) polyelectrolyte multilayers was investigated in a serum-containing medium for films built at different pHs and subsequently exposed to the culture medium. The buildup of the films and their stability was first investigated. Whereas film growth is linear at all pHs after a few layers have been deposited, the growth is much larger for the films built at basic pH and even more for those built at acidic pH. However, these latter
Acknowledgements
The authors wish to thank Fouzia Boulmedais for her assistance in the FTIR experiments. We also thank Prof. M. Rubner and Dr. S.Y. Yang from the MIT (Boston) for fruitful discussions. C.P. is indebted to the “Université Louis Pasteur” for financial support to setup the micropipette technique. This work was supported by the program “ACI Technologies pour la Santé” from the French Ministery of Research.
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