Biomacromolecules 2005, 6, 1689-1697
1689
Patterning Poly(organophosphazenes) for Selective Cell Adhesion Applications Eric W. Barrett,† Mwita V. B. Phelps,† Ricardo J. Silva,‡ Roger P. Gaumond,*,‡ and Harry R. Allcock*,† Departments of Chemistry, 104 Chemistry Building, and of Bioengineering, 205 Hallowell Building, The Pennsylvania State University, University Park, Pennsylvania 16802 Received December 21, 2004
Five polyphosphazenes with different hydrophilicites were synthesized and screened in vitro. The purpose was to identify unique types of polymeric substrates that distinctly favored or markedly prevented cellular adhesion. The SK-N-BE(2c) human neuroblastoma cell line, utilized for its electrogenic responses, was used to test this differential adhesion. In particular, the objective was to specifically culture this cell line in a highly selective pattern. Each candidate polymer was cast into films and plated with neuroblastoma cells for 3 days. The polyphosphazene materials which showed negative cellular adhesive properties (-CAPs) were poly[bis(trifluoroethoxy)phosphazene] (TFE) and poly[bis(methoxyethoxyethoxy)phosphazene] (MEEP). The polyphosphazenes which showed positive cellular adhesive properties (+CAPs) were poly[(methoxyethoxyethoxy)1.0(carboxylatophenoxy)1.0phosphazene] (PMCPP), poly[(methoxyethoxyethoxy)1.0(cinnamyloxy)1.0phosphazene] (PMCP), and poly[(methoxyethoxyethoxy)1.0(p-methylphenoxy)1.0phosphazene] (PMMP). To test cellular selectivity, films of -CAP and +CAP were copatterned onto glass substrates. The micropatterned films were plated with SK-N-BE(2c) neuroblastoma cells for one week. The results showed that neuroblastoma cells adhere selectively (over 60%) to the +CAP microfeatures. We also showed that multiple properties can be achieved with a single material and that we can use TFE as both a -CAP and an insulation layer and PMCP as a conductive +CAP layer. Introduction To understand the complex relationships that develop between cells as they integrate into higher order structures (tissues), researchers have been working to develop techniques to culture cell systems into specific reproducible patterns.1-9 The idea is to use specific materials to arrange cells into complex patterns with potential physiological characteristics and to study the behavior of the system. Out of the many physiological systems, electroactive cells and in particular neural-like cells are of particular interest because the electrical response can be recorded readily and utilized as a direct measure of activity in the system. In fact, the possibility to selectively plate cells in specific patterns has evolved into advanced applications such as biosensors, cellbased biochips, and patterned neural networks.1-9 The most important requirement for building functional circuits is the substrate, and specifically the utilization of materials that will either promote or prevent adhesion of the selected cells into the specific pattern. Materials for such applications should ideally be easy to fabricate, hydrolytically stable, nontoxic, chemically inert, synthetically reproducible, scalable, and inexpensive. To couple the pattern networks to microscopic observations, the material should also be transparent, and if electrogenic cellular activity is to be monitored, it is convenient for the material to be electrically conductive. * To whom correspondence should be addressed. † Department of Chemistry. ‡ Department of Bioengineering.
For over a decade, highly successful neuronal networks have been developed with good cellular adhesion control and direction.9 A wide variety of techniques and materials have been employed to deposit or fabricate intricate patterns that have marked differences in their surface hydrophilicites.9 Both bimolecular and synthetic substrates have been isolated or developed to fulfill the requirements for substrate patterning. Extracellular matrix proteins such as laminin, fibronectin, tenascin, and collagen are commonly used as cell-friendly materials. Also proteoglycans, glycosaminoglycans, and cell adhesion molecules (e.g., N-CAM, cardherins, and F-spondin) have been utilized.9 A major drawback for biomolecules is that they are hard to obtain, relatively expensive, and batch dependent. On the other hand, synthetic substrates can be reproducible, which is a potential advantage for cell patterning. Of the synthetic polymers studied, polylysine (PL) is a widely accepted cell-friendly and transparent substrate for cell culture applications. PL has been shown to allow patterned cell adhesion and growth.9-11 However, it is expensive and it is difficult to form stable long-term patterns because of its solubility in water under cell culture conditions.9 Poly(ethylene glycol) (PEG) can form a very stable hydrogel suitable for long-term biological applications if processed properly. For example, functionalized PEG such as PEG-diacrylate can be covalently cross-linked by exposure to UV light. It is the preferred cell-unfriendly polymer, being transparent, inexpensive, and biocompatible.9,11 The combination of PL and PEG has been used extensively for cell
10.1021/bm049193z CCC: $30.25 © 2005 American Chemical Society Published on Web 03/17/2005
1690
Biomacromolecules, Vol. 6, No. 3, 2005
Barrett et al.
Figure 1. General polyphosphazene structure.
patterning.9 A major limitation for this system is that PL dissolves in cell culture media within a few days and patterns are inherently lost, and PEG needs to be treated to remain stable. The most extensively used systems for cell patterning are based on lithography (hard and soft) of monolayer surface chemistry and self-assembled monolayers (SAMs).9 In monolayer surface chemistry (including SAMs),12,13 a molecular thin film of material is covalently attached or coordinated to the surface of a substrate. Some materials that are typically used include polymeric oligomers and peptides or biomolecules.9 SAMs have the additional feature of molecular alignment in that each individual molecule is arranged in an orderly manner with respect to the other molecules.12,13 A great deal of effort has also been focused on multilayered monolayer systems.9 In this technique, sequential monolayers are deposited or patterned on top of one another. However, because of oligomer entanglement, each additional monolayer is likely to be less uniform due to imperfections in the previous monolayer. Monolayers are typically 10-30 Å thick, making them difficult to see optically. They can, however, be characterized easily by elipsometry, XPS, contact angle measurements, AFM, etc.9 The extreme thinness of the film may lead to easy spreading of the biofilm being deposited by an adhesive cell into the boundary regions of the adhesive pattern. Monolayers that are coordinated to a substrate run a higher risk of detachment over time. Covalently attached SAMs can potentially improve long-term stability and have been well characterized. It is difficult to determine how well assembled and ordered they actually are. The outer ends of the monolayer monomers may be buried or tangled within the SAM layer. XPS is widely used to characterize the surface chemistry of SAMs to get the elemental, elemental state, and atomic binding characteristics in particular. Even with this technique, it can be difficult to determine the degree of entanglement. Polyphosphazenes form a broad class of inorganicorganic polymers with a phosphorus-nitrogen backbone and two organic side groups covalently attached to each phosphorus (Figure 1).14 These polymers are synthesized by replacement of the chlorine atoms in poly(dichlorophosphazene) by organic groups.14 The final material properties are determined, to a large degree, by the physical and chemical properties of the side-group structures.14 By changing the side-group composition and ratio, the opportunity exists to create a range of polyphosphazenes with characteristics that vary from poor surface cell adhesion to good cell adhesion. Various micropatterning techniques can then be used to pattern regions of interest with such materials. In this work various polyphosphazenes were developed to promote or prevent cellular adhesion, and both photolithography and microcontact printing were studied as
Figure 2. Mature differentiated SK-N-BE(2c) cell cluster grown over a cell-adhesive polyphosphazene, 4.
candidate methods to pattern the materials. The SK-N-BE(2c) human neuroblastoma cell line was selected as the subject of research for various reasons. First, it is a neurallike cell line, and neural cell patterning is one of the main goals of this research. This cell line is also electrogenic,15,16 making it an ideal subject for cell-based biosensors. It has been shown to exhibit poor cellular adhesion on glass but is very adherent to various polymeric materials. Microcontact printing and photolithography are of particular interest for our applications because they possess certain advantages over monolayer systems. The primary reason is the bulk thickness, which can be obtained using lithography (micrometers, not angstroms). This allows for simple verification and increased long-term patterned stability. Background The SK-N-BE(2c) cell line was established by Biedler, in November 1972, from a bone marrow biopsy of a 2-yearold male patient.17 The cell line tends to grow in tight aggregates, consisting of cells with short neuritelike processes. It was found that the cells prefer tissue culture polystyrene rather than glass for attachment and that, when grown over specific polymer surfaces, they replicate very well with an average population doubling time of 27 h. If left unattended, the SK-N-BE(2c) will proliferate indefinitely. However, retinoic acid (RA) inhibits cell proliferation and induces morphological differentiation.18 All these characteristics are well preserved, and both the morphology and the doubling time are consistent with the experiments performed in our program (Figure 2). In recent decades, polyphosphazenes have been used in an increasing number of biomedical-related applications. Some examples include live pancreatic or liver cell entrapment,19 active enzyme entrapment,20 nerve guide conduits,21-23 and substrates for cultured osteoblast cells.24 For these and other reasons, polyphosphazenes have been used as a group of polymers that can be tailored to generate the required physical, chemical, and biological properties to promote or prevent cellular adhesion. Polyphosphazenes can be tailored to possess physical and chemical properties similar to those of a variety of FDAapproved polymers, such as PLGA25 and PEG.9 Synthesized polyphosphazenes are produced with batch-to-batch unifor-
Patterning Polyphosphazenes for Cell Adhesion
Biomacromolecules, Vol. 6, No. 3, 2005 1691
Figure 3. Structure of the synthesized polyphosphazenes.
mity, unlike natural biopolymers such as collagen or alginate which are obtained from animal or other natural sources and vary from one sample to another. Several researchers have shown that the wettability of a substrate influences cell adhesion and that an intermediate contact angle is required for mammalian cell adhesion.26-28 Thus, a mixture of hydrophobic with other more hydrophilic side groups is required for good cell adhesion. For example p-cresoxy side groups linked to a phosphazene chain can generate high hydrophobicities, whereas a polymer fully substituted with methoxyethoxyethoxy (MEE) is hydrophilic. When a phosphazene chain is cosubstituted with both MEE and p-cresoxy (50% ratio), a material with excellent cellular adhesion is produced. Following this trend, various compounds with good hydrophilic or hydrophobic properties were cosubstituted on a polyphosphazene chain and examined for stability, patternability, and differential cell adhesiveness. Experimental Design With this research, we had three main goals: (a) to generate selective cell adhesion by changing the side groups of polyphosphazenes, (b) to fabricate stable polymeric devices using micropatterned polyphosphazenes, (c) to produce stable patterned devices for long-term cell selectivity. To test the cell adhesive properties of the polymers, cells were cultured over the different materials, and selectivity was analyzed. For stability and patternability, UV photolithography and microcontact printing were used to pattern the polyphosphazene materials into microhydrogel arrays, and the arrays were kept in cell culture media for over a month to test hydrolytic stability. To test for cell selectivity, a dual polyphosphazene polymer system was created, and cell-friendly and -unfriendly polymer films were patterned together. Preferential cellular adhesion between the two polymers was followed for up to one month. Materials and Methods Polymers Selected. Several hydrophobic side groups linked to a polyphosphazene chain were found to promote good cellular adhesion if cosubstituted with MEE side
groups. These include phenoxy, benzyloxy, other arylalkoxy groups, p-cresoxy, ethylphenoxy, and other alkylphenoxy groups. With this in mind, three hydrophobic and two hydrophilic side groups were selected: These are the hydrophobic trifluoroethoxy, p-methylphenoxy, and cinnamyloxy groups and the hydrophilic methoxyethoxyethoxy and carboxylatophenoxy units. The hydrophilic MEE side group was selected because, when covalently bonded to the phosphazene backbone, it gives a water-soluble, transparent solution. Following exposure of the solid to ultraviolet (UV) radiation or γ radiation, polyphosphazene 2 cross-links covalently and forms a very stable hydrogel.29-31 Cinnamyloxy was chosen as a co side group because this species is especially photosensitive to UV radiation.30 Exposure to UV radiation induces 2 + 2 cycloaddition, which cross-links the polymer.32 Cinnamyloxy is also very hydrophobic compared to MEE. By adjusting the cosubstitution ratios of these two side groups on the polyphosphazene backbone, both the hydrophilicity and the UV exposure time can be controlled. All polyphosphazene candidates (Figure 3) were synthesized by a well-established macromolecular substitution approach.14 Each polymer was characterized by multinuclear NMR (Bruker AMX-360) to confirm the structure and sidegroup ratio (Table 1). Molecular weights were determined using gel permeation chromatography (GPC) (HewlettPackard HP 1090 equipped with an HP-1047A refractive index detector and Phenomenex Phenogel 10 µm analytical columns calibrated using polystyrene standards) (Table 2). Thin films (
