Papers

AVS2004 Session BI-FrM: "Active" - Dynamic Biointerfaces

Friday, November 19, 2004 8:20 AM in Room 210D

Friday Morning

Start	Invited?	Item
8:20 AM		BI-FrM-1 The Effect of Cell Detachment Method on the Identity and Quantity of Residual ECM Proteins Retained at Surfaces H.E. Canavan, X. Cheng, B.D. Ratner, D.G. Castner (University of Washington) Treatment of tissue culture polystyrene (TCPS) with poly(n-isopropylacrylamide) (pNIPAM) has been developed as a technique for the harvest of intact cell monolayers. Although low-temperature liftoff from pNIPAM is known to be less damaging to cells than traditional cell removal methods, little is known about the effects these techniques have on the underlying Extracellular Matrix (ECM). Recently, we demonstrated that although immunoassay of ECM components show that low-temperature liftoff removes the majority of the ECM concurrently with the cells, XPS and SIMS results reveal that some protein does remain at the pNIPAM surface. In this work, we further examine the effect that low-temperature liftoff from pNIPAM and traditional cell removal methods have on the ECM. Using XPS, we compare the relative amount of ECM remaining at culture surfaces after cell removal by the different methods. Using SIMS, MALDI, and immunostaining, we identify the individual proteins left behind. Finally, LDH assay is used to ascertain the viability of the residual ECM left behind by each cell removal method. We find that in addition to its dramatic effects on cell viability and morphology, trypsin removes much of the underlying ECM and often adsorbs to the surface itself, drastically reducing the adhesion of new cells. Although mechanical dissociation of the cell layer is less damaging to the underlying ECM, harvest via this method results in incomplete cell layers with partially damaged appearance. Of these techniques, only low-temperature liftoff from the pNIPAM surface harvests a complete cell monolayer while leaving behind ECM proteins capable of promoting new cell adhesion.
8:40 AM		BI-FrM-2 Thermally Responsive Surfaces Formed by Plasma Polymerisation of N-Isopropyl Acrylamide N.A. Bullett, R.A. Talib, S.L. McArthur, R.D. Short, A.G. Shard (University of Sheffield, UK) Temperature responsive surface coatings show great potential for a number of novel applications, such as 'smart' cell culture substrates for the control of cell attachment and detachment. Surface grafted N-isopropyl acrylamide (NIPAAm) has been shown to be suitable for this purpose, and the possibility of producing these coatings by deposition from plasma has been recently demonstrated by Pan et al.¹ We find that, although it is possible to produce surfaces that demonstrate a temperature-dependent transition (as determined by contact angle goniometry and ellipsometry), there is a significant risk of delamination or solubilisation of the plasma polymers when they are in contact with water. In this work we demonstrate the importance of substrate temperature and discharge power on the properties of plasma polymerized NIPAAm. X-ray photoelectron spectroscopy (XPS) analyses were used to examine the composition of the deposited films, demonstrating that the high power deposits contain less characteristic functional groups (notably amides), indicating greater fragmentation of the monomer, and a high degree of cross-linking, whilst the low power deposit closely resembles conventionally polymerised NIPAAm. However, the very low power plasma polymers were shown, by XPS analysis and ellipsometry, to be unstable to rinsing with distilled water. The effect of substrate temperature during deposition was also investigated. It was found that higher substrate temperatures produced a more stable film at lower plasma powers. Useful films are therefore only produced within a narrow parameter space. The effect of using pulsed discharges and co-monomers was also investigated. We demonstrate that these plasma polymers can be patterned and used for cellular co-culture. ¹ Pan YV, Wesley RA, Luginbuhl R, Denton DD, Ratner BD; Biomacromolecules 2001, 2, 32-36.
9:00 AM		BI-FrM-3 Using Enzymes to Switch Surface Properties R.V. Ulijn (University of Manchester, UK); M.R. Alexander, F.J.M. Rutten (University of Nottingham, UK); J.E. Gough, F. Carabine, J.L. Rutherford (University of Manchester, UK) We study synthetic surfaces that change their properties upon biochemical stimuli (i.e. an enzymatic reactions). Surfaces that can be tuned (switched) to either bind or resist (biological) molecules or cells are desirable for a number of applications in the biomedical sciences. Enzymes are ideal tools for such surface engineering because they are highly selective and truly compatible with biology. We demonstrated that a peptide-hydrolyzing enzyme (chymotrypsin) could be employed to significantly alter the wettability of modified glass surfaces by hydrolysis of surface bound di-peptides. Here, we describe the use of XPS and ToF-SIMS analysis to characterise the changes in surface chemistry achieved using this approach. The methodology was applied in switching the attachment of cells to surfaces. A number of surfaces have been identified that are â?~stickyâ?T (promoting cell adhesion) or â?~non-stickyâ?T to certain cells. By using our biochemically responsive surfaces we demonstrate for the first time the possibility of switching between these two surface properties and therefore to switch cell adhesion in real time.
9:20 AM		BI-FrM-4 Conformational Mechanics of Surface Grafted Stimulus-Responsive Polypeptides A. Valiaev, D.W. Lim, N. Abu-Lail, A. Chilkoti, S. Zauscher (Duke University) Stimulus-responsive macromolecules have attracted significant research interest due to their potential applications in drug delivery, molecular motors, and nanoscale actuation devices. ELPs are stimuli-responsive biomacromolecules that undergo an inverse phase transition triggered by changes in solvent temperature, pH, or ionic strength. Associated with this transition is a significant conformational collapse and change in surface energy. To date, most research on ELPs has focused on the investigation of bulk properties and their aggregation behavior in solution. Our research is the first, we believe, to provide detailed insight into the mechanisms of elasticity and conformational mechanics of ELPs immobilized as ensembles on surfaces and on the single molecule level. First we performed single-molecule force spectroscopic experiments on elastin-like polypeptides (ELPs) that shed light on their time-dependent structural changes, physico-chemical and mechanical properties. We also present results obtained with a quartz crystal microbalance (QCM), cantilever deflection measurements, and adhesion force spectroscopy on surface immobilized ELP ensembles. QCM enabled us to study the effective mass change and the change in the mechanical dissipation behavior when ELPs collapse and swell as waters of hydrophobic hydration are released or consumed by the surface grafted protein. Experiments with micro-cantilevers decorated on one side with covalently grafted ELPs showed that a phase transition induces a considerable surface stress, leading to significant cantilever bending. Our approach promises to yield simple and sensitive bioanalytical devices, because cantilever bending can be easily measured. Lastly, adhesion force spectroscopy enabled us to quantify surface energetic changes associated with the phase transition behavior of surface grafted ELP ensembles.
9:40 AM		BI-FrM-5 pH Induced Conformational Behaviour of Polyelectrolytes in Bulk Solution and Grafted to Surfaces: Neutron Reflectometry and Fluorescence Studies M. Geoghegan, L. Ruiz-Perez, A.J. Parnell, J.R. Howse, A.J. Pryke, C.J. Crook, P. Topham, S.J. Martin, A.J. Ryan, R.A.L. Jones (Univ. of Sheffield, UK); A. Menelle (Lab. Lön Brillouin, France); J.R.P. Webster (Rutherford Appleton Lab, UK); I. Soutar, L. Swanson (Univ. of Sheffield, UK) We present neutron reflectometry results on (deuterated) water-swollen poly[diethylamino)ethyl methacrylate] (PDEAMA, a polybase) and polymethacrylic acid (PMAA) brushes grafted from silicon substrates using atom transfer radical polymerisation. The PDEAMA data are presently the more comprehensive and reveal that the expanded brush (low pH) is some 6 to 10 times thicker than the collapsed brush at high pH. Brush data will be compared with bulk solution data of the collapse transition of PMAA in water measured by a variety of fluorescence techniques: steady state energy transfer, lifetime, and time-resolved fluorescence anisotropy measurements, which enable a correlation of this single polymer in solution collapse transition with the confinement-influenced transition which occurs when the polymers are tethered to a surface.