Construction and characterization of solid-supported lipid bilayers to investigate cell-surface interactions

Investigation of cell-cell/biomolecule and cell-surface interactions are essential to understand basic mechanisms such as cell signaling, attachment, proliferation and differentiation. Getting insight to these mechanisms also elucidates the topics such as the development and improvement of effective medical systems. Supported lipid bilayers (SLBs) when functionalized with bioactive molecules that are responsible for specific biological interactions, allow long-term experiments for many practical applications such as investigation of the basic features of biological membranes, construction of biosensors, drug screening platforms, cancer cell detection platforms and investigation of the cell–cell/biomolecule, cell-surface interactions. These biomimetic lipid bilayer platforms allow the usage of surface-sensitive characterization techniques such as atomic force spectroscopy (AFM), surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). The aim of the thesis is to construct functional SLB platform to investigate cell-surface interactions and to monitor whole process in real time via QCM-D system. In this way, it is possible to investigate changes in cell-surface interactions (surface characteristics, cell type and time dependent) that can not be distinguished under the microscope. SLB surfaces were functionalized with RGD, and osteocalcin mimetic (OSN) peptides and their interaction with osteoblast cells were evaluated. RGD is the binding motif of various extracellular matrix (ECM) proteins so it is expected to promote cell adhesion whereas OSN is known to promote biomineralization rather than cell attachment so chosen as control. Similar platforms were used in parallel in cell culture environment for comparison. The first part of the thesis focused on the construction of an SLB platform in single step. Most of the model lipid membrane studies on gold-coated sensors involve the usage of various surface-modification strategies to rupture liposomes and induce lipid bilayer formation since liposomes with polar surfaces do not interact with bare, hydrophobic gold. For this purpose, it was aimed to form SLB on gold sensor surfaces without further modification by incorporating a thiol-modified phospholipid, 1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol (DPPTE) into phosphatidylcholine (PC) based liposomes. The binding kinetics of liposomes with different DPPTE ratio (0.01 to 100% mol/mol) and diameters were monitored by QCM-D. The dissipation change per frequency change, i.e., acoustic ratio, which is evaluated as a degree of the viscoelasticity, considerably decreased with the presence of DPPTE (from 162.3 GHz−1 for flattened PC liposomes to ca. 89.5 GHz−1 for 100% DPPTE liposomes) when compared to the results of two reference rigid monolayers and two viscoelastic layers. To assess the quality of SLB platform, the interpretation of QCM-D data was also complemented with Surface Plasmon Resonance. While QCM-D is sensitive to the liquid content within the layers, SPR provides liquid-independent results and thus the results of these techniques are compared to make more realistic interpretations. The optimum thiolated-lipid ratio (1%, lower thiol ratio and higher rigidity) was then used to determine the dry-lipid mass deposition, the water content and the thickness values of the SLB via viscoelastic modelling. Further, surface characterization studies were performed by Atomic Force Microscopy with high spatial resolution. The results suggested that model membrane was almost continuous with minimum defects but showed more dissipative/soft nature compared to an ideal bilayer due to partially fused liposomes/overlapped lipid bilayers/multilayer islands. These local elevations distorted the planarity and led the increase of overall membrane thickness to ∼7.0 nm. However, the construction of SLB on gold in a single step has been successfully accomplished. In the second part of the thesis, the aim was to obtain insight about how to evaluate cell – surface interactions in QCM-D system. QCM-D is one of the powerful techniques, which allow real time, quantitative and noninvasive analysis of the interaction of different cell types with various modified surfaces. For this purpose, different surfaces were prepared without using SLB system and their interaction with the cells for 18 h were interpreted. First, the dynamic adhesion behavior of human fetal osteoblastic bone (hfOB) cell lines was monitored on untreated and hydrophilically treated gold sensor surfaces as reference substrates. Adhesion was also observed under light microscopy to facilitate the evaluation. Cells increased their surface contact area and spread more on hydrophilic surfaces, and showed distinct profile with an increased rigidity at the cell-surface interfacial layer, which is assigned to extracellular matrix remodeling. Further, the adhesion strength and kinetics were characterized on cell adhesive (poly-L-lysine and fibronectin) and repellent (bovine serum albumin) surfaces. The overall results indicated that protein-mediated specific interactions contributed mostly to the dissipation changes (ΔD) or acoustic ratio (ΔD/Δf). Finally, the potential of QCM-D to distinguish healthy and cancerous cells were evaluated by comparing the results of hfOB cells with that of SaOS-2 (osteosarcoma) cancerous cells. Cancerous cells interacted more strongly with the surfaces and showed more viscoelastic characteristic than the healthy cells. In the final part of the thesis, SLB activation and functionalization with RGD and OSN peptides and examination of the interactions of these systems with cells via QCM-D were aimed. First, the stability of lipid bilayers in various medium conditions were evaluated. The structural integrity was conserved until fifth day in PBS, but hole formations started to form from the first day and increase their number and size in time under serum-supplemented medium conditions. SLBs enriched with different lipids (cholesterol, PS, DOTAP) increased the stability of SLB to pure PC. Lateral mobility of the lipid bilayer was also demonstrated via FRAP (Fluorescence Recovery after Photobleaching) method. After the SLB characterization for both stability and fluidity, the kinetics of peptide attachment to SLBs were compared to their nonspecific adsorption onto gold surfaces and the characteristic of resultant layers were analyzed via QCM-D. The results indicated that conjugation to SLB provide higher structural flexibility than passive adsorption. When cells were incubated with these surfaces, it was seen that low density of mobile RGD peptides on SLB platforms preserved their biological activity and promoted cell adhesion more efficiently than high number of immobile peptides on the gold surfaces. Cell attachment and spread on RGD-fSLB surfaces were also confirmed in cell culture. Limited number of cells were attached on bare SLB surface and they remained in rounded morphology. Cells did not spread well on OSN-coated surfaces, and acoustic ratio measured on each OSN layers probably did not originate from specific adhesion of the cells, instead the negative charge of OSN could provide electrostatic interactions between surface and biomolecules on the cell membrane, thus promoting nonspecific protein and cell attachment. In conclusion, nonfouling SLB surfaces were successfully functionalized and constructed fSLB platforms were shown to have the potential for real time and label-free monitoring of the specific cell – surface interactions via QCM-D.