Fluid Lipid Multilayer Stabilization by Tetraethyl Orthosilicate for Underwater AFM Characterization and Cell Culture Ap
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Fluid Lipid Multilayer Stabilization by Tetraethyl Orthosilicate for Underwater AFM Characterization and Cell Culture Applications Aubrey E. Kusi-Appiah1, Troy W. Lowry1,2, Nicholas Vafai1, David H. Van Winkle2, Steven Lenhert*1,3 1
Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370, USA. Department of Physics, Florida State University, Tallahassee, FL 32306-4370, USA. 3 Integrative NanoScience Institute, Florida State University, Tallahassee, FL 32306, USA * Corresponding author 2
ABSTRACT Stabilization of surface supported fluid lipid multilayers for underwater characterization is an essential step in making them useful for scalable cell culture applications such as high throughput screening. To this end, we used tetraethyl orthosilicate (TEOS), recently shown to stabilize fluid lipid films while maintaining their fluidity and functionality under water, to stabilize lipid multilayer micropatterns of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The treated multilayers were immersed under water and successfully imaged by atomic force microscopy (AFM), a difficult feat to perform on fluid lipid multilayers without TEOS treatment. The treated lipid multilayer showed an average swelling of approximately 18% in water but remained stable during the imaging process. The TEOS-treated lipid multilayers also proved compatible with cell culture as HeLa, MDCK, and HEK cell types all adhered and grew in high numbers over the multilayers. The results obtained here open the door to the use of fluid lipid multilayers in biotechnology applications such as microarray based high throughput cell assays. INTRODUCTION Fluid phospholipids have demonstrated their usefulness in biotechnology and biomimetic applications such as cell modeling and drug delivery [1-3]. However, their exploitation has been limited to low throughput applications due to the instability of supported lipid multilayers (SLM) to aqueous immersion. This instability can occur as a result of dissolution of the phospholipids at the air-water-lipid interface, causing the lipids to be carried along with the solution (Figure 1b shows the fluorescence image of the destruction of lipid multilayer patterns when immersed in cell culture media). Attempts have been made to stabilize SLM patterns by reducing the humidity of the immersion environment and using hydrophobic surfaces like poly(methyl methacrylate) [4]. While these attempts have been successful for immersion of the SLMs in simple buffers, immersion under high-protein-content media has remained a challenge. Recent investigations into this problem have yielded a technique for lipid multilayer stabilization that involves the exposure of SLMs to vapors of tetraethyl orthosilicate (TEOS), a precursor for the synthesis of ordered mesoporous silica [5], resulting in the formation of highly ordered nanostructured silica-phospholipid hybrids [6]. These hybrids show long term stability in air, as well as in water, and demonstrate resistance to destruction during aqueous immersion. This o
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