Nilufer I. Nurinova Dr. David Lo Dr. Victor G. J. Rodgers 08/20/2009 Electrostatic Analysis Approach for Measuring Zeta Potential of M-cells In Mucosal Epithelia Nilufer I. Nurinova Dr. David Lo Dr. Victor G. J. Rodgers 08/20/2009

Outline Motivations Overview Rationale Objectives Experimental methods Data collection and analysis Conclusion Future work Acknowledgements

Motivations Characterize electrostatic behavior of intestinal epithelia Better procedure may be achieved in vaccine delivery protocol

Overview M-cells are major component of follicle-associated epithelium and found in [1] : Peyer's Patches of gut-associated lymphoid tissue (GALT) Mucosal-associated lymphoid tissue (MALT) sites outside the gastrointestinal tract In the gastrointestinal tract, M-cells play an important role in transport of antigen from the lumen of the small intestine to mucosal lymphoid tissues where initiation and processing of immune responses occur [1] Mary Petzke, MD-PhD Harvard Medical School and Children’s Hospital, 2000 Images provided by David Lo, UCR, 2009

Mucosal M-cell Targeting M-cells’ surface lack of brush border and glycocalyx which usually serve as a barrier in intestinal epithelia [1] Understanding the mechanism by which M-cells sample antigens can provide insight to mucosal vaccination methods 5 µm [1] Mary Petzke, MD-PhD Harvard Medical School and Children’s Hospital , 2000 Images are courtesy of Dr, David Lo, Biomedical Sciences Dept. UC Riverside, CA

Selective Uptake by Mucosal Epithelium Specific bacteria (fixed and fluorescently labeled) taken up by Caco-2BBe epithelial cells, with uptake quantified by flow cytometry Enhanced by cytokine treatment, highly significant effect for S. Aureus and Yersinia Images are courtesy of David Lo, Biomedical Sciences Dept. UC Riverside, CA

Selective Uptake by Mucosal Epithelium However, another research analysis has been conducted with nanoparticles, such as Polystyrene latex beads and similar bacterial strains with varying physiochemical properties in order to induce the uptake by M-cells [1]. Results of particles treated in PBS buffer at physiological pH showed: Polystyrene latex beads showed highest ζ-potential approximately -107.9 mV S. Aureus showed ζ- potential around -31 mV Y. Enterolitica had ζ-potential around -19 mV 7 [1] Kaila Bennett , Bioengineering Dept. UC Riverside, CA 2009 7

Experimental Rationale Given some insight into electrostatic interaction between bacteria and mucosal epithelia, our research group aims to explore the electrostatic properties of M-cells Use streaming potential to relate to electrostatic properties Develop streaming potential device to determine values for M-cell cultured on membranes Compare device performance for protein loaded membranes in literature

Objectives Test the streaming potential measuring device for integrity with protein loaded on PES and CRC membranes Observe and collect data of streaming potential values to compare the results with preliminary studies Calculate zeta potential (ζ-potential) of protein loaded membranes using Helmholtz-Smoluchowski equation Modify the streaming potential measuring device for electrostatic analysis of intestinal epithelia Measure zeta potential of M-cells in intestinal mucosal epithelia

Common Factors Considered in Electrostatic Analysis pH Ionic strength Zeta potential Electrostatic potential distribution

Schematic Diagram of the Streaming Potential Device Courtesy of Yiheng Wang, Bioengineering Dept. UC Riverside, CA 2008

Membranes used in electrostatic test 30 kDa composite regenerated cellulose membrane (CRC) not charged not affected by pH Courtesy of Yiheng Wang, UCR, 2008 30 kDa polyethersulfone (PES) negatively charged at most pH conditions

Experimental Materials and Methods Set up of the streaming potential device Protein coated membranes are put in the middle of the device

Data Collection A piece of each CRC and PES membranes was soaked in Bovine Serum Albumin solution (BSA) solution and in nanopure water overnight prior to test A protein loaded membrane was put into the device for the streaming potential test Changing streaming potential was recorded with the changing hydraulic pressure applied on the device Zeta potential was calculated

ζ-Potential ζ-Potential was calculated through Helmholtz –Smoluchowski equation[1] ε0 - Permittivity of free space εr - Relative dielectric constant of the solvent µ - Viscosity of the solution Λ0 - Conductivity of the electrolyte solution [1] Nystrom et al. E.Streaming Potential as a tool in the characterization of ultrafiltration membranes. Colloids and Surfaces 1989, 36, 297-312

Zeta Potential Results and Analysis Membrane Coating ζ-potential (mV) – pH 7.0 pH 4.7 pH 10 CRC BSA+NaCl 19 + 5.86 8.6 + 0.83 _______ BSA+H2O 2.3 + 0.46 9.2 + 8.15 PES 30 + 19.04 16 + 1.46 11 + 5.13 23 + 6.48

Comparison to Preliminary Study Analysis CRC PES Yiineg Wang, Bioenginering Department, UC Riverside CA

Conclusion The test for the device integrity came out successful   The test for the device integrity came out successful Expected approximate values and readings were obtained in order to calculate zeta potential

Future Agenda Modifying and integrating the streaming potential device and methods in order to test zeta potential values of cultured intestinal mucosal epithelia Growing Caco2 cell culture from stomach mucosa Courtesy of Lo, David Biomedical Science Dept. UC Riverside, CA 2009

Acknowledgements NSF Dr. Victor G. J. Rodgers Dr. David Lo Mr. Devin McBride Ms. Kaila Bennett The B2K Group Mr. Jun Wang BRITE REU 2009 Personnel