1. 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

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

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

4. 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

5. 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

6. 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

7. 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 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

8. 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

9. 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

10. Common Factors Considered in Electrostatic AnalysispH
Ionic strength
Zeta potential
Electrostatic potential distribution

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

12. 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

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

14. 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

15. 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,

16. Zeta Potential Results and Analysis
Membrane
Coating
ζ-potential (mV) –
pH 7.0
pH 4.7
pH 10
CRC
BSA+NaCl
_______
BSA+H2O
PES

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

18. 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

19. 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

20. 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