High Throughput Methodologies for the Discovery of Materials Resistant to Biofilm Formation

1. High Throughput Methodologies for the Discovery of Materials Resistant to Biofilm Formation AVS – 12th of November Andrew Hook, Jing Yang, Chien-Yi Chang, Steve Atkinson, Paul Williams,Dan Anderson, Robert Langer, Martyn Davies,Morgan Alexander

2. Polymer micro arrays Group’s Micro Array Interests Materials for embryonic stem cell culture Anderson et al. Nature Biotech 2004 New bacteria resistant polymers Mei et al. Nature Materials Sept 2010 Yang et al. Biomaterials 2010 Saccharide arrays Staphylococcus aureus Scurr et al. Langmuir 2010 (in press) Uropathogenic E. coli Hook et al. in preparation 2010

3. We are searching for polymers that resist biofilm formation • Plasticware is widely used for medical devices • Catheters • Sutchers • Joint replacement • Contact lenses

4. Material Micro Arrays: Method • Screen for ‘Hits’; new materials with desirable biological response • Develop surface structure-function relationships to improve understanding and aid discovery Attempt to understand why cells respond to synthetic materials the way they do

5. Combinatorial polymeric library synthesised on-slide: • 24 x 24 polymer spot microarray • ca. 500 novel polymers in each array in triplicate • parallel assessment of all materials in one exposure experiment Polymeric Library as a microarray Anderson et al. Nat. Biotech. 2004

6. Piezo dosing of monomers

7. Development of a HT biological assay PBS wash twice, H2O rinse • Biofilms involved in 80% of hospital acquired infections • Highly resistant to antibiotic treatment • Screen for 3 clinical relevant bacterial strains • Pseudomonas aeruginosa (PA) • Staphylococcus aureus (SA) • UropathogenicEscherichia coli (UPEC) • Transformed with green fluorescing protein (GFP) GFP-bacteria Incubate at 37oC, 60 rpm for 72 hours OD600 ≈ 2 RMPI-1640 OD600 ≈ 0.01 Analyse with a GenePix 4200AL slide scanner Incubate at 37oC, 60 rpm for 24 hrs in AU Freshly printed slide

8. Multiple generation screening process Scale bar = 15 mm

9. High throughput surface characterisation (HTSC) Urquhart et al. Advanced Materials 2007

10. WCA and biofilm formation NO CORRELATION

11. XPS and biofilm formation NO CORRELATION

12. Explanatory variables X Predicted Y PLS Experimental Y Regression coefficient Responses Y Explanatory variable How to explore correlations in multivariate data (ToF SIMS): Partial least squares regression analysis The intensity of 455 ions from 576 polymer spots Urquhart, A. et al. Analytical Chemistry 2008.

13. ToF SIMS and PA01 loading A GOOD CORRELATION

14. Conclusions • Polymer microarrays coupled with a high throughput bacterial adhesion assay can rapidly identify materials that resist biofilm formation from screen to scale up. • Acrylate polymers show a wide range of bacterial adhesion. • The ToF SIMS spectra capture the important information that controls bacterial response (as it did for hES cells).

15. Acknowledgements Funding: BBSRC, MRC, EPSRC, EMDA and the Wellcome Trust Nottingham: Andrew Urquhart (now Strathclyde), Michael Taylor (now UCLan), Frank Rutten (now Keele), Paul Roach (now Keele), Jing Yang, Andrew Hook, Paul Williams and Martyn Davies MIT: Ying Mei,Robert Langer and Daniel Anderson.

16. Acknowledgements • Funding: BBSRC, EPSRC, MRC, Wellcome Trust, EMDA, • Collaborators: Rob Short, James Bradley, Nikolaj Gadegaard, Terry Parker, Felicity Rose, Lee Buttery, Mike Modo, Kevin Shakesheff, Steve Howdle. • My research Division the LBSA

17. Additional information

18. PLS of ToF SIMS and bacterial loading Peak list built in IonSpec The number of latent variables is selected via the cross validation step using RMSECV: 10 for SA and PA but 20 for UPEC. The software used is Eigenvector. The R2 value for an x=y line is 0.78 for PAO1, 0.81 for Staphylococcus and 0.6 for UPEC.