Smart Surfaces for the Control of Bacterial Attachment and Biofilm Accumulation

1. CBME @ UNM Smart Surfaces for the Control of Bacterial Attachment and Biofilm Accumulation Linnea K. Ista and Gabriel P. Lopez Center for Biomedical Engineering Department of Chemical and Nuclear Engineering The University of New Mexico Smart Coatings Symposium 15 February, 2006 Orlando, Florida The University of New Mexico

2. Bacterial attachment and biofilm formation: • The accumulation of bacteria, bacterial metabolites and small organics on a surface • Specialized ecological niche • Separate developmental form of bacteria • Resistant to antimicrobial agents, desiccation, • Increased nutritional concentration • Increased genetic exchange O’Toole et al, Annu. Rev. Microbiol. 2000, 59:49-79

3. To promote adhesion: Antibiotic production Fermentation Bioremediation Biosensing applications To deter adhesion: Medical implants Ship hulls/oil platforms Environmental systems Non specific adsorption in assays Why do we want to control biofilm formation/bacterial adhesion? • To enable reversible attachment and detachment • Preconcentration • Fundamental studies of biofilm development

4. 2500 2000 1500 Cells mm-2 1000 500 0 CF3 CH3 EG6 COOH Surface How can we control biofilm formation/bacterial adhesion? Surfaces that resist bacterial attachment: Ista, et al, FEMS Microbiol. Lett. 1996. 149:59-63

5. How can we control biofilm formation/bacterial adhesion? Surfaces that resist bacterial attachment: Surfaces with biocidal activity:

6. How can we control biofilm formation/bacterial adhesion? Surfaces that resist bacterial attachment: Surfaces with biocidal activity: Fouling release surfaces: Ista and Lopez, J. Industr. Microbiol. Biotechnol, 1998. 20:121-125

7. C H C H 2 C O n N H C H ~45OC 25°C C H C H 3 3 Smart polymers as fouling release surfaces Exploit interfacial phase transitions for fouling release Poly (N-isopropylacrylamide) PNIPAAM Solubility phase transition ~32oC

8. C H C H 2 C O n N H C H ~45OC 25°C C H C H 3 3 Smart polymers as fouling release surfaces Exploit interfacial phase transitions for fouling release Poly(N-isopropylacrylamide) PNIPAAM Solubility phase transition ~32oC

9. Attachment/detachment studies Sample Samples incubated in bacterial suspension at incubation temperature (2-72 hr) Sample rinsed with deionized H2O at incubation temperature Count number of attached cells; 60x, phase contrast Rinse with 60 mL release temperature buffer, followed by a rinse in deionized H2O Count number of attached cells; 60x, phase contrast

10. What happened? Cobetia marina(formerly Halomonas marina) ATCC 25374 • Gram negative • Obligately aerobic • Marine source -Pacific • Motile by gliding (mostly) or flagella Ista, et al, Appl. Eviron. Microbiol 1999. 65:1603-1609 Incubation temp. 37OC; release 4oC

11. What happened? Staphylococcus epidermidisATCC 14990 No release upon transition when attached at 37oC and rinsed at 4oC! • Gram positive • Facultative anaerobe • Skin bacteria- medically relevant • Non motile

12. S A u X + HS X Why? Attachment to SAMs • Self Assembled Monolayers of ω-substituted alkanethiolates on gold • Well ordered surface • Can express a single or multiple moieties on the surface • Can do chemical modification on the surface • Gold can be made any thickness, allowing for a variety of analytical techniques

13. Why? Attachment to SAMs C. marina S. epidermidis

14. Changing attachment/detachment temperature regime results in removal of S. epidermidis 10mm 10mm S. epdermidis Incubation at 25oC; 72 hours S. epidermidis After rinsing with 37oC PBS

15. Both newly attached cells and biofilms can be released using this method C. marina attach at 37oC; Release at 4oC S. epidermidis attach at 25oC; release at 37oC

16. Exploring the effects of changing physicochemistry on fouling release using PNIPAAM grafted from SAMs Ista, et al, Langmuir, 2001 65:2552-2555

17. Exploring the effects of changing hydrophobicity on fouling release using PNIPAAM grafted from SAMs Atom transfer radical polymerization (Balamurguran, et al. Langmuir, 2003, 19:2545-2549 R-Br +[Cu(I)Br]L Advantages of ATRP • Free radical is formed only at the surface eliminating undesired reactions caused by solution free radicals • Surface coverage can be varied by altering the surface mole% of OH-terminated thiolate in the SAM • Length of polymer can be controlled by time of polymerization. • Room temperature polymerization • Slow reaction results in increased monodispersity

18. Changing hydrophobicity by changing the comonomer Copolymerization with tert-butyl acrylamide results in surfaces on which the overall hydrophobicity is higher. Change in θAw PNIPAAM: 60o to 42o P 21: 78o to 63o P 14: 81o to 60o With Drs. Sreelatha and Subramanian Balamurugaran

19. C. marina : attach 37oC; release 4OC Changes in temperature result in different attachment and detachment behavior C. marina : attach 25oC; release 4OC

20. Using “tunable” PNIPAAM to examine effect of changing hydrophobicity Mendez, et al, Langmuir, 2003. 19:8115-8116

21. Attachment to tunable PNIPAAM S. epidermidis attach 25oC C. marina : attach 37oC With Dr. Sergio Mendez

22. Release from tunable PNIPAAM S. epidermidis attach 25oC; Release 37oC C. marina : attach 37oC; release 4OC

23. Toward practical coatings:silica/smart polymer composites PNIPAAM Silica network With Dr. V.G. Rama Rao

24. Smart polymer-silica composites both resist attachment andpromoterelease C. marina: Attach 2 hr 37oC; release 4oC

25. Mixed oligo(ethylene glycol)/ methyl terminated sams – a new smart material for biofilm release? Prime and Whitesides, J. Amer. Chem. Soc. 1993, 115: 10714- 10721

26. Balamurugaran, et al, J. Amer. Chem Soc. 2005. 127:14548-14549 C. marina: Attach 2 hr 37oC; release 4oC

27. Acknowledgements • Sergio Mendez, Sreelatha Balamurugaran, Subramanian Balamurugaran, G.V. Rama Rao • Robin Simons • Office of Naval Research • Sandia National Labs

28. Thank you! Any questions?