Jeffrey Clark and Dr. Paul B. Savage, Chemistry and Biochemistry
Introduction
One current problem in medicine is the accumulation of biofilm on medical devices. Bacterial colonization of endotracheal tubes and urinary catheters is the leading cause of hospital-acquired infections. These infections contribute significantly to patient mortality rates and care costs1. Ceragenin-containing thin films have the potential to prevent these infections. Our lab and collaborators have recently shown that the ceragenin CSA-13 in thin films derived from polyurethane undergo sustained release and prevent bacterial colonization of a coated device2. Optimization of this technology has the potential to yield coatings for a variety of medical devices including endotracheal tubes and urinary catheters.
One of the problems we faced with polyurethane-based thin films was that we were unable to quantify how much CSA-13 was being released. By answering this question, we could determine whether the coatings eventually failed because they had released all of the sequestered CSA-13, or because not enough CSA-13 was able to escape the polymer for quantification.
Methods
To answer this question, I produced a solution with known concentrations of CSA-13 and Polyurethane dissolved in tetrahydrofuran (THF). I took a small portion of this solution and attempted to separate CSA-13 and polyurethane using column chromatography. The CSA-13 that came off the column was diluted and then quantified by comparison to a known sample using Liquid Chromatography-Mass Spectrometry. Using this technique, I was able to recover 10% of the expected amount of CSA-13. This could be due to at least two explanations. First, the CSA-13 could be inseparable from the polyurethane, arguing that the endotracheal tubes fail because CSA-13 is sequestered too tightly. Second, the CSA-13 could be retained on the column and so the data do not provide evidence for a conclusion.
A second method was used to further explore these possibilities. The polymer was advertised as insoluble in water, and so I extracted the CSA-13 from methylene chloride with five 2 ml portions of water. This method was then compared to a known sample using Liquid Chromatography-Mass Spectrometry and showed a recovery of 13% of the expected amount of CSA-13. To further increase the solubility of CSA-13 in water, I retried this extraction in dilute hydrochloric acid. CSA-13 has amine moieties that are easily protonated to become charged.
These charged regions of the molecule make it more soluble in water. Even with this additional help, only 20% of the expected CSA-13 could be recovered.
Conclusions
These results together argue that CSA-13 and polyurethane cannot be readily separated. Therefore, thin films made of CSA-13 and polyurethane only release a small portion of their CSA-13. The antimicrobial effects that prevent biofilm formation, therefore, are likely to be the result of an initial release of CSA-13. The eventual failure of these thin films is believed to be because of excessive sequestration of CSA-13.
Future Work
As I was carrying out this work, our lab was also testing thin films derived from a silicone medium. Ultimately, this medium has been shown to be much more effective in preventing biofilm formation on medical devices. Optimization of this technology is ongoing, and is currently being conducted in sheep.
These projects have looked at the effects of CSA-13 release from thin films. However, we also know that ceragenins are effective antimicrobial agents when covalently bound to surfaces. Furthermore, the ceragenin CSA-113 can be efficiently incorporated into polymer-brush coatings. Some of these brush coatings show potential reductions in biofilm on their own. In theory, a mixed polymer containing CSA-113 could produce a synergistic effect that would make these brush coatings even more effective. I am currently working on this project and hope to see it brought to fruition.
Sources
- Richards, M. J.; Edwards, J. R.; Culver, D. H.; Gaynes, R. P.; Crit. Care Med. 1999, 27, 887-892.
- Savage, P. B.; Nielson, J.; Lai, X.; Feng, Y.; Li, Y.; Nelson, G.; Linford, M. R.; Genberg, C.; Antibacterial Activities of Thin Films Containing Ceragenins. In Microbial Surfaces: Structure, Interactions, and Reactivity; Camesano, T. A.; Mello, C. M.; ACS Symposium Series 984; Oxford University Press; 65-78.