Toward Infection-Resistant Surfaces: Achieving High Antimicrobial Peptide Potency by Modulating the Functionality of Polymer Brush and Peptide

Bacterial infection associated with indwelling medical devices and implants is a major clinical issue, and the prevention or treatment of such infections is challenging. Antimicrobial coatings offer a significant step toward addressing this important clinical problem. Antimicrobial coatings based on tethered antimicrobial peptides (AMPs) on hydrophilic polymer brushes have been shown to be one of the most promising strategies to avoid bacterial colonization and have demonstrated broad spectrum activity. Optimal combinations of the functionality of the polymer-brush-tethered AMPs are essential to maintaining long-term AMP activity on the surface. However, there is limited knowledge currently available on this topic. Here we report the development of potent antimicrobial coatings on implant surfaces by elucidating the roles of polymer brush chemistry and peptide structure on the overall antimicrobial activity of the coatings. We screened several combinations of polymer brush coatings and AMPs constructed on nanoparticles, titanium surfaces, and quartz slides on their antimicrobial activity and bacterial adhesion against Gram-positive and Gram-negative bacteria. Highly efficient killing of planktonic bacteria by the antimicrobial coatings on nanoparticle surfaces, as well as potent killing of adhered bacteria in the case of coatings on titanium surfaces, was observed. Remarkably, the antimicrobial activity of AMP-conjugated brush coatings demonstrated a clear dependence on the polymer brush chemistry and peptide structure, and optimization of these parameters is critical to achieving infection-resistant surfaces. By analyzing the interaction of polymer-brush-tethered AMPs with model lipid membranes using circular dichroism spectroscopy, we determined that the polymer brush chemistry has an influence on the extent of secondary structure change of tethered peptides before and after interaction with biomembranes. The peptide structure also has an influence on the density of conjugated peptides on polymer brush coatings and the resultant wettability of the coatings, and both of these factors contributed to the antimicrobial activity and bacterial adhesion of the coatings. Overall, this work highlights the importance of optimizing the functionality of the polymer brush to achieve infection-resistant surfaces and presents important insight into the design criteria for the selection of polymers and AMPs toward the development of potent antimicrobial coating on implants

Ultrasound imaging.

<p>A. The mice were anesthetized with isoflurane and mounted on the heated imaging table with continuous monitoring of vital signs. After visualization of the bladder with the Vevo 700® small animal imaging platform the skin was perforated with a 30G needle. B. Ultrasound visualisation of normal mouse bladder in sagittal section with typical dimensions indicated (lumen dimensions 4.4×6.5 mm; wall thickness 0.25 mm).</p

Inoculation of tumor cells.

<p>A. Detection of the needle on the ultrasound screen. B. Perforation of the skin and abdominal wall muscles. C. Needle insertion into the bladder wall without penetration of the mucosa. D. Injection of PBS (50 µl) between the muscular layer and the mucosa. E. Guidance of second needle into the artificially created space. F. Injection of tumor cells suspended in Matrigel®.</p

Longitudinal imaging of xenograft growth.

<p>Tumor growth was measured at regular time intervals by: A. bioluminescence imaging, and B. ultrasound. C. Correlation of bioluminescence and xenograft volume for all three cell lines. D. H&E section of a representative UM-UC1 luc xenograft demonstrating invasive growth into the muscle (*) without invasion into adjacent organs. All tumors originated from the anterior bladder wall and often occupied most of the bladder lumen without infiltrating the posterior wall (**).</p

Ultrasound-guided intratumoral injection of treatment agents.

<p>A. The xenografts were visualized by ultrasound and either VSV (1.05×10⁷ pfu) dissolved in 25 µl PBS or PBS alone was injected through a 30G needle into the center of the tumor. B. 48 h after injection of VSV, all xenograft tumors showed positive staining for VSV-G around the injection site which correlated to TUNEL staining. C. VSV-G and TUNEL staining were negative after PBS injection alone.</p

Treatment of xenograft tumors by chemotherapy.

<p>A. Mice bearing UM-UC13 luc tumors showed a remarkable decrease in tumor volume after systemic therapy with a combination of gemcitabine and cisplatin starting on day #28 after inoculation, compared to PBS control (** = P<0.01). B. Xenograft perfusion was measured by injection and ultrasound imaging of non-targeted microbubbles in UM-UC13 luc xenografts before and 5 days after administration of control agent (PBS; left panel) or systemic chemotherapy (gemcitabine/cisplatin; right panel). Perfusion was quantified as contrast percent area. Representative single results out of 4 measured animals per group are shown.</p

Intramural injection of agarose.

<p>The sagittal H&E section of a whole murine bladder demonstrates a layer of gel strictly in the lamina propria between mucosa and the muscularis propria.</p

Percutaneous tumor cell injection – procedure, complications and growth.

<p>Percutaneous tumor cell injection – procedure, complications and growth.</p