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Prevention of pathogenic colonization on medical devices over a long period of time remains a great challenge, especially in high-nutrient environment that accelerates the production of biomass leading to biofouling of the devices. Since biofouling and the subsequent pathogen colonization is inevitable, an alternative strategy using non-pathogenic bacteria as living guards against pathogenic colonization on medical devices attracts increasing interest. Crucial to the success of this strategy is to pre-establish a high coverage and stable biofilm of benign bacteria on the surface. Silicone elastomers are one of the most widely used materials in biomedical devices. This dissertation presents the studies aiming to biofunctionalize the silicone surfaces to enhance the formation of non-pathogenic biofilms that reduce the colonization by pathogenic bacteria. The non-pathogenic Escherichia coli 83972 is sluggish to establish bladder colonization. Pre-establishing high coverage and stable E. coli 83972 biofilms on silicone catheter surfaces is promising to improve the bacterial duration in bladder as well as simplify the tedious inoculation protocol. We use a high affinity propynylphenyl mannoside (PPh-Man) ligand for binding to the non-pathogenic E. coli, which is pre-conjugated to the amino-terminated poly(amido amine) (PAMAM) dendrimers that are covalently attached to activated silicone substrates. The coverage, stability and bacterial interference efficacy of non-pathogenic biofilms on the modified silicone substrates were evaluated. As a result, 94 % non-pathogenic bacteria were retained on the modified silicone substrates under >0.5 Pa shear stress. Synthesis of a series of mannoside derivatives varying the aromatic glycosidic linkage, including CF3-BP-Man, C2F5-BP-Man, CF3-TP-Man, CH3-BP-Man, CF3-PPh-Man and CH3-PPh-Man with an OEG linker terminated with an azido group for surface immobilization via the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC). These linkers were selected based on the reported X-ray structure-based design, and optimization of biaryl mannoside FimH inhibitors. To quantify the equilibrium binding affinity (IC50) of FimH to these mannosides, a method based on flow cytometry (FCM) was established. Mono-disperse polystyrene microspheres (~10 µm diameter) were covalently modified with mannoside, and their density was determined. These mannoside-coated particles were used for bacterial-particle incubation assay. This method greatly improved the efficiency and reproducibility over the reported method based using eukaryotic cell as target for measurement and comparison of the binding affinity of mannoside derivatives. An efficient method by using CO2 plasma to activate the catheter and functionalize the catheter inner lumen with G5 PAMAM dendrimers was developed. The efficacy of coatings was evaluated by radioactive isotopic iodine-125 labeling analysis. As a result, the PAMAM coverage on catheter is (94 ± 8) % and the PAMAM density per cm cather was (2.82 ± 0.24) × 1012 PAMAM/cm. The highest 125I-PAMAM coating was found in the innermost chamber. The best condition for CO2 plasma is 45 seconds in a low-power mode. The long-term stability result indicated more than 90 % of PAMAM dendrimers retained on the catheter rather than in the artificial urine solution for up to 15 days at room temperature statically. Our bioconjugation tool for surface and colloid functionalization is mostly based on the CuAAC reaction. The current Cu(I)-tris(triazolylmethyl)-amine catalysts still have the limitation of relatively low efficiency, oxidative degradation, and ligand dissociation in biological conditions. Contributing to address these issues, we measured kobs, KD and V0 for twenty one Cu(I) catalysts. Tris(triazolylmethyl)amines remained to be the best co-catalyst for CuAAC, but they were weaker ligands of Cu(I) compared to common biological ligands. More importantly, the polynuclear copper(I)-ligand-alkyne complexes were found crucial for CuAAC reaction that was directly observed by electrospray-ionization mass spectrometry (ESI-MS). Significant air oxidation still occurred in the presence of ligands, leading to rapid degradation of a histidine-containing peptide as a model of proteins. We present here a simple approach to reduce the air oxidation of the peptide, based on the use of oligo(ethylene glycol) chains tethered to the Cu(I) ligand for sacrificial protection of the ligand and the biomolecules from being oxidized by the oxy radicals generated from the copper center during CuAAC reaction. I also initiated the project on the development of fluorous silica nanospheres as carriers of perfluoroalkanes as ultrasound imaging agents and drug molecules with a fluorous tag for controlled release. The use of fluorous nanospheres should allow the loading of low-boiling point perfluoropropane (PFP) and perfluorobutane (PFB) that can be released and imaged by applying low frequency ultrasound (LFUS), and the fluorous-tagged cargo molecules be ejected with a temporal and spatial control.



Biofilm, Mannoside, Catheter, PAMAM, CuAAC