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theses

Bacterial biofilms build up in the sputum of cystic fibrosis patients, causing chronic respiratory infections and eventual lung failure. This mucus layer contains several bacterial species including gram-negative Pseudomonas aeruginosa and gram-positive Staphylococcus aureus, which are hard to eliminate, as multi-drug resistant strains, such as MRSA, develop. Three significant antimicrobial drugs known to fight cystic fibrosis lung infections include polymyxin B (PB), tobramycin (TB), and cathelicidin LL-37. Encapsulating such antimicrobials in self-assembling, amphiphilic polyelectrolyte surfactants (PSs) is hypothesized to enhance mucus penetration and biofilm eradication.
Numerous PS-PB, PS-TB and PS-LL37 formulations were prepared, varying the poly(alkylacrylic acid) backbones, the graft percentage of the Jeffamine® pendant chains, and the drug to polymer charge ratio. These “GRAPLON” nanomedicines were then characterized based upon their hydrodynamic diameter, zeta potential and release kinetics. Optimal formulations were aerosolized via a 3-jet Collision nebulizer, and aerosol droplet size distributions were determined for proper pulmonary delivery. Finally, the antimicrobial and anti-biofilm activities of particular formulations against clinical isolates from cystic fibrosis patient sputum samples were examined, and their minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs) were determined.
The “GRAPLON” nanomedicine, PMAA-g-10%J:TB, exhibited the most potential as an effective antimicrobial agent to eradicate cystic fibrosis lung infections. Complexes with PMAA-g-1%J and PPAA-g-1%J appeared to aggregate, as exhibited by their large hydrodynamic diameters. This was due to the nature of the PSs having less Jeffamine® graft, or fewer ethylene oxide groups, which typically resist aggregation. Nanoparticle formulations encapsulating LL-37 produced large and polydispersed solutions. The high molecular weight and amphiprotic nature of LL-37 may have caused weaker binding to the PSs, leading to mutual self-association, rather than complexation. LL-37 controlled release studies further exemplified the challenges with this peptide, as the concentrations of its membrane dialysis assay samples were below the limit of HPLC detection.
Small and stable complexes, less than 200 nm in hydrodynamic diameter and greater than |5| mV in zeta potential, were observed with the formulations PMAA-g-10%J:PB, PMAA-g-5%J:PB, PMAA-g-10%J:TB and PMAA-g-5%J:TB, with PSs dialyzed in 1X PBS. PMAA-g-10%J:TB exhibited a controlled release over the first 6 hours, demonstrating the release kinetics of the Higuchi model. PMAA-g-10%J:PB aerosolized easily, without mechanical damage, and its aerosol size distribution exhibited droplet sizes appropriate for pulmonary delivery, or under 5 μm in diameter.
Antimicrobial and anti-biofilm activity studies utilized nanoparticles conjugated with PMAA-g-10%J due to its high density of Jeffamine®, which is a copolymer of ethylene oxide and propylene oxide, and is hypothesized to confer particle stabilization and mucus penetration. As expected, the MICs of drugs were retained following PS encapsulation into nanoparticles. PMAA-g-10%J:TB consistently exhibited greater antimicrobial activity against the clinical isolate and commercial P. aeruginosa strains, as compared to PMAA-g-10%J:PB; therefore, the MBECs of tobramycin were investigated. As hypothesized, the MBECs of clinical isolate samples treated with PMAA-g-10%J:TB were less than or equal to the MBECs of analogous samples treated with free tobramycin. The greater anti-biofilm activity of PS-TB was due to the PS’s high detergency and membrane permeability, which enhanced mucus penetration. Therefore, due to its consistently small and stable nanoparticle formations, its Higuchi-like release kinetics, and its consistently high antimicrobial and anti-biofilm activities, PMAA-g-10%J:TB was concluded as the most optimal “GRAPLON” nanomedicine for cystic fibrosis treatment.

ETD_10386

M.S.

Includes bibliographical references

doi:10.7282/t3-wyca-d222

ETD graduate

The author owns the copyright to this work.