Introduction: Over 70% of cystic fibrosis patients are affected by persistent lung infections due to biofilms – clusters of bacteria encased in a self-produced extracellular matrix formed from DNA, polysaccharides, and proteins. A key factor in the persistence of these infections is the highly viscous nature of cystic fibrosis mucus. Combined, the mucus and biofilm act as drug barriers that prevent otherwise effective antimicrobials from eradicating the infection. In particular, the activity of cationic antimicrobials, such as tobramycin, is hindered by their interaction with negatively charged compounds of the extracellular matrix. Nanoparticles provide a valuable opportunity to overcome current limitations by enhancing mucus penetration, protection from degradation, and controlled release. For this application, we have developed polyelectrolyte surfactants that are designed to self-assemble with cationic antimicrobials into nanoparticles and to facilitate transport across mucus and biofilm drug delivery barriers. Specifically, we focus on poly(methylacrylic acid) (PMAA) and poly(propylacrylic acid) (PPAA) backbones, as they have shown stable encapsulation of cationic cargoes. Additionally, amphiphilic chains, such as Jeffamine M-2070, are capable improving particle stability, protection from the reticuloendothelial system, and enhanced sputum penetration.
Materials and
Methods: These surface-active polymers are composed of poly(alkylacrylic acid) (PAAA) backbones grafted via carbodiimide coupling with polyetheramine pendent chains (Jeffamine® M-2070). Nanocomplexes were formed via self-assembly between these polyelectrolyte surfactants and the cationic antimicrobials tobramycin, polymyxin B and colistin. Nanocomplex characterization included size, colloidal stability, release rate, and binding efficiency. Their ability to travel across a cystic fibrosis sputum mode l was studied using a transwell system and a cystic fibrosis mucus model. The antimicrobial activity of nanoparticle formulations was then evaluated against planktonic Pseudomonas aeruginosa cultures and their biofilms through common microtiter plate assays. Finally, their biodistribution was also studied following nebulized and systemic delivery in vivo.
Results, Conclusions, and Discussions: In the absence of a molecular cargo, each of the polymers forms nanoparticles of approximately 100 nm. When combined with cationic antimicrobials, all formulations exhibit particle size averages between 150-250 nm. Increasing graft density resulted in slightly larger size distributions explained by the decrease in linear charge density of the polymer backbone. Size distributions showed no significant change after 4 weeks, demonstrating their colloidal stability. Most polyelectrolyte surfactant:antimicrobial formulations showed binding efficiencies over 90%, with the exception of PPAA-g-1%J:tobramycin, for which the binding efficiency was 73%. PMAA-based formulations displayed slower drug release compared to free drug and PPAA-based nanoparticles. It was also observed that antimicrobials were released faster from formulations with lower graft density. The diffusivity of select nanoparticle formulations was evaluated using a cystic fibrosis mucus model that resembles the composition and viscosity profile of clinical samples. It was observed that nanoparticles enhance drug diffusion across the mucus model, likely by protecting the drug cargo from interactions with DNA and other negatively charged compounds. Poly(methylacrylic acid) (PMAA) based nanoparticles also showed a ≥2-fold increase of antimicrobial activity against planktonic cultures of four clinical isolates and retained activity against biofilms. In preparation for in vivo studies, we used a commercially available nebulizer to evaluate the aerosolization stability of our nanoparticles. We found that formulations remain in nanoparticle form and retain their antimicrobial activity. These findings demonstrate the potential use of polyelectrolyte surfactant nanoparticles to enhance antimicrobial treatment of cystic fibrosis lung infections via pulmonary delivery.
