RESUMO
Pseudomonas aeruginosa (PsA) is an opportunistic bacterial pathogen that causes life-threatening infections in individuals with compromised immune systems and exacerbates health concerns for those with cystic fibrosis (CF). PsA rapidly develops antibiotic resistance; thus, novel therapeutics are urgently needed to effectively combat this pathogen. Previously, we have shown that a novel cationic Zinc (II) porphyrin (ZnPor) has potent bactericidal activity against planktonic and biofilm-associated PsA cells, and disassembles the biofilm matrix via interactions with eDNA In the present study, we report that ZnPor caused a significant decrease in PsA populations in mouse lungs within an in vivo model of PsA pulmonary infection. Additionally, when combined with an obligately lytic phage PEV2, ZnPor at its minimum inhibitory concentration (MIC) displayed synergy against PsA in an established in vitro lung model resulting in greater protection of H441 lung cells versus either treatment alone. Concentrations above the minimum bactericidal concentration (MBC) of ZnPor were not toxic to H441 cells; however, no synergy was observed. This dose-dependent response is likely due to ZnPor's antiviral activity, reported herein. Together, these findings show the utility of ZnPor alone, and its synergy with PEV2, which could be a tunable combination used in the treatment of antibiotic-resistant infections.
RESUMO
Silver nanoparticles (AgNPs) are being employed in numerous consumer goods and applications; however, they are renowned for inducing negative cellular consequences including toxicity, oxidative stress, and an inflammatory response. Nanotoxicological outcomes are dependent on numerous factors, including physicochemical, biological, and environmental influences. Currently, NP safety evaluations are carried out in both cell-based in vitro and animal in vivo models, with poor correlation between these mechanisms. These discrepancies highlight the need for enhanced exposure environments, which retain the advantages of in vitro models but incorporate critical in vivo influences, such as fluid dynamics. This study characterized the effects of dynamic flow on AgNP behavior, cellular interactions, and oxidative stress within both adherent alveolar (A549) and suspension monocyte (U937) models. This study determined that the presence of physiologically relevant flow resulted in substantial modifications to AgNP cellular interactions and subsequent oxidative stress, as assessed via reactive oxygen species (ROS), glutathione levels, p53, NFκB, and secretion of pro-inflammatory cytokines. Within the adherent model, dynamic flow reduced AgNP deposition and oxidative stress markers by roughly 20%. However, due to increased frequency of contact, the suspension U937 cells were associated with higher NP interactions and intracellular stress under fluid flow exposure conditions. For example, the increased AgNP association resulted in a 50% increase in intracellular ROS and p53 levels. This work highlights the potential of modified in vitro systems to improve analysis of AgNP dosimetry and safety evaluations, including oxidative stress assessments.
RESUMO
The incorporation of engineered nanoparticles (NPs) into everyday consumer goods, products, and applications has given rise to the field of nanotoxicology, which evaluates the safety of NPs within biological environments. The unique physicochemical properties of NPs have made this an insurmountable challenge, as their reactivity and variable behavior have given rise to discrepancies between standard cell-based in vitro and animal in vivo models. In this study, enhanced in vitro models were generated that retained the advantages of traditional cell cultures, but incorporated the modifications of (1) inclusion of an activated immune element and (2) the presence of physiologically-relevant dynamic flow. Following verification that the human alveolar epithelial and macrophage (A549/U937) co-culture could be successfully sustained under both static and dynamic conditions, these cultures, in addition to a standard A549 static model, were challenged with 10 nm citrate coated silver NPs (AgNPs). This work identified a reshaping of the AgNP-cellular interface and differential biological responses following exposure. The presence of dynamic flow modified cellular morphology and reduced AgNP deposition by approximately 20% over the static exposure environments. Cellular toxicity and stress endpoints, including reactive oxygen species, heat shock protein 70, and secretion of pro-inflammatory cytokines, were found to vary as a function of both cellular composition and flow conditions; with activated macrophages and fluid flow both mitigating the severity of AgNP-dependent bioeffects. This work highlights the possibility of enhanced in vitro systems to assess the safety of engineered NPs and demonstrates their effectiveness in elucidating novel NP-cellular interactions and toxicological profiles.