Selective Endocytic Uptake of Targeted Liposomes Occurs within a Narrow Range of Liposome Diameters.

Cell surface receptors facilitate signaling and nutrient uptake. These processes are dynamic, requiring receptors to be actively recycled by endocytosis. Due to their differential expression in disease states, receptors are often the target of drug-carrier particles, which are adorned with ligands that bind specifically to receptors. These targeted particles are taken into the cell by multiple routes of internalization, where the best-characterized pathway is clathrin-mediated endocytosis. Most studies of particle uptake have utilized bulk assays rather than observing individual endocytic events. As a result, the detailed mechanisms of particle uptake remain obscure. To address this gap, we employed a live-cell imaging approach to study the uptake of individual liposomes as they interact with clathrin-coated structures. By tracking individual internalization events, we find that the size of liposomes rather than the density of the ligands on their surfaces primarily determines their probability of uptake. Interestingly, targeting has the greatest impact on endocytosis of liposomes of intermediate diameters, with the smallest and largest liposomes being internalized or excluded, respectively, regardless of whether they are targeted. These findings, which highlight a previously unexplored limitation of targeted delivery, can be used to design more effective drug carriers.

Selective endocytic uptake of targeted liposomes occurs within a narrow range of liposome diameter.

Cell surface receptors facilitate signaling and nutrient uptake. These processes are dynamic, requiring receptors to be actively recycled by endocytosis. Due to their differential expression in disease states, receptors are often the target of drug-carrier particles, which are adorned with ligands that bind specifically to receptors. These targeted particles are taken into the cell by multiple routes of internalization, where the best-characterized pathway is clathrin-mediated endocytosis. Most studies of particle uptake have utilized bulk assays, rather than observing individual endocytic events. As a result, the detailed mechanisms of particle uptake remain obscure. To address this gap, we have employed a live-cell imaging approach to study the uptake of individual liposomes as they interact with clathrin-coated structures. By tracking individual internalization events, we find that the size of liposomes, rather than the density of the ligands on their surfaces, primarily determines their probability of uptake. Interestingly, targeting has the greatest impact on endocytosis of liposomes of intermediate diameters, with the smallest and largest liposomes being internalized or excluded, respectively, regardless of whether they are targeted. These findings, which highlight a previously unexplored limitation of targeted delivery, can be used to design more effective drug carriers.

Steric pressure between glycosylated transmembrane proteins inhibits internalization by endocytosis.

Clathrin-mediated endocytosis is essential for the removal of transmembrane proteins from the plasma membrane in all eukaryotic cells. Many transmembrane proteins are glycosylated. These proteins collectively comprise the glycocalyx, a sugar-rich layer at the cell surface, which is responsible for intercellular adhesion and recognition. Previous work has suggested that glycosylation of transmembrane proteins reduces their removal from the plasma membrane by endocytosis. However, the mechanism responsible for this effect remains unknown. To study the impact of glycosylation on endocytosis, we replaced the ectodomain of the transferrin receptor, a well-studied transmembrane protein that undergoes clathrin-mediated endocytosis, with the ectodomain of MUC1, which is highly glycosylated. When we expressed this transmembrane fusion protein in mammalian epithelial cells, we found that its recruitment to endocytic structures was substantially reduced in comparison to a version of the protein that lacked the MUC1 ectodomain. This reduction could not be explained by a loss of mobility on the cell surface or changes in endocytic dynamics. Instead, we found that the bulky MUC1 ectodomain presented a steric barrier to endocytosis. Specifically, the peptide backbone of the ectodomain and its glycosylation each made steric contributions, which drove comparable reductions in endocytosis. These results suggest that glycosylation constitutes a biophysical signal for retention of transmembrane proteins at the plasma membrane. This mechanism could be modulated in multiple disease states that exploit the glycocalyx, from cancer to atherosclerosis.

Investigation of an XDR-Acinetobacter baumannii ST2 outbreak in an intensive care unit of a Lebanese tertiary care hospital.

Aim: We sought to investigate the genetic epidemiological relatedness of carbapenem-resistant Acinetobacter baumannii (CRAB) strains of a suspected outbreak in a Lebanese tertiary care hospital to implement necessary infection prevention and control measures. Methods: Twenty-eight nonduplicate CRAB isolates detected among hospitalized patients between January 2016 and July 2017 were studied by real-time polymerase chain reaction (PCR), pulsed-field gel electrophoresis and multilocus sequence typing analyses. Results: Twenty-seven isolates harbored blaOXA-23, of which one also carried blaNDM-1. The isolates distributed temporally in two presumably episodes were stratified by pulsed-field gel electrophoresis into many clusters. Although several clones have become endemic in the hospital, we have rapidly implemented appropriate infection prevention and control measures, achieving full eradication from August 2017 to November 2019. Conclusion: We have successfully investigated and controlled a polyclonal outbreak of OXA-23 producing ST2 CRAB.

Molecular mechanisms of antimicrobial resistance in Acinetobacter baumannii, with a special focus on its epidemiology in Lebanon.

Acinetobacter baumannii is an opportunistic bacterium involved in several types of infection with high mortality and morbidity, especially in intensive care units. Treatment of these infections remains a challenge due to the worldwide emergence of broad-spectrum resistance to many antibiotics. Following the implementation of molecular techniques to study A. baumannii outbreaks, it has been shown that they are mainly caused by specific clones such as international clones I, II and III. The present work aims to review the available data on the mechanisms underlying antimicrobial resistance in A. baumannii, with a special focus on the molecular epidemiology of this species in Lebanon.