Determining the effects of PEI adsorption on the permeability of 1,2-dipalmitoylphosphatidylcholine/bis(monoacylglycero)phosphate membranes under osmotic stress

Clark, Scott; Lee, Keel Yong; Lee, Hoyoung; Khetan, Jawahar; Kim, Hyeon Chang; Choi, Yeon-Seok; Shin, Kwanwoo; Won, You‐Yeon

doi:10.1016/j.actbio.2017.10.027

Cited by 22 publications

(18 citation statements)

References 34 publications

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“…As endosomal escape efficiencies of delivery systems in general are routinely extraordinarily low (approximately 1%), 61 SEE assays help to elucidate trends and help suggest when modifications to delivery systems would be advantageous. [33][34][35][36] Lipid bilayer leakage assays 61,62 can be used to similarly reveal endosomal escape capabilities and when engineering may be needed but notably, they do not completely mimic biological contexts. 21 This is imperative to note as endosomal escape is truly the bottleneck of delivery, 22 and the lack of easy assays to evaluate structure-activity relationships hinder delivery vehicle optimization and progress.…”

Section: Discussionmentioning

confidence: 99%

Tracking exogenous intracellular casp‐3 using split GFP

et al. 2020

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Cytosolic protein delivery promises diverse applications from therapeutics, to genetic modification and precision research tools. To achieve effective cellular and subcellular delivery, approaches that allow protein visualization and accurate localization with greater sensitivity are essential. Fluorescently tagging proteins allows detection, tracking and visualization in cellulo. However, undesired consequences from fluorophores or fluorescent protein tags, such as nonspecific interactions and high background or perturbation to native protein's size and structure, are frequently observed, or more troublingly, overlooked. Distinguishing cytosolically released molecules from those that are endosomally entrapped upon cellular uptake is particularly challenging and is often complicated by the inherent pH-sensitive and hydrophobic properties of the fluorophore. Monitoring localization is more complex in delivery of proteins with inherent protein-modifying activities like proteases, transacetylases, kinases, etc. Proteases are among the toughest cargos due to their inherent propensity for self-proteolysis. To implement a reliable, but functionally silent, tagging technology in a protease, we have developed a caspase-3 variant tagged with the 11th strand of GFP that retains both enzymatic activity and structural characteristics of wild-type caspase-3. Only in the presence of cytosolic GFP strands 1-10 will the tagged caspase-3 generate fluorescence to signal a non-endosomal location. This methodology facilitates easy screening of cytosolic vs. endosomallyentrapped proteins due to low probabilities for false positive results, and further, allows tracking of the resultant cargo's translocation. The development of this tagged casp-3 cytosolic reporter lays the foundation to probe caspase therapeutic properties, charge-property relationships governing successful escape, and the precise number of caspases required for apoptotic cell death.

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Section: Discussionmentioning

confidence: 99%

Tracking exogenous intracellular casp‐3 using split GFP

et al. 2020

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“…In this respect, a paper was published very recently by Clark et al who studied the interaction of PEI with endosomal lipids under osmotic stress using synthetic monolayers and vesicles. 56 Moreover, it would be fascinating not only to study the intercellular differences in membrane composition, but also intracellular differences such as different membrane composition resulting from different uptake pathways within the same cell type. This information could lead us towards identifying types of endosomes that are less likely to form these leaks, thereby giving proton sponge-based rupture a better chance at success.…”

Section: Endosomal Leakinessmentioning

confidence: 99%

Endosomal Size and Membrane Leakiness Influence Proton Sponge-Based Rupture of Endosomal Vesicles

et al. 2018

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In gene therapy, endosomal escape represents a major bottleneck since nanoparticles often remain entrapped inside endosomes and are trafficked toward the lysosomes for degradation. A detailed understanding of the endosomal barrier would be beneficial for developing rational strategies to improve transfection and endosomal escape. By visualizing individual endosomal escape events in live cells, we obtain insight into mechanistic factors that influence proton sponge-based endosomal escape. In a comparative study, we found that HeLa cells treated with JetPEI/pDNA polyplexes have a 3.5-fold increased endosomal escape frequency compared to ARPE-19 cells. We found that endosomal size has a major impact on the escape capacity. The smaller HeLa endosomes are more easily ruptured by the proton sponge effect than the larger ARPE-19 endosomes, a finding supported by a mathematical model based on the underlying physical principles. Still, it remains intriguing that even in the small HeLa endosomes, <10% of the polyplex-containing endosomes show endosomal escape. Further experiments revealed that the membrane of polyplex-containing endosomes becomes leaky to small compounds, preventing effective buildup of osmotic pressure, which in turn prevents endosomal rupture. Analysis of H1299 and A549 cells revealed that endosomal size determines endosomal escape efficiency when cells have comparable membrane leakiness. However, at high levels of membrane leakiness, buildup of osmotic pressure is no longer possible, regardless of endosomal size. Based on our findings that both endosomal size and membrane leakiness have a high impact on proton sponge-based endosomal rupture, we provide important clues toward further improvement of this escape strategy.

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“…A previous report by Iqbal et al [44] suggested that encapsulation of bacteria in the PDA film also inhibited bacterial proliferation and survival. In another study, positively charged polymers, such as PEI, can enhance permeability of negatively charged bacterial outer membranes [45,46]. It is considered that the PEI chemistry on the titanium surfaces alone can also inhibit bacterial activity [47].…”

Section: Discussionmentioning

confidence: 99%

Facile preparation of mussel-inspired antibiotic-decorated titanium surfaces with enhanced antibacterial activity for implant applications

Lee

Yang

et al. 2019

Applied Surface Science

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Titanium implants (Ti) have been widely used in several medical fields. In clinical practice, Ti can become contaminated with bacteria through a variety of mechanisms. This contamination can lead to implant failure and serious infections. In this study, we aimed to develop a new, hybrid Ti with good biocompatibility and antibacterial properties by immobilizing ceftazidime (CFT) onto the Ti surface through polydopamine (PDA) and polyethyleneimine (PEI) chemistry. Hybrid Ti was confirmed by assessing the cell proliferation of human adipose-derived stem cells using a cell counting. The biofilm formation across the Ti surface of two bacterial strains associated with nosocomial infections, Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus, was evaluated by scanning electron microscopy. The viability of the bacteria exposed to Ti surface was evaluated by cell counting. Our results clearly demonstrate that the bacterial biofilm formation as well as bacterial viability was significantly reduced on the hybrid Ti as compared to the control, Ti alone. Collectively, the Ti surface was successfully modified to form the hybrid Ti exhibiting good biocompatibility and antibacterial properties through PDA, PEI, and CFT grafting. Within the limitations of this in vitro study, we conclude that the hybrid Ti may be useful for successful implant treatment.

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Determining the effects of PEI adsorption on the permeability of 1,2-dipalmitoylphosphatidylcholine/bis(monoacylglycero)phosphate membranes under osmotic stress

Cited by 22 publications

References 34 publications

Tracking exogenous intracellular casp‐3 using split GFP

Tracking exogenous intracellular casp‐3 using split GFP

Endosomal Size and Membrane Leakiness Influence Proton Sponge-Based Rupture of Endosomal Vesicles

Facile preparation of mussel-inspired antibiotic-decorated titanium surfaces with enhanced antibacterial activity for implant applications

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