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1. Dipolar Janus liposomes: formation, electrokinetic motion and self-assembly

   https://doi.org/10.1039/c9sm02254f  

   Liu, Zening; Cui, Jinyan; Zhan, Wei (March 2020, Soft Matter)

   Presented herein is the first report on dipolar Janus liposomes–liposomes that contain opposite surface charges decorating the two hemispheres of the same colloidal body. Such heterogeneous organization of surface charge is achieved through cholesterol-modulated lipid phase separation, which sorts anionic/cationic lipids into coexisting liquid-ordered/liquid-disordered domains. We present optimized experimental conditions to produce these liposomes in high yields, based on the gel-assisted hydration of ternary lipid systems consisting of cholesterol, 1,2-dipalmitoyl- sn-glycero -3-phosphocholine, and 1,2-dioleoyl- sn-glycero -3-phosphocholine. The size/charge distribution and domain configuration of these liposomes are characterized in detail by confocal fluorescence microscopy, nanosphere binding and zeta potential measurements. Using confocal fluorescence microscopy, we also follow the electrokinetic motion as well as the electrostatic self-assembly of these new dipolar Janus particles. 

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2. Intracellular phase separation of globular proteins facilitated by short cationic peptides

   https://doi.org/10.1038/s41467-022-35529-2  

   Yeong, Vivian; Wang, Jou-wen; Horn, Justin M.; Obermeyer, Allie C. (December 2022, Nature Communications)

   Abstract Phase separation provides intracellular organization and underlies a variety of cellular processes. These biomolecular condensates exhibit distinct physical and material properties. Current strategies for engineering condensate formation include using intrinsically disordered domains and altering protein surface charge by chemical supercharging or site-specific mutagenesis. We propose adding to this toolbox designer peptide tags that provide several potential advantages for engineering protein phase separation in bacteria. Herein, we demonstrate the use of short cationic peptide tags for sequestration of proteins of interest into bacterial condensates and provide a foundational study for their development as tools for condensate engineering. Using a panel of GFP variants, we demonstrate how cationic tag and globular domain charge contribute to intracellular phase separation inE. coliand observe that the tag can affect condensate disassembly at a given net charge near the phase separation boundary. We showcase the broad applicability of these tags by appending them onto enzymes and demonstrating that the sequestered enzymes remain catalytically active. 

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3. Cationic Liposomes as Vectors for Nucleic Acid and Hydrophobic Drug Therapeutics

   https://doi.org/10.3390/pharmaceutics13091365  

   Ewert, Kai K.; Scodeller, Pablo; Simón-Gracia, Lorena; Steffes, Victoria M.; Wonder, Emily A.; Teesalu, Tambet; Safinya, Cyrus R. (September 2021, Pharmaceutics)

   Cationic liposomes (CLs) are effective carriers of a variety of therapeutics. Their applications as vectors of nucleic acids (NAs), from long DNA and mRNA to short interfering RNA (siRNA), have been pursued for decades to realize the promise of gene therapy, with approvals of the siRNA therapeutic patisiran and two mRNA vaccines against COVID-19 as recent milestones. The long-term goal of developing optimized CL-based NA carriers for a broad range of medical applications requires a comprehensive understanding of the structure of these vectors and their interactions with cell membranes and components that lead to the release and activity of the NAs within the cell. Structure–activity relationships of lipids for CL-based NA and drug delivery must take into account that these lipids act not individually but as components of an assembly of many molecules. This review summarizes our current understanding of how the choice of the constituting lipids governs the structure of their CL–NA self-assemblies, which constitute distinct liquid crystalline phases, and the relation of these structures to their efficacy for delivery. In addition, we review progress toward CL–NA nanoparticles for targeted NA delivery in vivo and close with an outlook on CL-based carriers of hydrophobic drugs, which may eventually lead to combination therapies with NAs and drugs for cancer and other diseases. 

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4. Shake It Off! Acoustic Manipulation of Lipid Vesicles for Mass Spectrometric Analysis

   https://doi.org/10.1021/acs.analchem.3c01722  

   Taylor, Ashton N.; Huang, Yuqi; Sircher, Cheyenne; Khalife, Sandra; Bhethanabotla, Venkat; Evans-Nguyen, Theresa (September 2023, Analytical Chemistry)

   Analyzing lipid assemblies, including liposomes and extracellular vesicles (EVs), is challenging due to their size, diverse composition, and tendency to aggregate. Such vesicles form with a simple phospholipid bilayer membrane, and they play important roles in drug discovery and delivery. The use of mass spectrometry (MS) allows for broad analysis of lipids from different classes; however, their release from the higher order structural aggregates is typically achieved by chemical means. Mechanical disruption by high frequency surface acoustic waves (SAW) is presented as an appealing alternative to preparing lipid vesicles for MS sampling. In this work, SAWs used to disrupt liposomes allow for the direct analysis of their constituent lipids by employing SAW nebulization with corona discharge (CD) ionization. We explore the effects of duration, frequency, and incorporation of nonpolar lipids, including cholesterol, on the SAW’s ability to disrupt the liposome. We also report on the successful MS analysis of liposome-derived lipids along with cytochrome C in solution, thus demonstrating applications to aqueous samples and native MS conditions. 

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5. Direct label-free imaging of nanodomains in biomimetic and biological membranes by cryogenic electron microscopy

   https://doi.org/10.1073/pnas.2002200117  

   Heberle, Frederick A.; Doktorova, Milka; Scott, Haden L.; Skinkle, Allison D.; Waxham, M. Neal; Levental, Ilya (August 2020, Proceedings of the National Academy of Sciences)

   The nanoscale organization of biological membranes into structurally and compositionally distinct lateral domains is believed to be central to membrane function. The nature of this organization has remained elusive due to a lack of methods to directly probe nanoscopic membrane features. We show here that cryogenic electron microscopy (cryo-EM) can be used to directly image coexisting nanoscopic domains in synthetic and bioderived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying chain lengths, we demonstrate that cryo-EM can distinguish bilayer thickness differences as small as 0.5 Å, comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryo-EM images result from a complex interplay between the atomic distribution normal to the plane of the bilayer and imaging parameters. Simulations of phase-separated bilayers were used to predict two sources of contrast between coexisting ordered and disordered phases within a single liposome, namely differences in membrane thickness and molecular density. We observe both sources of contrast in biomimetic membranes composed of saturated lipids, unsaturated lipids, and cholesterol. When extended to isolated mammalian plasma membranes, cryo-EM reveals similar nanoscale lateral heterogeneities. The methods reported here for direct, probe-free imaging of nanodomains in unperturbed membranes open new avenues for investigation of nanoscopic membrane organization. 

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