Cellular membranes play host to a wide variety of morphologically and chemically complex processes. Although model membranes, like liposomes, are already widely used to reconstitute and study these processes, better tools are needed for making model bilayers that faithfully mimic cellular membranes. Existing methods for fabricating cell‐sized (µm) or organelle‐sized (tens to hundreds of nanometers) lipid vesicles have distinctly different requirements. Of particular note for biology, it remains challenging for any technique to efficiently encapsulate fragile cargo molecules or to generate liposomes with stable, asymmetric lipid leaflets within the bilayer. Here a tunable microfluidic device and protocol for fabricating liposomes with desired diameters ranging from ≈10 µm to ≈100 nm are described. Lipid vesicle size is templated by the simple inclusion of a polycarbonate filter within the microfluidic system and tuned with flow rate. It is shown that the vesicles made with this device are stable, unilamellar, lipid asymmetric, and capable of supporting transmembrane protein assembly, peripheral membrane protein binding, as well as soluble cargo encapsulation (including designer nanocages for biotechnology applications). These fabricated vesicles provide a new platform for studying the biophysically rich processes found within lipid–lipid and lipid–protein systems typically associated with cellular membranes.
Rapid Production and Purification of Dye-Loaded Liposomes by Electrodialysis-Driven Depletion
Liposomes are spherical-shaped vesicles that enclose an aqueous milieu surrounded by bilayer or multilayer membranes formed by self-assembly of lipid molecules. They are intensively exploited as either model membranes for fundamental studies or as vehicles for delivery of active substances in vivo and in vitro. Irrespective of the method adopted for production of loaded liposomes, obtaining the final purified product is often achieved by employing multiple, time consuming steps. To alleviate this problem, we propose a simplified approach for concomitant production and purification of loaded liposomes by exploiting the Electrodialysis-Driven Depletion of charged molecules from solutions. Our investigations show that electrically-driven migration of charged detergent and dye molecules from solutions that include natural or synthetic lipid mixtures leads to rapid self-assembly of loaded, purified liposomes, as inferred from microscopy and fluorescence spectroscopy assessments. In addition, the same procedure was successfully applied for incorporating PEGylated lipids into the membranes for the purpose of enabling long-circulation times needed for potential in vivo applications. Dynamic Light Scattering analyses and comparison of electrically-formed liposomes with liposomes produced by sonication or extrusion suggest potential use for numerous in vitro and in vivo applications.
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- Award ID(s):
- 1554166
- PAR ID:
- 10405669
- Date Published:
- Journal Name:
- Membranes
- Volume:
- 11
- Issue:
- 6
- ISSN:
- 2077-0375
- Page Range / eLocation ID:
- 417
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract -
null (Ed.)The cell membrane is a protective barrier whose configuration determines the exchange both between intracellular and extracellular regions and within the cell itself. Consequently, characterizing membrane properties and interactions is essential for advancements in topics such as limiting nanoparticle cytotoxicity. Characterization is often accomplished by recreating model membranes that approximate the structure of cellular membranes in a controlled environment, formed using self-assembly principles. The selected method for membrane creation influences the properties of the membrane assembly, including their response to electric fields used for characterizing transmembrane exchanges. When these self-assembled model membranes are combined with electrophysiology, it is possible to exploit their non-physiological mechanics to enable additional measurements of membrane interactions and phenomena. This review describes several common model membranes including liposomes, pore-spanning membranes, solid supported membranes, and emulsion-based membranes, emphasizing their varying structure due to the selected mode of production. Next, electrophysiology techniques that exploit these structures are discussed, including conductance measurements, electrowetting and electrocompression analysis, and electroimpedance spectroscopy. The focus of this review is linking each membrane assembly technique to the properties of the resulting membrane, discussing how these properties enable alternative electrophysiological approaches to measuring membrane characteristics and interactions.more » « less
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Targeted delivery of drugs or other therapeutic agents through internal or external triggers has been used to control and accelerate the release from liposomal carriers in a number of studies, but relatively few utilize energy of therapeutic X-rays as a trigger. We have synthesized liposomes that are triggered by ionizing radiation (RTLs) to release their therapeutic payload. These liposomes are composed of natural egg phosphatidylethanolamine (PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG-2000), and the mean size of the RTL was in the range of 114 to 133 nm, as measured by nanoparticle tracking analysis (NTA). The trigger mechanism is the organic halogen, chloral hydrate, which is known to generate free protons upon exposure to ionizing radiation. Once protons are liberated, a drop in internal pH of the liposome promotes destabilization of the lipid bilayer and escape of the liposomal contents. In proof of principle studies, we assessed RTL radiation-release of fluorescent tracers upon exposure to a low pH extracellular environment or exposure to X-ray irradiation. Biodistribution imaging before and after irradiation demonstrated a preferential uptake and release of the liposomes and their cargo at the site of local tumor irradiation. Finally, a potent metabolite of the commonly used chemotherapy irinotecan, SN-38, was loaded into RTL along with near infrared (NIR) fluorescent dyes for imaging studies and measuring tumor cell cytotoxicity alone or combined with radiation exposure, in vitro and in vivo. Fully loaded RTLs were found to increase tumor cell killing with radiation in vitro and enhance tumor growth delay in vivo after three IV injections combined with three, 5 Gy local tumor radiation exposures compared to either treatment modality alone.more » « less
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Cell-based therapies have the potential to transform the treatment of many diseases. One of the key challenges relating to cell therapies is to modify the cell surface with molecules to modulate cell functions such as targeting, adhesion, migration, and cell–cell interactions, or to deliver drug cargos. Noncovalent insertion of lipid-based amphiphilic molecules on the cell surface is a rapid and nontoxic approach for modifying cells with a variety of bioactive molecules without affecting the cellular functions and viability. A wide variety of lipid amphiphiles, including proteins/peptides, carbohydrates, oligonucleotides, drugs, and synthetic polymers have been designed to spontaneously anchor on the plasma membranes. These molecules typically contain a functional component, a spacer, and a long chain diacyl lipid. Though these molecular constructs appeared to be stably tethered on cell surfaces both in vitro and in vivo under static situations, their stability under mechanical stress (e.g., in the blood flow) remains unclear. Using diacyl lipid-polyethylene glycol (lipo-PEG) conjugates as model amphiphiles, here we report the effect of molecular structures on the amphiphile stability on cell surface under mechanical stress. We analyzed the retention kinetics of lipo-PEGs on erythrocytes in vitro and in vivo and found that under mechanical stress, both the molecular structures of lipid and the PEG spacer have a profound effect on the membrane retention of membrane-anchored amphiphiles. Our findings highlight the importance of molecular design on the dynamic stability of membrane-anchored amphiphiles.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/membranes11060417
