Articoli di riviste: "Giant unilamellar vesicle (GUVs)"

1

Litschel, Thomas, e Petra Schwille. "Protein Reconstitution Inside Giant Unilamellar Vesicles". Annual Review of Biophysics 50, n. 1 (6 maggio 2021): 525–48. http://dx.doi.org/10.1146/annurev-biophys-100620-114132.

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Abstract (sommario):

Giant unilamellar vesicles (GUVs) have gained great popularity as mimicries for cellular membranes. As their sizes are comfortably above the optical resolution limit, and their lipid composition is easily controlled, they are ideal for quantitative light microscopic investigation of dynamic processes in and on membranes. However, reconstitution of functional proteins into the lumen or the GUV membrane itself has proven technically challenging. In recent years, a selection of techniques has been introduced that tremendously improve GUV-assay development and enable the precise investigation of protein–membrane interactions under well-controlled conditions. Moreover, due to these methodological advances, GUVs are considered important candidates as protocells in bottom-up synthetic biology. In this review, we discuss the state of the art of the most important vesicle production and protein encapsulation methods and highlight some key protein systems whose functional reconstitution has advanced the field.

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Wu, Hao, Zhong-Can Ou-Yang e Rudolf Podgornik. "A Note on Vestigial Osmotic Pressure". Membranes 13, n. 3 (14 marzo 2023): 332. http://dx.doi.org/10.3390/membranes13030332.

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Recent experiments have indicated that at least a part of the osmotic pressure across the giant unilamellar vesicle (GUV) membrane was balanced by the rapid formation of the monodisperse daughter vesicles inside the GUVs through an endocytosis-like process. Therefore, we investigated a possible osmotic role played by these daughter vesicles for the maintenance of osmotic regulation in the GUVs and, by extension, in living cells. We highlighted a mechanism whereby the daughter vesicles acted as osmotically active solutes (osmoticants), contributing an extra vestigial osmotic pressure component across the membrane of the parent vesicle, and we showed that the consequences were consistent with experimental observations. Our results highlight the significance of osmotic regulation in cellular processes, such as fission/fusion, endocytosis, and exocytosis.

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Mardešić, Ivan, Zvonimir Boban e Marija Raguz. "Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films with a Focus on Vesicles with High Cholesterol Content". Membranes 14, n. 4 (27 marzo 2024): 79. http://dx.doi.org/10.3390/membranes14040079.

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Giant unilamellar vesicles (GUVs) are membrane models used to study membrane properties. Electroformation is one of the methods used to produce GUVs. During electroformation protocol, dry lipid film is formed. The drying of the lipid film induces the cholesterol (Chol) demixing artifact, in which Chol forms anhydrous crystals which do not participate in the formation of vesicles. This leads to a lower Chol concentration in the vesicle bilayers compared to the Chol concentration in the initial lipid solution. To address this problem, we propose a novel electroformation protocol that includes rapid solvent exchange (RSE), plasma cleaning, and spin-coating methods to produce GUVs. We tested the protocol, focusing on vesicles with a high Chol content using different spin-coating durations and vesicle type deposition. Additionally, we compared the novel protocol using completely dry lipid film. The optimal spin-coating duration for vesicles created from the phosphatidylcholine/Chol mixture was 30 s. Multilamellar vesicles (MLVs), large unilamellar vesicles (LUVs) obtained by the extrusion of MLVs through 100 nm membrane pores and LUVs obtained by extrusion of previously obtained LUVs through 50 nm membrane pores, were deposited on an electrode for 1.5/1 Chol/phosphatidylcholine (POPC) lipid mixture, and the results were compared. Electroformation using all three deposited vesicle types resulted in a high GUV yield, but the deposition of LUVs obtained by the extrusion of MLVs through 100 nm membrane pores provided the most reproducible results. Using the deposition of these LUVs, we produced high yield GUVs for six different Chol concentrations (from 0% to 71.4%). Using a protocol that included dry lipid film GUVs resulted in lower yields and induced the Chol demixing artifact, proving that the lipid film should never be subjected to drying when the Chol content is high.

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Boban, Zvonimir, Ivan Mardešić, Sanja Perinović Jozić, Josipa Šumanovac, Witold Karol Subczynski e Marija Raguz. "Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films Formed by Vesicle Fusion". Membranes 13, n. 3 (18 marzo 2023): 352. http://dx.doi.org/10.3390/membranes13030352.

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Giant unilamellar vesicles (GUVs) are artificial membrane models which are of special interest to researchers because of their similarity in size to eukaryotic cells. The most commonly used method for GUVs production is electroformation. However, the traditional electroformation protocol involves a step in which the organic solvent is completely evaporated, leaving behind a dry lipid film. This leads to artifactual demixing of cholesterol (Chol) in the form of anhydrous crystals. These crystals do not participate in the formation of the lipid bilayer, resulting in a decrease of Chol concentration in the bilayer compared to the initial lipid solution. We propose a novel electroformation protocol which addresses this issue by combining the rapid solvent exchange, plasma cleaning and spin-coating techniques to produce GUVs from damp lipid films in a fast and reproducible manner. We have tested the protocol efficiency using 1/1 phosphatidylcholine/Chol and 1/1/1 phosphatidylcholine/sphingomyelin/Chol lipid mixtures and managed to produce a GUV population of an average diameter around 40 µm, with many GUVs being larger than 100 µm. Additionally, compared to protocols that include the dry film step, the sizes and quality of vesicles determined from fluorescence microscopy images were similar or better, confirming the benefits of our protocol in that regard as well.

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Tan, Cherng-Wen Darren, Magdalena Schöller e Eva-Kathrin Ehmoser. "Rapid Multi-Well Evaluation of Assorted Materials for Hydrogel-Assisted Giant Unilamellar Vesicle Production: Empowering Bottom-Up Synthetic Biology". Gels 11, n. 1 (2 gennaio 2025): 29. https://doi.org/10.3390/gels11010029.

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Giant unilamellar vesicles (GUVs) are versatile cell models in biomedical and environmental research. Of the various GUV production methods, hydrogel-assisted GUV production is most easily implemented in a typical biological laboratory. To date, agarose, polyvinyl alcohol, cross-linked dextran-PEG, polyacrylamide, and starch hydrogels have been used to produce GUVs. Some leach and contaminate the GUVs, while others require handling toxic material or specialised chemistry, thus limiting their use by novices. Alternative hydrogel materials could address these issues or even offer novel advantages. To facilitate discovery, we replaced the manual spreading of reagents with controlled drop-casting in glass Petri dishes and polystyrene multi-well plates, allowing us to rapidly screen up to 96 GUV-production formulations simultaneously. Exploiting this, we rapidly evaluated assorted biomedical hydrogels, including PEG-DA, cross-linked hyaluronic acid, Matrigel, and cross-linked DNA. All of these alternatives successfully produced GUVs. In the process, we also developed a treatment for recycling agarose and polyvinyl alcohol hydrogels for GUV production, and successfully encapsulated porcine liver esterase (PLE-GUVs). PLE-GUVs offer a novel method of GUV labelling and tracing, which emulates the calcein-AM staining behaviour of cells. Our results highlight the utility of our protocol for potentiating substrate material discovery, as well as protocol and product development.

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6

Sirbu, Dumitru, Lingli Zeng, Paul G. Waddell, Nikolai V. Tkachenko, Stanley W. Botchway e Andrew C. Benniston. "Voltage-induced fluorescence lifetime imaging of a BODIPY derivative in giant unilamellar vesicles as potential neuron membrane mimics". Chemical Communications 57, n. 94 (2021): 12631–34. http://dx.doi.org/10.1039/d1cc03756k.

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Voltage induced lifetime imaging (VILI) is described using a BODIPY dye and Giant Unilamellar Vesicles (GUVs) as neuron membrane mimics. Fluorescence lifetime modulation was shown to map to a change in voltage across the GUV.

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Boban, Zvonimir, Ivan Mardešić, Witold Karol Subczynski, Dražan Jozić e Marija Raguz. "Optimization of Giant Unilamellar Vesicle Electroformation for Phosphatidylcholine/Sphingomyelin/Cholesterol Ternary Mixtures". Membranes 12, n. 5 (16 maggio 2022): 525. http://dx.doi.org/10.3390/membranes12050525.

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Artificial vesicles are important tools in membrane research because they enable studying membrane properties in controlled conditions. Giant unilamellar vesicles (GUVs) are specially interesting due to their similarity in size to eukaryotic cells. We focus on optimization of GUV production from phosphatidylcholine/sphingomyelin/cholesterol mixtures using the electroformation method. This mixture has been extensively researched lately due to its relevance for the formation of lipid rafts. We measured the effect of voltage, frequency, lipid film thickness, and cholesterol (Chol) concentration on electroformation successfulness using spin-coating for reproducible lipid film deposition. Special attention is given to the effect of Chol concentrations above the phospholipid bilayer saturation threshold. Such high concentrations are of interest to groups studying the role of Chol in the fiber cell plasma membranes of the eye lens or development of atherosclerosis. Utilizing atomic force and fluorescence microscopy, we found the optimal lipid film thickness to be around 30 nm, and the best frequency–voltage combinations in the range of 2–6 V and 10–100 Hz. Increasing the Chol content, we observed a decrease in GUV yield and size. However, the effect was much less pronounced when the optimal lipid film thickness was used. The results underline the need for simultaneous optimization of both electrical parameters and thickness in order to produce high-quality GUVs for experimental research.

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Yandrapalli, Naresh, Tina Seemann e Tom Robinson. "On-Chip Inverted Emulsion Method for Fast Giant Vesicle Production, Handling, and Analysis". Micromachines 11, n. 3 (10 marzo 2020): 285. http://dx.doi.org/10.3390/mi11030285.

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Liposomes and giant unilamellar vesicles (GUVs) in particular are excellent compartments for constructing artificial cells. Traditionally, their use requires bench-top vesicle growth, followed by experimentation under a microscope. Such steps are time-consuming and can lead to loss of vesicles when they are transferred to an observation chamber. To overcome these issues, we present an integrated microfluidic chip which combines GUV formation, trapping, and multiple separate experiments in the same device. First, we optimized the buffer conditions to maximize both the yield and the subsequent trapping of the vesicles in micro-posts. Captured GUVs were monodisperse with specific size of 18 ± 4 µm in diameter. Next, we introduce a two-layer design with integrated valves which allows fast solution exchange in less than 20 s and on separate sub-populations of the trapped vesicles. We demonstrate that multiple experiments can be performed in a single chip with both membrane transport and permeabilization assays. In conclusion, we have developed a versatile all-in-one microfluidic chip with capabilities to produce and perform multiple experiments on a single batch of vesicles using low sample volumes. We expect this device will be highly advantageous for bottom-up synthetic biology where rapid encapsulation and visualization is required for enzymatic reactions.

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9

Billah, Md Masum, Samiron Kumar Saha, Md Mamun Or Rashid, Farzana Hossain e Masahito Yamazaki. "Effect of osmotic pressure on pore formation in lipid bilayers by the antimicrobial peptide magainin 2". Physical Chemistry Chemical Physics 24, n. 11 (2022): 6716–31. http://dx.doi.org/10.1039/d1cp05764b.

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Osmotic pressure (Π) induces membrane tension σ in giant unilamellar vesicles (GUVs), which elevates rate constant kp for antimicrobial peptide magainin 2-induced pore formation in GUVs. The process of its pore evolution in GUVs with σ was revealed.

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Jennings, Christopher S., Jeremy S. Rossman, Braeden A. Hourihan, Ross J. Marshall, Ross S. Forgan e Barry A. Blight. "Immobilising giant unilamellar vesicles with zirconium metal–organic framework anchors". Soft Matter 17, n. 8 (2021): 2024–27. http://dx.doi.org/10.1039/d0sm02188a.

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A straightforward procedure for immobilising giant unilamellar vesicles (GUVs) using zircomium metal-organic frameworks as the anchroing medium is presented. Using this method GUVs can be immoblised and visualised for hours.

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11

Wang, Xinmao, Yangruizi Zhang, Maobin Xie, Zhibiao Wang e Hai Qiao. "Temperature-Promoted Giant Unilamellar Vesicle (GUV) Aggregation: A Way of Multicellular Formation". Current Issues in Molecular Biology 45, n. 5 (26 aprile 2023): 3757–71. http://dx.doi.org/10.3390/cimb45050242.

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The evolution of unicellular to multicellular life is considered to be an important step in the origin of life, and it is crucial to study the influence of environmental factors on this process through cell models in the laboratory. In this paper, we used giant unilamellar vesicles (GUVs) as a cell model to investigate the relationship between environmental temperature changes and the evolution of unicellular to multicellular life. The zeta potential of GUVs and the conformation of the headgroup of phospholipid molecules at different temperatures were examined using phase analysis light scattering (PALS) and attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), respectively. In addition, the effect of increasing temperature on the aggregation of GUVs was further investigated in ionic solutions, and the possible mechanisms involved were explored. The results showed that increasing temperature reduced the repulsive forces between cells models and promoted their aggregation. This study could effectively contribute to our understanding of the evolution of primitive unicellular to multicellular life.

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Wiemann, Jared T., Zhiqiang Shen, Huilin Ye, Ying Li e Yan Yu. "Membrane poration, wrinkling, and compression: deformations of lipid vesicles induced by amphiphilic Janus nanoparticles". Nanoscale 12, n. 39 (2020): 20326–36. http://dx.doi.org/10.1039/d0nr05355d.

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13

Lefrançois, Pauline, Bertrand Goudeau e Stéphane Arbault. "Electroformation of phospholipid giant unilamellar vesicles in physiological phosphate buffer". Integrative Biology 10, n. 7 (2018): 429–34. http://dx.doi.org/10.1039/c8ib00074c.

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Albanese, Paola, Simone Cataldini, Chloe Z. J. Ren, Nadia Valletti, Jlenia Brunetti, Jack L. Y. Chen e Federico Rossi. "Light-Switchable Membrane Permeability in Giant Unilamellar Vesicles". Pharmaceutics 14, n. 12 (12 dicembre 2022): 2777. http://dx.doi.org/10.3390/pharmaceutics14122777.

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In this work, giant unilamellar vesicles (GUVs) were synthesized by blending the natural phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with a photoswitchable amphiphile (1) that undergoes photoisomerization upon irradiation with UV-A (E to Z) and blue (Z to E) light. The mixed vesicles showed marked changes in behavior in response to UV light, including changes in morphology and the opening of pores. The fine control of membrane permeability with consequent cargo release could be attained by modulating either the UV irradiation intensity or the membrane composition. As a proof of concept, the photocontrolled release of sucrose from mixed GUVs is demonstrated using microscopy (phase contrast) and confocal studies. The permeability of the GUVs to sucrose could be increased to ~4 × 10–2 μm/s when the system was illuminated by UV light. With respect to previously reported systems (entirely composed of synthetic amphiphiles), our findings demonstrate the potential of photosensitive GUVs that are mainly composed of natural lipids to be used in medical and biomedical applications, such as targeted drug delivery and localized topical treatments.

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Karal, Mohammad Abu Sayem, Nadia Akter Mokta, Victor Levadny, Marina Belaya, Marzuk Ahmed, Md Kabir Ahamed e Shareef Ahammed. "Effects of cholesterol on the size distribution and bending modulus of lipid vesicles". PLOS ONE 17, n. 1 (28 gennaio 2022): e0263119. http://dx.doi.org/10.1371/journal.pone.0263119.

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The influence of cholesterol fraction in the membranes of giant unilamellar vesicles (GUVs) on their size distributions and bending moduli has been investigated. The membranes of GUVs were synthesized by a mixture of two elements: electrically neutral lipid 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol and also a mixture of three elements: electrically charged lipid 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), DOPC and cholesterol. The size distributions of GUVs have been presented by a set of histograms. The classical lognormal distribution is well fitted to the histograms, from where the average size of vesicle is obtained. The increase of cholesterol content in the membranes of GUVs increases the average size of vesicles in the population. Using the framework of Helmholtz free energy of the system, the theory developed by us is extended to explain the experimental results. The theory determines the influence of cholesterol on the bending modulus of membranes from the fitting of the proper histograms. The increase of cholesterol in GUVs increases both the average size of vesicles in population and the bending modulus of membranes.

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Souissi, Mariem, Julien Pernier, Olivier Rossier, Gregory Giannone, Christophe Le Clainche, Emmanuèle Helfer e Kheya Sengupta. "Integrin-Functionalised Giant Unilamellar Vesicles via Gel-Assisted Formation: Good Practices and Pitfalls". International Journal of Molecular Sciences 22, n. 12 (13 giugno 2021): 6335. http://dx.doi.org/10.3390/ijms22126335.

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Giant unilamellar vesicles (GUV) are powerful tools to explore physics and biochemistry of the cell membrane in controlled conditions. For example, GUVs were extensively used to probe cell adhesion, but often using non-physiological linkers, due to the difficulty of incorporating transmembrane adhesion proteins into model membranes. Here we describe a new protocol for making GUVs incorporating the transmembrane protein integrin using gel-assisted swelling. We report an optimised protocol, enumerating the pitfalls encountered and precautions to be taken to maintain the robustness of the protocol. We characterise intermediate steps of small proteoliposome formation and the final formed GUVs. We show that the integrin molecules are successfully incorporated and are functional.

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Maktabi, Sepehr, Jeffrey W. Schertzer e Paul R. Chiarot. "Dewetting-induced formation and mechanical properties of synthetic bacterial outer membrane models (GUVs) with controlled inner-leaflet lipid composition". Soft Matter 15, n. 19 (2019): 3938–48. http://dx.doi.org/10.1039/c9sm00223e.

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Carvalho, Denise, Ana Rodrigues, Vera Faustino, Diana Pinho, Elisabete Castanheira e Rui Lima. "Microfluidic Deformability Study of an Innovative Blood Analogue Fluid Based on Giant Unilamellar Vesicles". Journal of Functional Biomaterials 9, n. 4 (4 dicembre 2018): 70. http://dx.doi.org/10.3390/jfb9040070.

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Blood analogues have long been a topic of interest in biofluid mechanics due to the safety and ethical issues involved in the collection and handling of blood samples. Although the current blood analogue fluids can adequately mimic the rheological properties of blood from a macroscopic point of view, at the microscopic level blood analogues need further development and improvement. In this work, an innovative blood analogue containing giant unilamellar vesicles (GUVs) was developed to mimic the flow behavior of red blood cells (RBCs). A natural lipid mixture, soybean lecithin, was used for the GUVs preparation, and three different lipid concentrations were tested (1 × 10−3 M, 2 × 10−3 M and 4 × 10−3 M). GUV solutions were prepared by thin film hydration with a buffer, followed by extrusion. It was found that GUVs present diameters between 5 and 7 µm which are close to the size of human RBCs. Experimental flow studies of three different GUV solutions were performed in a hyperbolic-shaped microchannel in order to measure the GUVs deformability when subjected to a homogeneous extensional flow. The result of the deformation index (DI) of the GUVs was about 0.5, which is in good agreement with the human RBC’s DI. Hence, the GUVs developed in this study are a promising way to mimic the mechanical properties of the RBCs and to further develop particulate blood analogues with flow properties closer to those of real blood.

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Zartner, Luisa, Moritz S. Muthwill, Ionel Adrian Dinu, Cora-Ann Schoenenberger e Cornelia G. Palivan. "The rise of bio-inspired polymer compartments responding to pathology-related signals". Journal of Materials Chemistry B 8, n. 29 (2020): 6252–70. http://dx.doi.org/10.1039/d0tb00475h.

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Self-organized nano- and microscale polymer compartments such as polymersomes, giant unilamellar vesicles (GUVs), polyion complex vesicles (PICsomes) and layer-by-layer (LbL) capsules have increasing potential in many sensing applications.

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Haller, Barbara, Kerstin Göpfrich, Martin Schröter, Jan-Willi Janiesch, Ilia Platzman e Joachim P. Spatz. "Charge-controlled microfluidic formation of lipid-based single- and multicompartment systems". Lab on a Chip 18, n. 17 (2018): 2665–74. http://dx.doi.org/10.1039/c8lc00582f.

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Witt, Hannes, Marian Vache, Andrea Cordes e Andreas Janshoff. "Detachment of giant liposomes – coupling of receptor mobility and membrane shape". Soft Matter 16, n. 27 (2020): 6424–33. http://dx.doi.org/10.1039/d0sm00863j.

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We combine detachment experiments of giant unilamellar vesicles (GUVs) and membrane coated glass beads with theoretical considerations to study the impact of receptor mobility of adhesive glycolipids.

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Bao, Peng, Daniel A. Paterson, Sally A. Peyman, J. Cliff Jones, Jonathan A. T. Sandoe, Helen F. Gleeson, Stephen D. Evans e Richard J. Bushby. "Production of giant unilamellar vesicles and encapsulation of lyotropic nematic liquid crystals". Soft Matter 17, n. 8 (2021): 2234–41. http://dx.doi.org/10.1039/d0sm01684e.

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We describe a modified microfluidic method for making Giant Unilamellar Vesicles (GUVs) via water/octanol-lipid/water double emulsion droplets and encapsulation of nematic lyotropic liquid crystals (LNLCs).

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McDonough, Jake, Trevor A. Paratore, Hannah M. Ketelhohn, Bella C. DeCilio, Alonzo H. Ross e Arne Gericke. "Engineering Phosphatidylserine Containing Asymmetric Giant Unilamellar Vesicles". Membranes 14, n. 9 (23 agosto 2024): 181. http://dx.doi.org/10.3390/membranes14090181.

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The plasma membrane lipid distribution is asymmetric, with several anionic lipid species located in its inner leaflet. Among these, phosphatidylserine (PS) plays a crucial role in various important physiological functions. Over the last decade several methods have been developed that allow for the fabrication of large or giant unilamellar vesicles (GUVs) with an asymmetric lipid composition. Investigating the physicochemical properties of PS in such asymmetric lipid bilayers and studying its interactions with proteins necessitates the reliable fabrication of asymmetric GUVs (aGUVs) with a high degree of asymmetry that exhibit PS in the outer leaflet so that the interaction with peptides and proteins can be studied. Despite progress, achieving aGUVs with well-defined PS asymmetry remains challenging. Recently, a Ca2+-initiated hemifusion method has been introduced, utilizing the fusion of symmetric GUVs (sGUVs) with a supported lipid bilayer (SLB) for the fabrication of aGUVs. We extend this approach to create aGUVs with PS in the outer bilayer leaflet. Comparing the degree of asymmetry between aGUVs obtained via Ca2+ or Mg2+ initiated hemifusion of a phosphatidylcholine (PC) sGUVwith a PC/PS-supported lipid bilayer, we observe for both bivalent cations a significant number of aGUVs with near-complete asymmetry. The degree of asymmetry distribution is narrower for physiological salt conditions than at lower ionic strengths. While Ca2+ clusters PS in the SLB, macroscopic domain formation is absent in the presence of Mg2+. However, the clustering of PS upon the addition of Ca2+ is apparently too slow to have a negative effect on the quality of the obtained aGUVs. We introduce a data filtering method to select aGUVs that are best suited for further investigation.

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Mardešić, Ivan, Zvonimir Boban e Marija Raguz. "Electroformation of Giant Unilamellar Vesicles from Damp Films in Conditions Involving High Cholesterol Contents, Charged Lipids, and Saline Solutions". Membranes 14, n. 10 (12 ottobre 2024): 215. http://dx.doi.org/10.3390/membranes14100215.

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Abstract (sommario):

Giant unilamellar vesicles (GUVs) are frequently used as membrane models in studies of membrane properties. They are most often produced using the electroformation method. However, there are a number of parameters that can influence the success of the procedure. Some of the most common conditions that have been shown to have a negative effect on GUV electroformation are the presence of high cholesterol (Chol) concentrations, the use of mixtures containing charged lipids, and the solutions with an elevated ionic strength. High Chol concentrations are problematic for the traditional electroformation protocol as it involves the formation of a dry lipid film by complete evaporation of the organic solvent from the lipid mixture. During drying, anhydrous Chol crystals form. They are not involved in the formation of the lipid bilayer, resulting in a lower Chol concentration in the vesicle bilayer compared to the original lipid mixture. Motivated primarily by the issue of artifactual Chol demixing, we have modified the electroformation protocol by incorporating the techniques of rapid solvent exchange (RSE), ultrasonication, plasma cleaning, and spin-coating for reproducible production of GUVs from damp lipid films. Aside from decreasing Chol demixing, we have shown that the method can also be used to produce GUVs from lipid mixtures with charged lipids and in ionic solutions used as internal solutions. A high yield of GUVs was obtained for Chol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) samples with mixing ratios ranging from 0 to 2.5. We also succeeded in preparing GUVs from mixtures containing up to 60 mol% of the charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and in NaCl solutions with low ionic strength (<25 mM).

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Wang, Xuejing, Liangfei Tian, Hang Du, Mei Li, Wei Mu, Bruce W. Drinkwater, Xiaojun Han e Stephen Mann. "Chemical communication in spatially organized protocell colonies and protocell/living cell micro-arrays". Chemical Science 10, n. 41 (2019): 9446–53. http://dx.doi.org/10.1039/c9sc04522h.

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Abstract (sommario):

Arrays of giant unilamellar vesicles (GUVs) with controllable geometries and occupancies are prepared by acoustic trapping and used to implement chemical signaling in protocell colonies and protocell/living cell consortia.

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Karamdad, K., R. V. Law, J. M. Seddon, N. J. Brooks e O. Ces. "Studying the effects of asymmetry on the bending rigidity of lipid membranes formed by microfluidics". Chemical Communications 52, n. 30 (2016): 5277–80. http://dx.doi.org/10.1039/c5cc10307j.

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In this article we detail a robust high-throughput microfluidic platform capable of fabricating either symmetric or asymmetric giant unilamellar vesicles (GUVs) and characterise the mechanical properties of their membranes.

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Sarmento, M. J., S. N. Pinto, A. Coutinho, M. Prieto e F. Fernandes. "Accurate quantification of inter-domain partition coefficients in GUVs exhibiting lipid phase coexistence". RSC Advances 6, n. 71 (2016): 66641–49. http://dx.doi.org/10.1039/c6ra13170k.

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Giant unilamellar vesicles (GUVs) with phase coexistence allow for the recovery of inter-domain partition coefficients (K_p) of fluorescent molecules through comparison of fluorescence intensities in each phase.

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Boban, Zvonimir, Ivan Mardešić, Witold Karol Subczynski e Marija Raguz. "Giant Unilamellar Vesicle Electroformation: What to Use, What to Avoid, and How to Quantify the Results". Membranes 11, n. 11 (7 novembre 2021): 860. http://dx.doi.org/10.3390/membranes11110860.

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Abstract (sommario):

Since its inception more than thirty years ago, electroformation has become the most commonly used method for growing giant unilamellar vesicles (GUVs). Although the method seems quite straightforward at first, researchers must consider the interplay of a large number of parameters, different lipid compositions, and internal solutions in order to avoid artifactual results or reproducibility problems. These issues motivated us to write a short review of the most recent methodological developments and possible pitfalls. Additionally, since traditional manual analysis can lead to biased results, we have included a discussion on methods for automatic analysis of GUVs. Finally, we discuss possible improvements in the preparation of GUVs containing high cholesterol contents in order to avoid the formation of artifactual cholesterol crystals. We intend this review to be a reference for those trying to decide what parameters to use as well as an overview providing insight into problems not yet addressed or solved.

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Rideau, Emeline, Frederik R. Wurm e Katharina Landfester. "Giant polymersomes from non-assisted film hydration of phosphate-based block copolymers". Polymer Chemistry 9, n. 44 (2018): 5385–94. http://dx.doi.org/10.1039/c8py00992a.

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Polybutadiene-block-poly(ethyl ethylene phosphate) can reproducibly self-assemble in large number into giant unilamellar vesicles (GUVs) by non-assisted film hydration, representing a stepping stone for better liposomes – substitutes towards the generation of artificial cells.

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Sych, Taras, Thomas Schubert, Romain Vauchelles, Josef Madl, Ramin Omidvar, Roland Thuenauer, Ludovic Richert, Yves Mély e Winfried Römer. "GUV-AP: multifunctional FIJI-based tool for quantitative image analysis of Giant Unilamellar Vesicles". Bioinformatics 35, n. 13 (26 novembre 2018): 2340–42. http://dx.doi.org/10.1093/bioinformatics/bty962.

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Abstract Motivation Giant Unilamellar Vesicles (GUVs) are widely used synthetic membrane systems that mimic native membranes and cellular processes. Various fluorescence imaging techniques can be employed for their characterization. In order to guarantee a fast and unbiased analysis of imaging data, the development of automated recognition and processing steps is required. Results We developed a fast and versatile Fiji-based macro for the analysis of digital microscopy images of GUVs. This macro was designed to investigate membrane dye incorporation and protein binding to membranes. Moreover, we propose a fluorescence intensity-based method to quantitatively assess protein binding. Availability and implementation The ImageJ distribution package FIJI is freely available online: https://imagej.net/Fiji. The macro file GUV-AP.ijm is available at https://github.com/AG-Roemer/GUV-AP. Supplementary information Supplementary data are available at Bioinformatics online.

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Bhuiyan, Md Tariqul Islam, Mohammad Abu Sayem Karal, Urbi Shyamolima Orchi, Nazia Ahmed, Md Moniruzzaman, Md Kabir Ahamed e Md Masum Billah. "Probability and kinetics of rupture and electrofusion in giant unilamellar vesicles under various frequencies of direct current pulses". PLOS ONE 19, n. 6 (10 giugno 2024): e0304345. http://dx.doi.org/10.1371/journal.pone.0304345.

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Irreversible electroporation induces permanent permeabilization of lipid membranes of vesicles, resulting in vesicle rupture upon the application of a pulsed electric field. Electrofusion is a phenomenon wherein neighboring vesicles can be induced to fuse by exposing them to a pulsed electric field. We focus how the frequency of direct current (DC) pulses of electric field impacts rupture and electrofusion in cell-sized giant unilamellar vesicles (GUVs) prepared in a physiological buffer. The average time, probability, and kinetics of rupture and electrofusion in GUVs have been explored at frequency 500, 800, 1050, and 1250 Hz. The average time of rupture of many ‘single GUVs’ decreases with the increase in frequency, whereas electrofusion shows the opposite trend. At 500 Hz, the rupture probability stands at 0.45 ± 0.02, while the electrofusion probability is 0.71 ± 0.01. However, at 1250 Hz, the rupture probability increases to 0.69 ± 0.03, whereas the electrofusion probability decreases to 0.46 ± 0.03. Furthermore, when considering kinetics, at 500 Hz, the rate constant of rupture is (0.8 ± 0.1)×10−2 s-1, and the rate constant of fusion is (2.4 ± 0.1)×10−2 s-1. In contrast, at 1250 Hz, the rate constant of rupture is (2.3 ± 0.8)×10−2 s-1, and the rate constant of electrofusion is (1.0 ± 0.1)×10−2 s-1. These results are discussed by considering the electrical model of the lipid bilayer and the energy barrier of a prepore.

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Zhang, Yiting, Haruto Obuchi e Taro Toyota. "A Practical Guide to Preparation and Applications of Giant Unilamellar Vesicles Formed via Centrifugation of Water-in-Oil Emulsion Droplets". Membranes 13, n. 4 (18 aprile 2023): 440. http://dx.doi.org/10.3390/membranes13040440.

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Abstract (sommario):

Giant vesicles (GVs), which are closed lipid bilayer membranes with a diameter of more than 1 μm, have attracted attention not only as model cell membranes but also for the construction of artificial cells. For encapsulating water-soluble materials and/or water-dispersible particles or functionalizing membrane proteins and/or other synthesized amphiphiles, giant unilamellar vesicles (GUVs) have been applied in various fields, such as supramolecular chemistry, soft matter physics, life sciences, and bioengineering. In this review, we focus on a preparation technique for GUVs that encapsulate water-soluble materials and/or water-dispersible particles. It is based on the centrifugation of a water-in-oil emulsion layered on water and does not require special equipment other than a centrifuge, which makes it the first choice for laboratory use. Furthermore, we review recent studies on GUV-based artificial cells prepared using this technique and discuss their future applications.

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Bartelt, S. M., E. Chervyachkova, J. Steinkühler, J. Ricken, R. Wieneke, R. Tampé, R. Dimova e S. V. Wegner. "Dynamic blue light-switchable protein patterns on giant unilamellar vesicles". Chemical Communications 54, n. 8 (2018): 948–51. http://dx.doi.org/10.1039/c7cc08758f.

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Hasan, Sharif, Mohammad Abu Sayem Karal, Salma Akter, Marzuk Ahmed, Md Kabir Ahamed e Shareef Ahammed. "Influence of sugar concentration on the vesicle compactness, deformation and membrane poration induced by anionic nanoparticles". PLOS ONE 17, n. 9 (29 settembre 2022): e0275478. http://dx.doi.org/10.1371/journal.pone.0275478.

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Sugar plays a vital role in the structural and functional characteristics of cells. Hence, the interaction of NPs with cell membranes in the presence of sugar concentrations is important for medicinal and pharmacological innovations. This study integrated three tools: giant unilamellar vesicles (GUVs), anionic magnetite nanoparticles (NPs), and sugar concentrations, to understand a simplified mechanism for interactions between the vesicle membranes and NPs under various sugar concentrations. We focused on changing the sugar concentration in aqueous solution; more precisely, sucrose inside the GUVs and glucose outside with equal osmolarity. 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were used to prepare the charged membranes of 40mole%DOPG/60mole%DOPC-GUVs, whereas only DOPC was used to prepare the neutral membranes. Phase contrast fluorescence microscopy shows that the adherence of 18 nm magnetite NPs with anionic charge depends on the sugar concentration. The alterations of GUVs induced by the NPs are characterized in terms of i) vesicle compactness, ii) deformation, and iii) membrane poration. The presence of sugar provides additional structural stability to the GUVs and reduces the effects of the NPs with respect to these parameters; more precisely, the higher the sugar concentration, the smaller the alteration induced by the NPs. The differences in NPs effects are explained by the change in the type of interaction between sugar molecules and lipid membranes, namely enthalpy and entropy-driven interaction, respectively. In addition, such alterations are influenced by the surface charge density of the lipid bilayer. The surface pressure of membranes due to the adsorption of NPs is responsible for inducing the poration in membranes. The differences in deformation and poration in charged and neutral GUVs under various sugar concentrations are discussed based on the structure of the head of lipid molecules.

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Garten, Matthias, Lars D. Mosgaard, Thomas Bornschlögl, Stéphane Dieudonné, Patricia Bassereau e Gilman E. S. Toombes. "Whole-GUV patch-clamping". Proceedings of the National Academy of Sciences 114, n. 2 (21 dicembre 2016): 328–33. http://dx.doi.org/10.1073/pnas.1609142114.

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Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiologically high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiology, but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the “whole-GUV” patch-clamp configuration were identified and resolved. First, unless the patch pipette and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipette. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipette surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compositions. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiological range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane composition, tension, and shape.

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Wadud, Md Abdul, Mohammad Abu Sayem Karal, Md Moniruzzaman e Md Mamun Or Rashid. "Effects of membrane potentials on the electroporation of giant unilamellar vesicles". PLOS ONE 18, n. 9 (12 settembre 2023): e0291496. http://dx.doi.org/10.1371/journal.pone.0291496.

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Living organisms maintain a resting membrane potential, which plays an important role in various biophysical and biological processes. In the context of medical applications, irreversible electroporation (IRE) is a non-thermal and minimally invasive technique that utilizes precisely controlled electric field pulses of micro- to millisecond durations to effectively ablate cancer and tumor cells. Previous studies on IRE-induced rupture of cell-mimetic giant unilamellar vesicles (GUVs) have primarily been conducted in the absence of membrane potentials. In this study, we investigated the electroporation of GUVs, including parameters such as the rate constant of rupture and the probability of rupture, in the presence of various negative membrane potentials. The membranes of GUVs were prepared using lipids and channel forming proteins. As the membrane potential increased from 0 to −90 mV, the rate constant of rupture showed a significant increase from (7.5 ± 1.6)×10−3 to (35.6 ± 5.5)×10−3 s-1. The corresponding probability of rupture also exhibited a notable increase from 0.40 ± 0.05 to 0.68 ± 0.05. To estimate the pore edge tension, the electric tension-dependent logarithm of the rate constant was fitted with the Arrhenius equation for different membrane potentials. The presence of membrane potential did not lead to any significant changes in the pore edge tension. The increase in electroporation is reasonably explained by the decrease in the prepore free energy barrier. The choice of buffer used in GUVs can significantly influence the kinetics of electroporation. This study provides valuable insights that can contribute to the application of electroporation techniques in the biomedical field.

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Basu, Amrita, Pabitra Maity, Prasanta Karmakar e Sanat Karmakar. "Preparation of Giant Unilamellar Vesicles and Solid Supported Bilayer from Large Unilamellar Vesicles: Model Biological Membranes". Journal of Surface Science and Technology 32, n. 3-4 (5 aprile 2017): 85. http://dx.doi.org/10.18311/jsst/2016/7753.

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Abstract (sommario):

Giant Unilamellar Vesicles (GUV) and supported planar membranes are excellent model biological systems for studying the structure and functions of membranes. We have prepared GUV from Large Unilamellar Vesicles (LUV) using electroformation and Supported planar Lipid Bilayer (SLB) by vesicle fusion method. LUV was prepared using an extrusion method and was characterized using Dynamic Light Scattering (DLS) and zeta potential measurements. The techniques for obtaining GUV as well as SLB from LUV have been demonstrated. We have directly observed the formation of GUV under phase contrast microscopy. This study will provide some insights into the physico-chemical properties of both nano and micron size vesicles. We believe that this method could be extremely useful for reconstituting various bio-molecules in GUV. We have presented one example where an antimicrobial peptide NK-2 was reconstituted in GUV prepared from LUV. SLB formation was monitored and characterized using Atomic Force Microscopy (AFM).

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Abucayon, Erwin G., Mangala Rao, Gary R. Matyas e Carl R. Alving. "QS21-Initiated Fusion of Liposomal Small Unilamellar Vesicles to Form ALFQ Results in Concentration of Most of the Monophosphoryl Lipid A, QS21, and Cholesterol in Giant Unilamellar Vesicles". Pharmaceutics 15, n. 9 (26 agosto 2023): 2212. http://dx.doi.org/10.3390/pharmaceutics15092212.

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Army Liposome Formulation with QS21 (ALFQ), a vaccine adjuvant preparation, comprises liposomes containing saturated phospholipids, with 55 mol% cholesterol relative to the phospholipids, and two adjuvants, monophosphoryl lipid A (MPLA) and QS21 saponin. A unique feature of ALFQ is the formation of giant unilamellar vesicles (GUVs) having diameters >1.0 µm, due to a remarkable fusion event initiated during the addition of QS21 to nanoliposomes containing MPLA and 55 mol% cholesterol relative to the total phospholipids. This results in a polydisperse size distribution of ALFQ particles, with diameters ranging from ~50 nm to ~30,000 nm. The purpose of this work was to gain insights into the unique fusion reaction of nanovesicles leading to GUVs induced by QS21. This fusion reaction was probed by comparing the lipid compositions and structures of vesicles purified from ALFQ, which were >1 µm (i.e., GUVs) and the smaller vesicles with diameter <1 µm. Here, we demonstrate that after differential centrifugation, cholesterol, MPLA, and QS21 in the liposomal phospholipid bilayers were present mainly in GUVs (in the pellet). Presumably, this occurred by rapid lateral diffusion during the transition from nanosize to microsize particles. While liposomal phospholipid recoveries by weight in the pellet and supernatant were 44% and 36%, respectively, higher percentages by weight of the cholesterol (~88%), MPLA (94%), and QS21 (96%) were recovered in the pellet containing GUVs, and ≤10% of these individual liposomal constituents were recovered in the supernatant. Despite the polydispersity of ALFQ, most of the cholesterol, and almost all of the adjuvant molecules, were present in the GUVs. We hypothesize that the binding of QS21 to cholesterol caused new structural nanodomains, and subsequent interleaflet coupling in the lipid bilayer might have initiated the fusion process, leading to creation of GUVs. However, the polar regions of MPLA and QS21 together have a “sugar lawn” of ten sugars, the hydrophilicity of which might have provided a driving force for rapid lateral diffusion and concentration of the MPLA and QS21 in the GUVs.

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Oka, M., T. Tanaka, S. Furuike e M. Yamazaki. "Physical Properties of GUVs containing PEG-lipids". Seibutsu Butsuri 39, supplement (1999): S97. http://dx.doi.org/10.2142/biophys.39.s97_1.

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Sarkar, Malay Kumar, Mohammad Abu Sayem Karal, Marzuk Ahmed, Md Kabir Ahamed, Shareef Ahammed, Sabrina Sharmin e Sayed Ul Alam Shibly. "Effects of osmotic pressure on the irreversible electroporation in giant lipid vesicles". PLOS ONE 16, n. 5 (14 maggio 2021): e0251690. http://dx.doi.org/10.1371/journal.pone.0251690.

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Abstract (sommario):

Irreversible electroporation (IRE) is a nonthermal tumor/cell ablation technique in which a series of high-voltage short pulses are used. As a new approach, we aimed to investigate the rupture of giant unilamellar vesicles (GUVs) using the IRE technique under different osmotic pressures (Π), and estimated the membrane tension due to Π. Two categories of GUVs were used in this study. One was prepared with a mixture of dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylcholine (DOPC) and cholesterol (chol) for obtaining more biological relevance while other with a mixture of DOPG and DOPC, with specific molar ratios. We determined the rate constant (kp) of rupture of DOPG/DOPC/chol (46/39/15)-GUVs and DOPG/DOPC (40/60)-GUVs induced by constant electric tension (σc) under different Π. The σc dependent kp values were fitted with a theoretical equation, and the corresponding membrane tension (σoseq) at swelling equilibrium under Π was estimated. The estimated membrane tension agreed well with the theoretical calculation within the experimental error. Interestingly, the values of σoseq were almost same for both types of synthesized GUVs under same osmotic pressure. We also examined the sucrose leakage, due to large osmotic pressure-induced pore formation, from the inside of DOPG/DOPC/chol(46/39/15)-GUVs. The estimated membrane tension due to large Π at which sucrose leaked out was very similar to the electric tension at which GUVs were ruptured without Π. We explained the σc and Π induced pore formation in the lipid membranes of GUVs.

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Mushenheim, Peter C., Joel S. Pendery, Douglas B. Weibel, Saverio E. Spagnolie e Nicholas L. Abbott. "Straining soft colloids in aqueous nematic liquid crystals". Proceedings of the National Academy of Sciences 113, n. 20 (2 maggio 2016): 5564–69. http://dx.doi.org/10.1073/pnas.1600836113.

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Liquid crystals (LCs), because of their long-range molecular ordering, are anisotropic, elastic fluids. Herein, we report that elastic stresses imparted by nematic LCs can dynamically shape soft colloids and tune their physical properties. Specifically, we use giant unilamellar vesicles (GUVs) as soft colloids and explore the interplay of mechanical strain when the GUVs are confined within aqueous chromonic LC phases. Accompanying thermal quenching from isotropic to LC phases, we observe the elasticity of the LC phases to transform initially spherical GUVs (diameters of 2–50 µm) into two distinct populations of GUVs with spindle-like shapes and aspect ratios as large as 10. Large GUVs are strained to a small extent (R/r < 1.54, where R and r are the major and minor radii, respectively), consistent with an LC elasticity-induced expansion of lipid membrane surface area of up to 3% and conservation of the internal GUV volume. Small GUVs, in contrast, form highly elongated spindles (1.54 < R/r < 10) that arise from an efflux of LCs from the GUVs during the shape transformation, consistent with LC-induced straining of the membrane leading to transient membrane pore formation. A thermodynamic analysis of both populations of GUVs reveals that the final shapes adopted by these soft colloids are dominated by a competition between the LC elasticity and an energy (∼0.01 mN/m) associated with the GUV–LC interface. Overall, these results provide insight into the coupling of strain in soft materials and suggest previously unidentified designs of LC-based responsive and reconfigurable materials.

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Sessa, Lucia, Simona Concilio, Peter Walde, Tom Robinson, Petra S. Dittrich, Amalia Porta, Barbara Panunzi, Ugo Caruso e Stefano Piotto. "Study of the Interaction of a Novel Semi-Synthetic Peptide with Model Lipid Membranes". Membranes 10, n. 10 (19 ottobre 2020): 294. http://dx.doi.org/10.3390/membranes10100294.

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Abstract (sommario):

Most linear peptides directly interact with membranes, but the mechanisms of interaction are far from being completely understood. Here, we present an investigation of the membrane interactions of a designed peptide containing a non-natural, synthetic amino acid. We selected a nonapeptide that is reported to interact with phospholipid membranes, ALYLAIRKR, abbreviated as ALY. We designed a modified peptide (azoALY) by substituting the tyrosine residue of ALY with an antimicrobial azobenzene-bearing amino acid. Both of the peptides were examined for their ability to interact with model membranes, assessing the penetration of phospholipid monolayers, and leakage across the bilayer of large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs). The latter was performed in a microfluidic device in order to study the kinetics of leakage of entrapped calcein from the vesicles at the single vesicle level. Both types of vesicles were prepared from a 9:1 (mol/mol) mixture of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1′-rac-glycerol). Calcein leakage from the vesicles was more pronounced at a low concentration in the case of azoALY than for ALY. Increased vesicle membrane disturbance in the presence of azoALY was also evident from an enzymatic assay with LUVs and entrapped horseradish peroxidase. Molecular dynamics simulations of ALY and azoALY in an anionic POPC/POPG model bilayer showed that ALY peptide only interacts with the lipid head groups. In contrast, azoALY penetrates the hydrophobic core of the bilayers causing a stronger membrane perturbation as compared to ALY, in qualitative agreement with the experimental results from the leakage assays.

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Mora, Nestor Lopez, Yue Gao, M. Gertrude Gutierrez, Justin Peruzzi, Ivan Bakker, Ruud J. R. W. Peters, Bianka Siewert et al. "Evaluation of dextran(ethylene glycol) hydrogel films for giant unilamellar lipid vesicle production and their application for the encapsulation of polymersomes". Soft Matter 13, n. 33 (2017): 5580–88. http://dx.doi.org/10.1039/c7sm00551b.

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Bąk, Krzysztof M., Bartjan van Kolck, Krystyna Maslowska-Jarzyna, Panagiota Papadopoulou, Alexander Kros e Michał J. Chmielewski. "Oxyanion transport across lipid bilayers: direct measurements in large and giant unilamellar vesicles". Chemical Communications 56, n. 36 (2020): 4910–13. http://dx.doi.org/10.1039/c9cc09888g.

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Ikeda, Atsushi, Tomohiro Hida, Tatsuya Iizuka, Manami Tsukamoto, Jun-ichi Kikuchi e Kazuma Yasuhara. "Dynamic behaviour of giant unilamellar vesicles induced by the uptake of [70]fullerene". Chem. Commun. 50, n. 11 (2014): 1288–91. http://dx.doi.org/10.1039/c3cc47711h.

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Karal, Mohammad Abu Sayem, Tawfika Nasrin, Marzuk Ahmed, Md Kabir Ahamed, Shareef Ahammed, Salma Akter, Sharif Hasan e Zaid Bin Mahbub. "A new purification technique to obtain specific size distribution of giant lipid vesicles using dual filtration". PLOS ONE 16, n. 7 (29 luglio 2021): e0254930. http://dx.doi.org/10.1371/journal.pone.0254930.

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Abstract (sommario):

A new purification technique is developed for obtaining distribution of giant unilamellar vesicles (GUVs) within a specific range of sizes using dual filtration. The GUVs were prepared using well known natural swelling method. For filtration, different combinations of polycarbonate membranes were implemented in filter holders. In our experiment, the combinations of membranes were selected with corresponding pore sizes–(i) 12 and 10 μm, (ii) 12 and 8 μm, and (iii) 10 and 8 μm. By these filtration arrangements, obtained GUVs size distribution were in the ranges of 6−26 μm, 5–38 μm and 5–30 μm, respectively. In comparison, the size distribution range was much higher for single filtration technique, for example, 6−59 μm GUVs found for a membrane with 12 μm pores. Using this technique, the water-soluble fluorescent probe, calcein, can be removed from the suspension of GUVs successfully. The size distributions were analyzed with lognormal distribution. The skewness became smaller (narrow size distribution) when a dual filtration was used instead of single filtration. The mode of the size distribution obtained in dual filtration was also smaller to that of single filtration. By continuing this process of purification for a second time, the GUVs size distribution became even narrower. After using an extra filtration with dual filtration, two different size distributions of GUVs were obtained at a time. This experimental observation suggests that different size specific distributions of GUVs can be obtained easily, even if GUVs are prepared by different other methods.

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Sachdev, Shaurya, Aswin Muralidharan, Dipendra K. Choudhary, Dayinta L. Perrier, Lea Rems, Michiel T. Kreutzer e Pouyan E. Boukany. "DNA translocation to giant unilamellar vesicles during electroporation is independent of DNA size". Soft Matter 15, n. 45 (2019): 9187–94. http://dx.doi.org/10.1039/c9sm01274e.

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Abstract (sommario):

DNA delivery into GUVs during electroporation is governed by bulk electrophoretic mobility implying a mechanism in which DNA molecules enter in their coiled conformation, as opposed to stochastic threading, through electro-pores.

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Olety, Balaji, Sarah L. Veatch e Akira Ono. "Phosphatidylinositol-(4,5)-Bisphosphate Acyl Chains Differentiate Membrane Binding of HIV-1 Gag from That of the Phospholipase Cδ1 Pleckstrin Homology Domain". Journal of Virology 89, n. 15 (20 maggio 2015): 7861–73. http://dx.doi.org/10.1128/jvi.00794-15.

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Abstract (sommario):

ABSTRACTHIV-1 Gag, which drives virion assembly, interacts with a plasma membrane (PM)-specific phosphoinositide, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. While cellular acidic phospholipid-binding proteins/domains, such as the PI(4,5)P2-specific pleckstrin homology domain of phospholipase Cδ1 (PHPLCδ1), mediate headgroup-specific interactions with corresponding phospholipids, the exact nature of the Gag-PI(4,5)P2interaction remains undetermined. In this study, we used giant unilamellar vesicles (GUVs) to examine how PI(4,5)P2with unsaturated or saturated acyl chains affect membrane binding of PHPLCδ1and Gag. Both unsaturated dioleoyl-PI(4,5)P2[DO-PI(4,5)P2] and saturated dipalmitoyl-PI(4,5)P2[DP-PI(4,5)P2] successfully recruited PHPLCδ1to membranes of single-phase GUVs. In contrast, DO-PI(4,5)P2but not DP-PI(4,5)P2recruited Gag to GUVs, indicating that PI(4,5)P2acyl chains contribute to stable membrane binding of Gag. GUVs containing PI(4,5)P2, cholesterol, and dipalmitoyl phosphatidylserine separated into two coexisting phases: one was a liquid phase, and the other appeared to be a phosphatidylserine-enriched gel phase. In these vesicles, the liquid phase recruited PHPLCδ1regardless of PI(4,5)P2acyl chains. Likewise, Gag bound to the liquid phase when PI(4,5)P2had DO-acyl chains. DP-PI(4,5)P2-containing GUVs showed no detectable Gag binding to the liquid phase. Unexpectedly, however, DP-PI(4,5)P2still promoted recruitment of Gag, but not PHPLCδ1, to the dipalmitoyl-phosphatidylserine-enriched gel phase of these GUVs. Altogether, these results revealed different roles for PI(4,5)P2acyl chains in membrane binding of two PI(4,5)P2-binding proteins, Gag and PHPLCδ1. Notably, we observed that nonmyristylated Gag retains the preference for PI(4,5)P2containing an unsaturated acyl chain over DP-PI(4,5)P2, suggesting that Gag sensitivity to PI(4,5)P2acyl chain saturation is determined directly by the matrix-PI(4,5)P2interaction, rather than indirectly by a myristate-dependent mechanism.IMPORTANCEBinding of HIV-1 Gag to the plasma membrane is promoted by its interaction with a plasma membrane-localized phospholipid, PI(4,5)P2. Many cellular proteins are also recruited to the plasma membrane via PI(4,5)P2-interacting domains represented by PHPLCδ1. However, differences and/or similarities between these host proteins and viral Gag protein in the nature of their PI(4,5)P2interactions, especially in the context of membrane binding, remain to be determined. Using a novel giant unilamellar vesicle-based system, we found that PI(4,5)P2with an unsaturated acyl chain recruited PHPLCδ1and Gag similarly, whereas PI(4,5)P2with saturated acyl chains either recruited PHPLCδ1but not Gag or sorted these proteins to different phases of vesicles. To our knowledge, this is the first study to show that PI(4,5)P2acyl chains differentially modulate membrane binding of PI(4,5)P2-binding proteins. Since Gag membrane binding is essential for progeny virion production, the PI(4,5)P2acyl chain property may serve as a potential target for anti-HIV therapeutic strategies.

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Faizi, Hammad A., Rumiana Dimova e Petia M. Vlahovska. "Transient Electrodeformation of Giant Unilamellar Vesicles (GUVS) to Probe Membrane Viscosity". Biophysical Journal 118, n. 3 (febbraio 2020): 322a. http://dx.doi.org/10.1016/j.bpj.2019.11.1808.

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Vallejo, D., S. H. Lee, D. Lee, C. Zhang, C. Rapier, S. D. Chessler e A. P. Lee. "Cell-sized lipid vesicles for cell-cell synaptic therapies". TECHNOLOGY 05, n. 04 (dicembre 2017): 201–13. http://dx.doi.org/10.1142/s233954781750011x.

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Abstract (sommario):

Cell-sized lipid vesicles (CLVs) have shown great promise for therapeutic and artificial cell applications, but their fragility and short shelf life has hindered widespread adoption and commercial viability. We present a method to circumvent the storage limitations of CLVs such as giant unilamellar vesicles (GUVs) and single-compartment multisomes (SCMs) by storing them in a double emulsion precursor form. The double emulsions can be stored for at least 8 months and readily converted into either GUVs or SCMs at any time. In this study, we investigate the interfacial parameters responsible for this morphological change, and we also demonstrate the therapeutic potential of CLVs by utilizing them to present a transmembrane protein, neuroligin-2, to pancreatic [Formula: see text]-cells, forming cell-cell synapses that stimulate insulin secretion and cellular growth.

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