Evaluation of dextran(ethylene glycol) hydrogel films for giant unilamellar lipid vesicle production and their application for the encapsulation of polymersomes
Abstract:Giant Unilamellar Vesicles (GUVs) prepared from phospholipids are becoming popular membrane model systems for use in biophysical studies. The quality, size and yield of GUVs depend on the preparation method used to obtain them. In this study, hydrogels consisting of dextran polymers crosslinked by poly(ethylene glycol) (DexPEG) were used as hydrophilic frameworks for the preparation of vesicle suspensions under physiological ionic strength conditions. A comparative study was conducted using hydrogels with vari… Show more
“…The corresponding intensities of the donors are shown in panel C. Representative images of GUVs are shown on panel D. The emission of the donors was recorded at 515 nm. (2020) 10:3087 | https://doi.org/10.1038/s41598-020-59926-z www.nature.com/scientificreports www.nature.com/scientificreports/ DexPEG substrates allows for the growth of GUVs under physiological conditions in good yields 59 . The lipid film was deposited on DexPEG substrates and hydrated at room temperature with phosphate buffer solution (PBS, pH = 7.4), containing CaCl 2 (1 mM), MgCl 2 (0.5 mM) and sucrose (200 mM).…”
We have employed a model system, inspired by SnARe proteins, to facilitate membrane fusion between Giant Unilamellar Vesicles (GUVs) and Large Unilamellar Vesicles (LUVs) under physiological conditions. in this system, two synthetic lipopeptide constructs comprising the coiled-coil heterodimerforming peptides K 4 , (KiAALKe) 4 , or e 4 , (eiAALeK) 4 , a peG spacer of variable length, and a cholesterol moiety to anchor the peptides into the liposome membrane replace the natural SnARe proteins. GUVs are functionalized with one of the lipopeptide constructs and the fusion process is triggered by adding LUVs bearing the complementary lipopeptide. Dual-colour time lapse fluorescence microscopy was used to visualize lipid-and content-mixing. Using conventional confocal microscopy, lipid mixing was observed on the lipid bilayer of individual GUVs. in addition to lipid-mixing, content-mixing assays showed a low efficiency due to clustering of K 4-functionalized LUVs on the GUVs target membranes. We showed that, through the use of the non-ionic surfactant Tween 20, content-mixing between GUVs and LUVs could be improved, meaning this system has the potential to be employed for drug delivery in biological systems.
“…The corresponding intensities of the donors are shown in panel C. Representative images of GUVs are shown on panel D. The emission of the donors was recorded at 515 nm. (2020) 10:3087 | https://doi.org/10.1038/s41598-020-59926-z www.nature.com/scientificreports www.nature.com/scientificreports/ DexPEG substrates allows for the growth of GUVs under physiological conditions in good yields 59 . The lipid film was deposited on DexPEG substrates and hydrated at room temperature with phosphate buffer solution (PBS, pH = 7.4), containing CaCl 2 (1 mM), MgCl 2 (0.5 mM) and sucrose (200 mM).…”
We have employed a model system, inspired by SnARe proteins, to facilitate membrane fusion between Giant Unilamellar Vesicles (GUVs) and Large Unilamellar Vesicles (LUVs) under physiological conditions. in this system, two synthetic lipopeptide constructs comprising the coiled-coil heterodimerforming peptides K 4 , (KiAALKe) 4 , or e 4 , (eiAALeK) 4 , a peG spacer of variable length, and a cholesterol moiety to anchor the peptides into the liposome membrane replace the natural SnARe proteins. GUVs are functionalized with one of the lipopeptide constructs and the fusion process is triggered by adding LUVs bearing the complementary lipopeptide. Dual-colour time lapse fluorescence microscopy was used to visualize lipid-and content-mixing. Using conventional confocal microscopy, lipid mixing was observed on the lipid bilayer of individual GUVs. in addition to lipid-mixing, content-mixing assays showed a low efficiency due to clustering of K 4-functionalized LUVs on the GUVs target membranes. We showed that, through the use of the non-ionic surfactant Tween 20, content-mixing between GUVs and LUVs could be improved, meaning this system has the potential to be employed for drug delivery in biological systems.
“…LacY GUVs were prepared from LacY LUVs in a similar way to the partial dehydration and electroformation method ( 16), but the electroswelling step was replaced by the use of DexPEG hydrogels for GUV generation, which has been proven to be compatible with the use of anionic lipids and physiological ionic strength conditions (35,46) required to preserve biological protein activity in the lipid membrane. For instance, lack of functionality in bacteriorhodopsin was found when the reconstitution was performed with the partial dehydration and electroswelling method just in water (16).…”
Section: Guv Preparation and Lacy Reconstitution In Guvsmentioning
Cellular life relies on membranes, which provide a resilient and adaptive cell boundary. Many essential processes depend upon the ease with which the membrane is able to deform and bend, features that can be characterized by the bending rigidity. Quantitative investigations of such mechanical properties of biological membranes have primarily been undertaken in solely lipid bilayers and frequently in the absence of buffers. In contrast, much less is known about the influence of integral membrane proteins on bending rigidity under physiological conditions. We focus on an exemplar member of the ubiquitous major facilitator superfamily of transporters and assess the influence of lactose permease on the bending rigidity of lipid bilayers. Fluctuation analysis of giant unilamellar vesicles (GUVs) is a useful means to measure bending rigidity. We find that using a hydrogel substrate produces GUVs that are well suited to fluctuation analysis. Moreover, the hydrogel method is amenable to both physiological salt concentrations and anionic lipids, which are important to mimic key aspects of the native lactose permease membrane. Varying the fraction of the anionic lipid in the lipid mixture DOPC/DOPE/DOPG allows us to assess the dependence of membrane bending rigidity on the topology and concentration of an integral membrane protein in the lipid bilayer of GUVs. The bending rigidity gradually increases with the incorporation of lactose permease, but there is no further increase with greater amounts of the protein in the membrane.
“…Products with DS values of 0.10 to 0.26 were obtained as calculated from the 1 H NMR signals of maleimide and glucosidic protons . Moreover, N , N ′‐diisopropylcarbodiimide (DIC) was used to perform the esterification of dextran with N ‐maleoyl β‐alanine in DMSO . Catalytic amounts of 4‐(dimethylamino) pyridinium 4‐toluenesulfonate (DPTS) were added to the reaction mixture ( Table 3 , Figure 6 ).…”
Section: Synthesis Of Polysaccharide Maleimido Derivativesmentioning
confidence: 99%
“…The drop‐casting on thiolated glass slides yielded hydrogel films, which were coated with phospholipid solutions to produce giant unilamellar vesicles (GUVs) upon film rehydration. The study is interesting for membrane model systems in biophysics allowing the encapsulation of biomolecular cargos and particles . The authors believe that GUVs can be used to design cell models providing insights into cellular structure in context with the origin of life.…”
In context with facile and efficient syntheses of functional polymeric materials, the combination of polysaccharides and functional moieties based on renewable resources is a sustainable and valuable approach. This review presents alternatives to prominent click reactions utilizing biopolymer derivatives with furfuryl and maleimide groups. On the one hand, the cross‐linking by Diels–Alder reaction of these polymers enables the synthesis of novel materials in the fields of self‐healing polymers, tissue engineering, and drug delivery. On the other hand, thiol‐ene click reactions allow their conjugation to complex (bio)molecules. Different synthetic strategies are reviewed and the applicability of functional materials is evaluated.
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